strategies for improved escherichia coli bioprocessing ...810686/fulltext01.pdf · process...

Strategies for improved Escherichia coli bioprocessing performance

Johan Jarmander Tekn. Lic.

KTH Royal Institute of Technology Stockholm 2015

©Johan Jarmander 2015 KTH Royal Institute of Technology School of Biotechnology AlbaNova University Center 106 91 Stockholm Sweden Printed by Universitetsservice US-AB 2015 ISBN 978-91-7595-523-0 TRITA-BIO Report 2015:9 ISSN 1654-2312

“I see my path, but I do not know where it leads. Not knowing where I’m going is what inspires me to travel it.”

- Rosalia de Castro

Abstract Escherichia coli has a proven track record for successful production of anything from small molecules like organic acids to large therapeutic proteins, and has thus important applications in both R&D and commercial production. The versatility of this organism in combination with the accumulated knowledge of its genome, metabolism and physiology, has allowed for development of specialty strains capable of performing very specific tasks, opening up opportunities within new areas. The work of this thesis has been devoted to alter membrane transport proteins and the regulation of these, in order for E. coli to find further application within two such important areas. The first area was vaccine development, where it was investigated if E. coli could be a natural vehicle for live vaccine production. The hypothesis was that the introduction and manipulation of a protein surface translocation system from pathogenic E. coli would result in stable expression levels of Salmonella subunit antigens on the surface of laboratory E. coli. While different antigen combinations were successfully expressed on the surface of E. coli, larger proteins were affected by proteolysis, which manipulation of cultivation conditions could reduce, but not eliminate completely. The surface expressed antigens were further capable of inducing proinflammatory responses in epithelial cells. The second area was biorefining. By altering the regulation of sugar assimilation, it was hypothesized that simultaneous uptake of the sugars present in lignocellulose hydrolyzates could be achieved, thereby improving the yield and productivity of important bio-based chemicals. The dual-layered catabolite repression was identified and successfully removed in the engineered E. coli, and the compound (R)-3-hydroxybutyric acid was produced from simultaneous assimilation of glucose, xylose and arabinose. Keywords: E. coli, Salmonella, surface expression, autotransport, AIDA-I, lignocellulose, glucose, xylose, arabinose, simultaneous uptake, 3HB

Sammanfattning Inom bioteknik är bakterier viktiga organismer som används till att producera allt från kemikalier och plaster till tvättmedelsenzymer och läkemedel. En av de bakterier som används mest, både inom forskning och industri, är E. coli. Denna bakterie finns i både sjukdomsalstrande och ofarliga varianter. Till exempel ingår en ofarlig variant av E. coli i människans normala tarmflora, och det är de ofarliga varianterna som används av forskare och företag. E. coli upptäcktes redan 1885 och idag är kännedomen om dess fysiologi, ämnesomsättning, arvsmassa med mera omfattande. Detta gör att det är möjligt att modifiera denna bakterie till att utföra uppgifter och producera molekyler som den normalt sett inte gör. I denna avhandling har E. coli celler modifierats för att utföra två specifika uppgifter, och på så sätt bredda användningsområdet för denna organism inom bioteknik. Det första användningsområdet är vaccinutveckling där uppgiften var att få E. coli celler att presentera proteiner från Salmonella på cellytan. Detta i syfte att utveckla ett E. coli-baserat vaccin mot just Salmonella. Flera olika Salmonella proteiner kunde framgångsrikt presenteras på cellytan hos E. coli i denna avhandling, och dessa gav dessutom upphov till ett proinflammatoriskt svar i mänskliga celler. Det upptäcktes dock att stora proteiner bröts ner och inte presenterades i sin kompletta form på cellytan. Detta problem kunde minskas genom förändrade cellodlings-betingelser, men inte elimineras. Det andra användningsområdet är bioraffinering där uppgiften var att få E. coli celler att ta upp och växa på en kolkälla bestående av en blandning av de socker som normalt återfinns i växtmaterial. I bioraffinaderier används bakterier för producera kemikalier och bränslen från växtmaterial, och för att kunna övergå till en biobased samhällsekonomi där bioraffinaderier ersätter oljeraffinaderier är det viktigt att alla socker i växtmaterialet kan tas upp och omvandlas till en produkt. För att åstadkomma simultant upptag av olika sockerarter i E. coli så eliminerades det regulatoriska system som styr i vilken ordning olika socker tas upp. E. coli celler som framgångsrikt tog upp flera olika socker samtidigt kunde sedan också användas till att producera kemikalien hydoxybutyrsyra från en blandad sockerlösning.

List of publications This thesis is based on the following publications, which are referred to in the text by their roman numerals: I. Jarmander J, Gustavsson M, Do T-H, Samuelson P, Larsson G (2012): A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli. Microbial Cell Factories 11:118 II. Jarmander J, Janoschek L, Lundh S, Larsson G, Gustavsson M (2014): Process optimization for increased yield of surface-expressed protein in Escherichia coli. Bioprocess and Biosystems Engineering 37:1685-1693 III. Jarmander J, Hallström B M, Larsson G (2014): Simultaneous uptake of lignocellulose-based monosaccharides by Escherichia coli. Biotechnology and Bioengineering 111:1108-1115 IV. Jarmander J, Belotserkovsky J, Sjöberg G, Guevara-Martínez M, Pérez-Zabaleta M, Quillaguamán J, Larsson G (2015): Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli. Microbial Cell Factories 14:51 V. Gustavsson M, Jarmander J, Arvestad L, Larsson G (2015): Extended signal peptides in autotransporters are associated with large passenger proteins. Manuscript

Contributions of papers I. Wrote the manuscript and performed the majority of the experiments together with M. Gustavsson. II. Contributed to the manuscript and was together with M. Gustavsson responsible for the original concept, the planning and the execution of the majority of the experiments. III. Wrote the manuscript and performed the majority of the experiments. IV. Wrote the manuscript and performed the majority of the experiments. V. Contributed to the manuscript and the experiments.

Table of contents

1 INTRODUCTION .......................................................................................... 1 1.1 Outline of this thesis ................................................................................ 1 1.2 Escherichia coli ........................................................................................ 2 1.3 E. coli and production of biopharmaceuticals ...................................... 3 1.3.1 Protein production through bioprocessing ............................................. 3 1.3.1.1 Industrial enzymes ................................................................................. 4 1.3.1.2 Therapeutic proteins ............................................................................. 4 1.3.2 Production organisms ............................................................................. 5 1.3.3 The market of E. coli produced biopharmaceuticals .............................. 6 1.3.4 The importance of post-translational modifications ............................... 8 1.3.5 Future potential for E. coli ....................................................................... 9 1.3.5.1 PEGylated therapeutics ......................................................................... 9 1.3.5.2 Antibody fragments ............................................................................. 10 1.3.5.3 Vaccines ............................................................................................. 11 1.3.5.3.1 Live attenuated vaccines ......................................................................... 12 1.3.5.3.2 Inactivated vaccines ................................................................................ 13 1.3.5.3.3 Subunit vaccines ..................................................................................... 13 1.3.5.3.4 Recombinant vaccines ............................................................................ 14 1.3.5.3.5 Cell-based delivery systems .................................................................... 14 1.3.5.3.6 Surface expression in E. coli .................................................................... 16 1.3.5.3.7 Autotransport .......................................................................................... 17 1.3.5.3.8 Translocation mechanism and folding ..................................................... 19 1.3.5.3.9 The adhesin involved in diffuse adherence .............................................. 20 1.3.5.3.10 Proteolysis of surface expressed proteins ............................................. 21 1.4 E. coli and production of chemicals and fuels .................................... 22 1.4.1 Returning to a bio-based economy ...................................................... 22 1.4.1.1 Dependency on fossil resources .......................................................... 22 1.4.1.2 Petroleum price fluctuation .................................................................. 24 1.4.1.3 Government directives ........................................................................ 25 1.4.2 Production organisms ........................................................................... 26 1.4.2.1 Tools to design improved organisms ................................................... 27 1.4.3 Current Industrial bioprocesses and products ..................................... 28 1.4.3.1 Fuels ................................................................................................... 28 1.4.3.1.1 Ethanol ................................................................................................... 28 1.4.3.1.2 Butanol ................................................................................................... 28 1.4.3.1.3 Diesel ...................................................................................................... 29 1.4.3.2 Chemicals ........................................................................................... 29 1.4.3.2.1 Acetic acid .............................................................................................. 30 1.4.3.2.2 Lactic acid .............................................................................................. 31 1.4.3.2.3 Polyhydroxyalkanoates ............................................................................ 32

1.4.3.2.4 1,3-Propanediol ...................................................................................... 32 1.4.3.2.5 Succinic acid .......................................................................................... 33 1.4.4 The vision of a biorefinery ..................................................................... 33 1.4.4.1 Biorefinery classification ...................................................................... 34 1.4.4.2 Lignocellulose, a cruder raw material ................................................... 35 1.4.4.3 Lignocellulose sugar composition ........................................................ 36 1.4.4.4 Pretreatment of lignocellulose .............................................................. 37 1.4.4.5 Obstacles to lignocellulose biorefineries .............................................. 38 1.4.4.5.1 Inhibitory components ............................................................................ 38 1.4.4.5.2 Co-assimilation of glucose and pentoses ................................................ 39 1.4.5 Regulation of sugar uptake in E. coli .................................................... 40 1.4.5.1 Catabolite repression and inducer exclusion ........................................ 40 1.4.5.2 Regulation of pentose uptake and metabolism .................................... 42

2 PRESENT INVESTIGATION ...................................................................... 45 2.1 Aim .......................................................................................................... 45 2.2 E. coli live vaccines (papers I, II and V) ................................................ 46 2.2.1 Previous results ..................................................................................... 46 2.2.2 An improved surface expression vector (Paper I) ................................. 47 2.2.3 Influence of cultivation conditions (Paper II) ......................................... 52 2.2.4 Engineered Salmonella proteins ........................................................... 55 2.2.5 Proinflammatory response from Salmonella proteins ........................... 57 2.2.6 Surface expression of large passenger proteins .................................. 59 2.2.7 The signal peptide and passenger size (Paper V) ................................. 60 2.2.8 Summary of surface expression results ................................................ 63 2.3 Lignocellulose biorefineries (Papers III and IV) ................................... 64 2.3.1 Simultaneous uptake of sugars (Paper III) ............................................ 64 2.3.2 Production of (R)-3-hydroxybutyric acid (Paper IV) .............................. 67 2.3.3 The benefit of nitrogen depletion .......................................................... 73 2.3.4 Strategies to further improve production of 3HB .................................. 73 2.4 Concluding remarks .............................................................................. 77

3 ACKNOWLEDGEMENTS ........................................................................... 81

4 ABBREVIATIONS ....................................................................................... 85

5 REFERENCES ............................................................................................ 89

Johan Jarmander | 1

1 Introduction 1.1 Outline of this thesis The thesis will begin by describing the current state of the art in bio-based production of therapeutic proteins, chemicals and fuels, and the role that Escherichia coli plays in production of these. Emerging opportunities within new areas are recognized and the topic of the present investigation is the development of E. coli cells with novel, specific abilities, allowing bioprocessing with this organism to be expanded within two such important areas. The first area is vaccine development, where it was hypothesized that Salmonella antigens could be expressed on the surface of laboratory E. coli in order to create a combined production and delivery system of low-cost, well-defined live vaccines. The idea was that the cells would help in inducing a stronger immune response and prolonging the half-life of the expressed antigens, compared to if they would have been distributed as subunit vaccines. The second area is biorefining, where it was investigated if simultaneous uptake of the sugars present in lignocellulose hydrolyzates could be achieved in E. coli. Since current production organisms cannot simultaneously consume lignocellulose sugars, the hypothesis was that such an E. coli could act as a platform strain for production of important bio-based chemicals, thereby improving the yield and productivity.

2 | Strategies for improved Escherichia coli bioprocessing performance

1.2 Escherichia coli The Gram-negative, facultative anaerobic bacterium E. coli that was discovered in 1885 by Theodor Escherich [1] has a long history of laboratory use and is today considered “the” model organism of biotechnology [2]. It was in E. coli that DNA cloning was developed, and once it was realized that functional recombinant proteins could be produced, the potential of this bacterial production system was unveiled [3-8]. Since then, E. coli has been used for production of a multitude of compounds, ranging from small organic acids to large complex proteins. Today, it is used both within R&D and commercial production, and the reason that E. coli still is so popular for a variety of tasks can be explained with the fact that it has a number of distinct advantages:

1) E. coli cells can divide as fast as in 20 minutes under optimal growth conditions, compared to 1-2 h for Saccharomyces cerevisiae, and up to 24 h for mammalian cells [9].

2) In contrast to many other organisms, E. coli is capable of synthesizing all essential amino acids, purines, pyrimidines and vitamins. This means that it can grow on inexpensive minimal salt medium supplemented with a simple carbon source [10].

3) E. coli grows well in a broad span of cultivation conditions, such as temperature and pH. For example mammalian cells are very sensitive in this regard.

4) The E. coli genome is well characterized. It was one of the first genomes to be sequenced almost 20 years ago [11], and it can be readily altered to change cell abilities, as well as to express heterologous proteins [5].

5) There is a vast amount of accumulated knowledge on E. coli physiology and metabolism.

6) Most laboratory E. coli strains, including the widely used K-12 and BL21, are harmless and lack virulence characteristics [12].

7) High cell density cultures are easily achieved [13], subsequently making high protein productivities achievable.

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Taken together, this gives that E. coli is used for a variety of tasks, such as molecular cloning, high throughput protein screening, bioremediation and commercial production of proteins and chemicals. The scope of this thesis is however limited to the role E. coli plays in production of proteins (particularly those with therapeutic applications), and chemicals.

Figure 1: Escherichia coli: this facultative anaerobic, Gram-negative, rod-shaped bacterium is here shown in a scanning electron microscopy image.

1.3 E. coli and production of biopharmaceuticals 1.3.1 Protein production through bioprocessing Proteins that are commercially produced through bioprocessing include industrial enzymes, diagnostic and therapeutic proteins, as well as proteins for research applications. Industrial enzymes and therapeutic proteins are the two main product categories and these are further discussed below while the others will not be covered in this thesis.

200 nm


1.3.1.1 Industrial enzymes Industrial enzymes are predominantly hydrolases and isomerases directed towards detergent, food and starch processing industries. They are therefore mostly low-cost commodities that the production organisms need to be able to produce in high titers in order to achieve economical processes [14]. This can be exemplified by detergent protease, which in the year of 2000 was estimated to be produced at 100 million tons at a value of 3 € per kg [14]. E. coli is not particularly well-suited for production of these high volume proteins, as one of the disadvantages with this organism is that it is difficult to produce proteins to high concentration, and that they tend to aggregate in intracellular inclusion bodes [15, 16]. Instead, there are other organisms that excel at this task, two examples being Bacillus bacteria and Aspergillus fungi [17]. The advantage these organisms hold over E. coli is that they naturally produce and secrete several industrially important enzymes to the medium. Through overexpression strategies, high titers can further be reached. This enables Bacillus and Aspergillus to be utilized for commercial production of low cost/large volume industrial enzymes. Examples of enzymes produced by these organisms are proteases and amylases by Bacillus [18, 19], and glucoamylases, pectinases and cellulases by Aspergillus [14, 17]. Although E. coli often is not the preferred choice for production of industrial enzymes, there still exist processes where this organism is being used. One example is in the hydrolysis of penicillin G to 6-aminopenicillanic acid, which is catalyzed by immobilized penicillin amidase that is homologously produced in E. coli [14, 17, 20]. 1.3.1.2 Therapeutic proteins Proteins have been considered important drugs since early 1900s, but their original production required harvesting of plant or animal material, which was expensive, time consuming, and not always possible [21]. Production of therapeutic proteins through bioprocessing is a faster method that also can be done at a lower cost. Therapeutic proteins are different from industrial enzymes since a majority of them are of eukaryotic origin and are produced

Johan Jarmander | 5

by recombinant techniques, while further being much more valuable than enzymes. Biopharmaceutical production processes are consequently not as sensitive to parameters such as yield and productivity, but a demand on the production organism is that it easily can incorporate, alter and express foreign DNA. As recombinant DNA technology originally was developed in E. coli, it has resulted in that this organism historically has been the most extensively used for production of recombinant therapeutic proteins [22]. The first recombinant biopharmaceutical to reach the commercial market was E. coli produced recombinant insulin, sold by Eli Lily and Genentech in a joint venture, which was approved by the U.S. Food and Drug Administration (FDA) in 1982 [23]. 1.3.2 Production organisms The pharmaceutical industry has constantly strived to increase its assortment of protein therapeutics, and the desire to produce more structurally complex proteins has led to the development of additional production organism. The reason for this is that E. coli lacks the capability of performing many of the post-translational modifications (PTMs) required by these proteins. Today, the most widely used production organisms are [22, 24-26]:

• E. coli • Yeast (primarily S. cerevisiae and Pichia pastoris) • Chinese hamster ovary (CHO) cells • Human cells

The current biopharmaceutical portfolio includes everything from hormones and growth factors to immunotherapy products and vaccines [27]. This diversity in origin, size, action etc. between products gives that there is no single organism that is suitable for production of all of them. The choice of production organism for a new protein therapeutic instead has to be made on a case-to-case basis, where the requirements of the specific protein have to be carefully evaluated against the capabilities of the different organisms.


