Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation.
The previously constructed strain Corynebacterium glutamicumilvNM13 with acetohydroxy acid synthase, resistant to inhibition by all three branched-chain amino acids (L-valine, L-isoleucine and L-leucine), was used as a basis to develop a new type of valine producer by genetic engineering. The main strategy was to modulate expression of the genes involved in the biosynthesis of branched-chain amino acids. The activity of the promoters P-ilvD (dihydroxyacid dehydratase) and P-ilvE (transaminase) was up-modulated and the activity of the promoters P-ilvA (threonine deaminase) and P-leuA (isopropylmalate synthase) was down-modulated by site-directed mutagenesis. A constructed weak promoter of ilvA (or leuA), which was introduced into the C. glutamicum chromosome via a gene-replacement technique reduced the biosynthetic rate of isoleucine (or leucine), which lowered the mutant growth rate and increased valine production. Overexpression of ilvD and ilvE driven by the strong mutant promoters P-ilvDM7 and P-ilvEM6 resulted in an even higher level of valine production. Thus, the strain C. glutamicum ilvNM13 DeltapanB P-ilvAM1CG P-ilvDM7 P-ilvEM6, having all mutations constructed within the chromosome, produced 136 mM valine in a 48-h cultivation.
Holátko
Jirí
J
Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague 4, Czech Republic.
Elisáková
Veronika
V
Prouza
Marek
M
Sobotka
Miroslav
M
Nesvera
Jan
J
Pátek
Miroslav
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D003470:Culture Media; D005818:Genetic Engineering / Q000379:methods*; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D007532:Isoleucine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D013913:Threonine Dehydratase / Q000235:genetics / Q000378:metabolism; D000637:Transaminases / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000096:biosynthesis*
D001426:Bacterial Proteins; D003470:Culture Media; D007532:Isoleucine; D000637:Transaminases; D006836:Hydro-Lyases; C020052:dihydroxyacid dehydratase; D013913:Threonine Dehydratase; D014633:Valine