Impact of glucose uptake rate on recombinant

protein production in Escherichia coli

Emma Bäcklund

M. Sc.

Royal Institute of Technology

Stockholm 2011

© Emma Bäcklund

Stockholm, 2011

School of Biotechnology

Royal Institute of Technology

SE-106 91 Stockholm

Sweden

Printed at AJ E-print AB

Oxtorgsgatan 9

SE-111 57 Stockholm

Sweden

ISBN 978-91-7415-994-3

ISSN 1654-2312

TRITA-BIO Report 2011:18

©Emma Bäcklund (2011). Impact of glucose uptake rate on recombinant protein production in Escherichia coli. School of Biotechnology, Royal Institute of Technology (KTH), Albanova University Center, Stockholm, Sweden.

ABSTRACT

Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli. E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures. The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation. For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm. The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z. Keywords: AIDA-autotransporter, Escherichia coli, fed-batch, glucose uptake rate, integral membrane proteins, outer membrane proteins, periplasmic retention, phosphotransferase system, recombinant proteins, specific growth rate, surface expression.

LIST OF PUBLICATIONS

The thesis is based on the following papers, referred to in the text by their Roman

numerals:

I. Bäcklund E, Markland K, Larsson G (2008). Cell engineering of Escherichia coli

allows high cell density accumulation without fed-batch process control. Bioprocess

and Biosystems Engineering 31:11-20

II. Bäcklund E, Reeks D, Markland K, Weir N, Bowering L, Larsson G (2008).

Fedbatch design for periplasmic product retention in Escherichia coli. Journal of

Biotechnology 135:358-365

III. Bäcklund E, Ignatuschenko M, Larsson G (2011). Suppressing glucose uptake

and acetic acid production increases membrane protein overexpression in Escherichia

coli. Accepted for publication in Microbial Cell Factories.

IV. Gustavsson M, Bäcklund E, Larsson G (2011). Optimisation of surface

expression using the AIDA autotransporter. Manuscript.

CONTENTS

1  INTRODUCTION ................................................................................................................................... 1 

1.1  ADAPTIVE RESPONSES TO CHANGES IN GROWTH RATE ................................................................ 3 1.2  THE MEMBRANE STRUCTURE OF E. COLI ...................................................................................... 4 1.3  CONTROL OF GLUCOSE UPTAKE RATE .......................................................................................... 6 

1.3.1  Cultivation techniques ......................................................................................................... 6 1.3.2  Glucose uptake .................................................................................................................... 8 1.3.3  Acetic acid formation – a result of high glucose uptake rate ............................................ 10 1.3.4  Reduction of acetate formation ......................................................................................... 11 

1.4  LIMITING FACTORS IN RECOMBINANT PROTEIN PRODUCTION .................................................... 12 1.4.1  Cytoplasmic production .................................................................................................... 14 1.4.2  Periplasmic production ..................................................................................................... 21 1.4.3  Production in the inner membrane .................................................................................... 25 1.4.4  Surface display of proteins in E. coli ................................................................................ 27 1.4.5  Extracellular production ................................................................................................... 33 

2  PRESENT INVESTIGATION ............................................................................................................ 34 2.1  A CELLULAR ALTERNATIVE TO FED-BATCH CULTURES (I, III) ................................................... 35 

2.1.1  Strain evaluation (I) .......................................................................................................... 35 2.1.2  Production of integral membrane proteins by the PTS-mutants (III) ............................... 41 

2.2  IMPACT OF FEED-RATE ON PROCESSES WITH OTHER PRODUCT LOCALISATIONS THAN THE CYTOPLASM (II, IV) ............................................................................................................................. 46 

2.2.1  Leakage of periplasmic products in relation to the glucose uptake rate (II) .................... 46 2.2.2  Optimisation of surface expression using the AIDA autotransporter (IV) ........................ 52 

3  CONCLUDING REMARKS ............................................................................................................... 58 4  ABBREVIATIONS ............................................................................................................................... 62 5  ACKNOWLEDGMENT ...................................................................................................................... 64 6  REFERENCES ...................................................................................................................................... 65 

1

1 INTRODUCTION

Since the first announcement on microbial production of a protein of human origin,

insulin, was made in 1978 by researchers at the company Genentech (Genentech,

press release, 1978), recombinant protein production has become a routine business.

Today, it is a common strategy for production of many high-value proteins used in for

example various medical applications (e.g., vaccines, recombinant factor VIII for

treatment of haemophilia, insulin for treatment of diabetes and tissue-plasminogen

activator against stroke). The principle behind this technology is relatively simple: a

foreign gene, encoding a target protein of interest, can be introduced into a host cell

that will use its own cellular machinery to translate the gene into the desired protein

product. However, this important breakthrough would never have been realised

without the pioneering work made in the early seventies on how to isolate and amplify

genes (or DNA) and then insert them into specific genetic locations to create

transgenic organisms (Cohen, et al., 1973; Lobban and Kaiser, 1973; Morrow, et al.,

1974), hence forming the ground for what we today know as recombinant DNA

technology. Although Escherichia coli (E. coli) was used for expression of insulin in

this first example, the use of host cells is not restricted to bacteria such as E. coli but

many other prokaryotic (e.g., Bacillus) and eukaryotic (yeast, insect and mammalian)

cells can be used as well. Regardless of the specific host cell type used, the

productivity is influenced by environmental conditions (e.g., temperature, pH,

availability of oxygen and nutrients) as well as by genetic factors (e.g., the

gene/protein itself, the promoter strength, mRNA stability, codon usage, gene copy

numbers, availability of co-factors and various helper systems that aid in the

expression). Thus, in the end, process optimisation boils down to finding the

appropriate combination of environmental conditions and genetic factors that will

result in the highest amount of active protein.

The focus when producing recombinant proteins in the pharmaceutical industry today

is generally either on i) large-scale production of particular commercial protein

products or on ii) small scale high-throughput production (HTP) of proteins that are

used either for structural and functional studies or for high-throughput screening

(HTS) against potential drug leads in the drug development process. E. coli, with its

ability to grow rapidly to high cell densities on inexpensive substrates, its well-

2

characterised genetics and the availability of numerous cloning vectors and mutant

host strains, is an attractive host for production of recombinant proteins (Schmidt,

2004).

The goal for the large-scale industrial production of a protein is to achieve a high total

productivity in order to produce a large amount of the protein in a cost efficient

manner. Operator supervised bioreactors, which offer a high level of control and

regulation of the process are used and parameters like temperature, pH and dissolved

oxygen tension (DOT) are measured and regulated. The fed-batch technique is usually

applied, where a glucose feed is continuously added to the bioreactor during the

cultivation, making it possible to control the glucose uptake rate of the cells. The fed-

batch technique enables the establishment of high cell densities, since growth rate and

by-product formation are controlled.

The goal for high-throughput production of proteins is to achieve “sufficient”

amounts of soluble proteins for functional or structural studies. This production

usually relies on unsupervised batch cultivation in small scale, most commonly micro

titre plates or shake flasks. The cells grow exponentially until limitations related to

e.g., oxygen availability or by-product formation arises. The level of control of the

environmental conditions is low in this type of small scale shaken systems and the

conditions changes rapidly as a consequence of the exponential biomass increase. For

practical reasons, the fed-batch technology is not applicable for control of the glucose

uptake rate in such systems, and there consequently is a need for other innovative

strategies for controlling the glucose uptake rate; especially as the glucose uptake rate

has been shown to have an impact on product-associated parameters such as specific

productivity, solubility and proteolysis of the recombinant protein in earlier studies

(Boström, et al., 2005; Ryan, et al., 1996; Sandén, et al., 2005; Sandén, et al., 2002).

In general, recombinant proteins are preferably produced in active, soluble forms. One

problem with production of recombinant proteins in E. coli is that the overexpressed

proteins are not always properly folded and then associate into mainly non-active and

insoluble aggregates, which are termed inclusion bodies. Proteins in inclusion bodies

may regain activity after a refolding process. Refolding is, however, a complicated

3

process, and the process has to be optimised for each protein in question (Hauke, et

al., 1998) and is therefore not an applicable strategy in HTP applications. However,

some proteins fold properly if they are secreted to the periplasm. Other “difficult-to-

express” proteins such as membrane proteins and toxic proteins are also preferably

transported to other parts of the cell than the cytoplasm. In order to understand the

impact of the glucose uptake rate on these kinds of processes where the product is

localised to other parts of the cell than the cytoplasm, further investigations are

needed.

1.1 Adaptive responses to changes in growth rate

The growth rate of cells varies depending on the growth conditions, i.e. the growth

rate is influenced by factors such as substrate concentration, growth medium,

temperature, pH and the supply of oxygen. In general, fast growing cells contain more

DNA, RNA, ribosomes, proteins, phospholipids and cell wall material, and tend to

increase in size (Lengeler and Postma, 1999). Cells respond to carbon or amino acid

limitation by inhibited RNA and protein synthesis. Also the DNA replication as well

as the biosynthesis of carbohydrates, phospholipids and cell wall constituents is

inhibited and the cell size decreases. This set of responses, with a tight coupling

between growth rate, ribosomal synthesis and cell size is referred to as the stringent

response and is mediated by the production of the alarmone guanosine tetraphosphate

(ppGpp). E. coli uses two different pathways to produce ppGpp. The lack of amino

acids results in the binding of uncharged tRNA to the ribosome, which activates the

RelA enzyme leading to the formation of ppGpp. The lack of carbon, on the other

hand, leads to the activation of the alternative pathway for ppGpp formation involving

SpoT (Lengeler and Postma, 1999). The ppGpp bind to the RNA polymerase core

enzyme, which affects the expression of a plethora of genes. In general, genes

involved in cell proliferation and growth are negatively regulated by ppGpp, whereas

genes involved in maintenance and stress defence are positively regulated

(Magnusson, et al., 2005). The starvation sigma subunit, σS, accumulates in the cell

whenever the growth rate is lowered and not only when the growth ceases. The

synthesis of σS is positively controlled by ppGpp (Booth, 1999).

4

1.2 The membrane structure of E. coli

The lipid structure of the membranes depends on the glucose uptake rate (Shokri, et

al., 2002). However, before discussing this dependency, an overview of the basic

structures of the membranes will be given. The basic unit of a membrane is a bilayer

that is formed by phospholipids organized in two layers with their polar head groups

along the two surfaces and the acyl chains forming the nonpolar domain between.

Membranes are dynamic with movement both across and in the plane of the bilayer.

The bilayer serves as matrix and support for many proteins that are involved in

transmembrane processes, including translocation of proteins and other molecules

across membranes (Dowhan, 1997). “The fluid mosaic model” (Singer and Nicolson,

1972) has been used for many years for describing the nature of the membranes. The

membranes proteins are in this model more or less viewed as icebergs floating in a sea

of lipids. However, in the last years, this view has been shifting and the importance of

transient, specialized regions called membrane rafts, which are enriched in special

lipids or proteins has become clear (Luckey, 2008).

Figure 1. Model of Escherichia coli cell envelope. The cell has a two-membrane structure composed of the cytoplasmic and the outer membrane. The periplasm, the space between theses membranes, contains the cell wall made of peptidoglycans.

Outer Membrane

Inner Membrane

Periplasm

Pore

LPS

Lipoprotein

Peptidoglycan

Phospholipid

Protein

5

The cell envelope of gram-negative bacteria, to which E. coli belongs, is a two-

membrane structure of cytoplasmic and outer membrane (Fig 1). The space between

the membranes is the periplasm where a thin cell wall consisting of peptidoglycan is

situated. The cell wall gives shape and rigidity to the cell, and prevents the cell from

lysing in dilute environments. The outer membrane contains lipopolysaccharide

(LPS), a structure that is not found in the cytoplasmic membrane.

Escherichia coli membranes contain three major phospholipids:

phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). PE,

which is the major phospholipid, constitutes roughly 75 % of the total phospholipid

content, PG 18% and CL 5 % (Cronan and O.Rock, 1996). The phospholipid

composition of the cytoplasmic and outer membrane is similar, but with a slight

enrichment of PE in the outer membrane (Nikaido, 1996). PE is zwitterionic and does

not carry a net charge at physiological pH, while PG and CL are anionic.

Phospholipids contain both saturated and unsaturated fatty acids as well as cyclic fatty

acids, which are formed by methylation of unsaturated ones. E. coli adjusts the fatty

acid composition of its phospholipids in response to growth temperature in order to

preserve a more or less constant degree of membrane fluidity. The proportion of

unsaturated fatty acids increases as the temperature decreases and vice versa for the

saturated ones. This change results in more or less constant fluidity of the membranes

since the melting point of lipids decreases as the proportion of unsaturated fatty acids

increases (Neidhardt, et al., 1990). The total amount of phospholipids does not vary

with the growth rate but the composition of the phospholipids does (Cronan and

O.Rock, 1996). PG reaches a maximum at a specific growth rate of 0.3 h-1 (Shokri, et

al., 2002) and this growth rate is associated with a high permeability of the

membrane. The membrane becomes more rigid as the growth rate declines (Shokri, et

al., 2003).

6

1.3 Control of glucose uptake rate

1.3.1 Cultivation techniques

Batch

In a batch process all substrate components are available at high enough

concentrations to make the reaction rate unrestricted with respect to substrate

concentration (Fig 2). The biomass concentration increases exponentially, i.e. the

specific growth rate (μ) is constant at μmax, until some factor e.g., by-product

concentration, oxygen supply or low substrate concentration reduces the specific

growth rate.

Oxygen limitation and the formation of the by-product acetic acid limit the usefulness

of the batch technique. The batch technique is however the easiest cultivation method

and it is used when proteins are produced either for functional and structural studies

or for HTS. Further, screening for new production methods in the industrial process

development is usually performed in batch mode.

Fed-batch

The fed-batch technology is a common industrial method for recombinant protein

production. One substrate component, usually glucose, is added to the bioreactor at a

rate so that its concentration is growth rate limiting and thus the growth rate can be

controlled via the feed (Fig 2). The condition for growth rate limitation in a fed-batch

process is:

Figure 2. The difference between batch and fed-batch cultivation. F= feed rate of limiting substrate (l h-1), Si=concentration of the substrate (g l-1)

!"#$%#

&'()*# !+,-.'()*#

7

F/V(t)*Si < qs,max X(t)

Where F (l h-1) is the feed rate, V (l) the cultivation volume, Si (g l-1) the concentration

of the substrate in the feed solution, qs,max (g g-1 h-1) the maximal specific consumption

rate of the limiting substrate and X (g l-1) the cell mass at that time point (Enfors and

Häggström, 2000). There are two main reasons to apply the fed-batch technique. First,

the substrate limitation offers a tool for reaction rate control to avoid engineering

limitations with respect to cooling and oxygen transfer. Secondly, the substrate

limitation also permits a sort of metabolic control by which overflow metabolism,

resulting in formation of acetic acid, can be avoided (Enfors and Häggström, 2000).

The fed-batch technique makes is possible to obtain high cell densities and thus to

reach a high total productivity.

The feed profile can be designed in different ways. A constant feed results in a

continuously decreasing growth rate of the cells since the amount of substrate per cell

decreases as a function of time. An exponential feed results in a constant growth rate

of the cells, since each cell receives the same amount of substrate during the whole

cultivation. By using exponential feed-profiles, exponential growth can be

maintained, but at a lower rate than the maximal rate (μmax). A steady state with

respect to substrate concentration and growth rate is established in exponential fed-

batch cultures.

Fed-batch cultivations may be started as batch cultures and the feed of substrate is

then started when the initial substrate is depleted. In the industry, however, it is

common to start the feed of substrate directly after the inoculation of the reactor.

Usually, an exponential feed is used until the oxygen limitation of the reactor is

reached. To further increase the biomass, a constant feed is then applied, which results

in gradually reduced specific growth rates of the cells.

8

1.3.2 Glucose uptake

Diffusion of carbohydrates through the outer membrane occurs mainly through the

outer membrane channel forming proteins OmpC, OmpF and LamB (Nikaido, 1996).

The phosphotransferase system (PTS) transports carbohydrates such as glucose,

fructose and mannose from the periplasm to the cytoplasm. The PTS system is

composed of both soluble proteins and proteins that are integrated in the cytoplasmic

membrane (Fig 3). Enzyme I (EI) and phosphohistidine carrier protein (HPr) are

general PTS proteins, while the enzymes IIs (EIIs) are sugar specific. EIIGlc is specific

for glucose and EIIMan for mannose. At least one of the EII-domains is bound to the

membrane. The glucose EII consists of the parts IIAGlc and IICBGlc where the latter is

membrane bound, while the mannose EII consists of the IIABMan and the membrane

bound parts IICMan and IIDMan (Fig 3). A series of reactions are involved when the

sugar molecule enters the cytoplasm. A phosphate group is transferred from

phosphoenolpyruvate (PEP) to EI and further to HPr. The phosphate group is then

transferred to the EIIA domains and further to the EIIB/EIICB domains. These

domains perform phosphorylation of the incoming sugar molecule (Postma, et al.,

1996).