1.3.3 The market of E. coli produced biopharmaceuticals Of the 246 biopharmaceutical products that have been approved for commercial production in the United States (U.S.) or the European Union (EU) between 1982 and 2014, 71 (equivalent to 29%, figure 2 A), have been produced in E. coli [26]. Only CHO cell produced pharmaceuticals have a bigger market share of 33%. However, even if E. coli historically has been one of the most widely used production systems, in the last five-year period there has been a decrease in the number of E. coli derived products (figure 2 B). Only 10 of the 54 biopharmaceuticals that were approved between 2010 and 2014 utilize E. coli (table 1). This translates into 19%, to compare with 24% for the period of 2004-2013 [28], and 29% for 1982-2014 (figure 2 A). While E. coli still was the second most used organism between 2010-2014 (although not by much), this reveals a negative trend over the last decade. One explanation to that E. coli seems to become a less attractive choice to other biopharmaceutical production systems is the strong dominance of monoclonal antibodies (mABs) on the market, for which there were 17 (30%) approved products between 2010 and 2014 [26]. Monoclonal antibodies are complex proteins that are made up of several peptide chains, and contain both disulfide bridges and glycosylated motifs [29], two PTMs that cannot innately be made by E. coli in its cytoplasm. These proteins are therefore very difficult to produce in E. coli, a task that has not been successfully achieved yet [30]. Instead, they are produced in mammalian cells, and specifically CHO cell lines have been used [26].

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Figure 2: The number of biopharmaceuticals approved for sale by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) in different expression systems between 1982 and 2014 (A), and between 2010 and 2014 (B) [26].

71

41

81

6

39

8

Num

ber o

f app

rova

ls

0

20

40

60

80

100

E. coli Yeast CHO Human Misc. mammalian Others

A

109

19

2

9

5Num

ber o

f app

rova

ls

0

5

10

15

20

25

E. coli Yeast CHO Human Misc. mammalian Others

B


Table 1: E. coli produced biopharmaceuticals approved for commercial production by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) between 2010 and 2014 [26]. G-CSF = Granulocyte colony stimulating factor, GLP-2 = Glucagon-like peptide-2, r = recombinant, rh = recombinant human.

1.3.4 The importance of post-translational modifications It is generally the fact that E. coli does not perform any eukaryotic PTMs that have made companies seek alternative production systems. If the therapeutic of choice does not contain any PTMs, E. coli is often the preferred host since mammalian cells are slow growing, sensitive to bacterial infections and shear stress, and require highly complex growth media [31]. The biopharmaceutical companies are also often at the mercy of the growth medium supplier, as the exact formula rarely is disclosed, thus withholding them total control of their own processes. In addition to this, the downstream processing of mammalian cultures is usually more costly to ensure removal of viruses [22]. However, for those eukaryotic proteins that have them, the PTMs tend to be important because they influence many characteristics such as folding, biological activity, stability and immunogenicity. Glycosylation [32] is the most widespread of these and approximately 50% of all human proteins are

Product name Substance Company Approved

Afrezza rh insulin MannKind 2014

Myalept rh leptin AstraZeneca 2014

Krystexxa PEGylated r urate oxidase BioMarin 2014

Grastofil rh G-CSF Apotex 2013

Lonquex PEGylated rh G-CSF Teva Pharmaceuticals 2013

Bexsero Subunit vaccine Novartis 2013

Voraxaze r carboxypeptidase BTG International 2012

Granix rh G-CSF Teva/Cephalon 2012

Gattex/Revestive rh GLP-2 analog NPS Pharma 2012

Nivestim rh G-CSF Hospira 2010

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affected by this PTM [33]. Other PTMs that are important in the context of therapeutic proteins are disulfide bridge formation and proteolytic maturation [34]. Innately, E. coli performs neither of these in its cytoplasm, and despite that the amino acid sequence of a recombinant protein that has been produced in E. coli is identical to that of the eukaryotic wild type, missing PTMs might result in lower, or no activity at all. This is however not always the case, and the impact of missing PTMs needs to individually evaluated for each potential protein therapeutic. Recombinant human interleukin-2 [35] and recombinant human interferon-β [26, 34] are two examples of therapeutic glycoproteins that are produced in E. coli and does not display any diminished biological activity, despite that they are lacking the glycosylation motifs. They do nonetheless have shorter half-lives than the wild type proteins. 1.3.5 Future potential for E. coli Although E. coli seems to become a less and less attractive choice to other biopharmaceutical production systems, there is extensive research devoted to further developing this organism and addressing its shortcomings, as well as to adapt therapeutics to make them easier to produce. This could potentially give E. coli a growing use as a production organism in the future. This section assesses some of the most interesting new developments in production of recombinant therapeutics by E. coli. 1.3.5.1 PEGylated therapeutics To compensate for the fact that E. coli does not perform any eukaryotic PTMs, a synthetic PTM called PEGylation has been developed [36]. PEGylation is a process in which polyethylene glycol (PEG) units are attached to a protein in vitro (figure 3). It has been shown to provide several advantageous traits, primarily prolonged half-life, but also increased solubility, reduced immunogenicity and resistance to proteolysis [37]. Today, there are several therapeutics on the market that utilize PEGylation, mainly in the categories of human growth hormone, colony stimulation factors, interferons, enzymes and antibody (AB) fragments [26]. Two of the E. coli produced biopharmaceuticals approved for commercial production since


2010 are further PEGylated: Krystexxa and Lonquex (table 1). PEGylation is thus a promising modification that has contributed to expanding the portfolio of E. coli produced biopharmaceuticals. However only time will tell if it becomes the success that keeps E. coli an attractive organism for biopharmaceutical production in the future.

Figure 3: PEGylation is a synthetic posttranslational modification where polyethylene glycol (PEG) units are coupled to a protein through a linking group (e.g. NH, NH2, OH, SH and COOH) [36]. PEGylation can have several positive effects on the protein, primarily increased half-life. 1.3.5.2 Antibody fragments A product category of great potential for E. coli is AB production. Over 30% of the approved biopharmaceuticals in the last 5 years belong to this category, and almost all of them were mABs produced in CHO cells. However, since mammalian cell systems are expensive and time-consuming to use, there has been a desire to change to a more simple production system, but due to their complexity and requirement of correct glycosylation, mABs are currently not possible to produce in E. coli. Therefore, the possibility of producing smaller antigen-binding units has been investigated [26]. The focus has been on production of the antigen-binding fragment (Fab) [38] of an AB, as well as single-chain variable fragments (scFvs) (figure 4) [39]. Both of these can be produced in E. coli, and they can penetrate tissues and tumors more easily than mABs. AB fragments can thus reach targets such as cryptotopes, which mABs, due to their size, cannot [40-42]. The negative aspects of AB fragments are decreased half-life and possibly lower stability, which however can be improved through PEGylation [43]. AB fragments can further not induce effector functions in the immune system, as the required Fc domain is not present, which limits their use to

Protein

PEGLinker

Johan Jarmander | 11

treatments where this effect is nonessential. As of January 2014, three Fabs were available on the market: ReoPro (Eli Lilly), Lucentis (Genentech) and Cimzia (UCB Pharma) [26, 44]. It is yet too early to realize the impact of AB fragments on the biopharmaceutical market, but it appears to be a promising future product category for E. coli.

Figure 4: An IgG antibody is built up of two heavy chains (dark green), each with its attached light chain (light green). The heavy chains contain three constant domains (CH1-CH3) and one variable domain (VH), while the light chains contain one constant (CL), and one variable domain (VL). The antigen-binding fragment (Fab) is made up of the CH1, VH, CL and VL domains, while the single-chain variable fragment (scFv) consists of the linked VH and VL domains [38, 39]. 1.3.5.3 Vaccines Vaccine production is another area in which E. coli has the potential to be used as the production, and also the delivery system. Vaccines can be classified as either active or passive, where active vaccines are intended to stimulate the immune system and passive vaccines are AB solutions given around the time of a known or potential infection [45]. It is the active vaccines that will be discussed here and these can further be broadly divided into three sub-classes: live attenuated, inactivated (killed) and subunit vaccines [45, 46]. Table 2 lists the bacterial and viral vaccines that so far have been approved from human use.

CH1

CH2

CH3

VH

CL

VL

Heavychain

Lightchain

IgG (~ 150 kDa)

CH1

VH

CL

VL

Fab (~ 55 kDa)

VH

VL

scFv (~ 30 kDa)

Fc


Table 2: Bacterial and viral vaccines that have been approved for human use [26, 47]. Legend: p = protein, ps = polysaccharide, psc = polysaccharide conjugate, r = recombinant. BCG = Bacillus Calmette-Guérin.

1.3.5.3.1 Live attenuated vaccines Live attenuated vaccines consist of viruses or bacteria that have been attenuated in their pathogenicity, which traditionally has been achieved through repeated culturing [46]. Since these vaccines still are alive, they can potentially replicate in the host [45]. Live vaccines are among the most potent as the immune response that they generate is practically identical to that produced by natural infections. These vaccines are however associated with several potential risks. For one, the genetic mutations responsible for

Live attenuated Inactivated Subunit

Bacter

ial

Vaccine type

Tuberculosis (BCG) Bordetella pertussis Bordetella pertussis (acellular)

Salmonella typhi (r) Bacillus anthracis Cholera (partly r p)

Cholera Diphtheria (toxid)

Haemophilus influenzae type b (ps)

Neisseria meningitidis (ps)

Neisseria meningitidis (psc)

Neisseria meningitidis (partly r p)

Salmonella typhi Vi (ps)

Streptococcus pneumoniae (ps)

Streptococcus pneumoniae (psc)

Tetanus (toxoid)

Viral

Bacter

ial

Influenza (r) Polio Hepatitis B (r p)

Measles Rabies Human papillomavirus (r p)

Mumps Influenza Influenza (r p)

Polio Hepatitis A

Rotavirus

Rubella

Vaccinia

Yellow fever

Viral


the attenuation were generally not characterized, which increases the chance of reversion into the pathogenic form [47]. There is also always a risk of patient side effects, ranging from headache to anaphylactic reactions and even death, depending on the vaccine in question [48]. 1.3.5.3.2 Inactivated vaccines Inactivated vaccines consist of cultivated pathogens that traditionally have been killed through heat or chemical treatment (usually formalin) [46]. Inactivated vaccines are safer than live attenuated vaccines, but care needs to be taken so that the pathogen is effectively killed without completely destroying the antigens [45]. These vaccines are typically weaker than live attenuated vaccines and generally needs to be administered in larger doses and more frequently to be effective [49]. Their immunogenicity may also need to be enhanced by an adjuvant system [45]. Because of the requirements put forward by regulatory bodies such as FDA and World Health Organization (WHO) on exact vaccine definitions, it may be harder to get live attenuated and inactivated vaccines that have been prepared by traditional methodology accepted for use in the future [45, 50]. 1.3.5.3.3 Subunit vaccines In subunit vaccines, only a part of the pathogen is used to induce an immune response [51]. This can be a peptide, a protein, a polysaccharide or even a DNA segment. Subunit vaccines have the advantage that they are clearly defined and offer a high level of safety since they cannot replicate in the host [45]. The negative aspect of subunit vaccines is that they are not very potent and often require an adjuvant and multiple doses. Typical adjuvants are aluminum salts, for example in administration of the vaccines against human papillomavirus (HPV) and hepatitis B [50, 52]. However, the mechanism by which aluminum salts enhance the immunogenicity of the vaccines has not fully elucidated, and the investigation of new adjuvants is a key area in current vaccine development. A method that has been used to enhance the effect of polysaccharide subunit vaccines is to conjugate the vaccine to a carrier protein, which leads to increased immunogenicity and a prolonged


immune response [53]. Conjugate subunit polysaccharide vaccines on the market are directed towards Neisseria meningitidis and Streptococcus pneumoniae (table 2) [47]. 1.3.5.3.4 Recombinant vaccines Seven of the vaccines that have been approved for human use are produced by recombinant technology (table 2) [26, 54]. These are directed against Salmonella typhi, Neisseria meningitidis, Cholera, Influenza (two vaccines), HPV and hepatitis B. Two of them are live vaccines: the one against S. typhi, consisting of live recombinant cells no longer causing illness, and also one of the influenza vaccines, consisting of attenuated viruses that only replicate in the throat and not in the lungs. Against influenza, there also exists a recombinant subunit vaccine, containing several recombinant proteins that are produced in insect cells [55]. The HPV and hepatitis B vaccines are also subunit vaccines, where virus-like particles are recombinantly produced in yeast or insect cells [47, 54]. The cholera vaccine in turn is only partly recombinant as it consists of both recombinant cholera toxin subunit B, produced in Vibrio cholera, as well as inactivated cells [26]. The newest recombinant vaccine is the one against Neisseria meningitidis (approved in 2013), which contains several proteins that are produced in E. coli [56]. 1.3.5.3.5 Cell-based delivery systems In order to arrive at simpler and less costly production processes of vaccines, live cells have been investigated as combined production and delivery systems of subunit vaccines [45, 47]. The idea is that the organism presents the antigen on its cell surface, a method known as surface expression or surface display, and its inherent immunological properties enhance the immune response, much like an adjuvant or carrier protein (figure 5) [57, 58]. Bacteria that have been investigated as vectors for display of subunit vaccines are attenuated or non-pathogenic strains of primarily Mycobacterium (BCG), Staphylococcus, Salmonella and E. coli [59].


E. coli was the first organism for which surface expression was demonstrated in the years of 1986-1987 [60-62]. A wide range of proteins has been expressed on the E. coli cell surface since then, including some antigenic proteins [45]. Examples of these are: type-I poliovirus coat fragments, hepatitis B antigens, AB fragments and the Leishmania major glycoprotein gp63 [63-66]. Because of the vast knowledge that has been gathered on surface expression in E. coli, this organism is a promising candidate to use for delivery of subunit vaccines. The lipopolysaccharides found in the outer membrane of E. coli can further help to provoke a stronger immune response [67]. E. coli live vaccines have the advantage that they can be administered orally or nasally, and also that they can be produced at a much lower cost than traditional vaccines, due to reduced purification costs. This enables such vaccines to be used primarily for veterinary purpose in large-scale immunization of animals, where it is vital that the vaccine is inexpensive and easy to distribute. Since the vaccine can be delivered orally, it can simply be mixed with animal feed. A specific application for E. coli live vaccines is in vaccination against Salmonella among chickens. Especially the Salmonella enterica serovar Enteritidis (S. Enteritidis, [68]) is of interest, since it is a major cause of Salmonella infections [69] and is the only human pathogen to regularly infect chicken eggs [70], often without the hen showing any symptoms [71]. It is thus important to prevent infection of eggs by S. Enteritidis, and this can potentially be achieved by the development of an E. coli live vaccine in which S. Enteritidis subunit antigens are expressed on the cell surface. Expression systems developed for E. coli (such as the one described in this thesis), might even be possible to transfer to Salmonella spp. to enable the development of live attenuated Salmonella vaccine vehicles that would confer protection to S. Enteritidis.


