Lysine Production

بسم الله الرحمن الرحیم قل سیروا فی الارض فانظرو کیف بدا الخلق سوره مبارکه عنکبوت - آیه شریفه 20

جمهوری اسلامی ایران انستیتو پاستور ایران Fermentative production of lysine by Corynebacterium glutamicum ATCC 21799 بخش پایلوت بیوتکنولوژی

Lysine Applications : 1. Food & dietary supplement (66%).

2. M edicine, 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 glutamicum Lysine Glucose Oxygen Ammonia Minerals & Vitamins

Corynebacterium glutamicum Gram-positive, non-pathogenic, fast growing soil bacterium Pathways leading to amino acids are much simpler in their regulation when compared to E. Coli: -- very few enzymes are feedback controlled -- no isoenzymes detected -- genome sequenced Problems: 1) vector system are far from ideal (E.coli vectors will not work) 2) 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: C orynebacterium glutamicum ATCC 21799

Nl,

Example of Amino Acid Production Pathway

Glutamate Aspartate Phosphoenolpyruvate Anaplerotic reactions CO 2 CO 2 NH 4 + Acetyl-CoA Glyoxylate Isocitrate

Aspartate family Split pathway to diaminopimelate (DAP) Lysine production

L-Lysine biosynthetic pathways in prokaryotes

C. glutamicum uses succinylase and dehydrogenase variant One enzyme controlled: aspartate kinase! Aspartate family Split pathway to diaminopimelate (DAP) Lysine production

[NH 4 + ] determines flux partitioning high NH 4 + : 50-70% via dehydrogenase low NH 4 + : only succinylase C. glutamicum uses succinylase and dehydrogenase variant Lysine production

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

What 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 engineering

Improve yield and productivity

Expand range of substrates consumed

Improve cellular properties

Fraction of substrate converted to product Volumetric rate of product synthesis

Process Overview Emphasis on complete metabolic networks rather than on individual reactions Two defining steps: Synthesis: Applied molecular biology Analysis: Engineering component

Engineering an organism to produce a specific product. Once an organism is chosen, how do we modify it? 1. Channel nutrients down existing metabolic pathways that produce the desired product. 2. If option 1 is not possible, use recombinant DNA to actually change genome and create new metabolic pathways

Channeling nutrients down specific pathways Goal Restrict or add excess of specified nutrients for the purpose of inhibiting unwanted reactions and driving forward desired reactions Problem Nature has designed organisms with strict metabolic regulating mechanisms. Solution Knowledge of major and minor metabolic pathways aid in determining what variables/nutrients can be changed and how to go 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.

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