Figure 3. A model for PTS mediated uptake of carbohydrates. The scheme shows the general enzymes of the PTS: Enzyme I (EI), Phosphohistidine carrier protein (HPr) and two Enzymes II (EII). P indicates phosphorylation of the various enzymes and the sugar molecules. Modified from (Postma et al. 1996).

9

Glucose is transported over the cytoplasmic membrane mainly through the glucose

and mannose specific PTS. GalP and the Mgl-system, proteins that are normally

involved in the transport of galactose, may however also transport glucose into the

cytoplasm. These proteins can be induced and used for glucose transport during

glucose limiting conditions. Glucose that is transported into the cytoplasm by the

Mgl-system or GalP is phosphorylated by glucokinase, encoded by the gene glk

(Gosset, 2005).

Glucose uptake is also controlled by the repressor protein Mlc. The phoshorylated

form of enzyme IICBGlc dominates in the absence of glucose. In this situation Mlc

binds upstream of the ptsG promoter and works as a repressor. Addition of glucose to

the medium results in dephosphorylation of enzyme IICBGlc. Mlc binds to the

dephoshorylated enzyme IICBGlc and is thereby sequestered away from the operator

making transcription of ptsG possible. Mlc is, as described above, thus capable of

binding both DNA and proteins (Plumbridge, 2002).

The PTS system also has an important role in catabolite repression, which results in

inhibition of gene expression and/or protein activity by the presence of a rapidly

metabolisable carbon source in the medium. Glucose is the preferred carbon source

for E. coli and decreased concentration of glucose in the medium results in

accumulation of the phoshorylated form of enzyme IIAGlc and IICBGlc. The

phosphorylated form of enzyme IIAGlc activates adenylate cyclase (AC), the enzyme

that converts ATP to cAMP, resulting in increased levels of cAMP. The cAMP binds

the product of the crp locus, termed the cAMP receptor protein (CRP). The cAMP-

CRP complex causes the induction of catabolite-repressed genes allowing uptake of

other sugars. The uptake of substrates is also regulated by another mechanism. The

unphosphorylated form of IIAGlc dominates when the glucose concentration in the

medium is high. When enzyme IIAGlc is in the unphosphorylated form, it can

inactivate several transport systems for non-PTS carbon sources, by binding to the

transporters. This mechanism is called inducer exclusion (Lengeler and Postma,

1999).

10

1.3.3 Acetic acid formation – a result of high glucose uptake rate

Aerobic growth of E. coli on excess of glucose leads to the excretion of partially

oxidised metabolites, mostly in the form of acetic acid. This phenomenon is called

overflow metabolism and occurs if the glucose concentration exceeds a critical value.

This value is approximately 20-30 mg l-1 glucose and corresponds to a specific growth

rate of approximately 0.30 h-1 in minimal medium (Enfors and Häggström, 2000;

Meyer, et al., 1984). The reason for the overflow metabolism is not clear but it may be

a result of an imbalance between the glycolysis and the TCA-cycle or of saturation of

the TCA-cycle or the electron transport chain (Lee, 1996).

The main route for acetate production is from Acetyl-CoA through the enzymes

phosphotransacetylase (Pta) and acetatekinase (Ack) (Fig 4). This route generates

ATP. The other route for acetate production is directly from pyruvate by pyruvate

oxidase B (PoxB), but it plays a minor role (Wolfe, 2005).

Most E. coli strains have the ability to assimilate acetate. This is primarily done by the

enzyme AMP-forming acetyl CoA-synthetase (AMP-ACS) (Wolfe, 2005). This route

is utilized when acetate is the carbon source or when there is a need to reabsorb

acetate that has been excreted as a consequence of overflow metabolism (Wolfe,

2005).

Figure 4. Simplified view of acetate formation in E. coli. Acetate is formed either by the Pta-Ack route or by the PoxB route.

Glucose!

Acetyl-CoA!

Pyruvate!

PEP!

Acetate!Acetyl-P!

ATP!

CO2!poxB!

pta! ack!

CO2!

NADH!

11

Acetate production is undesirable in recombinant protein production since it reduces

bacterial growth even at concentrations as low as 0.5 g l-1 (Nakano, et al., 1997).

Further, acetate formation reduces the yield of biomass and the production of

recombinant proteins has also been shown to be negatively affected (Jensen and

Carlsen, 1990; Turner, et al., 1994).

Acetate production depends on the bacterial strain, and it is known that E. coli B-

strains do not accumulate as much acetic acid as E. coli K12 strains. It has been

suggested that B-strains have a more active glyoxylate shunt than K12-strains, which

results in the direction of the pyruvate into biosynthetic precursors, e.g., succinate and

in the reduction of acetic acid (Phue and Shiloach, 2004; van de Walle and Shiloach,

1998). The lower accumulation of acetic acid in B-strains has also been explained by

differences in the transcription level of the acetate production genes (Phue and

Shiloach, 2004).

1.3.4 Reduction of acetate formation

The most commonly used approach in order to decrease the acetate production is the

fed-batch technique. Another strategy is to use genetic modification. The targets

genes are then i) genes that code for proteins involved in the uptake of glucose or ii)

genes that code for enzymes that are active in pathways resulting in acetic acid.

Using an E. coli strain with a mutation in ptsG, the gene coding for EIIGlc, resulted in

20-40% decrease in the specific acetate yield in comparison to the wild type. Other

consequences of the mutation were an increased biomass, from 13 to 19 g l-1 and an

increase in recombinant protein concentration exceeding 50% (Chou, et al., 1994).

Mlc overproducing strains were constructed by mutations in the promoter region of

the chromosomal mlc gene. This strain showed a reduced acetate accumulation, and as

a consequence of this, the pH of the media was higher in comparison to the wild type.

The effect of the mutation was also observed as an increase in the OD600 from 5 to 8 in

comparison to the wild type (Cho, et al., 2005).

12

Another approach that has been used in order to decrease the accumulation of acetic

acid is to replace the PTS system by the galactose permease. The galP gene is

normally repressed when E. coli grows on glucose. Its transcription is however

increased in PTS mutant strains. In this study, the chromosomal galP promoter was

replaced by the trc promoter, making induction with IPTG (isopropyl β-D-

thiogalactopyranoside) possible. The acetate concentration was decreased from 2,8 to

0,39 g l -1 and the GFP formation was increased four times in comparison to the wild

type (De Anda, et al., 2006).

Mutant strains, which lack the enzymes that form acetic acid, have also been

constructed and the excretion of acetate in these strains has been reduced (Bauer, et

al., 1990). Recently a triple BL21 mutant lacking all known acetate production genes

(ackA-pta-poxB) was constructed. Interestingly, this strain still accumulates acetic

acid, which indicates that there are other alternative acetate production pathways in

the cell (Phue, et al., 2010).

1.4 Limiting factors in recombinant protein production

The maintenance of a plasmid and high-level expression of the target protein

represents a metabolic burden to the cell. Production of a recombinant protein can

trigger stress responses in the cell that often resembles the cellular responses to

environmental stress such as heat shock and stringent response (Sørensen and

Mortensen, 2005). The extent of the stress responses is determined by the rates of

transcription and translation, but stress can also emerge from the specific properties of

the recombinant protein, i.e. misfolding, which results in the degradation of the

recombinant product and in inclusion body formation (Hoffmann and Rinas, 2004).

Production of recombinant proteins can also lead to ribosome destruction (Dong, et

al., 1995) and in severe cases to cell-death (Miroux and Walker, 1996).

Generally, the goal in recombinant protein production is not only to maximise the

amount of recombinant protein but also to achieve a protein with a high quality, i.e. a

protein in an active, soluble and pure form. However, some proteins cannot be

produced as active, soluble entities in the cytoplasm and must therefore be directed to

other compartments of the cell. The advantages and disadvantages associated with

13

expression in each compartment are summarised in table 1. Overexpression in the

outer membrane has the purpose of producing whole cells with recombinant proteins

expressed at the surface, and differs in that context from the production in the other

compartments, where the goal is to produce recombinant proteins for isolation and

purification.

Compartment Advantages Disadvantages

Cytosol (soluble protein) Higher protein yield No S-S bond formation

More complex purification

Many proteases

Cytosol (inclusion

bodies)

High protein yield

Protection from proteases

Reduced toxicity

Inclusion bodies are easy to isolate

Solubilisation and refolding needed

Lower yield

Higher cost

Refolding not always possible

Inner membrane (limited

to production of

membrane proteins)

The product can be purified after

detergent extraction

Low yield

Limited space in the membrane

Accumulation of product may be toxic to

the cell

Periplasm Fewer proteases

Formation of S-S bonds

N-terminus authenticity

Simpler purification if selective release

of product possible

Inclusion bodies may form

Inefficient transport

Outer membrane (limited

to surface expression of

proteins)

On-cell protein characterisation possible

No purification of protein is needed

Increased stability of product

Whole cells can simply be removed

Limited space in the membrane

Transport not always possible

Extracellular Less extensive proteolysis

Easier purification

N-terminus authenticity

Reduced toxicity

Transport not always possible

Diluted product

Table 1. Advantages and disadvantages with production of recombinant proteins in different compartments of the cell.

14

1.4.1 Cytoplasmic production

The goal for recombinant protein production in the cytoplasm is either to obtain

soluble proteins or to direct the product into inclusion bodies. The advantage with the

cytoplasmic production is the high yield of product. The cytoplasm is a reducing

environment, inhibiting the formation of disulphide bridges in the protein, a problem

that might be circumvented by secretion to the periplasm. Other disadvantages

associated with cytoplasmic production is the need for cell disruption, the complex

purification from a mixture of endogenous proteins and the higher amounts of

proteases in comparison to the periplasm. In some cases, cytoplasmic overexpression

of a gene results in the presence of one extra N-terminal methionine in the product

(Adams, 1968; Moerschell, et al., 1990), which may have negative impact on the

stability and solubility of the product (Chaudhiri, et al., 1999). That the product ends

up as inclusion bodies may for proteins that are easy to refold actually be beneficial.

Proteins in inclusion bodies are easy to separate form the cell debris after cell

disruption, they are mostly inactive and therefore not toxic to the cell, and they are

generally protected from degradation by proteases and are relatively pure (Jürgen, et

al., 2010; Makrides, 1996). However, when using this strategy, in vitro refolding is

needed to achieve a correctly folded product. There is however no guarantee that

proteins in inclusion bodies will regain activity or that the refolding will result in a

high yield. Refolding and purification from inclusion bodies is usually more

expensive and time consuming than purification of soluble proteins (Sorensen and

Mortensen, 2005). The renaturation conditions, such as temperature, buffer, pH and

ionic strength must be optimized for every protein (Hauke, et al., 1998) and using the

refolding strategy in HTP applications is therefore not an option.

Folding

The newly synthesized protein either folds independently or needs to be assisted by

molecular chaperones to attain a correct and biologically active structure. The

molecular chaperones are ”proteins that help the folding of other proteins, usually

through cycles of binding and release, without forming part of their final structure”

(Young, et al., 2004). The chaperones favor on-pathway folding by shielding

interactive surfaces from each other as well as from the solvent, and by accelerating

rate-limiting steps. Chaperones are also invaluable in protein secretion and

15

translocation where their function is to prevent folding of the target protein, thereby

keeping them in a translocation-competent state. Chaperones are also involved in the

process of disaggregation of already formed aggregates (Baneyx and Mujacic, 2004).

Figure 5. Chaperone-assisted folding in the cytoplasm. The nascent polypeptide first encounters TF or DnaK/DnaJ as it exits the ribosome. After release, the folding intermediate either reaches its native conformation or is transferred to GroEL/GroES for further folding assistance. Partially folded proteins may also be stabilized by the holding chaperones IbpA/B, Hsp31 and Hsp33 until folding chaperones are available. The disaggregating chaperone ClpB promotes disaggregation of unfolded proteins and cooperates with the folding chaperones to reactive them. 1

The molecular chaperones can be divided into three different groups based on their

function, the folding (e.g., DnaK and GroEL), the holding (e.g., IbpB) and the

disaggregating (e.g., ClpB) chaperones (Fig 5). Foldases constitute another important

group of chaperones and their role is to accelerate rate-limiting steps in the folding 1 Reprinted by permission from Macmillan Publishers Ltd: [Nature Biotechnology], (Francois Baneyx and Mirna Mujacic, 2004, Recombinant protein folding and misfolding in Escherichia coli, Nature Biotechnology, Vol. 22, No 11, p 1399-1408), Copyright (2004)

16

pathways. The peptidyl-prolyl isomerases (PPIases) increases the rate of cis-trans

isomerization of peptide bonds involving proline residues and the thiol disulfide

oxidoreductases, known as the Dsb proteins, catalyse the formation and shuffling of

disulphide bridges in the periplasm (Baneyx and Mujacic, 2004).

The heat shock response in E. coli is regulated by σ32 and it is induced by a large

variety of stress factors including temperature shift, metabolic harmful substances and

protein misfolding and aggregation. Major heat shock proteins are proteases and

molecular chaperones including DnaK/DnaJ/GrpE and GroEL/GroES (Arséne, et al.,

2000).

Proteolysis

Proteolysis and folding are tightly connected processes in the cell. The degradation of

misfolded proteins guarantees that abnormal polypeptides do not accumulate within

the cell and conserves the cellular resources by the recycling of amino acids. The

ATP-dependent cytoplasmic protease Lon and the Clp family of proteases are induced

by heat shock (Gross, 1996).

One strategy that can be used in order to avoid degradation of the recombinant

product is utilizing protease deficient mutants. Using such mutants is however often

associated with reductions of the growth rate and it is not known if the cell

compensates for the loss of one protease by increasing the concentration of another

(Martínez-Alonso, et al., 2010). One exception is however the strain BL21 that is

deficient of both Lon and OmpT but still exhibits good growth characteristics

(Sørensen and Mortensen, 2005). However, in strains devoid of the Lon protease, the

aggregation of over-produced proteins has shown to be enhanced (Corchero, et al.,

1996). For proteins that are easy to refold, the deposition of them into inclusion

bodies can be an alternative superior strategy for avoiding degradation from proteases

(Hauke, et al., 1998).

17

Recovery and degradation of aggregated proteins

Protein aggregates accumulate when the cell’s capacity for folding, unfolding and

degradation is exceeded, which is usually a consequence of stress or of

overexpression of recombinant proteins (Sørensen and Mortensen, 2005). The

molecular chaperones DnaK and GroEL not only mediate proper de novo folding of

proteins but are also involved in degradation of abnormal polypeptides by targeting

them to proteases such as Lon and ClpP (Martínez-Alonso, et al., 2010). The holding

chaperones IbpA and IbpB stabilize partly unfolded proteins without actively

promoting their refolding and are often found tightly associated to the target proteins

in inclusion bodies (Allen, et al., 1992). The disaggregating chaperone ClpB

cooperates with DnaK and GroEL in reversing protein aggregation (Weibezahn, et al.,

2004).

Strategies to improve specific productivity and solubility

The goal in recombinant protein production is to obtain as much as possible of high

quality product. A high total productivity can be achieved either by scaling up the

cultivation volume, by increasing the biomass or by increasing the specific

productivity (the productivity per cell). Promoter strength, inducer concentration, the

plasmid copy number and the specific growth rate of the producing cells are some

factors that have been shown to influence the specific productivity, but also the

solubility of the product (Sandén, et al., 2005; Sandén, et al., 2002; Swartz, 2001;

Tegel, et al., 2011; Terpe, 2006). When aiming at producing soluble proteins, the

strategy is in general to slow down the synthesis rate of the protein. Examples of such

strategies include using weaker promoters (Tegel, et al., 2011), using lower inducer

concentrations (Sandén, et al., 2005) and to lower the specific growth rate of the

producing cells (Sandén, et al., 2005). However, the opposite strategy, to use high

synthesis rates during the production, increases the specific productivity (Ryan, et al.,

1996; Sandén, et al., 2002). To find the best production conditions in order to obtain a

large amount of soluble protein therefore relies on finding the correct balance between

a high specific productivity and a high solubility.

