meta engg

8/2/2019 Meta Engg

1/43

3G1 Introduction to Biosciences (for engineers) 2010-2011

Lectures 10-12. Metabolic Engineering

Objectives:

Basic understanding of cellular metabolism, metabolic control,

metabolic engineering strategies and examples

8/2/2019 Meta Engg

2/43


Metabolic Engineering

Bailey, J. A. (1991) Science 252, 1668-1675

8/2/2019 Meta Engg

3/43



Metabolic engineering can be defined as the optimization of genetic and

regulatory processes to increase the cells production of a certain substance

and to reduce waste production.

Cells are complex systems. Metabolic and biological networks are robust,

optimized for each organism through evolution. Genetic and regulatory

changes may have relevant effects in growth and viability of the selected

cells.

(e.g. Kohlstedt, M. et al., (2010) Metabolic fluxes and beyondsystems biology understanding

and engineering of microbial metabolism. Appl. Microb. Biotechnol. 88, 1065-1075 ).

Future: -- > Systems level understanding and rational engineering/optimization.

8/2/2019 Meta Engg

4/43



(e.g. Stephanopoulos, G. N., Aristidou, A. A., Nielsen, J. (1998). "Metabolic

Engineering: Principles and Methodologies". San Diego: Academic Press.

Metabolic Engineering courses. Basic Contents

- Introduction

- Review of Central Metabolism (Transport; Catabolism; Anabolism;

Biosynthesis of macromolecules/polymerization; Energetics).

- Mass, redox and energy balances

- Regulation of Metabolic Pathways (Enzymatic activities; Allosteric

mechanisms; Enzyme levels; Global regulation).

- Metabolic Engineering in Practice (Optimization of yield and

productivity of homologous and/or heterologous products;

Extension to a broader range of substrates and products;

degradation of toxic compounds; new metabolic pathways;

secondary metabolites).

- Metabolic Flux Analysis and Metabolic Control Analysis.

- Metabolic and Biological Networks. Modelling. --> Integration. -- >

Systems Biology.

8/2/2019 Meta Engg

5/43

L10. Cellular Metabolism. Fundamentals. Core molecular types

State-of-the-art (Mon, 8th Nov, 2pm. LR3. / Supervision: Tues, 9th Nov, 4pm. LT2)

L11. Metabolic Fluxes and Metabolic Control. Systems Biology

approaches towards Metabolic Engineering (Thu, 11th Nov, 3pm, LR4)

L12. Metabolic engineering in Practice. Strategies. Case studies

(advanced studies/patents) (Mon, 15th Nov. 2pm. LR3 / Superv: Tues, 16th Nov, 4pm. LT2)


Lectures 10-12. Metabolic Engineering

Schedule:

8/2/2019 Meta Engg

6/43

8/2/2019 Meta Engg

7/43

Metabolism and Metabolic

networks. Fundamentals

( Alberts, B. et al., (2008) Molecular Biology of the Cell,5th Edition. Garland Science ).

8/2/2019 Meta Engg

8/43

Metabolic Networks.Central metabolism at the core of biological networks

8/2/2019 Meta Engg

9/43Figure 2-32 Molecular Biology of the Cell( Garland Science 2008)

Metabolic networks and pathways.From subunits to macromolecules

8/2/2019 Meta Engg


Metabolic networks based on metabolic pathways

8/2/2019 Meta Engg


Metabolic pathways catalyzed by enzymes

8/2/2019 Meta Engg


Enzymes lower the activation energyof the catalyzed reaction

8/2/2019 Meta Engg


Existence of a main core of central metabolic networks

8/2/2019 Meta Engg


In Nature (and throughout evolution)

Different organisms -> Differences in Metabolic Networks(e.g. Photosynthesis Respiration. Complementary processes)

8/2/2019 Meta Engg


What makes a cell?Cellular components. Bacterial cell

8/2/2019 Meta Engg

16/43Table 2-3 Molecular Biology of the Cell( Garland Science 2008)

Bacterial vs mammalian cell.Differences in macromolecular composition

8/2/2019 Meta Engg


Metabolic networksresponsible for conversion of building blocksinto macromolecules/polymerization. How?