Figure 5: The principle of surface expression, where a naturally surface-occurring protein is exploited to co-express a recombinant protein. 1.3.5.3.6 Surface expression in E. coli Surface expression has been investigated for several organisms, including Gram-negative and Gram-positive bacteria, as well as for yeast [72-74]. The surface expression field has however previously been reviewed by the author [75] and this thesis is limited to surface expression in E. coli. Initial surface expression studies consisted of display of small peptide inserts in the outwards-facing loops of E. coli outer membrane proteins (Omps), such as OmpA, LamB and PhoE [60-62] (figure 6). This method was however mostly restricted to shorter peptides rather than folded, globular proteins, which would disrupt the function of the Omp [76]. Other proteins that have been the subject of surface expression studies are the flagella and fimbriae protein complexes. These were considered interesting since their principal building blocks, flagellin and fimbrillin, contain regions of high variability that seem unimportant for biological function. It was reasoned that these regions likely could be deleted and replaced by a recombinant insert, which also was also proven to be possible for E. coli [77, 78]. Like for Omps, flagella and fimbriae surface expression is however limited by the size of the insert, and a maximum length of 25-60 amino acids (aa) has been suggested [57, 58, 79].

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Figure 6: Initial surface expression experiments were focused on displaying recombinant peptides in the outwards-facing loops of outer membrane proteins, such as OmpA, LamB and PhoE [60-62]. Two protein families that have been suggested to handle surface expression of larger, globular proteins, are the ice-nucleation proteins (Inps) and the autotransporters (ATs). The Inp of Pseudomonas syringae has for example been used to express the Zymomonas mobilis levansucrase (46.7 kDa) on the surface of E. coli [80]. ATs also seem promising for surface expression applications in E. coli, as several proteins, mainly enzymes, have been successfully expressed from this system [81, 82]. 1.3.5.3.7 Autotransport ATs make up the Va subgroup of the type V secretion system and is with 8209 sequences listed in Pfam the largest family of secreted proteins in Gram-negative bacteria [82-84]. They are intricate multi-domain proteins that contain all required information for transport over both cell membranes. Their domains have distinct functions and are arranged in the following order (from the N-terminal): signal peptide (SP), passenger protein, autochaperone, α-helix and β-barrel domain (figure 7). The N-terminal SP directs the protein for transport over the inner membrane through the SEC system. The passenger protein, which varies between different ATs, is responsible for the function of the protein, and is typically replaced with the recombinant protein one wishes to express. The autochaperone is technically a part of the passenger, but

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this highly conserved region appears important for folding of the passenger protein, and is therefore often denoted as an individual domain [85]. The α-helix domain links the autochaperone to the β-barrel, which in turn becomes integrated into the outer cell membrane and is responsible for translocating the passenger over the outer membrane [86].

Figure 7: (Left) Domain arrangement of a typical autotransporter pre-protein with a N-terminal signal peptide, passenger protein, autochaperone, α-helix and β-barrel domain. (Right) Protein structures of the Hemoglobin-binding protease (Hbp) passenger, autochaperone, α-helix and β-barrel domains [87, 88] (PDB id 1WXR and 3AEH).

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1.3.5.3.8 Translocation mechanism and folding The mechanism, by which ATs travel from the cytoplasm to the cell exterior appears simple, but is a complex process that has not yet been fully elucidated. After translation in the cytoplasm, the SP is recognized by the SecB chaperone, which keeps the pre-peptide in an unfolded state and directs it to the Sec translocase (figure 8) [89]. The pre-peptide there gets transferred to the ATPase SecA, and through hydrolysis of Adenosine triphosphate (ATP), the protein is translocated though the SecYEG core [90]. Once in the periplasm, the SP is cleaved off by a signal peptidase [91] and it is likely that chaperones such as SurA, Skp and DegP interact with the protein to avoid premature folding [92, 93]. To pass the outer membrane, the C-terminal β-barrel domain inserts itself into the membrane, likely by interaction with the β-barrel assembly machinery (BAM) [94]. BAM recognize a motif in β-barrel proteins, but the mechanism by which it assists the membrane insertion of the β-barrel is not known [95]. Once the β-barrel has been inserted into the outer membrane, the passenger is translocated through the central pore. Based on the structure of the β-barrel, it seems that the passenger needs to be in an unfolded state prior to the translocation in order to fit in the narrow pore [96]. There are several theories for the actual translocation mechanism, of which the predominant is called the hairpin model [97, 98]. In this model, the passenger is pulled through the pore with its C-terminal end first, forming a hairpin at the cell surface. The issue with this model is what drives the translocation, but it has been suggested to be due to successive folding of the passenger in a C-to-N-terminal direction after it has reached the cell surface, starting at the autochaperone domain [99-102]. Correct folding of the autochaperone might thus be essential for translocation of the rest of the passenger protein. Once at the cell surface, the folded passenger either remains covalently attached to the β-barrel or is cleaved off [98]. Almost all structure-determined passengers fold in a distinct motif of right-handed β-helices (EstA is a rare exception [103]), and the functionality is often attained through loops or additional globular domains.


Figure 8: The secretion mechanism of autotransporters. (I) The SecB chaperone recognizes the signal peptide and keeps the pre-peptide in an unfolded state while directing it to the SEC translocon. (II) At the expense of ATP, the peptide is pushed through the SecYEG complex, and once in the periplasm, a signal peptidase cleaves off the signal peptide. (III) Chaperones keep the peptide in an unfolded state while it passes the periplasm, after which the β-barrel gets inserted into the outer membrane. (IV) The unfolded passenger can thereafter pass the membrane through the formed pore, and either stay covalently bound to the β-barrel or become cleaved off. Legend: green = signal peptide, orange = passenger protein, pink = α-helix, blue = β-barrel. 1.3.5.3.9 The adhesin involved in diffuse adherence The adhesin involved in diffuse adherence (AIDA-I) is one of the ATs that has been used for surface expression experiments [82]. It was first discovered in enteropathogenic E. coli in 1989, and has been transferred to laboratory strains [104]. Due to being E. coli native, it has been widely studied and used to express several different recombinant proteins [105-108].

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AIDA-I is synthesized as a 1286 aa long pre-peptide that becomes autoproteolyzed at the cell surface, yielding a 797 aa (79.5 kDa) adhesin passenger and a 440 aa (47.5 kDa) translocator domain called AIDAC [97, 109]. The proteolysis is likely catalyzed by two acidic aa, Glu897 and Asp878, and can be negated by a single aa mutation [110]. After it becomes proteolyzed, the adhesin stays associated to the cell surface, but heat treatment releases it into solution [111]. AIDA-I is further one of few glycosylated proteins found in E. coli, where O-linked heptose residues give the mature passenger a size of approximately 100 kDa [112, 113]. 1.3.5.3.10 Proteolysis of surface expressed proteins Recombinant proteins that have been expressed on the cell surface by autotransport have typically been detected by using antiserum or ABs specific towards the passenger protein [106, 114-116]. By the addition of a secondary, labeled AB, the surface expression results could be evaluated by western blot analysis, and it was in some cases revealed that the passenger proteins had been subjected to proteolytic degradation. Using passenger-specific ABs does however not provide the full picture of the proteolytic pattern, as any proteolysis that has taken place on the C-terminal side of the passenger will go unnoticed. This is illustrated in one study, where both the recombinant passenger and the AIDAC domain was detected on western blot [107]. From the western blot of the passenger, no proteolysis could be seen, but from the western blot of AIDAC, several smaller-sized bands were detected, thereby confirming that proteolysis in fact had taken place. To fully understand the proteolytic pattern of surface expressed proteins, it is thus imperative that detection is performed not only directly of the passenger, but also of domains located closer to the C-terminal end of the AT.


1.4 E. coli and production of chemicals and fuels 1.4.1 Returning to a bio-based economy Biotechnology was one of the technologies applied by the first chemical industries, and most bulk chemicals such as fuels and organic acids, were derived from biomass up until 1930 [117]. However, with the discovery of petroleum and subsequent development of petrochemistry in the middle of the 20th century, biomass usage declined rapidly. Renewable substrates could not compete in price with the more universal petroleum, and biological process were almost completely stopped being used [118]. In more recent years, biotechnological production processes have made a return, and this section addresses the driving forces behind this development. 1.4.1.1 Dependency on fossil resources The oil crises in 1973 and 1979 resulted in temporary concern regarding oil dependence and renewable substrates were then again considered as an alternative to fossil resources [117, 118]. It was however concluded that they were not competitive at the time, and the matter was forgotten once oil prices were reduced again. Recently, the issue has yet again been raised and there is a common awareness that petroleum is not an infinite resource. There has been much debate regarding the concept of peak oil [119], at which time oil production will reach its maximum, and thereafter only decrease, approximately following the shape of a bell curve. Several predictions have been made of this date that have come and passed, and as seen in figure 9, production has increased steadily since 1965 apart from a dip in the 1980s. As technological advancements has allowed for finding of new oil resources, as well as harvesting of unconventional deposits, the prediction of peak oil has proven difficult. It is currently estimated to occur before the year of 2030 [120, 121].


Figure 9: Total oil production worldwide between 1965 and 2013 [122]. Oil reserve to production (R/P) ratio is another important parameter to consider when assessing the longevity of petroleum. It estimates the amount of time oil reserves will last if production would continue in current rate [122]. This quotient is paradoxical because it too has increased, from 30 years in 1980, to 53 years in 2013 (figure 10). It is very difficult to speculate on how the R/P ratio will change in the future, as additional oil resources will be exploited, but the world production level will also change. Since 1965, production has increased with an average of 2% per year [122]. Nevertheless, this puts a clock on chemical and fuel industries as to when a switch to renewable substrates must have been made.

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Figure 10: Total calculated oil reserve to production (R/P) ratio worldwide between 1980 and 2013 [122]. 1.4.1.2 Petroleum price fluctuation Chemical and fuel industries are sensitive to changes in the price of crude oil, and since the turn of the millennium, this price has oscillated heavily (figure 11). Between 2000 and 2008, there was a steep price increase that was followed by a dip in 2009. After this dip, there was another steep increase and the price topped out at 112 US$ / barrel in 2011. Since then, the price has dropped and 2015 has so far seen a dramatic decrease as the crude oil price currently is below 50 US$. These swift changes reflect the ratio between global production and consumption, as well the economic and political health in oil consuming / producing countries. Because the crude oil price tends to fluctuate so heavily, this could be one incentive to move away from petroleum-based production processes.

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Figure 11: The yearly average price for OPEC crude oil between 1960 and 2015 (in US$ per barrel), recalculated to 2015’s money value. The price of 2015 is the average value between January and March [123]. 1.4.1.3 Government directives In later years, the notion of replacing fossil-based chemical and fuel production with biotechnological processes has received increased attention from the public, and also from policy makers [124]. This is in part due to the energy insecurity in relying on a dwindling resource, but primarily because of climate change concerns. When looking at how the increased concentration of greenhouse gases in the atmosphere has contributed to rising Earth’s radiative forcing (difference in sunlight absorbed and energy radiated back to space), it is evident that this is a valid concern (figure 12, [125]). As a result, both the EU and U.S. have implemented targets towards renewable energy production and consumption, which further has driven the development of industrial biotechnology. The EU Commission did in 2009 pass the Renewable Energy Directive (Directive 2009/28/EC) [126], which mandated that by 2020, 20% of the energy consumed in the EU should be produced from renewable substrates, and that 10% of all fuel usage in each member state should be of renewable origin [127]. In addition, there were

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criteria regarding the green house gas emission savings from biofuel usage. A related directive was established in the U.S. in 2007, called the Energy Independence and Security Act of 2007 [128], which addressed energy efficiency, biofuel development and automobile fuel economy. A specific target to produce a total of 136 billion liters of biofuels by 2022 was also included in the act.

Figure 12: Radiative forcing due to greenhouse gases in the atmosphere, measured by the U.S. agency NOAA (National Oceanic and Atmospheric Administration) between 1979 to 2013 [125]. Zero radiative forcing is defined as the value of year 1750, before the pre-industrial era. Legend: cyan = CO2, green = CH4, purple = N2O, yellow = CFC-12, blue = CFC-11, red = 15 minor gases. 1.4.2 Production organisms Like most industrial enzymes, chemicals and fuels are typically of low value, although chemicals can be divided into several subcategories and fine/specialty chemicals are for example worth substantially more than bulk chemicals and fuels. Nevertheless, this means that the bio-based production processes of these compounds consequently are shaped around the parameters yield, titer and productivity. The choice of organism to use for

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commercial production of a specific compound is thus primarily based on its ability to overproduce said compound. This has resulted in that chemical and fuel industries apply a variety of organisms in their bioprocesses, mainly different bacteria and fungi. The yeast S. cerevisiae is maybe the most famous producer, due to its dominant role in bioethanol production [129]. Other examples of production organisms are Aspergillus niger for citric acid [130], Lactobacillus bacteria for lactic acid [131] and Corynebacterium glutamicum for various amino acids [132]. Although E. coli generally is not as capable as many other production organisms, the vast knowledge of its genome, metabolism and physiology has led to that also this organism is being used in commercial production of chemicals. An important example is in production of succinic acid, a process that previously was based on petroleum resources [124], but now is produced from glucose in an engineered E. coli strain [133]. 1,3-Propanediol is another example of a product that is now being produced in E. coli [134]. 1.4.2.1 Tools to design improved organisms As yield, titer and productivity are such important factors in developing commercially viable bioprocesses, it is crucial that the bioconversion of substrate into product by the organism is maximized. Several research disciplines can be utilized for this purpose: metabolic engineering, systems biology, synthetic biology and evolutionary engineering. Together, they are collected under the term systems metabolic engineering [135]. By using the tools that these disciplines provide, the complex network of reactions within a cell can be modeled and engineered systematically and globally, in order to arrive at a redesigned and improved organism. However, models rarely accurately account for the intricate and stringent regulation of intracellular metabolite fluxes, and each potential modification that is derived from a model needs to be carefully evaluated in vivo. Some of these disciplines have been utilized in the preparatory work in this thesis, but they will not be discussed to any further detail here.


1.4.3 Current Industrial bioprocesses and products While there is an increasingly growing interest in developing commercial production processes that rely on renewable resources, these will not be implementable unless they are cost effective and their products are of equal quality as those derived from petroleum [124]. The following section describes some important industrial bioprocesses for production of fuels and chemicals where this has been achieved. 1.4.3.1 Fuels 1.4.3.1.1 Ethanol The two most common biofuels today are ethanol and diesel, which together account for 90% of the market [136]. Ethanol is the major product, and there are hundreds of production plants in operation that utilize corn or sugar cane starch as the substrate [124, 137]. The ethanol is produced through anaerobic fermentation, and S. cerevisiae is the dominant production organism [138]. The advantage of S. cerevisiae is high ethanol tolerance, which enables titers of more than 100 g/L of [139]. It is however a rather inflexible organism that cannot metabolize a wide range of carbohydrates. It has further only a moderate yield of ethanol from glucose compared to some other organisms. This has led to a search for alternative producers, such as Z. mobilis, which has a higher theoretical yield and productivity [140]. E. coli has also been investigated for ethanol production [141-143] but both these bacteria have downsides, such as lower ethanol tolerance and neither are yet commercially competitive [144]. 1.4.3.1.2 Butanol Considering that ethanol is a low energy fuel compared to gasoline [145], holding only 60% of its energy density, there is an ongoing search for more efficient biofuels. Butanol has been suggested as an alternative since it contains approximately 90% of the energy density of gasoline, a significant improvement. Established processes for butanol production do exist, which


utilize natural 1-butanol producing Clostridia species [146]. This process is called the acetone-butanol-ethanol (ABE) process, and produces these three products in the proportions of approximately 3:6:1 during fermentation [147]. The issue with this process is however the low titers achieved, making butanol yet too expensive as a fuel, restricting production to the chemical market [137]. 1.4.3.1.3 Diesel Diesel is mainly made up of fatty acid methyl/ethyl esters [148], and biodiesel is made through transesterification of predominantly plant oils [149]. The production process is a conventional chemical process that utilizes a renewable substrate, different from the bioprocesses discussed above. One interesting aspect of biodiesel production is that the transesterification produce glycerol as a byproduct, which can be used as a substrate in other bioprocesses [150]. Since microalgae are capable of producing lipids in high concentration and are relatively easy to cultivate, they have been considered a promising alternative to plant oil in biodiesel production [151]. E. coli has also been discussed for biodiesel production, but no bio-based production system is close to market implementation. 1.4.3.2 Chemicals Examples of industrially important chemicals that are produced through bioprocessing and now are available on the market can be seen in figure 13. These include acetic acid, lactic acid, polyhydroxybutyrate, 1,3-propanediol and succinic acid.