18

The strength of a promoter is determined by the relative frequency of transcription

initiation. This is affected by the affinity of the promoter sequence for the RNA-

polymerase but also by the transcription rate of the RNA-polymerase. The T7

promoter, which is a very common promoter, requires host strains coding for the T7

RNA polymerase (Terpe, 2006). The E. coli BL21(DE3) strain and its derivatives,

contain a lambda lysogen DE3 with the T7 RNA polymerase under the control of the

IPTG inducible lacUV5 promoter (Terpe, 2006). The T7RNA polymerase transcribes

RNA five times faster than the E. coli RNA-polymerase (Golomb and Chamberlin,

1974). In many cases the use of the T7 leads to high product accumulation, in some

cases as high as 40-50% of the total cell protein (Studier and Moffatt, 1986).

Although the high product accumulation is desirable, the use of strong promoters such

as T7, has its drawbacks. High-level overexpression of genes can cause ribosome

destruction (Dong, et al., 1995) and cell death (Miroux and Walker, 1996) and the use

of strong promoter systems often results in the inability of the cell to fold the target

protein properly (Baneyx, 1999). Further, production of mRNA is energy demanding

and increases the metabolic burden on the cell.

Tegel and co-workers (2010) studied the effect of promoter strength on

overexpression of proteins during batch cultivation in shake flasks. The total

production as well as the soluble fraction of protein epitope signature tags (PrESTs),

short regions of human proteins, was measured. The expression from the T7, the

lacUV5 and the trc promoter was compared. In general, production under the control

of the T7 promoter resulted in the largest total amount of target protein, whereas the

use of the lacUV5 promoter, the weakest promoter in the study, resulted in the lowest

amount of total protein. The weakest promoter generated the largest fraction of

soluble protein and vice versa, and generally, the fraction of soluble protein was small

using both T7 and trc. However, the amount of soluble protein was highest using T7

even though the soluble fraction was the lowest. This is explained by the much higher

total production using this promoter (Tegel, et al., 2011).

19

The production of recombinant β-galactosidase was studied with respect to the

specific growth rate at the time-point of induction (Sandén, et al., 2002). Induction

was performed at specific growth rates of 0.5 h-1 and 0.1 h-1, respectively. It was

shown that induction at the higher growth rate resulted in an almost 100 % higher

specific productivity. The amount of mRNA formed was the same at both occasions

and was therefore not the limiting factor. The ribosomal content, represented by the

rRNA amount, was five times lower at the low specific growth rate. At the high

specific growth rate, degradation of the ribosomes after induction, as a consequence

of the high product formation rate, was also observed. In the study, the conclusion

was drawn that translation, and not transcription, was the limiting factor in the protein

synthesis capacity. Ryan et al (1996) also showed that cells growing at a high specific

growth rate at the time of induction were more efficient in synthesizing the

recombinant protein. The synthesis rate also remained higher for fast growing cells

(Ryan, et al., 1996).

The concentration of IPTG and the feed rate of glucose during fed-batch cultivation

was shown to influence the quality of the recombinant product when production was

controlled from the lacUV5 promoter (Sandén, et al., 2005). The model proteins in

the study were maltose binding protein (MBP) and a mutated form of this protein that

was less stable and more prone to aggregation. Two different exponential feed

profiles were used which resulted in constant specific growth rates of 0.2 h-1 (low feed

rate) and 0.5 h-1 (high feed rate), respectively. Both the soluble and the insoluble

fractions of the proteins increased with the inducer concentration. Using the highest

concentration of inducer did not result in more soluble protein but in increased

formation of inclusion bodies. The lowest amount of soluble protein was achieved by

a high feed rate with the mutated, unstable form of the protein, in combination with a

high inducer concentration. A high feed rate in combination with a high inducer

concentration leads to a high syntheses rate of the protein. In this situation, the folding

machinery of the cell might get overloaded and is unable to stabilise and fold the

protein properly, which leads to increased proteolysis and aggregation.

Another factor that influences the efficiency of translation is the translation initiation

region (TIR) of the mRNA. The main elements of translation initiation in E. coli are

20

the Shine-Dalgarno sequence, the initiation codon that in E. coli is mostly AUG, and a

downstream region (DR). The SD is located 5-9 bases upstream of the initiation

codon and is important for the binding of the mRNA to 30S ribosomal subunit. The

sequence of the SD, in comparison to consensus sequence in E. coli, and its distance

to the initiation codon are factors that determines the translational efficiency. The

down stream region (DR) located immediately after the initiation codon also

influences the gene expression level (Stenström and Isaksson, 2002). Engineering the

translation initiation region of the mRNA is a promoter independent strategy of

influencing gene expression. However, manipulations in the DR will in many cases

change the amino acid sequence of the product (Baneyx, 1999).

A possible strategy for increasing the solubility of the recombinant proteins is by co-

expression of chaperones. There are several chaperones or chaperone sets that have

been selected for over production along with the recombinant target protein. The

beneficial effect of co-expression of chaperones is however unpredictable and is a

matter of trial and error, and there is no guarantee that the overexpression of the

chaperones improves the solubility of the recombinant product (Sorensen and

Mortensen, 2005). Co-expression of the chaperones DnaK and GroEL/ES might

instead trigger proteolytic activities in the cell, which results in reduced yield, stability

and quality of the recombinant protein (Martínez-Alonso, et al., 2010). The dual role

of the chaperones, acting both as folding modulators and as proteolytic enhancers

might partly explain the diverse results reported from co-expression studies

(Martínez-Alonso, et al., 2010). Moreover, overproduction of chaperones contributes

to the metabolic burden of the cell, which might explain the reduced yield of the

recombinant protein.

Examples of other strategies that can be used in order to increase the solubility

include cultivation at low temperature and the use of fusion tags (Sørensen and

Mortensen, 2005).

21

1.4.2 Periplasmic production

The transport to the periplasm

The main protein-translocation machinery in the inner membrane of E. coli is the Sec-

translocase, which transport proteins across or insert proteins in the inner membrane.

The substrates for the Sec-translocase all contain hydrophobic N-terminal regions.

Proteins that are destined for secretion have N-terminal signal sequences that are

processed by signal peptidase that removes the signal sequence after the translocation

and allows release and folding of the protein in the periplasm. Inner membrane

proteins have membrane-anchoring signals in their N-termini that most often remain

associated with the inserted protein. The Sec-translocon can facilitate transport across

or integration of proteins into the membrane in a co-translational or post-translational

manner (Fig 6). The co-translational pathway is mainly employed for inner membrane

proteins, while the secretory proteins mainly utilize the post-translational pathway.

The selection of pathway lies at the early stage of translation when the nascent peptide

emerges from the ribosomal exit tunnel. Very hydrophobic signals that emerge from

the tunnel are bound by the signal recognition particle (SRP), which takes the

complex of the ribosome and the nascent chain to the membrane-associated SRP-

receptor FtsY. Elongation of the chain and insertion of the protein into the membrane

via the translocon occurs simultaneously. The membrane protein YidC (not included

in Fig 6) has also been shown to play a role in the insertion of a subset of membrane

proteins via the translocon. If the signal sequence that emerges from the ribosomal

exit tunnel does not display a high level of hydrophobicity, it is bound by the trigger

factor (TF), which shields the nascent polypeptide from binding SRP. The newly

synthesised protein is maintained in an unfolded state by the cytoplasmic chaperone

SecB, which also targets the protein to the membrane bound ATPase SecA. Protein

translocation across the membrane occurs at the translocon (du Plessis, et al., 2011).

22

Proteins may also be transported through the inner membrane by the twin arginine

translocation (TAT) system. This system transports proteins having specific twin

arginine motifs in their signal peptides and the proteins are transported in a folded

state (Berks, et al., 2005).

Targeting recombinant proteins to the periplasm

Recombinant proteins can be targeted to the periplasm by fusing natural signal

sequences to their N-termini. Periplasmic expression is associated with several

advantages in comparison with cytoplasmic production (Table 1). These include; (i)

Figure 6. Schematic and simplified representation of protein targeting to the Sec-translocase. i) The post-translational pathway: SecB binds to preproteins with less hydrophobic signal sequences and targets the preprotein to SecA which is associated to the translocase SecYEG. The signal sequence is cleaved off at the periplasmic side by the signal peptidase. ii) The co-translational pathway: More hydrophobic signals are bound by the signal recognition particle (SRP) as they exit the ribosomal tunnel. SRP takes the complex of the ribosome and the nascent chain to the membrane-associated SRP-receptor FtsY. Elongation of the chain and insertion of the protein into the membrane via the translocon occurs simultaneously.

23

authentic N-termini of the proteins are obtained after removal of the signal peptide by

the leader peptidase, (ii) possible formation of disulphides in the periplasm due to the

presence of the Dsb machinery (iii) lower amount of proteases in the periplasm

compared to the cytoplasm (iv) simplified purification of the target protein if the

periplasmic proteins are selectively released by osmotic chock or other strategies v)

higher solubility of the product (Baneyx and Mujacic, 2004; Mergulhão, et al., 2005).

One difficulty with secretion is the inefficient export of the proteins across the inner

membrane. This may result in degradation or in inclusion body formation or in the

jamming of the membrane (Baneyx and Mujacic, 2004). Using signal sequences with

increased hydrophobicity may direct the preprotein to SRP-pathway and thus

eliminate toxic effects that might come from membrane jamming since the SRP-

pathway has a tighter coupling between translation and translocation than the SecB-

pathway (Wilson Bowers, et al., 2003).

The export capacity of the Sec-translocase was also studied by Mergulhão and

Monteiro (2004). Different expression levels of two human proinsulin fusion proteins

were accomplished by using different promoters and copy number plasmids. The

periplasmic amount of product was almost the same although a 7 to 11-fold difference

in the total expression level was obtained. The study shows that the translation level

does not affect the maximum translocation efficiency and that a high synthesis rate of

the product is only wasting the resources of the cell (Mergulhão and Monteiro, 2004).

Folding and degradation in the periplasm

The periplasmic chaperones are involved in the folding of the periplasmic proteins

and in the incorporation of proteins in the outer membrane. The Dsb proteins enable

the formation and reshuffling of disulphide bridges. SurA and FkpA are examples of

peptidyl-prolyl isomerases that are present in the periplasm (Moat, et al., 2002). Skp

is and example of a general periplasmic chaperone that binds its substrate in a cavity,

thereby protecting it from degradation (Walton, et al., 2009).

There are fewer proteases in the periplasm than in the cytoplasm. One example of a

periplasmic protease is DegP. DegP function both as a chaperone and as a protease

24

and is essential for the removal of misfolded and aggregated inner membrane and

periplasmic proteins. Synthesis of DegP is controlled by the σE regulon, which is

activated in response to unfolded proteins in the periplasm (Missiakas, et al., 1996).

In order to increase the solubility of the product, the co-expression of chaperones may

be efficient. In a study by Sonoda and co-workers (Sonoda, et al., 2011) this effect

was studied on production of a single-chain Fv antibody secreted to the periplasm. It

was found that the overexpression of the periplasmic chaperones Skp and of FkpA

greatly increased the solubility of the product. However, co-expression of both FkpA

and Skp had no synergistic effect. Further, co-expression of the cytoplasmic

chaperones also affected the binding activity of the antibody fragment in the

periplasm, especially the co-expression of DnaKJE.

Boström and co-workers (Boström, et al., 2005) studied the effect of the feed rate on

recombinant protein secretion and degradation. Secretion of a protein to the periplasm

resulted in less degradation and in the avoidance of the stringent response. It was also

found that accumulation of acetic acid was ten times lower at a high specific growth

rate when the product was secreted to the periplasm.

Leakage to the medium

Periplasmic products show a tendency to leak to the culture medium, which is not

desirable in most periplasmic processes. The tendency of leakage can however be

utilized in processes where the recombinant proteins are targeted to the extracellular

medium. There are a number of hypotheses in the literature concerning the E. coli

cell´s inability to retain periplasmic products/leak periplasmic products to the medium

(Shokri, et al., 2003). A common basis is that the structural integrity of the membrane,

such as protein and lipid composition, is crucial for determining the retention. An

altered composition of the membrane may result from genetic changes or from

environmental changes such as medium composition or the feed rate of glucose

(Shokri, et al., 2002).

25

Some E. coli mutants leak periplasmic proteins to the medium in a large extent. These

mutants generally lack structural elements of the membranes or the cell wall, for

example, LPS (Tamaki, et al., 1971) and murein lipoprotein (Lpp) (Nikaido, et al.,

1977). Lazzaroni et al (1981) showed that mutants with a changed composition of

outer membrane proteins (OMP), in this case a lower amount of OmpF, released more

periplasmic protein to the medium (Lazzaroni and Portalier, 1981).

Secretion of overexpressed proteins to the periplasm also influences leakage. Cells

that were secreting β-lactamase were more sensitive to detergents and they also had a

higher non-specific release of periplasmic proteins to the medium. Analysis of the

outer membrane protein composition showed that the amount of OmpA and OmpC

was lower (40-60%) in secreting cells (Georgiou and Shuler, 1988).

Some signal peptides seem, for unknown reasons, to enhance export of periplasmic

proteins to the medium. This was for example shown for insulin-like growth factor I

that was secreted by the use of the signal peptide from staphylococcal protein A. This

signal peptide has an extension in the N-terminus when compared to other signal

peptides (Abrahmsén, et al., 1986).

1.4.3 Production in the inner membrane

Many membrane proteins can be overexpressed in inclusion bodies in the cytoplasm

of E. coli, but their refolding into actively and functional proteins is difficult (Kiefer,

2003). The production thus relies on the expression in the cytoplasmic membrane

from where the protein can be purified after detergent extraction (Wagner, et al.,

2006). The accumulation of overexpressed proteins in the membrane is often toxic to

the cell, which results in low yield and low biomass formation (Wagner, et al., 2006).

The limited space in the cytoplasmic membrane, where the product can accumulate, is

one potential bottleneck in membrane protein overexpression. The formation of a high

biomass is thus of outermost importance for achieving high product yields (Wagner,

et al., 2008).

26

Membrane proteins are usually targeted via the Sec-pathway to the Sec-translocon

where they are inserted co-translationally into the membrane. Another potential

bottleneck in membrane protein overexpression is the saturation of Sec-translocon

(Essen, 2002). In a study by Wagner and co-workers (Wagner, et al., 2007), it was

shown that membrane overexpression resulted in cytoplasmic aggregates containing

the overexpressed proteins, chaperones and proteases. The aggregates also contained

precursors of periplasmic and outer membrane origin, which indicates jamming of the

Sec-translocon.

Co-expression of chaperones and cultivation at lower temperatures are common

strategies that are used in order to facilitate folding and to reduce the level of

inclusion body formation in membrane protein production. The CorA, the major

magnesium transporter form the bacterial inner membrane, was used as a model

protein (Chen, et al., 2003). In this study it was shown that lowering the cultivation

temperature below 37°C reduced the levels of inclusion bodies formed and that a

higher fraction of the CorA was incorporated in the membrane. The co-expression of

the cytoplasmic chaperones DnaK/DnaJ and GroEL/GroES also increased the

incorporation of CorA in the membrane. A conclusion from this study is that

aggregation, and thus the formation of inclusion bodies, can be prevented by either

reducing the protein synthesis rate or by increasing the cytosolic concentrations of

DnaK/J and GroEL/ES.

The engineering or selection of host strains that are well suited for membrane protein

overexpression is another possible approach for improving membrane protein

overexpression. Two derivatives of BL21(DE3), named C41(DE3) and C43(DE3),

were isolated for their ability to produce eukaryotic membrane proteins at elevated

levels without toxic effects (Miroux and Walker, 1996). Through subsequent genomic

and proteomic studies (Wagner, et al., 2008), it was shown that the original lacUV5

promoter of these strains, which is generally stronger than the original lac variant, had

reverted to the original wild type promoter. It was shown that the lower strength of the

lac promoter led to a lower transcription rate of the T7 RNA polymerase, and that this

had a positive effect on the cellular viability leading to higher total production levels.

27

1.4.4 Surface display of proteins in E. coli

Proteins that are displayed at the surface of the E. coli cell have to be translocated

through the inner membrane and pass the periplasm before they are inserted in the

outer membrane. Before discussing surface display systems in E. coli, a short

description of the outer membrane proteome and the insertion process of proteins in

the outer membrane will therefore be given.

