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Fermentative production of lysine by Corynebacterium glutamicum

ATCC 21799

بخش پایلوت بیوتکنولوژیبخش پایلوت بیوتکنولوژی

Amino acid Production Application Amount (tons/year) L-Glutamate fermentation flavor enhancer 1.000.000 L-Lysine fermentation/enzymatic feed additive 450.000 D,L-Methionine chemical feed additive 350.000 Glycine chemical sweetener/antoxidant 15.000 L-Threonine fermentation feed additive 15.000 L-Tryptophan fermentation feed additive L-Aspartate fermentation/enzymatic aspartame 10.000 L-Phenylalanine fermentation/enzymatic aspartame 10.000

Lysine Applications: 1. Food & dietary supplement (66%). 2. Medicine, cosmetics, chemicals (4%). 3. Feed : essential aminoacid for most mammals (30%). Production by: 1. Chemical synthesis (cheap, racemic mixture)

2. Extraction from proteins (optical pure, purification difficult). 3. Fermentation (optical pure, cheap substrates). 4. Immobilized enzymes (optical pure, down-stream proc. easy).

Corynebacterium Corynebacterium glutamicumglutamicum

Glucose

Oxygen

Ammonia

Minerals &Vitamins

Lysine

Corynebacterium glutamicumCorynebacterium glutamicum

Gram-positive, non-pathogenic, fast growing soil bacteriumGram-positive, non-pathogenic, fast growing soil bacterium Pathways leading to amino acids are much simpler in their Pathways leading to amino acids are much simpler in their regulation when compared to regulation when compared to E. Coli:E. Coli:

-- very few enzymes are feedback controlled-- very few enzymes are feedback controlled-- no isoenzymes detected -- no isoenzymes detected -- genome sequenced-- genome sequenced

Problems:Problems:

1) vector system are far from ideal (E.coli vectors will not 1) vector system are far from ideal (E.coli vectors will not work) work) 2) Transformation rates are low2) Transformation rates are low

TAXONOMY

• Phylum :Actinobacteria • class : Actinobacteria • subclass : Actinobacteridae • order : Actinomycetales• suborder : Corynebacterineae• family : Corynebacteriaceae• genus : Caseobacter• species : Micrococcus glutamicus, Kinoshita et al. 1958 • Strain: Corynebacterium glutamicum ATCC 21799

NlNl,,

Example of Amino Acid Production PathwayExample of Amino Acid Production Pathway

Glutamate

Aspartate

Anaplerotic reactionsPhosphoenolpyruvate

CO2

CO2

NH4+

Acetyl-CoA Glyoxylate

Isocitrate

Aspartate family Split pathway to diaminopimelate (DAP)

Lysine production

L-Lysine biosynthetic pathways in prokaryotes

Lysine production

C. glutamicum uses succinylase and dehydrogenase variantOne enzyme controlled: aspartate kinase!

Aspartate family Split pathway to diaminopimelate (DAP)

Lysine production

[NH4+] determines flux partitioning

high NH4+: 50-70% via dehydrogenase

low NH4+: only succinylase

C. glutamicum uses succinylase and dehydrogenase variant

Enzyme reactions important in the assimilation of ammonia. GS Enzyme reactions important in the assimilation of ammonia. GS = glutamine. synthetase; GOGAT: glutamate synthase; AS: = glutamine. synthetase; GOGAT: glutamate synthase; AS: asparagine synthetase; AAT: aspartateaminotransferaseasparagine synthetase; AAT: aspartateaminotransferase..

What is metabolic engineeringWhat is metabolic engineering??

Directed improvement of product formation or cellular properties through:

• Modification of existing biochemical reactions or • Introduction of new ones with recombinant DNA technology

Motivation for metabolic engineeringMotivation for metabolic engineering

Improve yield and productivityImprove yield and productivityExpand range of substrates Expand range of substrates

consumedconsumedImprove cellular propertiesImprove cellular properties

Fraction of substrate converted to product

Volumetric rate of product synthesis

Process OverviewProcess Overview

Emphasis on complete metabolic Emphasis on complete metabolic networks rather than on individual networks rather than on individual reactionsreactions

Two defining steps:Two defining steps:

Synthesis: Applied molecular biologySynthesis: Applied molecular biologyAnalysis: Engineering componentAnalysis: Engineering component

Engineering an organism to produce a Engineering an organism to produce a specific product.specific product.

Once an organism is chosen, how do we Once an organism is chosen, how do we modify it?modify it?

1. Channel nutrients down existing 1. Channel nutrients down existing metabolic pathways that produce the metabolic pathways that produce the desired product.desired product.2. If option 1 is not possible, use 2. If option 1 is not possible, use recombinant DNA to actually change recombinant DNA to actually change genome and create new metabolic genome and create new metabolic pathwayspathways

Channeling nutrients down specific Channeling nutrients down specific pathwayspathways

GoalGoalRestrict or add excess of specified nutrients for Restrict or add excess of specified nutrients for the purpose of inhibiting unwanted reactions the purpose of inhibiting unwanted reactions and driving forward desired reactionsand driving forward desired reactionsProblemProblemNature has designed organisms with strict Nature has designed organisms with strict metabolic regulating mechanisms.metabolic regulating mechanisms.SolutionSolutionKnowledge of major and minor metabolic Knowledge of major and minor metabolic pathways aid in determining what pathways aid in determining what variables/nutrients can be changed and how to variables/nutrients can be changed and how to go about changing themgo about changing them

The specific objectives are as follows:

1. To optimize the fermentation process for lysine production.

2. To optimize the downstream processing for lysine separation.

3. To prepare the techno-economic feasibility study of commercial lysine plant.

Thank you for your Thank you for your attentionattention