Figure 13: Chemicals that are currently commercially produced through bioprocessing from renewable resources. The examples illustrated here are acetic acid, lactic acid, polyhydroxybutyrate, 1,3-propanediol and succinic acid. Lactic acid is used for chemical synthesis of polylactic acid, while the polyhydroxybutyrate polymer is synthesized in vivo. 1,3-Propanediol is through condensation with terephthalic acid used to produce polytrimethylene terephthalate. Acetic acid and succinic acid are both building blocks for a variety of chemicals. 1.4.3.2.1 Acetic acid Acetic acid is an interesting commercial product due to its usability as a building block for other chemicals [124]. In 2008, 10.6 million tons were produced from petroleum [137]. Acetic acid is also made from alcohols, using acetic acid bacteria such as Acetobacter aceti [152]. Like in biodiesel production, this is not a true bioprocess in the sense that it does not involve the central catabolic metabolism. The enzymes performing the reaction are present in the outer part of the cell’s inner membrane, and the process can rather be described as a type of whole-cell biocatalysis.

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The acetic acid produced by this method is mixed with water and sold as vinegar [153]. The raw material in the process is often ethanol, which gives that the acetic acid produced by this method is more expensive than the one produced form petroleum [137]. In the year of 2000, the production of acetic acid for vinegar was estimated at 190 000 tons/year. An interesting recent development is production of acetic acid from sugar through anaerobic digestion by so called homoacetogen microorganisms [154, 155]. These microorganisms have a very high yield of acetic acid from sugar due to the fact that they do not produce any carbon dioxide as a by-product. In 2012, the company ZeaChem completed the construction of a production plant where this technology is applied to produce acetic acid from carbohydrates [156]. Interestingly, acetic acid is not the end product in the plant, as the acid is further reduced to ethanol, but the same technology can be used to produce acetic acid at a lower cost and more efficiently than in current bio-based processes. 1.4.3.2.2 Lactic acid Polylactic acid (PLA) is a successful attempt to introduce a biodegradable polyester produced from renewable resources on the market. Industrially, the monomer lactic acid is produced through fermentation of corn starch by bacteria from mainly the Lactobacillus genus [157]. Production of lactic acid through fermentation was in 2011 estimated at 370 000 tons/year [137], and the Ingeo brand PLA (made by NatureWorks) is an example of a successfully launched commercial product that has found several applications [158]. Recently, it was further demonstrated that PLA and the copolymer poly(3-hydroxybutyrate-co-lactate) can be produced directly in engineered E. coli from glucose [159, 160].


1.4.3.2.3 Polyhydroxyalkanoates Polyhydroxyalkanoates (PHA) is another group of biodegradable polyesters that are industrially produced by microorganisms from carbohydrates [161]. Polyhydroxybutyrate (PHB) is one of the most common PHAs and its production has been extensively studied, primarily in the natural producer Cupriavidus necator (formerly Ralstonia eutropha and Alcaligenes eutropha), but also in E. coli [162-164]. The technology was established in the 1970s when Imperial Chemical Industries developed bioprocesses for production of PHB and a PHB copolymer called poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in C. necator [165]. The polymers were branded under the name of Biopol and later distributed by the company Monsanto [166]. In 2001, Monsanto sold the rights to Biopol to Metabolix who has developed the process further [167]. Metabolix now sell several PHAs, such as poly(3-hydroxybutyrate-co-4-hydroxybutyrate), produced by C. necator from glucose [168]. Metabolix is however not alone on the market as there are several other active companies who produce PHA in pilot or industrial scale. 1.4.3.2.4 1,3-Propanediol 1,3-Propanediol (PDO) is a monomer that is used together with terephthalic acid to obtain the polyester polytrimethylene terephthalate (PTT) [169]. PTT has similar properties as polyethylene terephthalate (PET) and polybutylene terephthalate (PBT), and can be a potential bio-based alternative to these. Many organisms naturally produce PDO through fermentation of glycerol [170], but DuPont and Tate & Lyle have in a joint venture commercialized a process that utilize corn starch as the substrate and recombinant E. coli as the production organism in an aerobic process [134]. The E. coli strain has been heavily engineered and has had the glycerol pathway from S. cerevisiae, as well as the PDO pathway from Klebsiella pneumoniae, introduced [144]. DuPont and Tate & Lyle currently produce PDO at 45 000 tons/year [137]. The company Metabolic Explorer has announced that they soon will enter the market with a process based on glycerol.


1.4.3.2.5 Succinic acid Succinic acid is a molecule with great potential as it can serve as a building block for a variety of chemicals [171-173]. Currently, succinic acid is produced from petroleum at 30 000 tons/year [174], but the market potential for the bio-produced monomer was in 2010 estimated to be 245 000 tons/year, as this product is expected to be cheaper [175]. Anaerobic production of succinic acid has been extensively researched in several organisms, such as Anaerobiospirillum succiniciproducens [176], E. coli [177, 178] and C. glutamicum [179], for which the highest titer has been achieved at 146 g/L. Several companies have recently commercialized production of succinic acid from renewable substrates [137, 180]: Myriant and BioAmber with E. coli, Reverdia with yeast, and Succinity with Basfia succiniciproducens. There are also several processes for production of succinic acid derivatives in the pipeline. 1.4.4 The vision of a biorefinery With the desire to reduce oil dependency, a vision of replacing conventional petroleum refineries with so-called biorefineries has materialized [181]. The definition of the term biorefining has been vague [182], but the International Energy Agency (IEA) has defined it as: “Biorefining is the sustainable processing of biomass into a spectrum of biobased products and bioenergy” [183]. A fully developed biorefinery competing to replace its petrochemical counterpart thus require incorporation of technologies from different areas, such as chemistry, biochemical engineering, biology, biotechnology and agriculture [184, 185]. In order to achieve the required versatility in the biorefinery, it is further important that the products derived can be used in a broad range of applications. Examples of such platform chemicals are those listed by U.S. Department of Energy in 2004 to be the top value added chemicals from biomass [186]. In figure 14, a principle outline of the biorefinery concept is illustrated.


Figure 14: The biorefinery concept, where different biomasses can be transformed into a range of products through chemical and biotechnological principles in several steps. 1.4.4.1 Biorefinery classification Biorefineries can be classified depending on the type of biomass they utilize and the range of products they produce [187, 188]. Facilities that produce one primary product from a food resource have been classified as first-generation, or phase I, biorefineries [188-190]. Although, according to the IEA’s definition, they are not real biorefineries since they only produce one product. Most currently operational plants that utilize bioprocessing of renewable substrates fall into this category, such as those that produce biofuels from starch or plant oil.

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Phase II biorefineries have been described as more advanced facilities capable of producing several end products. In a sense, the ethanol plant of ZeaChem belongs to this category, as acetic acid, syngas and ethanol all are produced from cellulose, although ethanol is the main product [156]. Phase III biorefineries are plants that that can utilize different biomass sources for production of an array of end products [189, 190]. By another definition, phase II biorefineries are omitted and phase III biorefineries are instead referred to as second-generation biorefineries [191], and there have even been discussions regarding third-generation biorefineries that utilize algae as the feedstock [188, 192]. 1.4.4.2 Lignocellulose, a cruder raw material In order for biorefineries to be competitive with petroleum refineries, production costs need to be reduced to comparable levels. Since the raw material is a major expense item [193], substituting starch/glucose for a less costly, cruder biomass is a strategy to achieve this goal. Using food crops for production of chemicals and fuels is also controversial as these could be used to ensure the availability of food for a growing global population [194]. Much research has been devoted towards developing bioprocesses that build upon usage of cruder raw materials. One resource that is believed to have big potential is lignocellulose, due to its abundant availability worldwide [195] and comparatively low price [196], while still containing up to 80-85% carbohydrates [197]. Lignocellulose is a complex raw material that consists of cellulose and hemicellulose bound to lignin in a compact matrix [198], and the relative amount of these depend on the biomass source (table 3, [199]). It is the sugars in the cellulose and hemicellulose fractions that might be metabolized by organisms in bioprocesses [200], but lignin can also be a useful substrate in biorefineries, as it has the potential to used for synthesis of aromatic compounds [201].


Table 3: Cellulose, hemicellulose and lignin content in different lignocellulosic biomasses [199].

1.4.4.3 Lignocellulose sugar composition Cellulose is the dominant polymer in lignocellulose, and consists of linear glucan chains of β(1-4) linked D-glucose units [202]. Cellulose is therefore not very different from starch, which consists of branched glucans connect by both β(1-4) and β(1-6) bonds [203]. However, the composition of hemicellulose is very different from starch. It varies depending on the lignocellulose source, and includes several copolymers such as glucomannan, xylan and arabinoxylan. Glucomannan, with a backbone of β(1-4) linked D-glucose and D-mannose units, is the principal hemicellulose found in softwood, while in hardwood, xylan with a backbone of β(1-4) linked D-xylose units is dominant [204]. Arabinoxylan, a specific type of xylan substituted with L-arabinose units through O-2/O-3 linkages [205], is in turn the most common hemicellulose in cereal grains [206]. This gives that the sugar monomers one can expect to extract from lignocellulose will differ depending on the resource used.

Lignocellulose cellulose (%) hemicellulose (%) lignin (%)hardwood stem 40-55 24-40 18-25softwood stem 45-50 25-35 25-35nut shell 25-30 25-30 30-40corn cob 45 35 15grass 25-40 35-50 10-30wheat straw 30 50 15switchgrass 45 31 12leaf 15-20 80-85 0


1.4.4.4 Pretreatment of lignocellulose As lignin is responsible for providing plants with mechanical strength and rigidity [207], its interaction with cellulose and hemicellulose results in a dense and cross-linked biomass, impenetrable by cellulolytic and hemicellulolytic enzymes [208]. Unlike starch, lignocellulose therefore needs to be harshly pretreated before the sugar polymers can be enzymatically saccharified. The first step is often physical pretreatment by grinding or milling, in order to reduce the size of the biomass [199]. This is followed by another pretreatment step that ideally disrupts the lignin structure, as well as reduces the crystallinity of the carbohydrate polymers [209]. Enzymes should thereafter be able to penetrate the biomass and can be added to digest the hemicellulose and cellulose into their respective sugar monomers [210]. Several methods have been considered for the subsequent pretreatment process [211, 212], including: acid hydrolysis, base hydrolysis, organosolvation and steam explosion. Acid hydrolysis has been performed with e.g. HCl or H2SO4. Diluted acids in combination with an elevated temperature have been preferred over using a higher concentration, since concentrated acids tend to be corrosive, and would need to be recovered because of their high cost [213]. Base hydrolysis with reagents such as NH3 or NaOH has been done at ambient conditions, but needed much longer processing time than acid hydrolysis, and was in the scale of hours or days, rather than minutes [199]. Organosolvation has been done at high temperature with alcohols, ethers, ketones, phenols and more [214]. It has been effective for pretreatment of biomasses with high lignin content, and is less dependent on a physical pretreatment step than other methods [215, 216]. However, using volatile chemicals at a high temperature means that there is an inherent risk of fire or explosion, as well as environmental and health issues. In steam explosion, the lignocellulose has been treated with high-pressure steam at an elevated temperature for 1-10 minutes, which resulted in hydrolysis of the biomass [217]. Steam explosion requires low energy input and only a limited amount of chemicals, but may give rise to incomplete hydrolysis and increased generation of inhibitory compounds [215].


1.4.4.5 Obstacles to lignocellulose biorefineries In addition to that the lignocellulose needs to be pretreated, there are also other barriers to using lignocellulose-derived sugars in biorefineries. These are primarily that the pretreatment releases growth-inhibitory components, and that both the glucose and pentoses need to be assimilated by the production organisms. 1.4.4.5.1 Inhibitory components Various growth-inhibitory components are inevitably released when lignocellulose is pretreated, but the types and amounts depend on the feedstock and the pretreatment method used [218-220]. The compounds that have been identified to be inhibitory are primarily furan aldehydes (furfural and hydroxymethyl furfural [HMF]), aliphatic acids (mainly acetic acid, levulinic acid and formic acid) and phenolic compounds (figure 15) [221]. Furan aldehydes are formed directly from dehydration of hexoses and pentoses, furfural from pentoses, and HMF from hexoses [219]. Acetic acid is released from hemicellulose, which is substituted with acetyl groups, while levulinic acid and formic acid are degradation products from hexoses and pentoses. Phenolic derivatives are in turn formed from lignin [222]. As these compounds inhibit cell growth and/or product synthesis, strategies need to be developed to counteract their formation in lignocellulose hydrolyzates. One suggestion is to use mild pretreatment conditions, as this results in the formation of fewer inhibitors, but it would also give a decreased sugar yield and might therefore not be feasible in these yield sensitive processes [219]. Another possibility is to dilute the hydrolyzate, but this would give higher downstream processing costs as the product later needs to be concentrated [223]. Detoxification methods have also been discussed, but would add an additional processing step and thus increase production costs [224, 225]. Another strategy is to develop strains with increased resistance to inhibitors. This has been investigated for ethanol producing S. cerevisiae, Z. mobilis and E. coli [226], and it has been suggested that E. coli has higher tolerance to lignocellulose-derived inhibitors than other organisms, making it a good candidate for further development [227].


Figure 15: Growth inhibitors are formed from lignocellulosic biomass upon pretreatment. The main inhibitors that have been identified are furfural, hydroxymethyl furfural, acetic acid, levulinic acid, formic acid and phenols [219]. 1.4.4.5.2 Co-assimilation of glucose and pentoses Agricultural waste lignocellulose, such as straws, cobs and brans, have been considered attractive to use in next generation biorefineries because of their low lignin content (table 3, [228, 229]). Less lignin means that this type of biomass is softer than e.g. wood, and less harsh pretreatment is therefore needed. As agricultural lignocellulose is mostly made up of cellulose and arabinoxylan [206], this gives that pretreatment will yield a sugar mixture that consists of predominantly D-glucose, D-xylose and L-arabinose. For example, after dilute acid pretreatment and enzymatic saccharification of wheat straw, the relative sugar composition has been reported to be 55.6% D-glucose, 37.6% D-xylose and 4.1% L-arabinose (w/w), with other sugars making up the remaining 2.7% [230]. Approximately 40% of the total sugar content is thus made up of pentoses, which puts a new demand on the lignocellulose biorefinery: that also pentoses, in addition to glucose, needs

Pentoses Hexoses

Hemicellulose Cellulose Lignin

Acetic acid

Levulinic acidFormic acid

Phenols

Hydroxymethyl furfural (HMF)Furfural

Lignocellulosic biomass


be converted into the product. As biorefineries struggle to be cost-competitive, the efficiency in pentose usage will be a vital parameter in determining their future success [193]. There are several barriers to achieving efficient conversion of sugar mixtures to chemicals and fuels by bioprocessing. Wild type strains of S. cerevisiae are for example unable to consume both D-xylose and L-arabinose [231]. Most strains of E. coli appear to be able to metabolize pentoses, but sugar uptake in this organism is controlled by several layers of regulation that results in a sequential uptake chain, where glucose is preferably consumed first [232, 233]. Because of this, there has been much interest in trying to develop production strains capable of simultaneously consuming glucose and pentoses, primarily for anaerobic production of ethanol. For S. cerevisiae, co-fermentation of glucose and xylose has been studied, but the result has so far been either slow or sequential sugar uptake, with residual sugar left in the cultivation medium [234, 235]. For E. coli, co-assimilation of both xylose and arabinose with glucose has been investigated, but simultaneous uptake of all three monosaccharides are yet to be achieved from a single strain under aerobic conditions [236-238]. 1.4.5 Regulation of sugar uptake in E. coli In E. coli (and bacteria in general), certain sugars are preferably assimilated before others, creating a sugar uptake hierarchy. The regulation depends on the presence of specific sugars and is exerted on both gene and protein level. It can further be dived into at least two different layers, where a primary, global layer is controlled by glucose, and a secondary layer by arabinose. 1.4.5.1 Catabolite repression and inducer exclusion The global sugar uptake regulation by glucose is derived from the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), for which there is a coupled membrane transport and phosphorylation mechanism (figure 16) [239-241]. In the PEP:PTS, sugar uptake is driven by PEP, which transfers its phosphate through a chain of phosphorylation


events to the sugar. From PEP, the phosphate is transferred to the generic Enzyme I (EI), which further phosphorylates the also generic Histidine protein (HPr). From here, the phosphate is transferred to a carbohydrate-specific Enzyme II (EII), which in turn phosphorylates the sugar as it passes the membrane into the cell. EII consists of several domains, which often are separate proteins, as in the case of the glucose-specific EII that is divided into EIIA and EIICB. EIIAGlc is the regulatory protein that puts glucose at the top of the sugar hierarchy, and the regulation is controlled by the degree of phosphorylation of this enzyme. When glucose is available in excess in the cultivation medium, EIIAGlc is mainly present in its unphosphorylated form, due to the drainage of phosphate to glucose. When glucose is absent, phosphorylated EIIAGlc is instead dominant. EIIAGlc regulates sugar uptake by two different mechanisms. The first is called carbon catabolite repression (CCR), by which the expression of genes needed for utilization of secondary carbon sources is prevented [242]. Transcription of these genes requires activation from a complex between cyclic adenosine monophosphate (cAMP) and the catabolite activator protein (CAP), but adenylate cyclase (AC), which produces cAMP, requires activation from phosphorylated EIIAGlc. Thus, when glucose is present, very little cAMP will be produced, and gene expression will consequently be repressed. The second regulatory mechanism exerted by EIIAGlc is called inducer exclusion, and works in the way that the unphosphorylated form of the enzyme binds to, and sterically blocks the permeases and ATP-binding cassette (ABC) transporters of catabolite-repressed sugars, or in the case of glycerol, the glycerol kinase.