The outer membrane proteome

Murein lipoprotein exists in a large number of copies in each cell and anchors the

peptidoglycan layer to the outer membrane. OmpA, a monomeric protein that spans

the outer membrane, is also important for the stability of the cells since it is cross-

linked to the peptidoglycan layer (Moat, et al., 2002). The outer membrane contains

many porins, trimeric proteins forming transmembrane β-barrel pores in the outer

membrane that allow relatively unspecific passage of small hydrophilic molecules.

OmpF and OmpC are some examples of porins. The porins can represent as much as 2

% of the total protein content in E. coli, making porins some of the most abundant

proteins in terms of mass (Nikaido, 1996).

Maltoporin B (LamB) forms a channel in the outer membrane that specifically allows

passage of maltose and maltodextrins (Nikaido, 1996). LamB also contributes to the

ability of the bacteria to take up other sugars from the surroundings during glucose

limiting conditions (Death, et al., 1993).

OmpT is a protease present in the outer membrane. It cleaves peptides between two

consecutive basic amino acids (Kramer, et al., 2000). OmpT has been associated with

the degradation of many recombinant proteins (Baneyx and Georgiou, 1990). It is

stable under denaturating conditions and hence acts during purification and refolding

processes (White, et al., 1995).

28

Insertion of proteins in the outer membrane

A network of proteins is involved in the folding and insertion process of outer

membrane proteins. The β-barrel assembly machinery (BAM) complex and a number

of periplasmic chaperones, especially SurA, Skp and Deg P, play important roles in

this process (Fig 7). The insertion process is ATP-independent due to the lack of a

periplasmic ATP-pool (Knowles, et al., 2009).

SurA function both as a peptidyl-prolyl isomerase and as general chaperone. The

main role of SurA in OMP maturation is that of a general chaperone (Sklar, et al.,

2007). SurA has a high specificity for outer membrane proteins due to a specific

pattern of aromatic residues that is found frequently amongst this group of proteins.

SurA deficient mutants have a reduced amount of outer membrane proteins

(Hennecke, et al., 2005).

The chaperone Skp has a selectivity for denatured OMPs (Chen and Henning, 1996).

Skp deficient mutants have shown decreased levels of outer membranes proteins, for

example decreased amounts of LamB, OmpA and OmpF. The skp gene is located

downstream of BamA (one of the genes in the BAM complex) and both of these genes

are regulated by the σE stress response that, for example, is activated in response to

unfolded OMPs (Sklar, et al., 2007). Skp mediates transport of OmpA in an unfolded

state across the periplasm (Walton, et al., 2009).

The last periplasmic chaperone that has a large impact on OMP biogenesis is DegP.

This chaperone has both protease and general chaperone activity, and it was suggested

that there are two different pathways for periplasmic chaperone activity in OMP

maturation (Sklar, et al., 2007). SurA is the main chaperone during normal conditions

whereas Skp/DegP have a primary role of rescuing OMPs that have fallen off the

normal assembly pathway. However, under stress, or in the absence of SurA, the

importance of Skp/DegP is increased.

29

Figure 7. A model for the periplasmic chaperone-mediated biogenesis of OMPs in E. coli. The OMPs are transported to the periplasm by the Sec-pathway. Most OMPs are escorted through the periplasm by SurA while off-pathway OMPs are rescued by the DegP/Skp complex. The DegP/Skp complex can deliver OMPs back to SurA, directly to the BAM-complex or use the protease activity of DegP for degradation. Modified from (Sklar et al. 2007).

β-barrel assembly machinery (BAM) complex

The E. coli BAM-complex is composed of five domains; BamA, BamB, BamC, Bam

D and BamE (Fig 7). The names of these proteins have recently changed and the

former names were: BamA (YaeT, also called Omp85), BamB (YfgL), BamC (NlpB),

BamD (YifO) and BamE (SmpA). BamA is an integral membrane protein and the

other four proteins are lipoproteins that are localized to the inner leaflet of the outer

membrane. All of the five BAM-proteins are involved in the OMP biogenesis but

their specific roles in the process are not clear (Knowles, et al., 2009).

Surface display systems in E. coli

The display of recombinant proteins at the surface of the bacteria is useful in many

different biotechnological applications. It may be applied in the field of live-vaccine

development, peptide library screening, whole-cell biocatalysis, biosensor

development and bioadsorbents (Jose and Meyer, 2007). Surface expression of

proteins in bacteria commonly relies on a fusion between the target protein (the

passenger) and a carrier protein that usually is a cell surface protein naturally present

in the host. For examples of surface display systems that have been used in E. coli,

30

see table 2. A successful carrier, (i) has an efficient signal peptide that transports the

fusion protein through the inner membrane, (ii) has a strong anchoring structure so

that the targets stays attached to the surface, (iii) is compatible with the target protein,

meaning that stability of the carrier is not influenced by the target, and (iv) is resistant

against degradation by proteases. The passenger protein itself also affects the

translocation process. The folding structure of the passenger, such as the presence of

disulphide bridges, is one factor that influences the success of the surface display. The

size of the passenger, as well as the presence of certain amino acids in the passenger,

are also known to influence the process (Lee, et al., 2003).

Fusions to OMPs and lipoprotein

The first examples of recombinant surface display in E. coli was reported more than

two decades ago and relies on fusions between outer membrane proteins such as

OmpA, Lam B and OmpC and the passenger proteins. This type of display systems

has been used for display of different peptides but it is not suitable for display of large

proteins as the upper limit seems to lie between 60 and 70 amino acids (Charbit, et al.,

1988).

Table 2. Selected examples of surface display systems in E. coli. Advantages and disadvantages associated with each display system.

Display system Advantages Disadvantages

OMP Limited to transport of small

passengers

Lpp´-OmpA Transport of large passengers

Intermediate number of passenger

copies per cell

Difficult to transport proteins with S-S

bridges

Surface organelles High number of passenger copies per

cell

Limited to transport of small

passengers

Difficult to transport proteins with S-S

bridges

Autotransporters Transport of large passengers

High number of passenger copies per

cell

Dimerisation at the surface

In some cases difficult to transport

proteins with S-S bridges

31

The Lpp’OmpA hybrid display system is based on a fusion between the signal peptide

and the first nine amino acids of the E. coli lipoprotein (Lpp) and three or five

membrane spanning loops from the E. coli outer membrane protein A (OmpA). This

system is not that sensitive to the sizes of the passengers, but appears instead to be

sensitive to the presence of disulphide bridges in the passengers (Stathopoulos, et al.,

1996). Intermediate numbers, approximately 104 recombinant passengers, has been

reported to be expressed on the surface of the cell (Francisco, et al., 1993).

Fimbriae and other polymeric surface organelles

Surface structures like fimbriae and flagella are present at high numbers at the surface

of gram negative bacteria and are therefore attractive for display purposes. A large

variety of fimbriae proteins have been used for surface display of different peptides.

One drawback associated with this system is that there seems to be a restriction with

regard to passenger size and composition. The successfully transported peptides are

not larger than 10-30 amino acids and they are relatively hydrophilic. The formation

of disulphide bridges in the passengers also seems to be a critical factor for transport

(Klemm and Schembri, 2000).

Autotransporters

Pathogenic gram-negative bacteria transport various kinds of virulence factors to the

surface, or to the exterior of the cell, by the autotransporter pathway. The

autotransporter protein family represents a large and highly diverse group of proteins

that can be used as carriers for recombinant proteins. The passenger proteins that are

naturally transported vary in length from 20 to 400 kDa and are also highly variable in

sequence (Dautin and Bernstein, 2007). The autotransporters are synthesised as

precursor proteins with a cleavable signal peptide, an N-terminal passenger domain

and a C-terminal translocator domain composed of a α-helical linker region and a

pore-forming β-barrel part (Fig 8). The signal peptide directs the preprotein to the Sec

translocon and initiates the translocation process of the precursor protein across the

inner membrane. The signal peptide is cleaved off and the β-domain becomes

integrated in the OM. The α-helical linker is thought to form a hairpin structure in the

pore and pull the passenger domain towards the surface (Jose, 2006). The

autotransporters initially got their name because it was thought that they contained

32

everything that was needed for the transport and incorporation into the outer

membrane in their own sequence. Today, there are several co-existing models

describing how the passengers are actually transported to the surface of the cell. The

model described in figure 8 is the hairpin model, which is the “classical” model. One

of the newer models suggests the involvement of the BAM-complex in the

incorporation process (Dautin and Bernstein, 2007).

There are several examples in the literature where different autotransporters have

been used for surface expression of recombinant proteins in E. coli. The AIDA-I

(adhesin involved in diffuse adherence) autotransporter from E. coli and the IgA

protease from Neisseria gonorrhoeae are two of the most frequently applied

autotransporters for this purpose (Jose, et al., 2002; Jose, et al., 2009; Jose and Meyer,

2007; Jose and von Schwichow, 2004; Klauser, et al., 1990; Li, et al., 2008; Maurer,

et al., 1997).

Figure 8. The autotransporter system. A) Suggested mechanism for autotransport. The signal peptide is cleaved off following translocation across the inner membrane through the Sec-translocon. The pore-forming part of the translocation unit forms a pore in the outer membrane and pulls the passenger towards the surface of the cell. B) Structure of an autotransporter vector. The vector includes a signal peptide (SP), a passenger protein and a translocation unit that is composed of a linker region and a pore-forming part.

SP!

Passenger! Translocation unit (TU)!

"-helical linker region!

#-barrel pore!

cytoplasm!

periplasm!

medium!

IM!

OM!

MECHANISM FOR AUTOTRANSPORT!

N! C!

A

B

33

1.4.5 Extracellular production

Recombinant proteins can also be targeted to the medium, which offers the benefits of

low levels of proteolysis, simple detection and purification, a better folding

environment and the avoidance of the N-terminal methionine extension. Extracellular

production of toxic proteins minimizes their impact on the host and may represent the

only possible way for their production (Hannig and Makrides, 1998; Ni and Chen,

2009). The high dilution of the product may however be disadvantageous. One

strategy is to utilize leaky mutants that release periplasmic proteins to the medium.

Another strategy relies on import of secretion mechanisms, either lysis or whole

transport mechanisms, from pathogenic E. coli (Shokri, et al., 2003).

The haemolysin transport system is one candidate for transport of the recombinant

products to the medium. The proteins are translocated directly to the medium in a

protein channel that spans both of the membranes. The channel is composed of

HlyB/HlyD and TolC and the signal peptide of HlyA is fused to the product and

directs it to the protein channel. The proteins that form the channel also need to be co-

expressed which is one disadvantage with the system. However, transport of the

product by the haemolysin system does not compete with transport of endogenous

proteins, which is advantageous (Blight and Holland, 1994).

Periplasmic products can also be released to the medium by import of lysis

mechanisms. This strategy is based upon the action of colicin lysis proteins where the

colicin E1 lysis protein (kil) has been mostly used. The induction of the lysis protein

results in permeabilisation of the outer membrane and the subsequent release of

products (Miksch, et al., 1997).

The autotransporters can also be utilized for extracellular transport of recombinant

proteins since some autotransporters release their passengers to the medium (Jose and

Meyer, 2007).

34

2 PRESENT INVESTIGATION

With the completion of the human genome project (HUGO) during recent years, there

is today a large need for production of proteins, not only in order to determine their

structures and functions, but also as they may constitute proteins of large value for the

biopharmaceutical industry. However, since proteins are very diverse and different

from each other, it is virtually impossible to know how to produce a certain protein in

the most suitable way just by looking at the amino acid sequence. Thus, in order to

increase the probability of finding production conditions appropriate for a particular

protein, there is a need for an increased understanding of production strategies already

available as well as the establishment of new and better production methods.

Proteins that are used for research purposes are preferably produced in an active,

soluble form. Typically, the proteins are produced in small-scale cultivations using

batch technology. Unfortunately, this technology suffers from drawbacks related to

e.g., oxygen limitation, acetic acid production and low biomass, but is in spite of that

a frequently used method since it is so easy to perform. However, cells grow at their

maximal specific growth rate in a batch culture, which in many cases is not optimal

when aiming at producing active soluble proteins, as described earlier.

In the biopharmaceutical industry, the fed-batch technology is commonly used for

control of the glucose uptake rate. The glucose uptake rate, or the feed-profile, has in

the literature been described to influence parameters such as specific productivity,

solubility and proteolysis (Boström, et al., 2005; Ryan, et al., 1996; Sandén, et al.,

2005; Sandén, et al., 2002). Some proteins cannot be produced in an active form in

the cytoplasm and are therefore instead secreted to the periplasm, but this production

strategy is usually associated with low yields. Other “difficult-to-express” proteins,

e.g., proteins that are toxic to the cell, are also preferably transported to parts of the

cell other than the cytoplasm. This will not only provide another possibility for

successful protein production but also simplified purification as the number of

endogenous proteins are significantly less in the periplasm or outside the cell than in

the cytoplasm.

35

A compartment that has been neglected for long from the perspective of large-scale

protein production is the cell surface. Despite this, bacterial surface display systems

has during the last 20 years emerged as an important research tool, e.g., display of

peptide/protein libraries in various protein engineering efforts, display of protein

immunogens for the construction of live vaccines, for biocatalysis and

bioremediation, recently reviewed in (Daugherty, 2007; van Bloois, et al., 2011).

Potentially, bacterial display could be an attractive alternative to more established

large-scale protein production strategies, as it offers a simple purification scheme and

possibilities for on-cell protein characterisation. However, the utility of surface

display in this context (large scale protein production) has to be proven by further

investigations.

As an alternative to traditional fed-batch technique, we here describe an approach for

control of the glucose uptake based on mutant bacterial strains deficient in the

phosphotransferase system (PTS) (Paper I), and its impact on cell density, specific

productivity, acetic acid formation and oxygen consumption (Paper I & III),

Historically, there have been very few reports on the impact of the feed-rate on

processes with product localisations other than the cytoplasm. Thus, we also

investigated how the glucose feed-rate affects recombinant protein production in the

periplasm (Paper II) and when displayed on the bacterial surface (Paper IV).

2.1 A cellular alternative to fed-batch cultures (I, III)

2.1.1 Strain evaluation (I)

The aim of this part of the study was to find a method to control the glucose uptake

rate in small-scale batch cultivations where the traditional fed-batch technique is not

applicable. The strategy was to use strains with mutations in the phosphotransferase

system (PTS) that is involved in the glucose uptake. We reasoned that the mutants

would have lower growth rates due to the mutations and as a result of that also lower

oxygen consumption and decreased formation of acetic acid. Hopefully, this would

result in the establishment of higher cell densities and improved product yield, and the

strains would thus represent a novel, simple alternative to fed-batch cultures.

36

The strain AF1000, that originates from the E. coli K12 strain MC4100 (Casadaban,

1976; Sandén, et al., 2002) was used in this study and is referred to as wild type (WT)

strain. This strain lacks all constituents of the lactose operon and it grows on minimal

salt medium without any additional supplements. The three variants of this strain,

with mutations in genes coding for proteins belonging to the PTS system (Picon, et

al., 2005), that where used are presented in table 3.

The E. coli homologous enzyme β-galactosidase, which hydrolyses lactose into

monosaccharides, was used as a model protein since it is a stable protein and has a

high solubility even at high concentrations. Another advantage with the model protein

is the straightforward analysis for enzyme activity. The lacUV5 promoter, which is

theoretically not subjected to catabolite repression (Arditti, et al., 1973), controlled

the expression of the recombinant protein from a low copy number plasmid

(pACYC). AF1000 and its derivatives are lac negative and production from the

lacUV5 is thus constitutive. A system that could be induced by IPTG addition was

also constructed by insertion of the F´-factor (lacIq, lacY, lacA).

Cell-growth

First, we studied impact of the mutations on the growth rate of the mutant strains. All

strains were therefore grown in batch cultivations with unlimited access to glucose.

The cell density increased exponentially and the resulting growth rates were 0.78 h-1,

0.38 h-1 and 0.25 h-1 for PTSMan, PTSGlc and PTSGlcMan, respectively (Fig 9). This is to

be compared to the wild type, which had a specific growth rate of 0.72 h-1. This

growth rate can however not be considered as deviating from the one obtained for

Table 3. Strains with mutations in PTS. The mutations in the different strains are shown as well as the absent enzymes.

Original name

of strains

Name of the strain

in this work

Mutation in

gene cluster

Absent enzyme

PPA 668 PTSMan manX Enzyme IIABMan

PPA 652 PTSGlc ptsG Enzyme IICBGlc

PPA 689 PTSGlcMan manX and

ptsG

Enzyme IIABMan

Enzyme IICBGlc

37

PTSMan. This result was encouraging as mutations in the PTS system in a previous

publication had been shown to reduce the glucose uptake rate (Picon, et al., 2005).