8/2/2019 Meta Engg

18/43

Figure 2-36 Molecular Biology of the Cell( Garland Science 2008)

Coupling of catabolic and anabolic (biosynthetic) pathways

8/2/2019 Meta Engg

19/43


Adenosine triphosphate (ATP), energy carrier

8/2/2019 Meta Engg

20/43


Activated energy carriers link catabolism and anabolism

8/2/2019 Meta Engg

21/43


ATP, main energy carrier in cells

8/2/2019 Meta Engg

22/43

Catabolism and Anabolism.Definitions

Catabolism. Biochemical processes involved in the breakdown of organic

compounds, usually leading to the production of energy.

Anabolism. Metabolic processes involved in the synthesis of cell constituents

from simpler molecules such as organic and/or inorganic precursors. An

anabolic process usually requires energy.

(Stephanopoulos, G. N., Aristidou, A. A., Nielsen, J. (1998). "Metabolic

Engineering: Principles and Methodologies". San Diego: Academic Press)

8/2/2019 Meta Engg

23/43


Catabolism strategy.Storage of energy as activated carrier molecules

8/2/2019 Meta Engg

24/43


Main catabolic pathways.Glycolysis. From glucose to pyruvate. Outline

( Cytosol )

1

2

3

8/2/2019 Meta Engg

25/43

Glycolysis. Net result

D-[glucose] + 2 [NAD]+ + 2 [ADP] + 2 [P]i 2 [Pyruvate] + 2 [NADH] + 2 H+ + 2 [ATP]
http://en.wikipedia.org/wiki/File:Pyruvate2_wpmp.pnghttp://en.wikipedia.org/wiki/File:Biochem_reaction_arrow_foward_NNNN_horiz_med.pnghttp://en.wikipedia.org/wiki/File:D-glucose_wpmp.png

8/2/2019 Meta Engg

26/43

Figure 2-71b Molecular Biology of the Cell( Garland Science 2008)

Two pathways for the anaerobic breakdown of pyruvateI. Alcoholic fermentation

(not

NADH)

8/2/2019 Meta Engg

27/43

Figure 2-71a Molecular Biology of the Cell( Garland Science 2008)

II. Lactic fermentation

8/2/2019 Meta Engg

28/43


In the presence of oxygen, pyruvate is transported intothe mitochondria to yield acetyl-CoA and enters the

tricarboxylic acid (TCA) cycle. TCA (Krebs cycle) overview

Pyruvate(H3C-CO-COOH)

Pyruvate

(H3C-CO-COOH)

? (PDH complex)

CO2

( Mitochondrion )

NADH

8/2/2019 Meta Engg

29/43


Final stages of catabolism.Generation of ATP by oxidative phosphorylation.

P/O ratio and ATPsynthase determine total ATP per NADH

Chemiosmotic mechanism (Mitchell, 1960): n sites of proton H+ (e-) translocation and a separate F1Fo ATP synthase. Each

site translocates H+ (e-) generating a proton electrochemical gradient or proton motive force in volts which drives ATP synthesis.

(P/O ratio = f ( H+ (e-) translocated per site and ATP generated per H+ translocated ); -- > latest , see next slide -- > )

( P/O ratio. Number of ATP molecules synthesized by oxidative phosphorylation for each pairof electrons passing from a particular substrate, typically NADH, via a respiratory chain, to O2 ).

ATP synthase

8/2/2019 Meta Engg

30/43

ATP synthase: From sequence to the P/O ratio(different ATP/H+stoichiometries in different organisms)

Watt, I. N. et al., (2010)Proc Natl Acad Sci U S A. 2010 Sep 28;107,16823-16827

Comment on:

Ferguson SJ. (2010) ATP synthase: from sequence to ring size to the P/O ratio.

Proc Natl Acad Sci U S A. 2010 Sep 28;107,16755-16756.

http://www.ncbi.nlm.nih.gov/pubmed/20858734

!!