Figure 16: Carbon catabolite repression and inducer exclusion in E. coli, mediated by EIIAGlc of the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS). Unphosphorylated EIIAGlc, the primary isomer present at excess of glucose, inhibits uptake of non-PTS sugars by sterically blocking their respective permeases, ATP-binding cassette transporters, or kinases. When glucose is absent, phosphorylated EIIAGlc accumulate, thereby relieving the inducer exclusion effect. Phosphorylated EIIAGlc further activate Adenylate cyclase (AC) to produce cyclic adenosine monophosphate (cAMP), which together with the catabolite activator protein (CAP), active transcription of catabolite-repressed genes. 1.4.5.2 Regulation of pentose uptake and metabolism In addition to the regulatory effects exerted by EIIGlc, there is a second layer of sugar uptake regulation in E. coli that concerns uptake of xylose and arabinose. When present in the cultivation medium, xylose will bind the XylR protein and the complex activates transcription of the operons for xylose uptake and metabolism (figure 17) [243]. There is a similar mechanism that regulates transcription of the equivalent arabinose operons, where arabinose in complex with the AraC protein acts as the activator [244]. In addition to this, the arabinose-AraC complex has been suggested to repress

Pyruvate)

PEP)

P)

EI)

EI)

HPr)

HPr)

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+"

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EIIC)

Non4PTS)sugar)Non4PTS)sugar)


transcription of the xylose operons [232], meaning that when both xylose and arabinose is available in a cultivation medium, arabinose will be the preferred carbon source. In conclusion, when glucose, xylose and arabinose all are present, the sugar uptake pattern to be expected is glucose > arabinose > xylose.

Figure 17: Xylose-bound XylR positively regulates uptake of xylose by activating transcription of the xylFGH, xylR, xylAB and xylE operons [243]. Arabinose-bound AraC in turn regulates transcription of the araC, araBAD, araFGH and araE operons. The complex is believed to repress transcription of araC, while activating the other three operons [244]. In addition, Arabinose-AraC is suggested to repress the transcription of xylFGH, xylR, xylAB and xylE, thereby negating xylose uptake at the presence of arabinose [232].

xylFGH xylR xylAB xylE

Xylose XylR

araC araBAD araFGH araE

Arabinose AraC


2 Present investigation 2.1 Aim In the introduction, the state of the art in bio-based production of pharmaceuticals, as well as chemicals and fuels was presented. The current role of E. coli as a production system within these fields was further discussed, and possible opportunities for this organism within new areas was recognized. The aim of this thesis was to engineer E. coli towards specific tasks that allow bioprocessing with this organism to be expanded within two such important areas. The first area was vaccine development, where the possibility of using E. coli as a production and delivery system of subunit vaccines was investigated. This was done in order to provide a methodology for production of low-cost, well-defined live vaccines. The second area was biorefining, where the intention was to create an E. coli strain capable of aerobic production of chemicals from simultaneous assimilation of the lignocellulose sugars glucose, xylose and arabinose. As chemical production processes are yield sensitive, achieving consumption of all the available sugars is considered a key aspect in designing economical biorefineries based on lignocellulose substrates. The strategy to achieve these goals was focused upon the redesign of E. coli membrane transport proteins and their regulatory mechanisms. For production and delivery of subunit vaccines, this included the introduction and manipulation of a protein surface translocation system from pathogenic E. coli. For simultaneous uptake of glucose, xylose and arabinose, the efforts were directed towards overcoming the cellular mechanisms that regulate sugar uptake. The present investigation is organized so that vaccine development (papers I, II and V) is covered first, and biorefining (papers III and IV) second.


2.2 E. coli live vaccines (papers I, II and V) Since laboratory strains of E. coli have been extensively investigated for surface expression of recombinant proteins, they may be suitable for combined production and delivery of subunit vaccines. The idea is that the cell displays the subunit vaccine on its surface, and its inherent immunological properties enhance the immune response. The antigens that were expressed on the surface of E. coli in this work originate from S. Enteritidis. These were the fimbrial subunit SefA and the flagellar subunit H:gm, which both have been shown to be central in Salmonella infections [245-249]. The AIDA-I autotransporter was further chosen as the surface expression system, as it has been used to express a variety of recombinant proteins on the surface of laboratory strains of E. coli [105-108]. 2.2.1 Previous results SefA and H:gm were in previous research expressed on the surface of an E. coli K-12 strain lacking the OmpT protease (0:17ΔOmpT) [250]. The plasmid that was used (pDT1) had the following organization of the expression cassette (from the N-terminal, figure 18): the AIDA-I SP, a His6-tag, a multiple cloning site holding the recombinant protein, a linker consisting of the 54 C-terminal aa of the native AIDA-I passenger, and the AIDAC translocation domain. Transcription of the construct was controlled by the native aidA promoter. The autoproteolytic site for separating the passenger from the AIDAC domain was further absent, and the recombinant proteins were to stay covalently bound to AIDAC at the cell surface. Upon analysis, it could be concluded that both proteins were present in the outer membrane, but only the His6-tag of the smaller SefA (14.4 kDa) could be detected at the cell surface by an anti-His6-AB. The His6-tag of H:gm (53.1 kDa) had been cleaved off and the orientation of the this protein in the outer membrane was therefore uncertain. The aidA promoter was also considered inadequate as it is poorly understood and was difficult to


regulate. Consequently, the expression vector needed to be improved with increased control of expression, and a more sophisticated detection system to better evaluate surface expression results.

Figure 18: The initially used plasmid pDT1 had the following organization of the expression cassette: N-terminal signal peptide (SP), His6-tag (H), multiple cloning site with recombinant passenger, linker region (L), and AIDAC domain. Transcription was under the control of the native aidA promoter. 2.2.2 An improved surface expression vector (Paper I) To understand the fate of the H:gm subunit protein, a new expression vector was considered that had the following: 1) Inducible protein expression 2) Detection of both full-length and degraded proteins 3) A modular surface expression cassette This lead to the creation of pAIDA1 (figure 19), a plasmid based on the p15A origin of replication (ori) of pACYC184, which provided a low copy number of approximately 15 copies per cell [251]. A high copy number plasmid was avoided in order to ensure that the capacity of the SEC translocon would not be overloaded. Protein expression was chosen to be controlled by the well-known LacUV5 promoter, which is both inducible and titratable with Isopropyl β-D-1-thiogalactopyranoside (IPTG) in lacY negative E. coli strains [252]. The SP, linker, and AIDAC domain of pAIDA1 were identical to those of pDT1. However, in pAIDA1, a dual detection tag approach was applied where two tags flanked the passenger protein. On the N-terminal side of the passenger, there was a His6-tag, just as for pDT1, but on the C-terminal side, a Myc-tag [253] had been added. This would hopefully facilitate detection of proteins where the His6-tag had been cleaved off. In order to provide the alternative of cleaving off the surface expressed protein and quantify it through conventional protein analysis assays, the recognition sequences for human rhinovirus (HRV) 3C, and tobacco etch virus (TEV)

Passenger LSP H AIDAC


proteases were added in-between the detection tags and the passenger protein. The protease sites also functioned as spacer elements between the detection tags and the passenger. To facilitate future modifications of the surface expression cassette, specific restriction sites enclosed each component. The translated fusion protein of pAIDA1 at the cell surface is illustrated at the top of figure 19.

Figure 19: The re-designed, modular surface expression plasmid pAIDA1 translates a fusion protein with improved detectability of surface expressed proteins due to a dual tag approach where the passenger is flanked by two unique detection tags, His6 and Myc.

pLacUV5 His6 3C site Passenger Linker AIDAcTEV site Myc

p15A oripAIDA15560 bp

CmR

LacI

Surface expression cassette

SPHindIII SalINdeI KpnI SacI XbaI NotI

+NH3

His6-tag antibody

Myc-tag antibody

His6-tag

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Outer membrane


To evaluate the performance of the new vector, the AIDAC-SefA and AIDAC-H:gm fusion proteins were expressed by E. coli 0:17ΔOmpT. The cells were cultivated at 37°C and at an initial pH of 7.0. Induction was done with 200 μM IPTG at OD600 = 0.2 and the cells were harvested 4 h later. The cells were subsequently labeled with fluorescent ABs specific towards either the His6- or the Myc-tag, and surface expression levels were analyzed by flow cytometry (figure 20). AIDAC-SefA expressed from pAIDA1 resulted in an approximately 50% higher fluorescence signal for the anti-His6 AB, than when expressed from pDT1 (figure 20 A). When the fusion protein was expressed from pDT1, it appeared that only half the cell population expressed it on the surface, an effect possibly originating in heterogeneous activation of the constitutive aidA promoter. Using an anti-Myc AB also resulted in a clearly distinguishable peak for AIDAC-SefA, verifying that this tag could be detected. Its interaction with the AB appeared not to be hindered by the presence of SefA or other proteins at the cell surface. Next, surface expression of H:gm was analyzed (figure 20 B). As expected and in line with previous research, neither expression from pDT1 nor pAIDA1 gave rise to a fluorescence signal that was separated from the negative control when detected by the anti-His6 AB, indicating severe proteolysis of the His6-tag. Interesting was however that when the cells harboring pAIDA1 were labeled with the anti-Myc AB, this resulted in a strong fluorescent signal clearly distinguishable form the control. This verified that the Myc-tag was successfully expressed at the cell surface and that the degraded form of H:gm had the desired outwards orientation in the outer membrane.


Figure 20: Histograms of surface expressed SefA (A) and H:gm (B) from pDT1 (green) and pAIDA1 (blue), when detected with anti-His6 ABs (left) and anti-Myc ABs (right). The negative control consisted of cells lacking the surface expression construct (red). In addition to flow cytometry, western blot was performed of the soluble, inner membrane and outer membrane fractions of cells expressing AIDAC-SefA and AIDAC-H:gm (figure 21). The proteins were again detected by using labeled anti-His6 and anti-Myc ABs. From this experiment, it was concluded that both proteins were found exclusively in the outer membrane, indicating that the expression did not overload the SEC system. The anti-His6 blot (figure 21 left) further corresponded well with the results from the flow cytometry for both AIDAC-SefA and AIDAC-H:gm. A strong band was visible

100 102101 103

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for AIDAC-SefA at the expected size of 72 kDa, while only a very weak band was seen at the expected size of AIDAC-H:gm of 111 kDa. The anti-Myc blot in turn (figure 21 right) revealed that a degradation product was present for both fusion proteins at a size that was slightly smaller than that of the positive control at 58 kDa. Interestingly, both proteins seem to have been cleaved in the same position, close to the C-terminal end of the passenger protein. For SefA, the degradation was minor, but for H:gm, practically all of the fusion protein were present in the degraded form, which was in line with the flow cytometry analysis.

Figure 21: Anti-His6 (left) and anti-Myc (right) western blots of cellular fractions when expressing SefA and H:gm from pAIDA1. Legend: C = positive control in the form of the protein product of pAIDA1 without a passenger, S = soluble fraction, IM = inner membrane, and OM = outer membrane.

C S IM OM C S IM OM S IM OM C S IM OM C


2.2.3 Influence of cultivation conditions (Paper II) As H:gm, but also SefA to some extent, was proteolyzed when expressed on the cell surface at standard cultivation conditions (pH 7.0, 37°C, 200 μM IPTG), it was investigated if this degradation could be reduced. The hypothesis was that periplasmic proteases, such as DegP, could be responsible for the proteolytic effect. Since it has previously been shown that periplasmic proteases are inactive below pH 6 [254], reducing the periplasmic pH was therefore considered a promising strategy to increase the amount of surface expressed protein. As the periplasm has approximately the same pH as the surrounding environment, this was achieved by lowering the pH of the cultivation medium. In addition to pH, the effects of inducer concentration (IPTG) and temperature were also studied, as both of these influence protein synthesis rate, and may therefore also be important factors for surface expression results. The three factors pH, temperature and IPTG concentration were studied in a factorial design of experiments (DOE) in order to determine synergistic effects between them, in addition to their individual influence. The protein used in the study was SefA expressed from pAIDA1, as it had previously been successfully expressed in full-length on the cell surface, without being too affected by proteolysis. In a first run, three levels were tested for each factor (pH: 5.5, 6.5, 7.5; temp: 22, 32, 42 [°C]; IPTG: 10, 100, 1000 [μM]), which required 17 bioreactor experiments. Surface expression was analyzed by flow cytometry as described previously, and models were constructed for how the factors influenced specific growth rate (μ), surface expression of full length protein, and the ratio of full-length to degraded protein. The latter two models are shown in figure 22 A. These indicated clear trends where both the amount of full-length protein as well as the ratio of full-length to degraded protein increased with lowered pH and temperature. As expected, μ was modeled to be negatively affected when pH and temperature were decreased.


Figure 22: Models for how the anti-His6 fluorescence (full-length protein) and anti-His6/anti-Myc fluorescence (full-length to degraded protein) of AIDAC-SefA varies as a function of cultivation pH and temperature. In the initial experimental series (A), the levels used were: pH: 5.5, 6.5, 7.5; temp: 22, 32, 42 (°C); IPTG: 10, 100, 1000 (μM). The models for 1000 μM IPTG are shown in the figure. In the second series (B), the levels used were: pH: 5.5, 6.5, 7.5; temp: 22, 32, 42 (°C); IPTG: 10, 100, 1000 (μM). The models for 1250 μM IPTG are shown in the figure.

A His-tag fluorescence His-tag/Myc-tag fluorescence

B His-tag fluorescence His-tag/Myc-tag fluorescence


The models predicted no maximum for surface expression levels, and a second run of experiments were made where pH and temperature were decreased further, and the IPTG concentration increased (pH: 5.0, 5.5, 6.0; temp: 18, 27, 36 [°C]; IPTG: 50, 650, 1250 [μM]). From these experiments, new models were made that displayed the same trends as seen previously (figure 22 B). As the combination of low pH and low temperature led to slow cell growth (< 0.1 h-1 at 18°C and pH 5.0), it was not considered fruitful to decrease these parameters further. In addition to this, E. coli has been reported to be incapable of growth below pH 5 [255]. To conclude how cultivation conditions could be used to improve surface expression, the conditions found to be optimal (pH 5.0, 18°C, 1250 μM IPTG) were compared to the standard cultivation protocol used (pH 7.0, 37°C, 200 μM IPTG) (figure 23). The result was a significant increase in the fluorescence from both detection tags at optimized cultivation conditions. The anti-His6 fluorescence was four times higher than for the standard cultivation protocol, while the anti-Myc fluorescence increased threefold. The greater increase in fluorescence from the anti-His6 AB indicated that the relative amount of full-length protein increased with approximately one third.