Acetic acid and specific oxygen consumption rate

The next variable studied was the acetic acid formation. The wild type and PTSMan

produced acetic acid in proportion to the growth rate (Fig 10). The PTSGlc and

PTSGlcMan produced small amounts of acetic acid. The lower production of acetic acid

in PTS-mutants has been observed previously (Chou, et al., 1994).

A further step was to compare the mutants grown in batch with the wild type grown in

fed-batch. Exponential feed rates were therefore designed to theoretically give the

same growth rates for the wild type in fed-batch as the mutants had in batch. The

PTSMan had a specific growth rate approximately equal to that of the wild type,

making this type of comparison irrelevant to perform for this mutant.

The mutants had a specific oxygen consumption rate that was proportional to the

growth rate (data not shown). The oxygen consumption rate for the PTSGlcMan

cultivated in batch was almost identical to the WT cultivated in fed-batch. This

comparison was important to make in order to exclude the possibility that the

Figure 9. Biomass accumulation as a function of cultivation time. Comparison of the wild type cell (WT) and the mutants grown in batch cultivation. Circles:WT, squares: PTSMan, diamonds: PTSGlc, triangles: PTSGlcMan.

0

1

2

3

4

5

6

0 2 4 6 8 10 12 14 16

Cel

l dry

wei

ght

(g l

-1)

! = 0.78

! = 0.72

! = 0.38

! = 0.25

Cultivation time (h)

38

mutations resulted in increased respiration, since this would have had negative

impacts on the establishment of high cell densities.

The acetic acid accumulation was close to zero in the fed-batch cultivations with the

wild type as was it for the mutants in batch (data not shown). Batch cultivated mutants

are thus equal to fed-batch cultivated WT with respect to acetic acid formation and

oxygen consumption.

Product formation

The next parameter to evaluate was the specific product formation rate. The

constitutive system was initially used for production of the model protein β-

galactosidase. Figure 11 shows data from batch cultivations with the wild type and the

mutants. The PTSMan strain had the highest and most stable specific production rate.

The wild type also had a relatively high specific production rate in the beginning of

the cultivation, but it dropped to around 50% at the time of harvest. PTSGlc had an

initial low specific production rate that dropped even more as a function of time. The

PTSGlcMan had an even lower specific productivity (data not shown). By using cells

containing an F´-factor with lacIq, the production could be induced by addition of

0

200

400

600

800

1000

1200

0 2 4 6 8 10 12 14 16

HA

c (m

g L

-1)

Cultivation time (h)

Figure 10. Accumulation of acetic acid as a function of cultivation time. Comparison of the wild type cell (WT) and the mutants grown in batch cultivation. Circles: WT, squares: PTSMan, diamonds: PTSGlc, triangles: PTSGlcMan.

39

IPTG. The production was increased in the PTSGlc (as can be seen in figure 12) but

remained low in PTSGlcMan. The reason to why the production is so low in the double

mutant, PTSGlcMan, is not known. The low production might be a consequence of the

low synthesis rate of proteins in this mutant. It is also possible that the absence of the

two PTS enzymes, which are central for the glucose uptake in the cell, somehow

affects the transcription from the substrate-induced promoter lacUV5.

The last step of this evaluation was to compare the specific production rate of the

PTSGlc cultivated in batch with the WT cultivated in fed-batch (Fig 12). The

production was induced by addition of IPTG when the cells had a specific growth rate

of approximately 0.4 h-1. The specific production rate was approximately 50 U mg-1

h-1 after the induction in both strains. A reduction in the specific productivity was seen

at an earlier time-point for the PTSGlc than for the wild type. The total β-galactosidase

accumulation at the end of the cultivation was estimated to be 33% and 13% of the

total protein, for the wild type and PTSGlc, respectively.

Figure 11. Specific production rate of β-galactosidase during batch cultivation of the WT strain and the mutants as a function of the cultivation time. Circles: WT, squares: PTSMan, diamonds: PTSGlc.

0

10

20

30

40

50

60

70

5 6 7 8 9 10 11 12 13

qp (

U m

g-1

h-1

)

Cultivation time (h)

40

High cell density cultivation

As a reduction of the growth rate, by the use of the mutants, potentially would result

in higher cell densities in batch cultivations, we studied this. Therefore, the WT and

the PTSGlcMan were grown in a repetitive batch mode where new batches of glucose

were added as the glucose in the medium was consumed. This glucose addition

strategy was chosen in order to avoid negative effects from high glucose

concentrations (Lara, et al., 2008; Shiloach and Fass, 2005). The final cell mass

reached was 27 g l-1 for the wild type and 34 g l-1 for PTSGlcMan respectively (Fig 13).

The WT produced ten times more acetic acid during the cultivation than the mutant,

9000 mg l-1 compared to 900 mg l-1. It was noted that already at acetate levels of 500

mg l-1, the growth rate of the WT was affected, and at this time the cell concentration

was only 3 g l-1. The shaded area in figure 13A shows the part of the cultivation that is

principally not operational due to the high concentration of acetic acid. The PTSGlcMan

(Fig 13 B) has constant but lower growth rate, a much longer period. At the end of the

cultivation an increase in the acetic acid accumulation is observed. This acetic acid

probably originates from mixed acid fermentation, which is a result of insufficient

supply of oxygen to the fermentor.

Figure 12. Dry weight and specific production rate of β-galactosidase of the PTSGlc mutant grown in batch and the WT strain grown in fed-batch at the corresponding growth rate. Circles: WT (fed-batch), diamonds: PTSGlc (batch). Closed symbols: cell dry weight, open symbols: specific production rate.

0

5

10

15

20

25

30

-10

0

10

20

30

40

50

60

-4 -2 0 2 4 6 8 10 12

Cel

l dry

wei

ght

(g l

-1)

qp (U

mg

-1 h-1)

Time from induction (h)

41

2.1.2 Production of integral membrane proteins by the PTS-mutants (III)

A following logical step was to explore the possibility of using the mutants for

production of other proteins, especially proteins that are known to be difficult to

produce. Integral membrane proteins were chosen as model proteins since their

production is usually associated with low yield, toxicity and inclusion body formation

(Essen, 2002). A set of five different E. coli integral membrane-spanning proteins

(IMPs) with human homologues were selected as model proteins (Table 4) and were

produced with N-terminal His6 tags to facilitate purification. These proteins have

earlier been expressed using the T7 promoter with varying success in different strains

(Eshagi, et al., 2005), but in the present study the expression was controlled from the

araBAD (PBAD) promoter. The B-strain, BL21(DE3)(Novagen) that is commonly used

for production of membrane proteins was also included in the study. This strain has a

specific growth rate of 0,76 h-1 in minimal medium, which is approximately equal to

the specific growth rate of AF1000 (WT).

Figure 13. High cell density cultivations of A wild type (AF1000) and B PTSGlcMan mutant strain. Closed circles: specific growth rate which is also approximated by a solid straight line, open circles: acetic acid concentration, solid line: specific oxygen consumption rate, qO2. The shaded area in figure A indicates the interval of cultivation that is preferably avoided.

0

0,2

0,4

0,6

0,8

1

0

2000

4000

6000

8000

0 2 4 6 8 10 12

my (

h-1

), q

O2 (

g g

-1 h

-1)

HA

c (m

g l

-1)

Cultivation time (h)

!

HAc

qO2

A

-0,1

0

0,1

0,2

0,3

0,4

0

2000

4000

6000

8000

0 5 10 15 20 25m

y (

h-1

), q

O2 (

g g

-1h

-1)

HA

c (m

g l

-1)

Cultivation time (h)

!

qO2

HAc

B

42

Small-scale production

All strains that were included in the study were grown in minimal medium in micro

titre plates, and expression of the IMPs was induced at OD600 ≈ 1 by addition of L-

arabinose. All cells were harvested after the same time of expression. A dot blot (Fig

14) was used to show the expression of the targets. PTSGlcMan and PTSGlc produced

three out of the five proteins, as did the BL21(DE3) strain. The amounts of cells at the

time of harvest varied since the strains have different growth rates. A quantitative

comparison of the expression levels in the different strains is not therefore possible

from this dot blot.

Table 4. Membrane protein characteristics. The integral membrane proteins used in the study.

Target Function/Predicted function

EM03 Ammonium transporter

EM09 Predicted transporter

EM16 Chloride transporter

EM20 Multi drug efflux system protein

EM29 Intramembrane serine protease

!"#$ %!"#& %!"'( %!")# %!")&%

*+'###%

,-."/0%

,-.123%

,-.123"/0%

45)'67!$8%

Figure 14. Expression of membrane proteins. Dot blot of purified His-tagged proteins overexpressed in the following strains: AF1000, PTSMan, PTSGlc, PTSGlcMan and BL21 (DE3).

43

Medium scale production and quantification

In order to be able to compare the relative amounts of IMP´s and to assess the degree

of homogeneity of the target protein, the production of the EM03 target was scaled up

into shake flasks. The membrane fraction was subjected to IMAC resin in order to

purify the His6-tagged product. Expression of the target protein was verified by

Comassie-stained SDS-PAGE (Fig 15).

The samples were further subjected to analytical gel filtration to allow a quantitative

comparison of the expressed levels in the different strains. The gel filtration

chromatogram is shown in figure 16A where values for the same amounts of cells are

shown normalized so that the top peak (maximum) value equals 1. The chromatogram

shows that EM03 could be purified as a non-aggregate.

!""#

$"#

%$#

&'#

("#

'"#

!)#

!#####'#####(#####%######*#####&######+,#

-./01#

Figure 15. Verification of expression of EM03. SDS-PAGE analysis of IMAC-purified target EM03. 1 marker, 2 AF1000, 3 PTSMan, 4 PTSGlc, 5 PTSGlcMan, 6 BL21 (DE3).

44

The amounts of EM03 that were produced in the different strains were calculated

from the areas under the curves and the results are plotted in figure 16B. The

comparison shows that PTSGlc produced approximately twice as much protein as

PPA689 PTSGlcMan and BL21(DE3). AF1000 and PTSMan produced very small

Figure 16. Purification and quantification of the selected membrane protein. (A) Analytical gel filtration from medium-scale shake flask cultivations using the target protein, EM03. The chromatogram shows values for an equal OD6oo -value of cells in the different samples, normalized with respect to the highest top peak absorbance set equal to one. (B) Estimated peak areas of analytical gel filtration chromatograms from the production of the target EM03. The values are shown as calculated peak areas for an equal OD600 -value of cells in the different samples, normalized with respect to the largest top peak area set equal to one.

45

quantities of EM03 also in shake flasks, so that it was not possible to determine the

areas in a statistically relevant fashion.

It is obvious that the glucose uptake rate influences the amount of protein that can be

extracted in a soluble form from the membrane, but this is apparently not the only

factor since AF1000 and BL21 have the same growth rates but very different

production levels.

Acetic acid

As stated before, the by-product acetate has a negative impact on both the growth rate

and on the production of recombinant proteins (Eiterman and Altman, 2006; Jensen

and Carlsen, 1990; Koh, et al., 1992). It is difficult to separate the effect of the acetic

acid from the effect of the growth rate on the production, since a growth rate above a

certain threshold value leads to the excretion of acetic acid. In some studies, acetic

acid has therefore been added to the cultivation and its effect on growth rate and on

production of recombinant proteins has been studied. Jensen and Carlsen (1990)

showed that acetic acid concentrations of above 6.1 g l-1 resulted in decreased growth

rate but in another study (Nakano, et al., 1997) this effect was seen already at

concentrations of 500 mg l-1. In our study (I) AF1000 showed a reduction in growth

rate at concentrations of 500 mg l-1 and at this time the cell concentration was

approximately 3 g l-1.

Table 5. Yield of acetic acid per cell for the strains used in the study.

Strain HAc (mg/L) per OD600-unit

AF1000 158

PTSMan 147

PTSGlc 3

PTSGlcMan 2

BL21(DE3) 58

46

The BL21(DE3) strain has a high specific growth rate (approximately 0,76 h-1) which

is comparable to the specific growth rate of AF1000, but has a comparably low

production of acetic acid (Table 5). The production of the membrane protein target

EM03 is twice as high in PTSGlc as in PTSGlcMan and BL21(DE3) and very low in

AF1000 and PTSMan. The differences in the accumulation of acetic acid between the

strains might be one possible explanation for the different production levels.

However, this in not the only explanation since the PTSGlc and the PTSGlcMan has the

same production of acetic acid, but different levels of production of EM03. The

production level is probably a combination of the glucose uptake rate and the acetic

acid accumulation level. The BL21(DE3) strain is deficient in the proteases Lon and

OmpT, which might also explain the relatively high production level in this strain.

2.2 Impact of feed-rate on processes with other product localisations

than the cytoplasm (II, IV)

2.2.1 Leakage of periplasmic products in relation to the glucose uptake

rate (II)

Some proteins need to be transported to the periplasm in order to fold properly.

Leakage of periplasmic proteins to the medium is a problem associated with

periplasmic production provided that the product ending up in the medium is not

collected. The aim of the present study was to investigate how the glucose uptake rate

is correlated to the leakage/periplasmic retention of secreted products. The growth

rate was varied and its effect on leakage of two periplasmic products to the medium

was studied. The focus was also on how the amount and specificity of the outer

membrane proteins was affected by the growth rate and by recombinant protein

production. Could the amounts of these proteins be connected to leakage of

periplasmic products to the medium?

Cutinase, a lipolytic enzyme, from Fusarium solani pisi was used as one of the model

proteins (Mannesse, et al., 1995). Two Z domains, i.e., engineered forms of the B

domain of staphylococcal protein A, which is present in the cell wall of

Staphylococcus aureus, were fused to the N-terminus of the cutinase (Bandmann, et

47

al., 2000). The expression of the product was controlled by the constitutive

staphylococcal protein A promoter (Löfdahl, et al., 1983), and the signal peptide from

the same protein was used to direct the recombinant product to the periplasm. An

antibody fragment (Fab) was used as a second model protein, which was directed to

the periplasm by an OmpA signal sequence. The production of Fab was under control

of the tac promoter. ZZ-cutinase was expressed in the K12 strain 0:17 (Olsson and

Isaksson, 1979) and Fab in W3110 (ATCC 27325).

Specific productivity and leakage

The specific productivity of the model proteins followed the growth rate of the cells

and was higher at higher growth rates. This trend was also observed for the

production of β-galactosidase (I) and is in accordance with earlier findings (Sandén,

et al., 2002). Figure 17 shows the leakage of ZZ-cutinase, the quotient between

product activity in the medium and product activity in total, as a function of the

growth rate. A higher specific growth rate resulted in a higher leakage of periplasmic

product to the medium. The same trend was observed for the production of Fab (data

not shown). Leakage is defined as the selective passage of proteins through the outer

membrane, whilst maintaining a functional cell. This is distinguished from lysis,

which leads to disruption and cell-death, but that also results in release of periplasmic

products to the medium. The presence of protein in the medium corresponding to the

normal endogenous composition of E. coli proteins (here referred to as total protein)

is usually considered as an indication of lysis. Product activity and total protein in the

medium was measured in order to exclude that the product found in the medium was a

result of lysis. Not more than 4% of the total protein was found in the medium in the

batch cultivation with recombinant product formation and not more than 3% in all

other cultivations (data not shown). The leakage of the product to the medium was as

high as 20% in the batch cultivation, and 9 and 6% respectively in the fed-batch

cultivations. The main part of the ZZ-cutinase activity found in the medium was thus

a result of leakage and not of lysis.

48

Effects of glucose uptake rate and recombinant protein production on outer

membrane composition

In order to investigate the relationship between the content of outer membrane

proteins and leakage, the first step was to identify which proteins that were present in

the outer membranes during the cultivations. The outer membrane proteins were

therefore isolated from cultivations with different growth rates (both with and without

production of ZZ-cutinase). The most abundant proteins were identified as OmpA,

OmpF and LamB by N-terminal sequencing (Fig 18). The expression of the outer

membrane proteins involved in uptake of nutrients, LamB and OmpF, was dependent

on the specific growth rate. The titre of these proteins decreased as the growth rate

increased for both ZZ-cutinase and non-producing cells. However, the reduction was

more noticeable for the producing cells. No LamB was detected in the batch

cultivation, which was to some degree expected since Death and co-workers (Death,

et al., 1993) showed that the LamB protein is upregulated during glucose limitation.

The OmpA protein, which is thought to have a role in stabilizing the cell, seemed to

be more or less constant regardless of the growth rate. There was however a slight

tendency that the amount of the protein was lower in producing cells than in non-

producing cells at comparable specific growth rates.