8/2/2019 Meta Engg

31/43


Glycolysis and the TCA cycle provide the precursorsto synthesize relevant cellular components

(link to anabolism)

8/2/2019 Meta Engg

32/43


Glycolysis and the TCA cycle at the coreof central metabolic networks

8/2/2019 Meta Engg

33/43


Catabolism of fats and fatty acids.Sugars and fats catabolic pathways converge

at the level of acetyl-CoA

8/2/2019 Meta Engg

34/43


Anabolism. Activated carrier molecules/high energyintermediates lead to polymerization

(biosynthesis of macromolecules)

8/2/2019 Meta Engg

35/43


Anabolism. Activated carrier molecules lead tothe biosynthesis of the essential cellular macromolecules:

polysaccharides, proteins and nucleic acids

8/2/2019 Meta Engg

36/43

Table 2-5 Molecular Biology of the Cell( Garland Science 2008)

Activated carrier molecules/high energy intermediates

8/2/2019 Meta Engg

37/43

Figure 2-60a Molecular Biology of the Cell( Garland Science 2008)

Activated carrier NADPH essential in anabolic/biosynthetic pathways is generated in the

pentose-phosphate pathway

( Pentose-phosphate

pathway )

Pentoses

(5-carbon sugars)

(Biosynthesis

)

8/2/2019 Meta Engg

38/43

Metabolic Networks. Central metabolism. Summary

8/2/2019 Meta Engg

39/43

Cells are rich in complexity, with a central core of metabolic and biological

networks robust, together with specific networks optimized for each

organism through evolution ( = interaction with the environment and other

organisms).

Metabolic pathways and metabolic patterns may vary in different organisms

(some appearing in specific organisms only) (e.g.

photosynthesis/respiration; lactic/alcoholic fermentation).

In one specific organism, different environmental conditions (e.g. O2

availability) may lead to different metabolic patterns (e.g. respiratory vs.

respirofermentative metabolism) and/or distribution of metabolic fluxes.

Some organisms are able to respond to stressful conditions by triggering the

expression of specific pathways/networks and compounds (e.g. secondary

metabolism; complex organic compounds/antibiotics).

Metabolic Networks.Central metabolism and other specific metabolic networks

8/2/2019 Meta Engg

40/43


Further reading

Main texts

Molecular Biology of the Cell.

Alberts, Johnson, Walter, Lewis, Raff, Roberts

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4

Molecular Cell Biology

Lodish, Berk, Kaiser, Krieger, Scott, Bretscher, Ploegh, Matsudaira

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb

Intro. to Genetic Analysis

Griffiths, Wessler, Lewontin, Carroll

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=iga

All can be looked up on NCBI bookshelf.

http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=igahttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcbhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4

8/2/2019 Meta Engg

41/43

8/2/2019 Meta Engg

42/43

1. Name three activated carriers needed in anabolic reactions and where they

are synthesized.

2. Would a mammalian cell culture be a good system to produce bioethanol? Explain.

3. Possible reasons why some organisms are not able to assimilate specific sugars

(e.g. pentoses; disaccharides such as lactose). Any solution?

4. Net result of complete oxidation of glucose (100% respiratory metabolism).

Could the total ATP per glucose generated by a microorganism (e.g. yeast) be

different compared to animal cells? Explain.

5. Net result and possible phenotypes of complete catabolism of glucose

in absence of oxygen: a) In mammalian cells. b) In yeast.

Supervision L10. Examples of questions
http://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Books

8/2/2019 Meta Engg

43/43

6. Net result of catabolism of glucose by an anaerobe facultative organism

(e.g. Saccharomyces cerevisiae ,budding yeast, able to assimilate carbon by both

respiratory and fermentative pathways, i.e. respirofermentative metabolism)

under environmental conditions leading to: 25% of carbon being metabolized by

respiration; 75% by fermentation.

7. Main cause (and consequences) of appearance of lactic fermentation in animal cells.

Among the organisms and conditions mentioned in 4), 5) and 6) :

8. What organisms would be adequate for production of bioethanol/biofuels?

9. Which ones would be preferable as hosts for synthesis of biomass and

coupled-to-growth (type-1) products? (e.g. recombinant proteins).

10. Would an environmental medium containing sugars, fats, nitrogen sources, salts

and vitamins be a good substrate for biotechnological processes? Suggest

Supervision L10. Examples of questions
http://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Bookshttp://www.ncbi.nlm.nih.gov/sites/entrez?db=Books

meta engg

Documents