Figure 23: Histograms of surface expressed SefA from pAIDA1 when detected with an anti-His6 AB (left) and an anti-Myc AB (right). Surface expression at standard cultivation conditions (blue, pH 7.0, 37°C, 200 μM IPTG) was compared with optimized conditions (green, pH 5.0, 18°C, 1250 μM IPTG). The negative control was cells lacking the surface expression construct (red).

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While altered cultivation conditions resulted in increased surface expression levels and a reduction in proteolysis, the degraded protein product could still be detected also under optimal conditions, as seen by western blot analysis (figure 24). Using optimized cultivation conditions is thus a promising strategy to improve the surface expression yield, but is alone insufficient in eliminating proteolysis, at least for the protein SefA.

Figure 24: Anti-His6 western blots of outer membrane fractions from cells expressing SefA from pAIDA1 at different cultivation conditions. Each lane is a separate experiment run at the respective temperature, pH and IPTG concentration. C = positive control (AIDAC-SefA cultivated in shake flask at pH 7.0, 37°C and 200 μM IPTG). 2.2.4 Engineered Salmonella proteins In paper I, H:gm was heavily proteolyzed at the cell surface. This can be hypothesized to be related either to its relatively large size compared with SefA (14.4 kDa vs. 53.1 kDa), or to the fact that it folds prematurely, both of which likely leads to increased exposure time to proteases. In an attempt to address the proteolysis, a truncated version of H:gm called H:gmd (11.0 kDa, figure 25), only containing the serotype-specific region [256], was created and tested for surfaced expression from the pAIDA1 vector [257]. Since SefA has proven to have excellent surface translocation properties, a fusion between SefA and H:gmd (H:gmdSefA, 25.4 kDa) was also created in order to see if SefA could stimulate translocation of the truncated protein.

18°C 27°C 36°C

1250 1250 650 125050 50 C5.0 6.0 5.5 5.56.0 5.0

650 650 650 6501250 6505.0 5.5 6.0 5.55.5 5.5

C 1250 1250 50 1250650 50 C6.0 5.0 6.0 5.55.5 5.0

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Surface expression of AIDAC-H:gmd and AIDAC-H:gmdSefA was compared to AIDAC-SefA and AIDAC-H:gm by using flow cytometry and anti-His6/Myc ABs as described previously. For all fusion combinations, with the exception of AIDAC-H:gm, there was a strong fluorescence signal both when anti-His6 and anti-Myc ABs were used (figure 26). H:gmd and H:gmdSefA were thus successfully expressed in the outer membrane with the correct outwards orientation, and did not seem to be very affected by proteolysis.

Figure 25: Atomic model of S. Enteritidis flagellar subunit H:gm (53.1 kDa), which folds with the N- and C-terminals in close proximity of each other [258] (PDB id 1UCU). The truncated version H:gmd (11.0 kDa) is highlighted in cyan in the structure.


Figure 26: Histograms of surface expressed SefA (green), H:gm (cyan), H:gmd (blue) and H:gmdSefA (Purple) from pAIDA1, when detected with anti-His6 ABs (left) and anti-Myc ABs (right). The negative control consisted of cells lacking the surface expression construct (red). 2.2.5 Proinflammatory response from Salmonella proteins Having successfully detected SefA, H:gmd and the H:gmdSefA fusion on the surface of E. coli, it was of interest to investigate if cells expressing these proteins would provoke an elevated proinflammatory response as S. enterica fimbriae and flagellar proteins have previously been shown to induce an interleukin 8 (IL-8) response from the gut epithelial cell line HT-29 [259]. This was assessed in vitro by exposing HT-29 cells to E. coli that expressed the respective antigens, while measuring the release of IL-8 (figure 27). Surface

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0 100 101 102 1030 100 101 102 103

0 100 101 102 103 0 100 101 102 103

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expression of SefA, H:gm and H:gmd resulted in an elevated proinflammatory response when compared to non-infected HT-29 cells. Interestingly, there appeared to be no significant increase of IL-8 from surface expression of H:gmdSefA. It was further shown that the non-pathogenic E. coli strain that was used (0:17ΔOmpT) displayed a very low immunogenicity and was not able to stimulate production of IL-8 by itself. As a result, the IL-8 response of HT-29 cells infected by E. coli expressing the antigens was low, only slightly higher than twice the value for non-infected cells for the most potent protein SefA. It should further be noted that since H:gm is heavily proteolyzed at the cell surface, it is possible that a stronger proinflammatory response could have been achieved if the full-length protein had been successfully expressed.

Figure 27: IL-8 response when the gut epithelial cell line HT-29 was exposed to E. coli 0:17ΔOmpT expressing SefA, H:gm, H:gmdSefA and empty pAIDA1 vector on the cell surface. IL-8 levels were measured in the supernatant after 4 h and compared to the level of non-infected HT-29 cells. The results are shown as the mean values and the standard error of the mean.


2.2.6 Surface expression of large passenger proteins In addition to expression of the S. Enteritidis antigens SefA (14.4 kDa), H:gm (53.1 kDa), H:gmd (11.0 kDa) and H:gmdSefA (25.4 kDa), the pAIDA1 vector has also been used to express enzymes on the surface of E. coli. These enzymes the Arthrobacter citreus ω-transaminase (Ac-ω-TA, 53.2 kDa) [260] and two tyrosinases (tyr), one from Bacillus megaterium (34.2 kDa) and one from Rhizobium etli (67.2 kDa). The R. etli tyrosinase has also been expressed in a truncated version with a size of 35.9 kDa. While surface expression of enzymes is not within the scope of this thesis, the results of those experiments can still be compared to those for the Salmonella antigens, to get a more complete picture of the proteolytic problem. The surface expression results of all the passenger proteins that so far have been expressed from pAIDA1 are compared in figure 28. For each protein in the figure, the mass of the detection tags and the protease recognition sequences of pAIDA1 (in total 4.5 kDa) have been added to the protein size. This was done in order to plot the size of the peptide that actually is considered foreign to AIDA-I. The fluorescence from the His6- and Myc-tag detection has for each protein been normalized against SefA, which consequently has been given a relative fluorescence of 100% for both tags. From the figure, it is quite evident that anything larger than H:gmdSefA at 25.4 kDa (29.9 kDa with tags and protease sites) has been notoriously difficult to express on the cell surface. The relative His6-tag fluorescences of Ac-ω-TA, H:gm and the three tyrosinases are all close to zero, indicating severe proteolysis. The Myc-tag fluorescence of Ac-ω-TA is on par with H:gm, but for the tyrosinases, also the Myc-tag appears to be difficult to express on the cell surface. From this comparison, the conclusion can be drawn that proteolysis might be a common problem for large passenger proteins.


Figure 28: Relative anti-His6 (left) and anti-Myc (right) fluorescence of different passenger proteins as a function of size, modified from Gustavsson 2013 [261]. The fluorescent signals have been normalized against that of SefA to yield the relative values. Legend: Tyr = tyrosinase, Ac-ω-TA = A. citreus ω-transaminase. 2.2.7 The signal peptide and passenger size (Paper V) As larger proteins proved difficult to express on the cell surface from the pAIDA1 vector, this led to the desire to identify other ATs that might be better suited for surface expression of large recombinant proteins. Specifically those ATs that have very large native passengers were deemed interesting. A methodology was therefore needed to identify ATs with large passenger proteins in order to find suitable candidates. E. coli proteins that are destined for transport over the inner membrane by the SEC system contain a signal peptide (SP) that usually is 20-30 aa long [262]. SP sequences are not particularly conserved, but have the same general layout with three regions: a basic (N) region, a hydrophobic (H) region, and a polar (C) region [263]. The N and H regions are recognized by the SEC system, and the C region contains a signal peptidase site for cleavage of the SP after translocation to the periplasm. AT SPs generally follow the same theme, but a subgroup of ATs has extended signal peptides (ESPs) that are 50-60 aa long (figure 29) [98, 264, 265]. These contain the

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H:gmd

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H:gmAc-ω-TA

R. etli tyrR. etli core tyrB. megaterium tyr

H:gmd

SefA

H:gmdSefA

H:gm

Ac-ω-TA

R. etli tyrR. etli core tyr

B. megaterium tyr


same segments as regular SEC-dependent SPs, but are extended at the N-terminal with an additional region called the extended signal peptide region (ESPR), which in contrast to the regular SP, is highly conserved. The function of the ESPR is yet to be fully elucidated, but it has been suggested to play a role in translocation of the passenger over the outer membrane [266]. It has been hypothesized that ESPs are processed more slowly than regular SPs by the signal peptidase to prevent premature folding in the periplasm before the β-barrel has been integrated into the outer membrane. It has further been suggested that ATs with ESPs generally have larger passenger proteins, as these would be more reliant on the effect of the ESPR to achieve surface translocation [264].

Figure 29: The organization of extended signal peptides (ESPs) in autotransporters. In addition to the basic (N), hydrophobic (H) and polar (C) regions found in regular signal peptides, they have an N-terminal extension consisting of an additional N- and H region. This extension is referred to as the extended signal peptide region (ESPR). To investigate if ATs containing large passengers could be identified based on the presence of ESPs in their sequence, a hidden markov model (HMM) [267] for ESPs was constructed based on the 41 ESP-containing sequences reported by Henderson et al. [264]. In agreement with previous data [265], the resulting HMM logo displayed a conserved N-terminal region corresponding to the ESPR, and a variable C-terminal region, in line with what is expected from a regular SP (figure 30).

extended signal peptide region (ESPR) regular signal peptide

Basic HydrophobicBasic Hydrophobic Polar

Extended signal peptide (ESP)

N- -C


Figure 30: HMM-logo from the alignment of the 41 extended signal peptides reported by Henderson et al. [264]. The N-terminal part, corresponding to the extended signal peptide region, is more conserved than the C-terminal part that corresponds to the regular signal peptide. After the model had been validated, all ATs available at Pfam (8208 at the time) were downloaded and highly similar sequences (those with > 95% sequence identity) were removed. The remaining 2486 ATs were analyzed with the constructed HMM, and of these were 128 (5.1%) identified to have ESPs. To understand if the length of the signal peptide correlate to the protein size, the sequence length of both ESP- and non-ESP containing ATs were plotted (figure 31). As can be seen in the figure, ATs with regular SPs (figure 31 A) are found in a broad size spectrum with a peak around 1000 aa. For ATs with ESPs (figure 31 B), there is a clear difference as no AT in this group is smaller than 800 aa, and most are present in the 1000-1500 aa range. It could consequently be confirmed that there is a correlation between ESPs and larger protein size, in line with previous suggestions. ATs with ESPs may thus possibly be favorably used over those with regular SPs for surface expression of large recombinant inserts due to their inherent ability to translocate larger passenger proteins. In this context, it should be noted that the autotransporter used in this work, AIDA-I, do have an ESP and is over 1200 aa in length, so utilization of ESP-containing ATs does not guarantee success, but these results simplify the task of finding additional candidates that might be better suited for surface expression applications.

1.000.001.9

MLW1

1.000.001.9

N21.000.271.1

KTRAH

3

1.000.001.9

ILVCT

4

1.000.001.9

YF5

1.000.001.9

RKSN

6

1.000.001.9

LVTI

7

0.980.001.9

VIK

8

0.990.001.9

FWY

9

0.990.001.9

NSDC

10

1.000.001.9

RKHAEVQN

11

1.000.001.9

AVSITRKH

12

1.000.001.9

TRL

13

1.000.001.9

GNQRK

14

1.000.001.9

ATCGVMH

15

1.000.001.9

LWFVMC

16

1.000.001.9

VIQKM

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AV

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E21

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23

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0.910.001.9

KGERC

28

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KRSQVNC

29

0.980.001.9

STAKEP

30

0.990.001.9

GSCARTH

31

0.980.449.7

AVRGKNTDQ

32

0.630.011.9

RSNKDFEPQ

33

0.700.011.9

ARVSKNI

34

0.770.011.9

AQKVGRSHTC

35

0.820.011.9

SRKQT

36

0.860.011.9

ALIVTSKM

37

0.890.011.9

LASNR

38

0.930.001.9

VSLAIGRF

39

0.960.001.9

LKFAISVTR

40

0.970.001.9

KSAREQTPF

41

0.980.001.9

KRANSFPH

42

0.990.001.9

LAIVSTH

43

0.990.001.9

LVSIFMC

44

0.990.001.9

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1.000.001.9

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1.000.001.9

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ALIGSC

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ALVGFCW

52

1.000.001.9

VLAITGF

53

1.000.001.9

LSVGTIFMC

54

1.000.001.9

LATSIFVYM

55

1.000.001.9

PSATNQ

56

1.000.001.9

LGSANTQY

57

1.000.001.9

PLSITQN

58

1.000.001.9

AVTY

59

1.000.001.9

LFSQTDNYH

60

1.000.001.9

A61

1.000.001.9

AQSDNH

62

1.000.001.9

DGTENPQ

63

0.980.001.9

VILAM

64

0.990.000

AVKTSDNQ

654.14

0

2.07


Figure 31: Size distribution of the 2358 autotransporters containing regular signal peptides (A), and the 128 autotransporters identified by the HMM created in this work to have extended signal peptides (B). As all ATs have the same principal domain structure, the sequence length of the AT protein reflects the size of the passenger protein. 2.2.8 Summary of surface expression results In this part of the thesis, an improved surface expression vector was created and used to express S. Enteritidis proteins on the cell surface of E. coli. Cells expressing the subunit proteins were further able to provoke an elevated proinflammatory response in epithelial cells. Larger proteins, such as the flagellar subunit H:gm (53.1 kDa), were however subjected to heavy proteolysis, making this a prioritized issue to solve in future, as it cripples the potential of the surface expression system.

A B

Sequence length (aa)

Coun

t

Coun

t

Sequence length (aa)


2.3 Lignocellulose biorefineries (Papers III and IV) To implement biorefineries based on lignocellulosic substrates, one crucial aspect is to achieve complete and simultaneous conversion of all the available sugars into biomass and product. From agricultural lignocellulose, the sugars released after pretreatment are primarily glucose, xylose and arabinose. Since E. coli is capable of metabolize all of these, it is a promising organism to use for production of chemicals from this resource. However, wild type strains of E. coli take up the sugars sequentially because of regulatory control mechanisms operating at several levels. The net result is a diauxic growth pattern where glucose is assimilated first, followed by arabinose, and lastly xylose. 2.3.1 Simultaneous uptake of sugars (Paper III) To create a platform E. coli for aerobic production of chemicals from lignocellulose sugars, it was considered imperative to overcome the two-layered sugar uptake regulation. In order to do so, the first step was to investigate how the sugar assimilation pattern differed between E. coli strains. Therefore, four laboratory strains (W3110, BL21(DE3), 0:17 and AF1000) were cultivated on either D-glucose, D-xylose or L-arabinose as the sole carbon source in batch mode (table 4). Interestingly, no two strains behaved the same, and the wild type W3110 and BL21(DE3) were the only ones to rapidly consume all three monosaccharides. AF1000 were able to assimilate all three sugars, but uptake of arabinose was preceded by a lag phase of approximately 20 h. This was a surprising feat, as this strain carries the lethal mutation [araD139]B/r, which leads to accumulation of the toxic intermediate L-ribulose-5-phosphate when cultivated on arabinose [268]. Strain 0:17 in turn, assimilated glucose but not xylose or arabinose. Also the acetic acid production varied greatly between the strains.


Table 4: Growth of E. coli strains on D-glucose, D-xylose or L-arabinose as the sole carbon source. Legend: n.g. = no cell growth, + = confirmed acetic acid production, – = the acetic acid concentration was below the detection limit of the assay (< 40 mg L-1).