0 5 10 15 20

Cuti

nas

e ac

tivit

y (

med

ium

/tota

l)

DW (g l-1

)

µ=0.3 h-1

(batch)

µ=0.2 h-1

(exp. fedbatch)

µ=0.1 h-1

(exp. fedbatch)

0.20

0.15

0.10

0.05

0

Figure 17. Leakage of ZZ-cutinase to the medium in percent of the total production. The specific growth rates (h-1) are indicated in the figure.

49

!

0

100

200

300

400

500

600

Om

pF

(m

g l

-1

)

0

20

40

60

80

100

120

140

160

180

200

Lam

B (

mg l

-1

)

not

det

not

det

0

50

100

150

200

250

300

350

400

Om

pA

(m

g l

-1

)

0

100

200

300

400

500

600

700

800

900

1000

0,1 0,2 0,3 0,45 0,6

Specific growth rate (h-1 )

OM

P (

mg l

-1

)A

B

C

D

Figure 18. Outer membrane proteins isolated from cultivations with different growth rates (exponential feed profiles) without production (black bars) and during ZZ-cutinase production (white bars). Lam B was not detected in all cultivations, this is indicated by “not det” in the figure. A. OmpF (mg l-1) B. LamB (mg l-1 ) C. OmpA (mg l-1) D. Sum of OmpF, LamB and OmpA (mg l-1)

50

Figure 18D summarises the combined titre of proteins investigated. The combined

titre of outer membrane protein decreased as the specific growth rate increased for

both producing and non-producing cells. The overall titre of outer membrane proteins

was lower if a recombinant protein was produced. The total reduction was almost

60% with a high product formation rate at µ= 0.3 h-1.

It is reasonable to hypothesise that outer membrane protein accumulation influences

product retention. When studying the total titre of outer membrane proteins it was

clear that the titre was inversely proportional to the growth rate and that a lower titre

was found in producing cells. Secretion of a recombinant product results in an

increased transport of proteins through the inner membrane. Our model system

showed a specific productivity that was proportional to the specific growth rate,

allowing different levels of transport through the Sec translocase to be studied.

Depletion of outer membrane proteins, due to jamming and overloading of the sec

system, has earlier been proposed as possible reasons for periplasmic leakage

(Baneyx, 1999). In the present study, a higher growth rate, which is connected to a

higher transport through the Sec translocon, resulted in a higher leakage and in a

lower amount of outer membrane proteins. Inhibited transport of outer membrane

proteins due to the overloading of the Sec translocon is therefore a possible

explanation to our results.

Another approach that can be used when studying consequences of limited transport

through the Sec translocon is by altering the levels of the components of the Sec

translocon itself. Baars and co-workers (2008) used this approach and compared the

outer membrane proteome of cells depleted of SecE with the outer membrane

proteome of cells expressing normal levels of this protein. It was shown that the

amount of most outer membrane proteins was reduced upon SecE depletion.

However, the amount of OmpA was however unaffected, which is in accordance with

our findings (Baars, et al., 2008).

In a previous study (Shokri, et al., 2002), it was shown that the growth rate influenced

the composition of phospholipids and fatty acids in the membranes. The leakage of

the periplasmic product, in that study β-lactamase, showed a peak at a dilution rate

51

corresponding to a specific growth rate of 0.3 h-1. At this specific growth rate, there

was an increased amount of phosphatidyl glycerol and unsaturated fatty acids in the

membrane. The expression level of the product in that study was low and maybe not

enough for influencing the outer membrane composition. The growth rate intervals

investigated in these two studies were different and therefore a direct comparison was

difficult to do.

In the present study, we investigated how the composition of the outer membrane

proteins was connected to leakage of recombinant proteins. Our results suggest that

the permeability of the outer membrane is a function of both the lipid composition and

its protein content.

Figure 19 shows the retention of ZZ-cutinase, i.e. the amount of product that can be

harvested from the cell paste, as a function of cell accumulation. Interestingly, the

amount of product that can be retained within the cell is always the same and not a

function of the growth rate. This means that a higher specific growth rate, which

results in higher synthesis rate, will not result in more product in the periplasm of

each cell due to the elevated leakage.

0

50

100

150

200

250

300

0 5 10 15 20

Cu

tin

ase

acti

vit

y i

n c

ell

pel

let

(U m

l-1)

DW (g l-1

)

Figure 19. Periplasmic retention of ZZ-cutinase (cell pellet) as a function of dry weight (DW). The specific growth rates are indicated in the figure as follows: a specific growth rate of 0.3 h-1 (filled circles), a specific growth rate of 0.2 h-1 (filled triangles) and a specific growth rate of 0.1 h-1 (open squares), respectively.

52

2.2.2 Optimisation of surface expression using the AIDA autotransporter

(IV)

Although bacterial surface display has been used extensively in various research

applications, there are few studies focusing on its use as a large-scale protein

production tool. If surface-display technology is to be used in the context of large-

scale production, methods for cultivation of large quantities of cells whilst

maintaining a high surface expression is needed. Here, we investigated the influence

of different cultivation conditions and techniques, batch and fed-batch, as well as the

impact of two different cultivation media on the production of cell surface-displayed

recombinant proteins.

As a model protein, we used the staphylococcal protein A-derivative Z, which was

chosen because it is a small protein without disulphide bridges, and should

theoretically not be difficult to transport to the surface. Further, Z binds to the Fc

region of IgG, thus enabling flow cytometry-based verification and quantification of

surface expression of Z by using fluorescent IgGs. The vector that was used for the

surface display is based on the AIDA autotransporter with its own wild type promoter

aidA (Fig 20).

Impact of OmpT on surface expression level of Z

Z contains a potential cleavage site for the outer membrane protease OmpT. A first

step of this work was to assess if the potential cleavage site was accessible for OmpT.

Analysis of the AIDA-Z fusion protein expressed in strains with and without OmpT

(0:17 (Olsson and Isaksson, 1979) and 0:17ΔOmpT, respectively) revealed that both

strains expressed the fusion but only the OmpT negative strain yielded the full-length

Figure 20. The AIDA-vector containing the aidA promoter, the signal peptide (SP), Z (the passenger protein), a linker (L) and the β-barrel pore (AIDAc).

SP Z L AIDAC PaidA

53

protein (Fig21). The degradation product in 0:17 has approximately the same size as

AIDAc, a size that is expected if OmpT cleaves within the Z-domain.

Figure 21. Comparison of surface expression of Z in OmpT positive and OmpT negative E. coli 0:17. (A) Western blot of the isolated outer membrane fraction, using antiserum against AIDAc. Lane 1: Marker, 2: 0:17, 3: 0:17ΔOmpT, 4: 0:17 with empty vector, 5: 0:17ΔOmpT with empty vector, 6: 0:17 with vector containing Z, 7: 0:17ΔOmpT with vector containing Z. (B). Cells analysed by flow cytometry using human IgG linked to AlexaFluor488. Red: 0:17 with vector without passenger, Blue: 0:17 with vector containing Z, Green: 0:17ΔOmpT with vector containing Z. Flow cytometry analysis of the different clones confirmed that Z was accessible and

functional at the cell surface, since binding to fluorophore-labeled IgG was achieved.

The fluorescence intensity was, as expected from the western blot analysis, lower in

0:17 than in 0:17ΔOmpT. The strain 0:17ΔOmpT was thus chosen for the further

experiments. The effect of the temperature on the expression level was also

investigated and expression was found to be favoured by cultivation of the cells at

37°C.

Effects of growth medium and regulation of the aidA promoter

In order to evaluate the effect of the growth medium on the surface expression of Z

the cells were grown either in minimal medium with glucose as carbon source or in

Luria Broth (LB) medium. As can be seen in figure 22, the expression level was

higher in the minimal medium than in LB, which is in accordance with earlier

findings (Benz, et al., 2010). Glucose was added to the LB medium in order to assess

54

if the lower expression level was a result of the absence of an easily available carbon

source. This was not the case since the addition of glucose to the LB medium resulted

in even lower expression levels (Fig 22). The concentration of amino acids in the

medium was followed during the cultivation (data not shown). Some amino acids

were depleted but their depletion did not result in increased expression levels of Z.

Another idea that we had was that glucose starvation and the subsequent entrance into

the stationary phase would affect the level of expression. The cells were thus grown in

minimal salt medium containing glucose and were allowed to grow until the glucose

was consumed. It was shown that entry into the stationary phase reduced the surface

expression level of Z (data not shown). These data together with those from the

cultivations in different media indicate that the aidA promoter is not induced by

stringent response.

Nevertheless, cultivation in different media does result in different growth rates.

Going from minimal medium to LB medium and finally to LB medium supplemented

with glucose result in progressively higher growth rates. Since it appeared likely that

lower growth rates resulted in enhanced surface expression, the next logical step was

to investigate if further reductions of the growth rate would be beneficial.

Figure 22. Comparison of surface expression of Z in 0:17ΔOmpT grown in glucose minimal medium (grey), LB medium (white) and LB medium with 10 g l-1 glucose (black), respectively.

55

Effects of glucose uptake rate in minimal salt medium

Different fed-batch cultivations were performed in order to investigate the influence

of the glucose uptake rate/growth rate on expression. Three different growth rates

(μ=0.1, 0.2 and 0.4 h-1) were achieved by exponentially feeding glucose to the

reactors after an initial batch phase. Surface expression of Z was followed using flow

cytometry. There is a tendency that the expression level is higher at a higher growth

rate (Fig 23). The limited transport capacity of the Sec-translocon (Baars, et al., 2008;

Baneyx, 1999; Mergulhão and Monteiro, 2004) is a potential bottleneck in production

systems where the recombinant protein is localised to parts of the cell other than the

cytoplasm. Likewise, the insertion of proteins in the outer membrane, mediated by the

BAM-complex, is a potential bottleneck in surface display systems. The outer

membrane protein fraction was isolated from the cultivations with different growth

rates (data not shown) and no clear difference in outer membrane protein content

could be seen. In (II) those differences appeared clear. The expression level of Z by

the aidA promoter is probably not high enough for influencing transport and insertion

of endogenous proteins and the pattern of protein expression therefore probably

reflects the synthesis rate of the protein.

Fig 23. Cell growth and antibody-binding activity of the surface displayed Z. OD600 values (filled symbols) from the fed-batch cultivations with different growth profiles and mean fluorescence per cell (open symbols) analysed by flow cytometry. Circles μ=0.1 h-1, squares μ=0.2 h-1, triangles μ=0.4 h-1.

56

Optimisation of cultivation

The last step of this study was to develop an efficient process for cultivation of cells

with high-level expression of proteins at the surface. Expression was favoured by

cultivation in minimal medium and there was a trend that expression was higher at

higher growth rates (Fig 23). We therefore reasoned that a “repeated batch strategy”,

were the cells grow at the maximal specific growth rate and where new batches of

glucose are added as the glucose is consumed, would be suitable for obtaining cells

with high-level expression (Fig 24).

A furhter advantage of the repeated batch strategy is seen when looking at the

fluorescence peaks from the flow cytometer analysis (Fig 25). The peaks obtained

during the extended batch culture are narrower than during the fed-batch cultivations,

indicating that the bacterial population is more homogenous. The peak width can be

considered as a quality parameter for cells expressing proteins at their surface. As

discussed before, acetic acid accumulation, due to overflow metabolism, is a problem

when using the batch cultivation strategy. The acetic acid accumulation in strain

0:17ΔOmpT is however much lower than in AF1000, even though the strains are both

Figure 24. Cell growth, acetic acid formation and surface expression during repeated batch cultivation. (A) Cell growth measured as optical density (filled squares), dry weight (open squares), surface expression per cell measured by flow cytometry (circles, line). (B) Specific growth rate (μ, diamonds), acetic acid concentration in the medium (triangles, line) and dissolved oxygen (DOT, line).

57

K-12 derivatives and have approximately the same growth rates, and this explains

why the repetitive batch strategy can be used. In the former study (I) the cell mass

was 3 g l-1 when an inhibiting concentration of acetic acid, 500 mg l-1, was reached. In

the present study the cell mass was 8 g l-1 when the acetic acid reached this

concentration but the cells continued to grow without severe effects on the growth

rate until the cell mass reached approximately 20 g l-1. The acetic acid concentration

does not reach inhibiting concentrations until the oxygen limitation of the fermentor is

reached. At this point, coinciding with the DOT approaching zero, a sharp increase in

the acetic acid accumulation is observed. This additional acetic acid probably

originates from mixed acid fermentation, which is a result of insufficient supply of

oxygen to the fermentor. The final dry weight reached, by using the repetitive batch

process, was approximately 30 g l-1, and the surface expression level remained high

up to a cell mass of approximately 20 g l-1 (Fig 24). For a further increase in cell

mass, the fed-batch technology is needed, even though this results in a less

homogenous cell population.

Figure 25. Flow cytometry analysis of antibody-binding activity of surface displayed Z from two selected samples. Black (repeated batch) and grey (fed-batch, μ=0.4 h-1)

58

3 CONCLUDING REMARKS

The potential of E. coli as host for production of recombinant proteins has not yet

been fully exploited. There are several examples in the literature describing efforts to

overcome problems associated with heterologous protein production in this host. The

development of novel and better methods for production of recombinant proteins is

essential for the purpose of structural and functional studies, but also for the industrial

production of biopharmaceuticals. In this thesis, the impact of the glucose uptake rate

was studied with the objective to improve and optimise production of recombinant

proteins in E. coli. The glucose uptake rate was shown to influence the production of

the recombinant protein in all works included in this thesis. The impact of the glucose

uptake rate was however different depending on the product localisation and the

protein product in question.

As an alternative to traditional fed-batch technology, we here showed that mutant

strains deficient in the phosphotransferase system (PTS) could be used for control of

the glucose uptake rate (I). This allows for control of the specific growth rate at a

genetic level rather than by physical means (e.g., by feed rate, temperature). We

suggest that the mutants will be valuable tools in small-scale parallel recombinant

protein production where the fed-batch technology, for practical reasons, is not

applicable. The mutants were further applied for production of integral membrane

proteins (III) and were able to produce membrane proteins that were not possible to

produce by the corresponding wild type strain. Our results suggest that the mutants

could be useful not only for production of membrane proteins but also for other

“difficult-to-express” proteins, when production benefits from a lower synthesis rate

and when a high cell density is needed in order to obtain a “sufficient” amount of

protein.

Process development is quite often characterised by a screening stage performed

under batch conditions in small parallel scale, e.g., in microtitre plates or shake flasks.

Unfortunately, the level of control of the environmental conditions in such shaken

systems is low and the conditions also change rapidly as a consequence of the

exponential biomass-increase. In contrast, in the production scale, the environmental

59

conditions are strictly controlled, the fed-batch technology is usually applied and the

production takes place under lower growth rates in comparison to batch cultures.

Thus, the results obtained from small-scale cultivations do not necessarily apply in a

large-scale production format, which makes the scale up process more complex. By

using the PTS-mutants, screening can be performed under fed-batch-like conditions.

Potentially, this will limit the amount of work for the process development team and

hopefully increase the probability of selecting conditions that will work also in the

large-scale production process.

That there is a need for control of the glucose uptake rate in small-scale cultivation is

evidenced by the recent development of other innovative methods. One such method

is EnBase®, or enzyme-based-substrate-delivery, which has been developed as a

complement to the fed-batch technology (Panula-Perälä, et al., 2008; Siurkus, et al.,

2010). Glucose is released into the medium by enzymatic degradation of starch by the

enzyme glucoamylase and no external feed of glucose is required. The main benefit of

this method is that it results in significantly higher cell densities compared to

traditional shake-flasks cultures (Siurkus, et al., 2010). However, as EnBase® is a kit-

based method, it is expensive to use and since the ingredients of the different

components in the kit are not fully disclosed, using the EnBase® technology as a tool

in the process development might be more complicated.

One problem associated with periplasmic processes is that the product tends to leak to

the medium, resulting in decreased yields if the product in the medium is not used in

the down stream processing. Leakage of recombinant products to the medium was

clearly affected by the feed rate of glucose (II) since leakage increased with the feed

rate. Although a higher feed rate resulted in an increased specific productivity, the

amount of product inside the cells was constant. In order to retain the product inside

the cells, the induction should preferably take place when the feed rate is low, which

would not only decrease leakage but also improve the product quality (Boström, et al.,

2005; Sandén, et al., 2005). In this study, we also showed that the feed rate influenced

the outer membrane composition. The total amount of outer membrane proteins

decreased as the feed rate increased and further reductions in outer membrane protein

accumulation was seen when a recombinant product was secreted to the periplasm.

60

Although bacterial surface display has been used extensively in various research

applications, there are few studies focusing on its use as a large-scale protein

production tool. Here we applied the AIDA autotransporter for surface display of a

recombinant protein and studied protein expression under control of the aidA

promoter (IV). We showed that cultivation in minimal salt medium resulted in higher

expression levels, as did higher feed rates of glucose during fed-batch cultivation.

Batch cultivation with repeated additions of glucose was distinguished as the best

method to improve the surface expression homogeneity in the bacterial population. In

the future, it would be interesting to change the aidA promoter to an inducible and

tunable promoter. By varying the inducer concentration it will then be possible to

investigate if the expression level can be further increased and also potential

limitations regarding Sec-translocation and the insertion process of proteins in the

outer membrane can be studied. The space in the outer membrane where the

transporters accumulate is limited and it will be interesting to find out (i) how many

transporters that can actually be inserted in the membrane and (ii) how many

transporters that can be inserted without affecting the stability of the membrane.

Extracellular production of recombinant proteins is desirable since it allows easy

purification, an environment free of cell-associated proteolytic degradation and

minimized impact on the host cell from toxic proteins. Some autotransporters release

their passenger to the medium and may therefore be exploited for extracellular

production of recombinant proteins. It would be interesting to investigate how

efficient such systems would be in terms of productivity and protein quality. The

number of transporters that can be inserted in the membrane is, as discussed above,

limited and furthermore, the cell has to produce one transporter for each passenger,

which is disadvantageous. However, for some proteins the extra cellular medium

represents the only possible method for their production, and for production of these

proteins, the autotransporter-based excretion strategy might be promising.

Considering what has been presented in this thesis, the glucose uptake rate is an

important parameter in recombinant protein production. It does not only effect the

biomass that can be reached in a certain process but influences also parameters such

as protein quality, specific productivity and leakage of recombinant proteins to the

61

medium. Therefore, it is important to be able to control the glucose uptake rate also in

small-scale cultivation, and as shown in this thesis, this becomes possible by using

PTS-mutants.

62

4 ABBREVIATIONS

AC Adenylate cyclase Ack Acetate kinase AMP-ACS AMP-forming acetyl CoA-synthetase AIDA Adhesin involved in diffuse adherence AMP Adenosine monophosphate ATP Adenosine triphosphate BAM β-barrel assembly machinery cAMP Cyclic adenosine monophosphate CCR Carbon catabolite repression cDNA Complementary DNA CL Cardiolipin CRP cAMP receptor protein DNA Deoxyribonucleic acid DOT Dissolved oxygen tension DR Down stream region DSB Disulphide bond DW Dry weight (g l-1) E. coli Escherichia coli EI Enzyme I EII Enzyme II F Flow rate (L/h) Fab Antibody fragment HAc Acetic acid His6 Hexahistidine tag HPr Phosphohistidine carrier protein HTP High–throughput production HTS High-throughput screening HUGO The Human Genome Organisation IgG Immunoglobulin G IMAC Immobilized metal affinity chromatography IMP Integral membrane proteins IPTG Isopropyl-β-D-thiogalactopyranoside kDa kilo Dalton LamB Maltose outer membrane porin LB Luria broth Lpp Murein lipoprotein LPS Lipopolysaccaride MBP Maltose binding protein mRNA Messenger ribonucleic acid OD Optical density OMP Outer membrane protein PE Phosphatidyletanolamine PEP Phosphoenolpyruvate PG Phosphatidylglycerol PoxB Pyruvate oxidase B ppGpp Guanosine tetraphoshate

63

PPIase Peptidyl-prolyl isomerase PrEST Protein epitope signature tag Pta Phosphotransacetylase PTS Phosphotransferase system qp Specific productivity (g g-1 h-1) qs Specific substrate consumption rate (g g-1 h-1) RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid SD Shine Dalgarno sequence SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis Si Substrate concentration (g l-1) SRP Signal recognition particle TAT Twin arginine translocation TCA Tricarboxylic acid cycle TF Trigger factor TIR Translation initiation region tRNA Transfer ribonucleic acid V Cultivation volume (l) wt Wild type X Cell dry weight (g l-1) µ Specific growth rate (h-1) σ24 = σE Extra-cytoplasmic stress sigma factor σ32= σH Heat shock sigma factor σ38= σs Starvation/stationary phase sigma factor

64

5 ACKNOWLEDGMENT

Allting har ett slut, så även en doktorandtid, och jag skulle därför vilja ta tillfället i akt att tacka alla Er som på ett eller annat sätt stöttat mig på vägen till examen. Ett speciellt tack till: Vinnova, Vetenskapsrådet och KTH för finansiellt stöd. Professor Gen Larsson, för handledning och stöd genom åren, för att du alltid delar med dig av dina kunskaper och idéer samt för att du är så entusiastisk över vår forskning. Professor Pär Nordlund och Marina Ignatushchenko och övriga medarbetare på MBB, Karolinska Institutet. Tack för att jag fick möjlighet att komma till Ert labb och genomföra min studie. Det var både lärorikt, roligt och trevligt att arbeta tillsammans med er. Dominic Reeks, Neil Weir and Leigh Bowering at Celltech for great cooperation. Katrin för ett ypperligt samarbete i början av doktorandtiden. Allt blir så mycket lättare och roligare när man är två. Martin för att du fortsatte ytexpressionsprojektet när jag labbade på KI, samt för ett finfint samarbete därefter. Tack också för att du alltid ställer upp och löser mina datorrelaterade problem och för att du ritar så fina bilder. Din hjälp nu i “skrivartider” har verkligen varit värdefull. Patrik för handledning i autotransportprojektet och för ovärderlig hjälp med avhandlingen. Examensarbetarna Maria, Sofia och Cecilia. Det var givande att jobba tillsammans med er och tack för era bidrag till mina projekt. Andres för hjälp med proteinrening och för att du alltid tar dig tid att diskutera forskningsrelaterade frågor. Per-Åke Nygren och Per-Åke Löfdahl för kloningstips, John Löfblom för hjälp med FACS-analys. Alla nuvarande och före detta kollegor och vänner på avdelningen (ingen nämnd, ingen glömd) för trevligt sällskap och roliga stunder under årens lopp, samt tack för all hjälp och goda råd. Ett extra tack till familjen som alltid hjälper mig när det behövs. Pappa och Ida för att ni lyssnar och förstår. Irene, Sören, Ida och Tobias för att ni varit barnvakt så att jag kunnat fokusera på att skriva. Jakob och Ines för att ni sprider så mycket glädje omkring er. Och sist men inte minst, ett stort tack till dig Lars för ditt tålamod, för att du är en fantastisk “hemma-pappa” och för att du alltid finns där.

65

6 REFERENCES

Abrahmsén, L., Moks, T., Nilsson, B. and Uhlén, M. (1986) Secretion of heterologous gene products to the culture medium of Escherichia coli, Nucleic Acids Research, 14, 7487-7500.

Adams, J.M. (1968) On the Release of the Formyl Group from Nascent Protein, Journal of Molecular Biology, 33, 571-589.

Allen, S.P., Polazzi, J.O., Gierse, J.K. and Easton, A.M. (1992) Two Novel Heat Shock Genes Encoding Proteins Produced in Response to Heterologous Protein Expression in Escherichia coli, Journal of Bacteriology, 174, 6938-6947.

Arditti, R., Grodzicker, T. and Beckwith, J. (1973) Cyclic Adenosine Monophosphate-Independent Mutants of the Lactose Operon on Escherichia coli, Journal of Bacteriology, 114, 652-655.

Arséne, F., Tomoyasu, T. and Bukau, B. (2000) The heat shock response of Escherichia coli, International Journal of Food Microbiology, 55, 3-9.

Baars, L., Wagner, S., Wickström, D., Klepsch, M., Ytterberg, J., van Wijk, K.J. and de Gier, J.-W. (2008) Effects of SecE Depletion on the Inner and Outer Membrane Proteomes of Escherichia coli, Journal of Bacteriology, 190, 3505-3525.

Bandmann, N., Collet, E., Leijen, J., Uhlén, M., Veide, A. and Nygren, P.-Å. (2000) Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems, Journal of Biotechnology, 79, 161-172.

Baneyx, F. (1999) Recombinant protein expression in Escherichia coli, Current Opinion in Microbiology, 10, 411-421.

Baneyx, F. and Georgiou, G. (1990) In vivo degradation of secreted fusion proteins by the Escherichia coli outer membrane protease OmpT, Journal of Bacteriology, 172, 491-494.

Baneyx, F. and Mujacic, M. (2004) Recombinant protein folding and misfolding in Escherichia coli, Nature Biotechnology, 22, 1399-1408.

Bauer, K.A., Ben-Bassat, A., Dawson, M., De La Puente, V. and Neway, J.O. (1990) Improved Expression of Human Interleukin-2 in High-Cell-Density Fermentor Cultures of Escherichia coli K-12 by a Phosphotransacetylase Mutant, Applied and Environmental Microbiology, 56, 1296-1302.

Benz, I., van Alen, T., Bolte, J., Wörmann, M.E. and Schmidt, A.M. (2010) Modulation of transcription and characterization of the promoter organization of the autotransporter adhesin heptosyltransferase and the autotransporter adhesin AIDA-I, Microbiology, 156, 1155-1166.

Berks, B.C., Palmer, T. and Sargent, F. (2005) Protein targeting by the bacterial twin-arginine translocation (Tat) pathway, Current Opinion in Microbiology, 8, 174-181.

Blight, M.A. and Holland, B. (1994) Heterologous protein secretion and the versatile Escherichia coli haemolysin translocator, Tibtech, 12, 450-455.

Booth, I.R. (1999) Adaptation to Extreme Environments, In: Biology of the Prokaryotes, Eds: J W Lengeler, G Drews, H G Schlegel (Blackwell Science), 652-671.

Boström, M., Markland, K., Sandén, A.M., Hedhammar, M., Hober, S. and Larsson, G. (2005) Effect of substrate feed rate on recombinant protein secretion, degradation and inclusion body formation in Escherichia coli, Applied Microbiology and Biotechnology, 68, 82-90.

Casadaban, M.J. (1976) Transposition and Fusion of the lac Genes to Selected Promoters in Escherichia coli using Bacteriophage Lambda and Mu, Journal of Molecular Biology, 104, 541-555.

Charbit, A., Molla, A., Saurin, W. and Hofnung, M. (1988) Versatility of a vector for expressing foreign polypeptides at the surface of Gram-negative bacteria, Gene, 70, 181-189.

Chaudhiri, T.K., Horii, K., Yoda, T., Arai, M., Nagata, S., Terada, T., Uchiyama, H., Ikura, T., Tsumoto, K., Kataoka, H., Matsushima, M., Kuwajima, K. and Kumagai, I. (1999) Effect of the Extra N-terminal Methionine Residue on the Stability and Folding of Recombinant alpha-lactalbumin Expressed in Escherichia coli, Journal of Molecular Biology, 285, 1179-1194.

66

Chen, R. and Henning, U. (1996) A periplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins, Molecular Microbiology, 19, 1287-1294.

Chen, Y., Song, J., Sui, S.-f. and Wang, D.-N. (2003) DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli, Protein Expression and Purification, 32, 221-231.

Cho, S., Shin, D., Ji, G.E., Heu, S. and Ryu, S. (2005) High-level recombinant protein production by overexpression of Mlc in Escherichia coli, Journal of Biotechnology, 119, 197-203.

Chou, C.-H., Bennett, G.N. and San, K.-Y. (1994) Effect of Modified Glucose Uptake Using Genetic Engineering Techniques on High-Level Recombinant Protein Production in Escherichia coli Dense Cultures, Biotechnology and Bioengineering, 44, 952-960.

Cohen, S., N, Chang, A.C.Y., Boyer, H.W. and Helling, R.B. (1973) Construction of Biologically Functional Bacterial Plasmids In Vitro, Proceedings of the National Academy of Sciences of the United States of America, 70, 3240-3244.

Corchero, J.L., Viaplana, E., Benito, A. and Villaverde, A. (1996) The position of the heterologous domain can influence the solubiity and proteolysis of β-galactosidase fusion proteins in E. coli, Journal of Biotechnology, 48, 191-200.

Cronan, J.E., JR and O.Rock, C. (1996) Biosynthesis of Membrane Lipids, In Escherichia coli and Salmonella, Cellular and Molecular Biology, Ed: Neidhart F C (ASM Press), 612-636.

Daugherty, P.S. (2007) Protein engineering with bacterial display, Current Opinion in Structural Biology, 17, 474-480.

Dautin, N. and Bernstein, H.D. (2007) Protein Secretion in Gram-Negative Bacteria via the Autotransporter Pathway, The Annual Review of Microbiology, 61, 89-112.

De Anda, R., Lara, A.R., Hernández, V., Hernández-Montalvo, V., Gosset, G., Bolivar, F. and Ramirez, O.T. (2006) Replacement of the glucose phospotransferase system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate, Metabolic Engineering, 8, 281-290.

Death, A., Notley, L. and Ferenci, T. (1993) Derepression of LamB Protein Facilitates Outer membrane Permeation of Carbohydrates into Escherichia coli under Conditions of Nutrient Stress, Journal of Bacteriology, 1475-1483.

Dong, H., Nilsson, L. and Kurland, C.G. (1995) Gratuitous Overexpression of Genes in Escherichia coli Leads to Growth Inhibition and Ribosome Destruction, Journal of Bacteriology, 177, 1497-1504.

Dowhan, W. (1997) Molecular basis for membrane phospholipid diversity: Why Are There So Many Lipids?, Annual Review of Biochemistry, 66, 199-232.

du Plessis, D.J.F., Nouwen, N. and Driessen, A.J.M. (2011) The Sec translocase, Biochimica et Biophyisca Acta, 1808, 851-865.

Eiterman, M.A. and Altman, E. (2006) Overcoming acetate in Escherichia coli recombinant protein fermentations, TRENDS in Biotechnology, 24, 530-536.

Enfors, S.-O. and Häggström, L. (2000) Bioprocess technology fundamentals and applications Royal Institute of Technology (KTH), Stockholm.

Eshagi, S., Hedrén, M., Ignatushenko Abdel Nasser, M., Hammarberg, T., Thornell, A. and Nordlund, P. (2005) An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins, Protein Science, 14, 676-683.

Essen, L.-O. (2002) Structural genomics of "non-standard" proteins: a chance for membrane proteins?, Gene Function & Disease, 3, 39-48.

Francisco, J.A., Campbell, R., Iverson, B.L. and Georgiou, G. (1993) Production and flourescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface, Proceedings of the National Academy of Sciences of the United States of America, Biochemistry, 90, 10444-10448.

Genentech (press release, 1978) Available from:, http://www.gene.com/gene/news/press-releases/display.do?method=detail&id=4160, 2011-05-10.

67

Georgiou, G. and Shuler, M.L. (1988) Release of Periplasmic Enzymes and Other Physiological Effects of ß-lactamase Overproduction in Escherichia coli, Biotechnology and Bioengineering, 32, 741-748.

Golomb, M. and Chamberlin, M. (1974) Characterization of T7-specific Ribonucleic Acid Polymerase. IV. Resolution of the major in vitro transcripts by gel electrophoresis., The Journal of Biological Chemistry, 249, 2858-2863.

Gosset, G. (2005) Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system, Microbial Cell Factories, 4:14.

Gross, C.A. (1996) Function and Regulation of the Heat Shock Proteins, In: Escherichia coli and Salmonella, Cellular and molecular biology, Eds: Neidhart F C (ASM Press).

Hannig, G. and Makrides, S.C. (1998) Strategies for optimizing heterologous protein expression in Escherichia coli, Tibtech, 9, 54-60.

Hauke, L., Schwarz, E. and Rudolph, R. (1998) Advances in refolding of proteins produced in E. coli, Current Opinion in Biotechnology, 9, 497-501.

Hennecke, G., Nolte, J., Volkmer-Engert, R., Schneider-Mergener, J. and Behrens, S. (2005) The periplasmic chaperone SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recognition, The Journal of Biological Chemistry, 280, 23540-23548.

Hoffmann, F. and Rinas, U. (2004) Stress induced by recombinant protein production in Escherichia coli, In: Physiological Stress Responses in Bioprocesses, Advances in Biochemical Engineering/Biotechnology, 89, Ed Enfors S. O (Springer).