When cultivated on a mixture of all three sugars, the E. coli wild type W3110 verified the multi-layered sugar uptake regulation (figure 32 A). As seen in the figure, glucose was utilized first, followed by arabinose and lastly xylose, in line with the predicted behavior. When AF1000 that had been adapted to growth on arabinose (called AF1000ara) in turn was cultivated on the sugar mixture, it displayed the unexpected phenotype of co-assimilation of xylose and arabinose after glucose had been depleted in the cultivation medium (figure 32 B). This was considered interesting, and in an attempt to characterize this trait, whole genome sequencing was performed on AF1000 and AF1000ara. However, when aligned against the reference genome of the wild type W3110, there was no variation detected for either of the two strains in araC, the gene suggested to inhibit xylose uptake, nor in the promoter regions of the xylose uptake and metabolism operons. There was further no other genetic variation between AF1000 and AF1000ara, and the phenotypic difference could not be attributed to a mutation.

Growth rate Acetic acid Growth rate Acetic acid Growth rate Acetic acid

(h-1) + / - (h-1) + / - (h-1) + / -

W3110 0.64 + 0.54 - 0.50 +

BL21(DE3) 0.62 - 0.32 - 0.77 -

0:17 0.91 + n.g. n.g.

AF1000 0.75 + 0.56 + 0.58 +

D-glucose D-xylose L-arabinose

Strain


Figure 32: Sugar uptake patterns of the E. coli strains W3110 (A), AF1000ara (B), and PPA652ara (C) when cultivated on the mixture of glucose, xylose and arabinose in batch mode. Even if the reason for co-assimilation of xylose and arabinose by AF1000ara could not be determined, it was still considered a promising candidate strain for further development towards simultaneous utilization of glucose, xylose and arabinose. The strategy chosen to achieve this was to combine the phenotype of AF1000ara with deletion of the gene ptsG (encoding EIICBGlc, see figure 16), since such mutants have been shown to avoid catabolite-repression [269]. The reason for this is that without EIICBGlc, the phosphotransfer chain becomes disrupted, and EIIAGlc won’t be able to

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donate its phosphate further. EIIAGlc will thus exist primarily in its phosphorylated form, unable to exercise catabolite-repression and inducer exclusion. The combined effect was accomplished through adaptation to growth on arabinose by the E. coli strain PPA652 (AF1000 ΔptsG::Km [270]), and the resulting strain was called PPA652ara. The strains PPA652 and PPA652ara were further sequenced, but like for AF1000 and AF1000ara, no genetic variation was detected between the two. PPA652ara was thereafter cultivated on the glucose, xylose and arabinose mixture (figure 32 C), and was able to simultaneously assimilate all three sugars completely. The resulting specific growth rate was registered to be 0.59 h-1, compared to 0.38 h-1 when PPA652 was cultivated solely on glucose [270]. Co-assimilation of arabinose and xylose thus improved the growth rate by over 50%, although it was still lower than for AF1000 cultivated on glucose (0.75 h-1). Due the fast metabolic shift that was observed for AF1000 once a sugar depleted in the medium, its sequential uptake did not have a significant negative impact on processing time, and both AF1000 and PPA652ara consumed all sugars in approximately the same time (figure 32). The main advantage of PPA652ara was thus recognized to be in processes where sugar concentrations were to remain high throughout the process, or in fed-batch cultivations. 2.3.2 Production of (R)-3-hydroxybutyric acid (Paper IV) After the intended platform E. coli PPA652ara had been developed, the next step was to investigate how utilization of a mixture of glucose, xylose and arabinose affected chemical production. For this purpose, part of the pathway towards PHB was cloned from the halophilic bacterium Halomonas boliviensis [271] and put onto a plasmid that was transformed into PPA652ara and the reference strain AF1000. The idea was to produce the PHB monomer (R)-3-hydroxybutyric acid (3HB), a molecule that contains a chiral center, as well as a hydroxyl- and a carboxyl group [272, 273]. These features makes it an interesting compound that potentially can be used for synthesis of fine chemicals, pharmaceuticals and copolyesters [274].


To produce 3HB, the genes for acetoacetyl-CoA thiolase (called t3) and acetoacetyl-CoA reductase (rx) were put under the control of the lacUV5 promoter in a plasmid based on pACYC184 (figure 33). This would result in synthesis of 3-hydroxybutyryl-CoA from acetyl-CoA through an acetoacetyl-CoA intermediate [164]. In H. boliviensis, and other natural producers of PHB, a polymerase catalyzes the formation of PHB from 3-hydroxybutyryl-CoA, and this enzyme was therefore not introduced into the expression vector. The hypothesis was instead that 3-hydroxybutyryl-CoA would be converted into 3HB in the E. coli background, possibly by the action of an unspecific thioesterase, such as Thioesterase II (tesB), which previously has been overexpressed in E. coli to increase production of 3HB [275].

Figure 33: Genes t3 (acetoacetyl-CoA thiolase) and rx (acetoacetyl-CoA reductase) were cloned from H. boliviensis into a pACYC184-dervied plasmid called pJBG. The genes were cloned both in the order of t3-rx (pJBGT3RX, shown in figure) and rx-t3 (pJBGRXT3), and their expression were put under the control of the lacUV5 promoter. This introduced a pathway from acetyl-CoA to 3-hydroxybutyric acid into E. coli. Two plasmids were initially constructed, pJBGT3RX with the gene order t3-rx (illustrated in figure 33), and pJBGRXT3 with the gene order of rx-t3. AF1000 harboring pJBGT3RX and pJBGRXT3 was initially cultivated on glucose in order to investigate the impact of the gene order on 3HB production. The cells were induced with 200 μM IPTG at OD600 = 0.2 and the 3HB concentration was measured in the stationary phase. It could be concluded that 3HB was successfully produced and secreted to the cultivation medium from both constructs, and there appeared to be no major contribution from the gene order as both plasmids produced similar titers (0.18-0.19 g L-1). It was further verified that there was no accumulation of either 3HB or PHB in the cell pellet, and that only the R-enantiomer of 3HB was being produced.

LacUV5 t3 rx

Acetoacetyl-CoA reductaseAcetoacetyl-CoA thiolase

Acetoacetyl-CoA

NADP+/NAD+NADPH/NADH

3-Hydroxybutyryl-CoA

Thioesterase II(tesB)

3-Hydroxybutyric acid

Acetyl-CoA

CoASH CoASHGlc, Xyl, Ara

TCA-cycleBiomass

pJBGT3RX


Plasmid pJBGT3RX was chosen for the continued work and PPA652ara harboring this plasmid was cultivated on glucose, xylose and arabinose, both individually and as a mixture. Cultivation on the pentoses resulted in similar 3HB concentrations as for AF1000, but when glucose was being used, there was practically no accumulation of 3HB, likely related to the ptsG deletion carried by this strain. However, when the sugar mixture was used as the substrate, a 3HB titer of 0.17 g L-1 was reached. As fed-batch cultivations often are used in industrial processes, it was of importance to investigate the performance of PPA652ara-pJBGT3RX during carbon-limited conditions. Therefore, continuous cultivation of AF1000 and PPA652ara, both with and without the plasmid, was performed. As the carbon source, a mixture of glucose (8.20 g L-1), xylose (5.95 g L-1), and arabinose (0.85 g L-1) was used that had the same relative ratios as a pretreated wheat straw hydrolyzate [230]. When the strains were cultivated without the plasmid, they both started to accumulate sugars at approximately the same dilution rate (D), between 0.4 and 0.5 h-1 (figure 34). At lower dilution rates, all three sugars were assimilated by both strains, and AF1000 thus also allowed for simultaneous uptake to take place under those conditions. The reason for this is likely that the glucose flux over EIICGlc was too low to trigger catabolite repression and inducer exclusion, and that the intracellular arabinose concentration was not high enough to inhibit uptake of xylose. These results suggested that there was no significant advantage of using PPA652ara over the wild type strain during carbon-limited conditions, such as in a standard fed-batch process. In the experiments where AF1000 and PPA652ara harbored pJBGT3RX, the specific productivity of 3HB (q3HB) was almost zero, indicating that 3HB further cannot be produced in carbon-limited bioprocesses.


Figure 34: Sugar assimilation by AF1000 (A) and PPA652ara (B) at different dilution rates, when cultivated in continuous mode on a mixture of glucose (8.20 g L-1), xylose (5.95 g L-1), and arabinose (0.85 g L-1). A different production approach was therefore considered, in which the sugar mixture instead was made available in excess. The performance of PPA652ara and AF1000 harboring pJBGT3RX was therefore evaluated in non-limited batch mode, as well as during nitrogen depletion and in nitrogen-limited fed-batch mode. The resulting graphs for PPA652ara-pJBGT3RX are shown in figure 35, and the summarized results of both AF1000 and PPA65ara are given in table 5.

0.2 0.3 0.4 0.5 0.6 0.7 0.2 0.3 0.4 0.5 0.6 0.7

0.2 0.3 0.4 0.5 0.6 0.7 0.2 0.3 0.4 0.5 0.6 0.7

2468

0.51.01.52.0

246810

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HAc (

g L-1

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0.51.01.52.0

Glc,

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2468

CDW

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-1)

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Dilution rate (h-1)0.2 0.3 0.4 0.5 0.6 0.7

Dilution rate (h-1)0.2 0.3 0.4 0.5 0.6 0.7

Acetic acid (HAc)Cell dry weight (CDW) Glucose (Glc) Xylose (Xyl) Arabinose (Ara)Legend:


Figure 35: PPA652ara-pJBGT3RX cultivated in batch mode with all nutrients available in excess (A), during nitrogen depletion (B), and in nitrogen-limited fed-batch mode (C). The dotted lines in B and C indicate the starting point of the nitrogen depletion and feed phase.

2 4 6 8

2 4 6 8

2 4 6 8

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Acetic acid (HAc)3-hydroxybutyric acid (3HB)

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g-1 h

-1)

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NH3 (

g L-1

)

0.10.20.30.40.5

HAc,

3HB

(g L

-1)

0.20.40.60.81.01.2

Glc,

Xyl, A

ra (g

L-1

)

2468

10

CDW

(g L

-1)

12345

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HAc,

3HB

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-1)

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L-1

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10

CDW

(g L

-1)

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q 3HB

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g-1 h

-1)

20406080

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HAc,

3HB

(g L

-1)

0.20.40.60.81.01.2

Glc,

Xyl, A

ra (g

L-1

)

2468

10

CDW

(g L

-1)

12345

Glucose (Glc)Xylose (Xyl)Arabinose (Ara)

Ammonia (NH3)

A

B C

Legend:


From these experiments, it could be concluded that carbon excess was necessary to achieve a high specific productivity of 3HB, with average values of 25-68 mg g-1 h-1 in the different carbon excess cultivation modes. Regular batch mode seemed the least promising choice for production of 3HB, as most of the carbon was directed to cell mass rather than 3HB. Nitrogen depletion or limitation seemed more favorable strategies, especially depletion, due to a high yield of 3HB/CDW (1.38 g g-1 for PPA652ara, to compare with 0.06 g g-1 in unlimited batch mode). While nitrogen limitation resulted in an average q3HB that was approximately 20% greater than that of the depletion experiment for PPA652ara, it was considered the inferior process due to a significantly lower yield of 3HB/CDW. Choosing a lower feed rate can possibly alter this ratio, but it was clear that the one used in this experiment (equivalent to μ = 0.2 h-1) was set too high. It was further shown that AF1000 and PPA652ara behaved similarly during the different process modes and displayed the same trends. The major difference was the ability of PPA652ara to produce 3HB from simultaneous assimilation of glucose, xylose and arabinose, while AF1000 consumed only glucose. Table 5: AF1000 and PPA652ara harboring pJBGT3RX cultivated during different process modes. For the nitrogen-depleted, and nitrogen-limited fed-batch cultivations, the values in the table correspond to the depleted/limited phases only, excluding prior batch phases.

Process mode Sugar Strain Time CDW Sugar consumed 3HB HAc mean q3HB Y3HB/HAcY3HB/CDW Y3HB/Sugar

(h) (g L-1) (g L-1) (g L-1) (g L-1) (mg g-1 h-1) (g g-1) (g g-1) (g g-1)

Batch Glc AF1000 5.5 4.18 8.90 0.51 0.96 68 0.54 0.12 0.06

Batch Glc+Xyl+Ara PPA652ara 7.3 4.88 9.65 0.31 0.00 25 → ∞ 0.06 0.03

N-depletion Glc+Xyl+Ara AF1000 16.0 0.27 2.73 0.64 0.48 29 1.33 2.37 0.23

N-depletion Glc+Xyl+Ara PPA652ara 15.0 0.39 3.79 0.54 0.54 31 1.00 1.38 0.14

N-fedbatch Glc+Xyl+Ara AF1000 11.0 10.37 37.05 1.84 2.66 45 0.69 0.18 0.05

N-fedbatch Glc+Xyl+Ara PPA652ara 11.0 11.82 36.75 1.87 0.77 38 2.43 0.16 0.05


2.3.3 The benefit of nitrogen depletion The reason that nitrogen depletion resulted in both increased specific productivity of 3HB, as well as an increased yield of 3HB/CDW for PPA652ara, is likely due to a combination of different effects. For one, the acetoacetyl-CoA thiolase, which converts acetyl-CoA into acetoacetyl-CoA, is feedback regulated by CoA [276]. During exponential growth in batch, CoA levels are expected to be high, and acetoacetyl-CoA thiolase should thus be inhibited. However, when the cells are nitrogen starved, the flux through the TCA cycle is drastically decreased, and CoA will mostly be present as acetyl-CoA, rather than its free form. As a consequence, acetoacetyl-CoA thiolase becomes more active. In addition to this, nitrogen depletion probably results in accumulation of NADH and/or ATP, due to a continued carbon flux over the glycolysis, but reduced opportunities to capitalize on the produced energy. Both of these compounds are inhibitory to citrate synthase (CS) [277], the competing enzyme to acetoacetyl-CoA thiolase. Hence, nitrogen depletion should result in a less active CS, and a more active acetoacetyl-CoA thiolase, and consequently higher 3HB productivity. 2.3.4 Strategies to further improve production of 3HB In addition to nitrogen depletion and limitation, other strategies for improving the production of 3HB were also investigated. It was for example considered whether the availability of NADPH, the reducing agent hypothesized to be used by acetoacetyl-CoA reductase to produce 3-hydroxybutyryl-CoA, was a limiting factor. To investigate this, the E. coli gene zwf, encoding for glucose-6-phosphate dehydrogenase (G6PDH), was cloned into pBAD [278] and co-expressed with the enzymes of pJBGT3RX in the E. coli strain AF1000. It was believed that overexpression of G6PDH would lead to an increased intracellular concentration of NADPH, as this first enzyme of the pentose phosphate pathway produces NADPH and 6-phosphoglucono-lactone from NADP+ and glucose-6-phosphate. Another strategy that was applied was to deplete phosphorus in the cultivation medium, rather than nitrogen. This was done in order to investigate if production of 3HB would be stimulated also under these conditions, and if the outcome would differ from nitrogen depletion.


The results of these strategies are reported in figure 36. In figure 36 A, AF1000-pJBGT3RX is cultivated on glucose during nitrogen depletion, which acts as a reference cultivation. In figure 36 B, pJBGT3RX and pBAD-zwf are co-expressed in AF1000 cultivated on glucose during nitrogen depletion. In figure 36 C, AF1000-pJBGT3RX is cultivated on glucose during phosphorus depletion. As described previously, all cultivations were induced at an OD600 of 0.2 with 200 μM IPTG. For pBAD-zwf, induction was performed in tandem with pJBGT3RX by the addition of 20 mg L-1 of L-arabinose. For comparative reasons, the cultivations are followed for 7 h after the initiation of the depletion phase, since glucose exhausted in the phosphorus-limited experiment at this time point. For AF1000 overexpressing G6PDH (zwf), approximately 60% more 3HB and acetic acid were being produced in the nitrogen depletion phase, than for the reference culture (table 6). In addition, this construct further produced approximately 60% less cell mass. As a result, both the specific productivity of 3HB and the yield of 3HB/CDW increased when G6PDH was being overexpressed during nitrogen depletion. It thus appeared that overexpression of G6PDH resulted in elevated levels of NADPH in the cell, but it remains to be concluded by how much, and if the acetoacetyl-CoA reductase exhibits an increased catalytic rate because of this. It also remains to be determined if the increased NADPH levels further inhibits CS, which the improved yield of 3HB/CDW might suggest.