Jensen, E.B. and Carlsen, S. (1990) Production of Recombinant Human Growth Hormone in Escherichia coli: Expression of Different Precursors and Physiological Effects of Glucose, Acetate and Salts, Biotechnology and Bioengineering, 36, 1-11.

Jose, J. (2006) Autodisplay: efficient bacterial surface display of recombinant proteins, Applied Microbiology and Biotechnology, 69, 607-614.

Jose, J., Bernhardt, R. and Hannemann, F. (2002) Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E. coli Journal of Biotechnology, 95, 257-268.

Jose, J., Chung, J.-W., Jeon, B.-J., Maas, R.M., Nam, C.-H. and Pyun, J.-C. (2009) Escherichia coli with autodisplayed Z-domain of protein A for signal amplification of SPR biosensor, Biosensors and Bioelectronics, 24, 1324-1329.

Jose, J. and Meyer, T.F. (2007) The Autodisplay Story, from Discovery to Biotechnical and Biomedical Applications, Microbiology and Molecular Biology Reviews, 71, 600-619.

Jose, J. and von Schwichow, S. (2004) Autodisplay of Active Sorbitol Dehydrogenase (SDH) Yields a Whole Cell Biocatalyst for the Synthesis of Rare Sugars, ChemBioChem, 5, 491-499.

Jürgen, B., Breitenstein, A., Urlacher, V., Büttner, K., Hongying, L., Hecker, M., Schweder, T. and Neubauer, P. (2010) Quality control of Inclusions bodies in Escherichia coli, Microbial Cell Factories, 9:41.

Kiefer, H. (2003) In vitro folding of alpha-helical membrane proteins, Biochimica et Biophyisca Acta, 1610, 57-61.

Klauser, T., Pohlner, J. and Meyer, T.F. (1990) Extracellular transport of cholera toxin B subunit using Neisseria IgA protease β-domain: conformation-dependent outer membrane translocation, The EMBO Journal, 9, 1991-1999.

Klemm, P. and Schembri, M., A (2000) Fimbrial surface display systems in bacteria: from vaccines to random libraries, Microbiology, 146, 3025-3032.

Knowles, T.J., Scott-Tucker, A., Overduin, M. and Henderson, I.R. (2009) Membrane protein architects: the role of the BAM complex in outer membrane protein assembly, Nature Reviews Microbiology, 7, 206-214.

Koh, B.T., Nakashimada, U., Pfeiffer, M. and Yap, M.G.S. (1992) Comparision of acetate inhibition on growth of host and recombinant E. coli K12 strains, Biotechnology Letters, 14, 1115-1118.

68

Kramer, R.A., Zandwijken, D., Egmond, M.R. and Dekker, N. (2000) In vitro folding, purification and characterization of Escherichia coli outer membrane protease OmpT, European Journal of Biochemistry, 267, 885-893.

Lara, A.R., Caspeta, L., Gosset, G., Bolivar, F. and Ramirez, O.T. (2008) Utility of an Escherichia coli Strain Engineered in the Substrate Uptake System for Improved Culture Performance at High Glucose and Cell Concentrations: An alternative to Fed-Batch Cultures, Biotechnology and Bioengineering, 99, 893-901.

Lazzaroni, J.-C. and Portalier, R.C. (1981) Genetic and Biochemical Characterization of periplasmic-Leaky Mutants of Escherichia coli K-12, Journal of Bacteriology, 145, 1351-1358.

Lee, S.Y. (1996) High cell-density culture of Escherichia coli, Tibtech, 14, 98-105.

Lee, S.Y., Choi, J.H. and Xu, Z. (2003) Microbial cell-surface display TRENDS in Biotechnology, 21, 45-52.

Lengeler, J.W. and Postma, P.W. (1999) Global regulatory networks and signal transduction pathways, In: Biology of the Prokaryotes, Eds: J W Lengeler, G Drews, H Schlegel (Blackwell Science) 491-523.

Li, C., Zhu, Y., Benz, I., Schmidt, A., Chen, W., Mulchandani, A. and Qiao, C. (2008) Presentation of functional organophosphorus hydrolase fusions on the surface of Escherichia coli by the AIDA-I autotransporter pathway, Biotechnology and Bioengineering, 99, 485-490.

Lobban, P.E. and Kaiser, A.D. (1973) Enzymatic end-to end joining of DNA molecules, Journal of Molecular Biology, 78, 453-471.

Löfdahl, S., Guss, B., Uhlén, M., Philipson, L. and Lindberg, M. (1983) Gene for staphylococcal protein A, Proceedings of the National Academy of Sciences of the United States of America, Biochemistry, 80, 697-701.

Luckey, M. (2008), Membrane Structural Biology, chapter 1 (Cambridge University Press).

Magnusson, L.U., Farewell, A. and Thomas, N. (2005) ppGpp: a global regulator in Escherichia coli, TRENDS in Microbiology, 13, 236-242.

Makrides, S.C. (1996) Strategies for Achieving High-level Expression of genes in Escherichia coli, Micobiological Reviews, 60, 512-538.

Mannesse, M.L.M., Cox, R.C., Koops, B.C., Verheij, H.M., de Haas, G.H., Egmond, M.R., van der Hijden, H.T.W.M. and de Vlieg, J. (1995) Cutinase from Fusarium solani pisi Hydrolyzing Triglyceride Analogues. Effect of Acyl Chain Length and Position in the Subtrate Molecule on Activity and Enantioselectivity, Biochemistry, 34, 6400-6407.

Martínez-Alonso, M., García-Fruitós, E., Ferrer-Miralles, N., Rinas, U. and Villaverde, A. (2010) Side effects of chaperone gene co-expression in recombinant protein production, Microbial Cell Factories, 9:64.

Maurer, J., Jose, J. and Meyer, T.F. (1997) Autodisplay: One-component system for efficient surface display and release of soluble recombinant proteins from Escherichia coli, Journal of Bacteriology, 179, 794-804.

Mergulhão, F.J.M. and Monteiro, G.A. (2004) Secretion Capacity Limitations of the Sec Pathway in Escherichia coli, Journal of Microbiology and Biotechnology, 14, 128-133.

Mergulhão, F.J.M., Summers, D.K. and Monteiro, G.A. (2005) Recombinant protein secretion in Escherichia coli, Biotechnology Advances, 23, 177-202.

Meyer, H.-P., Leist, C. and Fiechter, A. (1984) Acetate formation in continuous culture of Escherichia coli K12 D1 on defined and complex media, Journal of Biotechnology, 1, 355-358.

Miksch, G., Neitzel, R., Fiedler, E., Friehs, K. and Flaschel, E. (1997) Extracellular production of a hybrid β-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors, Applied and Environmental Microbiology, 47, 120-126.

Miroux, B. and Walker, J.E. (1996) Over-production of Proteins in Escherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels, Journal of Molecular Biology, 260, 289-298.

69

Missiakas, D., Betton, J.-M. and Raina, S. (1996) New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH, Molecular Microbiology, 21, 871-884.

Moat, A.G., Foster, J.W. and Spector, M.P. (2002), Microbial Physiology, (Wiley-Liss)

Moerschell, R.P., Hosokawa, Y., Tsunasawa, S. and Sherman, F. (1990) The Specificities of Yeast Methionine Aminopeptidase and Acetylation of Amino-terminal Methionine in Vivo, The Journal of Biological Chemistry, 265, 19638-19643.

Morrow, J.F., Cohen, S., N, Chang, A.C.Y., Boyer, H.W., Goodman, H.M. and Helling, R.B. (1974) Preplication and Transcription of Eukaryotic DNA in Escherichia coli, Proceedings of the National Academy of Sciences of the United States of America, 71, 1743-1747.

Nakano, K., Rischke, M., Sato, S. and Märkl, H. (1997) Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor, Applied Microbiology and Biotechnology, 48, 597-601.

Neidhardt, F.C., Ingraham, J.L. and Schaechter, M. (1990), Physiology of the Bacterial Cell, A Molecular Approach, chapter 1 (Sinauer Associates).

Ni, Y. and Chen, R. (2009) Extracellular recombinant protein production from Escherichia coli, Biotechnology Letters, 31, 1661-1670.

Nikaido, H. (1996) Outer Membrane, In: Escherichia coli and Salmonella, Cellular and molecular biology, Eds: Neidhart F C (ASM Press), 29-47.

Nikaido, H., Bavoil, P. and Hiroa, Y. (1977) Outer membranes of gram-negative bacteria. XV. Transmembrane diffusion rates in lipoprotein-deficient mutants of Escherichia coli, Journal of Bacteriology, 132, 1045-1047.

Olsson, M.O. and Isaksson, L.A. (1979) Analysis of rpsD Mutations in Escherichia coli. I: Comparison of Mutants with Various Alterations in Ribosomal Protein S4, Molecular & General Genetics, 1, 251-257.

Panula-Perälä, J., Siurkus, J., Vasala, A., Wilmanowski, R., Casteleijn, M.G. and Neubauer, P. (2008) Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks, Microbial Cell Factories, 7:31.

Phue, J.-N., Lee, S.J., Kaufman, B., Alejandro, N. and Shiloach, J. (2010) Acetate accumulation through alternative metabolic pathways in ackA- pta- poxB - triple mutant in E. coli B (BL21), Biotechnology Letters, 32, 1897-1903.

Phue, J.-N. and Shiloach, J. (2004) Transcription levels of key metabolic genes are the cause fo the different glucose utilization pathways in E. coli B (BL21) and E. coli K (JM109), Journal of Biotechnology, 109, 21-30.

Picon, A., Texiera de Mattos, M.J. and Postma, P.W. (2005) Reducing the Glucose Uptake Rate in Escherichia coli Affects Growth Rate But Not Protein Production, Biotechnology and Bioengineering, 90, 191-200.

Plumbridge, J. (2002) Regulation of gene expression in the PTS in Escherichia coli: the role and interactions of Mlc, Current Opinion in Microbiology, 5, 187-193.

Postma, P., Lengeler, J. and Jacobsson, G. (1996) Phosphoenolpyruvate: carbohydrate phosphotransferase systems (PTS), In: Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology, Ed: Neidhardt F. C (ASM Press).

Ryan, W., Collier, P., Loredo, L., Pope, J. and Sachdev, R. (1996) Growth Kinetics of Escherichia coli and Expression of a recombinant Protein and Its Isoforms under Heat Shock Conditions, Biotechnology Progress, 12, 596-601.

Sandén, A.M., Boström, M., Markland, K. and Larsson, G. (2005) Solubility and Proteolysis of the Zb-MalE and Zb-MalE31 Proteins During Overproduction in Escherichia coli, Biotechnology and Bioengineering, 90, 239-247.

Sandén, A.M., Prytz, I., Tubulekas, I., Förberg, C., Le, H., Hektor, A., Neubauer, P., Pragai, Z., Harwood, C., Ward, A., Picon, A., Texiera de Mattos, J., Postma, P., Farewell, A., Nyström, T., Reeh,

70

S., Pedersen, S. and Larsson, G. (2002) Limiting Factors in Escherichia coli Fed-Batch Production of Recombinant Proteins, Biotechnology and Bioengineering, 81, 158-166.

Schmidt, F.R. (2004) Recombinant expression systems in the pharmaceutical industry, Applied Microbiology and Biotechnology, 65, 363-372.

Shiloach, J. and Fass, R. (2005) Growing E. coli to high cell density - A historical perspective on method development, Biotechnology Advances, 23, 345-357.

Shokri, A., Sandén, A.-M. and Larsson, G. (2002) Growth rate-dependent changes in Escherichia coli membrane structure and protein leakage, Applied Microbiology and Biotechnology, 58, 386-392.

Shokri, A., Sandén, A.-M. and Larsson, G. (2003) Cell and process design for targeting of recombinant proteins into the culture medium of Escherichia coli, Applied Microbiology and Biotechnology, 60, 654-664.

Singer, S.J. and Nicolson, G.L. (1972) The Fluid Mosaic Model of the Structure of Cell Membranes, Science, 175, 720-731.

Siurkus, J., Panula-Perälä, J., Horn, U., Kraft, M., Rimseliene, R. and Neubauer, P. (2010) Novel approach of high cell density recombinant bioprocess development: Optimasation and scale-up from microtitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures, Microbial Cell Factories, 9:35.

Sklar, J.G., Wu, T., Kahne, D. and Silhavy, T.J. (2007) Defining the roles of the periplasmic chaperones SurA, Skp and DegP in Escherichia coli, Genes and Development, 21, 2473-2484.

Sonoda, H., Kumada, Y., Katsuda, T. and Yamaji, H. (2011) Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli, Journal of Bioscience and Bioengineering, 111, 465-470.

Sorensen, H.P. and Mortensen, K.K. (2005) Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli, Microbial Cell Factories, 4:1.

Sørensen, H.P. and Mortensen, K.K. (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli, Journal of Biotechnology, 115, 113-128.

Stathopoulos, C., Georgiou, G. and Earhart, C., F (1996) Characterization of Escherichia coli expressing an Lpp´OmpA(46-159)-PhoA fusion protein localized in the outer membrane, Applied Microbiology and Biotechnology, 45, 112-119.

Stenström, M.C. and Isaksson, L.A. (2002) Influences on translation initiation and early elongation by the messenger RNA region flanking the initiation codon at the 3´side, Gene, 288, 1-8.

Studier, F.W. and Moffatt, B.A. (1986) Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-level Expression of Cloned Genes, Journal of Molecular Biology, 189, 113-130.

Swartz, J.R. (2001) Advances in Escherichia coli production of therapeutic proteins, Current Opinion in Biotechnology, 12, 195-201.

Tamaki, S., Sato, T. and Matsuhashi, M. (1971) Role of lipopolysaccharides in antibiotic resistance bacteriophage adsorption of Escherichia coli K-12, Journal of Bacteriology, 968-975.

Tegel, H., Ottosson, J. and Hober, S. (2011) Enhancing the protein production levels in Escherichia coli with a strong promoter, FEBS Journal, 278, 729-739.

Terpe, K. (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems, Applied Microbiology and Biotechnology, 72, 211-222.

Turner, C., Gregory, M.E. and Turner, M.K. (1994) A study of the effect of specific growth rate and acetate on recombinant protein production of Escherichia coli JM107, Biotechnology Letters, 16, 891-896.

van Bloois, E., Winter, R.T., Kolmar, H. and Fraaije, M.W. (2011) Decorating microbes: surface display of proteins on Escherichia coli, TRENDS in Biotechnology, 29, 79-86.

van de Walle, M. and Shiloach, J. (1998) Proposed Mechanism of Acetate Accumulation in Two Recombinant Escherichia coli Strains During High Density Fermentation, Biotechnology and Bioengineering, 57, 71-78.

71

Wagner, S., Baars, L., Ytterberg, J., Klussmeier, A., Wagner, C.S., Nord, O., Nygren, P.Å., van Wijk, K.J. and de Gier, J.-W. (2007) Consequences of Membrane Protein Overexpression in Escherichia coli, Molecular & Cellular Proteomics, 1527-1549.

Wagner, S., Klepsch, M.M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Högbom, M., van Wijk, K.J., Slotboom, D.J., Persson, J.O. and de Gier, J.-W. (2008) Tuning Escherichia coli for membrane protein overexpression, Proceedings of the National Academy of Sciences of the United States of America, 105, 14371-14376.

Wagner, S., Lerch Bader, M.L., Drew, D. and De Gier, J.W. (2006) Rationalizing membrane protein overexpression, TRENDS in Biotechnology, 24, 364-371.

Walton, T.A., Sandoval, C.M., Fowler, C.A., Pardi, A. and Sousa, M.C. (2009) The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains, Proceedings of the National Academy of Sciences of the United States of America, 106, 1771-1777.

Weibezahn, J., Bukau, B. and Mogk, A. (2004) Unscrambling an egg: protein disaggregation by AAA+ proteins, Microbial Cell Factories, 3:1.

White, C.B., Chen, Q., Kenyon, G.L. and Babbit, P.C. (1995) A novel activity of OmpT. Proteolysis under extreme denaturing conditions, The Journal of Biological Chemistry, 270, 12990-12994.

Wilson Bowers, C., Lau, F. and Silhavy, T.J. (2003) Secretion of LamB-LacZ by the Signal Recognition Particle Pathway of Escherichia coli, Journal of Bacteriology, 185, 5697-5705.

Wolfe, A.J. (2005) The acetate Switch, Microbiology and Molecular Biology Reviews, 69, 12-50.

Young, J.C., Agashe, V.R., Siegers, K. and Hartl, F.U. (2004) Pathways of chaperone-mediated protein folding in the cytosol, Nature Reviews Molecular Cell Biology, 5, 781-791.