Figure 36: AF1000-pJBGT3RX during nitrogen (A and B) and phosphorus (C) depletion. In (B), the additional plasmid pBAD-zwf is used. The dotted lines indicate the starting point of the depletion phases.

Specific production rate of 3HB (q3HB)

Acetic acid (HAc)3-hydroxybutyric acid (3HB)

Cell dry weight (CDW)

Glucose (Glc)Xylose (Xyl)Arabinose (Ara)

Ammonia (NH3)

5 105 10

5 10

5 10 5 10

5 10

5 105 10

0

50

100

150

0.20.40.60.81.01.2

246810

12345

Nitrogen depletion phase Nitrogen depletion phase

A B

5 10 5 10

0.10.20.30.40.5

q 3HB

(mg

g-1 h

-1)

0

50

100

150

NH3 (

g L-1

)

0.10.20.30.40.5

HAc,

3HB

(g L

-1)

0.20.40.60.81.01.2

Glc (

g L-1

)2468

10

CDW

(g L

-1)

12345

Time (h)0 5 10

Time (h)0 5 10

5 105 10

5 10

5 10

Phosphorus depletion phase

C

5 10

q 3HB

(mg

g-1 h

-1)

0

50

100

150

PO-3 4

(g L

-1)

0.10.20.30.40.5

HAc,

3HB

(g L

-1)

0.20.40.60.81.01.2

Glc (

g L-1

)

2468

10

CDW

(g L

-1)

12345

Time (h)0 5 10

Phosphate (PO4-3)

Legend:


Table 6: Production parameters of AF1000 harboring pJBGT3RX (and pBAD-zwf), cultivated on glucose during nitrogen and phosphorus depletion. The values in the table correspond the depleted phases only, excluding prior batch phases.

When AF1000-pJBGT3RX was cultivated during phosphorus depletion, final concentrations of both cell mass and 3HB were approximately 350% higher than during nitrogen depletion (table 6). The yield of 3HB/CDW was thus similar in both cultivations, but the q3HB were significantly higher during phosphorus depletion. The fact that the cell mass increased much more under phosphorus depletion is in line with previous data [279], as the cell has a lot of internal phosphate, mainly in the RNA, that can utilized for continued synthesis of DNA. It is however difficult to speculate on why the specific production rate of 3HB was greater during phosphorus depletion, and further investigation is needed in order to come to a conclusion. In addition to these effects, phosphorus depletion seemed to result in a lower acetic acid titer. This is also difficult to explain, but one possibility is that there likely is very little inorganic phosphate (Pi) available for the phosphotransacetylase/acetate kinase (pta/ackA) production route [280] during phosphorus depletion. Although this is not the only route to acetic acid, it could contribute to a reduced titer. In summary, both phosphorus depletion and overexpression of G6PDH seemed promising strategies to further increase the production of 3HB by recombinant E. coli. The next step is to combine these strategies with production of 3HB from glucose, xylose and arabinose by E. coli PPA652ara.

Process mode Plasmid Time CDW 3HB Sugar consumed HAc mean q3HB Y3HB/HAc Y3HB/CDW Y3HB/Sugar

(h) (g L-1) (g L-1) (g L-1) (g L-1) (mg g-1 h-1) (g g-1) (g g-1) (g g-1)

N-depletion pJBGT3RX 7.0 0.41 0.32 3.28 0.33 19 0.97 0.78 0.10

N-depletion pJBGT3RX + pBAD-zwf 7.0 0.26 0.52 4.02 0.51 35 1.02 2.00 0.13

P-depletion pJBGT3RX 7.0 1.47 1.13 7.65 0.22 63 5.14 0.77 0.15


2.4 Concluding remarks This investigation has focused on engineering E. coli towards two principally different, important applications. In the first part of the study, the possibility of using E. coli as a production and delivery system of subunit vaccines was investigated. The novel surface expression vector pAIDA1 with improved detectability of surface expressed proteins was therefore created and used to successfully express the Salmonella antigens SefA, H:gm, H:gmd and H:gmdSefA on the E. coli cell surface. To arrive at an E. coli based live vaccine against S. Enteritidis, two main obstacles were encountered in the study. The first obstacle was that the inflammatory response of epithelial cells exposed to the E. coli expressing the antigens was relatively low. Substituting the E. coli strain to a variant that has a more powerful inherent response can potentially solve this issue, but a range of strains would need to be tested as it is difficult to predict the outcome based on the genotype. The second obstacle was that surface expression of recombinant passenger proteins larger than 25-30 kDa with from AIDA-I system seemed to result in some degree of proteolysis. By lowering the pH and cultivation temperature, this effect could be reduced, but not eliminated. Proteolysis of the recombinant passenger is however not unique for this work and has been seen in most studies where larger proteins, such as enzymes, have been expressed by AT [106, 107, 114-116]. Since many of these studies did not utilize a method for C-terminal detection of the passenger protein, proteolytic effects were not always identified or considered a problem. With the added knowledge from this work, it can be concluded that proteolysis is indeed a major problem, and it should be a prioritized issue to solve, as it cripples the potential of autotransport-based surface expression systems. One can imagine several potential solutions to this problem, where the simplest might be to delete one or several periplasmic proteases from the expression strain, such as Mi, Tsp and Pitrilysin. However, the proteolysis is not necessarily the main problem, but rather the consequence of another underlying cause. For example, if the passenger protein starts to fold


prematurely in the periplasm, it will be sterically impossible for the protein to enter the small pore of the β-barrel domain. The resulting fate would inevitably be either aggregation of misfolded proteins in the periplasm or proteolysis by, for instance, the householding chaperone/protease DegP. Another possible solution to increase the amount of full-length protein at the cell surface might therefore be to overexpress periplasmic chaperones, such as SurA, Skp and DegP [92, 93], as these may assist in preventing premature folding of the passenger protein, and as a consequence, proteolysis. Seeing as the autochaperone domain of ATs has proven to be crucial for folding of the passenger, one completely different strategy could be to include a larger portion of the native passenger protein in the surface expression construct. Supporting this strategy is the fact that presence of the wild type passengers of the ATs Hbp and IcsA has promoted surface expression results of recombinant proteins [116, 281]. It could further be so that AIDA-I is unsuitable for surface expression of large recombinant proteins, despite the fact that it harbors a reasonably large passenger of approximately 80 kDa. If searching for an alternative candidate, one should look for ATs containing ESPs, as several of these harbors native passengers well over 100 kDa, possibly making them more suited for surface expression of large proteins. By the methodology presented in paper V, finding ESP-containing ATs should not be difficult. In the second part of the study, the platform E. coli strain PPA652ara was developed and used to produce the chiral molecule (R)-3HB from simultaneous assimilation D-glucose, D-xylose and L-arabinose. To the author’s knowledge, this is the first time that a chemical has been produced by a single microorganism strain from simultaneous uptake of these three sugars under aerobic conditions. Others have investigated simultaneous uptake of hexoses and pentoses by S. cerevisiae and E. coli, but the prevailing pattern has been sequential or slow uptake, often with high levels of residual sugars in the medium [236, 237, 282, 283]. In a case where simultaneous uptake of glucose, xylose and arabinose was achieved, it required a consortium of E. coli strains where each was specialized on the uptake of one substrate [238].


In this part of the study, it could be concluded that there was no clear advantage of using the developed E. coli PPA652ara over a wild type strain if the production process was carbon-limited. It was however also shown that 3HB could not be produced during carbon limitation, likely due to the fact the predominant amount of the available carbon was prioritized to cell mass under those conditions. It was instead realized that non-limiting sugar concentrations were necessary for 3HB to be produced from the mixture of glucose, xylose and arabinose, and that the highest specific productivities of 3HB were achieved when the availability of either nitrogen or phosphorus was restricted. During nitrogen depletion, using PPA652ara was clearly favorable, as it simultaneously assimilated all three monosaccharides, while the wild type would only consume glucose. If the highest specific productivity of 3HB that was achieved in this work (phosphorus depletion, q3HB = 63 mg g-1 h-1) is compared with the currently highest published volumetric productivity of 3HB in E. coli from glucose at 0.50 g L-1 h-1 [275], it can be calculated that less than 10 g L-1 of CDW is needed in order to achieve an equivalent volumetric productivity. Considering that E. coli easily can be cultivated to over 100 g L-1 [13], this should not be difficult to achieve. The results presented here are thus promising for development of production processes of 3HB from mixed sugars by E. coli. Further on, seeing as many of the platform chemicals that are of interest to produce in biorefineries are metabolites, it is possible that the conditions that resulted in the highest specific productivity of 3HB in this work also would be applicable for production of these. While the results on 3HB production from glucose, xylose and arabinose by E. coli are promising, much work remains to be done. For example, overexpression of the E. coli enzyme G6PDH resulted in increased specific productivity of 3HB during nitrogen depletion. It is therefore of interest to investigate if this mutation, combined with phosphorus depletion, results in a further improved process. A potential production process of 3HB also needs to be outlined, which due to the constraints for 3HB production, most likely would need to be divided into a growth and a production phase. The synthetic medium used in this work also needs to be replaced with an actual lignocellulose hydrolyzate, where the effect of inhibitory compounds on growth and production kinetics needs to be evaluated.


3 Acknowledgements Without the assistance and support of a great amount of people, this thesis would never have happened. For those who have been directly or indirectly involved in its creation, I express my sincerest gratitude. Tack Gen Larsson, min handledare och mentor som jämt haft tid för mig, oavsett hur upptagen du själv varit. Du har alltid visat ett stort förtroende för mig och min forskning, betydligt större än jag själv haft mestadels. Du har en magisk förmåga att vrida det mesta till något positivt, och efter ett möte med dig har jag alltid känt mig peppad att gå vidare, oavsett hur dystert det kändes innan! Tack Per-Åke Nygren och Stefan Ståhl som tilldelade mig ett excellensstipendium 2010. Detta blev startskottet på denna resa! Tack också Stefan för att du tog dig tid att förhandsgranska denna avhandling. Tack Formas och Sida för finansiering av projekten jag jobbat i. Tack Martin Gustavsson för ett nära och underhållande samarbete under (nästan) hela min doktorandtid och för ditt betydande bidrag till artikel I, II och V. Vi har haft många (o)seriösa diskussioner genom åren som livade upp stämningen på kontoret och i labbet! Tack även för din hjälp rörande de praktiska detaljerna runt disputationen och för din korrläsning av denna avhandling. Thank you Jaroslav Belotserkovsky for a fairly short but close collaboration that resulted in paper IV. Thank you also for the many fruitful discussion we have had, especially the non-scientific ones! Tack Gustav Sjöberg för hjälp med det mesta sedan du kom till vår avdelning. Allt från genomförande av experiment och tolkning av resultat till korrläsning av denna avhandling och ditt bidrag till artikel IV. Det är du som nu tar över stafettpinnen och jag är övertygad om att både du och gruppen går en lysande framtid till mötes.


Thank you Mónica Guevara-Martínez and Mariel Pérez-Zabaleta for your contagious enthusiasm and contributions to paper IV. It is rare to seen people work with the dedication that you do! Thank you Jorge Quillaguamán for your initiative to the collaboration between Universidad Mayor de San Simón and KTH, which has led to significant scientific, as well as cultural exchange. Thank you also for your contribution to paper IV. Tack Patrik Samuelson för all hjälp med klonings-relaterade forskningsproblem och för ditt bidrag till artikel I. Du var alltid villig att hjälpa till när jag stötte på patrull. Tack Andres Veide och Johan Norén för labbhjälp, givande diskussioner och trevliga luncher genom åren! Thank you Ernesto Gonzalez de Valdivia for your valuable assistance in the lab. Thank you Thi-Huyen Do, Ngoc Tan Tran and Nam Hai Truong for your work that led up to paper I. Thank you Lars Janoschek for a very productive internship and your contribution to paper II. Tack Björn Hallström för din ovärderliga hjälp med genom-analys och för ditt bidrag till artikel III. Tack Susanna Lundh för en kort men trevlig doktorandtid tillsammans! Tack även för ditt bidrag till artikel II. Tack Lars Arvestad för ditt bidrag till artikel IV. Thank you all diploma workers over the years for the contributions that you have made towards this thesis: Anna-Karin Andermo, Jorge Freitas, Joshua Dhivyan Gnanasekhar, David Hörnström and Karin Sjöberg Gällnö.


Thank you all PhD student colleges in the seminar course in industrial biotechnology, which have contributed to increasing the quality of paper III and IV. Tack John Löfblom och Filippa Fleetwood för hjälp med att köra Galliosen. Tack också John för att du tog dig tid att förhandsgranska min licentiatavhandling. Tack Henrik Hansson och Jan Kinnander för hjälp med HPLC-analys. Thank you Sverker Lundin, Manoj Kumar Valluru and Ida Bodlund for helping out with the lab course in cultivation technology. Thank you all colleagues at the division of industrial biotechnology, both past and present, for assistance, advice and providing a nice work environment! Tack Mamma, Pappa och Calle. Utan er hade jag aldrig varit där jag är idag! Och slutligen, tack Josefine Bergholtz. Hur skall jag kunna tacka dig nog? Du har varit ovärderlig under skrivartider, både för licentiat- och doktorsavhandling! Tack för att du har stått ut med mitt emellanåt väldigt intensiva jobbande och tagit hand om ALLT. Men framförallt, tack för att du är en del av mitt liv och för att vi har så roligt tillsammans!


4 Abbreviations 3HB 3-hydroxybutyric acid aa Amino acid AB Antibody ABC ATP-binding cassette AC Adenylate cyclase Ac-ω-TA Arthrobacter citreus ω-transaminase Ara Arabinose AT Autotransport ATP Adenosine triphosphate AIDA-I Adhesin involved in diffuse adherence BAM β-Barrel assembly machine BCG Bacillus Calmette-Guérin cAMP Cyclic adenosine monophosphate CAP Catabolite activator protein CCR Carbon catabolite repression CDW Cell dry weight CFC-11 Trichlorofluoromethane CFC-12 Dichlorodifluoromethane CHO cells Chinese hamster ovary cells CoA Coenzyme A CS Citrate synthase D Dilution rate DNA Deoxyribonucleic acid DOE Design of experiments E. coli Escherichia coli EI Enzyme I EII Enzyme II EMA European Medicines Agency ESP Extended signal peptide ESPR Extended signal peptide region Fab Fragment, antigen-binding Fc Fragment, crystallizable FDA Food and Drug Administration


G6PDH Glucose-6-phosphate dehydrogenase Glc Glucose HAc Acetic acid His6-tag Hexa histidine-tag HMF Hydroxymethyl furfural HMM Hidden markov model HPr Histidine protein HRV Human rhinovirus IEA International Energy Agency IgG Immunoglobulin G IL-8 Interleukin 8 Inp Ice-nucleation protein IPTG Isopropyl β-D-1-thiogalactopyranoside kDa Kilodalton mAB Monoclonal antibody NAD Nicotinamide adenine dinucleotide NADP Nicotinamide adenine dinucleotide phosphate NOAA National Oceanic and Atmospheric Administration OD600 Optical density at 600 nm Omp Outer membrane protein OPEC Organization of petroleum exporting countries PBT Polybutylene terephthalate PDO 1,3-Propanediol PEG Polyethylene glycol PEP Phosphoenolpyruvate PET Polyethylene terephthalate PHA Polyhydroxyalkanoate PHB Polyhydroxybutyrate Pfam Protein families database Pi Inorganic phosphate PLA Polylactic acid PTM Post-translational modification PTS Phosphotransferase system PTT Polytrimethylene terephthalate q3HB Specific productivity of 3-hydroxybutyric acid RNA Ribonucleic acid R/P ratio Reserve to production ratio


S. cerevisiae Saccharomyces cerevisiae scFv Single-chain fragment, variable S. Enteritidis Salmonella enterica serovar Enteritidis SP Signal peptide TCA cycle Tricarboxylic acid cycle TEV Tobacco etch virus Tyr Tyrosinase Xyl Xylose Y Yield μ Specific growth rate


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