RESUMO
L-valine biosynthesis was analysed by comparing different plasmids in pyruvate-dehydrogenase-deficient Corynebacterium glutamicum strains in order to achieve an optimal production strain. The plasmids contained different combinations of the genes ilvBNCDE encoding for the L-valine forming pathway. It was shown that overexpression of the ilvBN genes encoding acetolactate synthase is obligatory for efficient pyruvate conversion and to prevent L-alanine as a by-product. In contrast to earlier studies, overexpression of ilvE encoding transaminase B is favourable in pyruvate-dehydrogenase-negative strains. Its amplification enhanced L-valine formation and avoided extra- and intracellular accumulation of ketoisovalerate.
Assuntos
Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Complexo Piruvato Desidrogenase/genética , Valina/biossíntese , Alanina/metabolismo , Vias Biossintéticas , Deleção de Genes , Dosagem de Genes , Expressão Gênica , Família Multigênica , PlasmídeosRESUMO
Cofactor recycling is known to be crucial for amino acid synthesis. Hence, cofactor supply was now analyzed for L-valine to identify new targets for an improvement of production. The central carbon metabolism was analyzed by stoichiometric modeling to estimate the influence of cofactors and to quantify the theoretical yield of L-valine on glucose. Three different optimal routes for L-valine biosynthesis were identified by elementary mode (EM) analysis. The modes differed mainly in the manner of NADPH regeneration, substantiating that the cofactor supply may be crucial for efficient L-valine production. Although the isocitrate dehydrogenase as an NADPH source within the tricarboxylic acid cycle only enables an L-valine yield of Y(Val/Glc) = 0.5 mol L-valine/mol glucose (mol Val/mol Glc), the pentose phosphate pathway seems to be the most promising NADPH source. Based on the theoretical calculation of EMs, the gene encoding phosphoglucoisomerase (PGI) was deleted to achieve this EM with a theoretical yield Y(Val/Glc) = 0.86 mol Val/mol Glc during the production phase. The intracellular NADPH concentration was significantly increased in the PGI-deficient mutant. L-Valine yield increased from 0.49 +/- 0.13 to 0.67 +/- 0.03 mol Val/mol Glc, and, concomitantly, the formation of by-products such as pyruvate was reduced.
Assuntos
Corynebacterium glutamicum/metabolismo , Redes e Vias Metabólicas , Modelos Biológicos , NADP/metabolismo , Valina/metabolismo , Técnicas de Cultura de Células , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Meios de Cultura , Fermentação , Glucose-6-Fosfato Isomerase/genética , MetabolomaRESUMO
The effect of different amounts of supplemented L-isoleucine and pantothenate has been analysed with the auxotrophic strain Corynebacterium glutamicum DeltailvA DeltapanB, showing that the final biomass concentration of this preliminary L-valine production strain can be controlled by the amount of added L-isoleucine. One gramme cell dry weight is formed from 48 micromol L-isoleucine. Different amounts of available pantothenate affect the intracellular pyruvate concentration. By limiting pantothenate supplementation from 0.8 to 0.1 microM, a 35-fold increase of cytoplasmic pyruvate up to 14.2 mM can be observed, resulting in the increased formation of L-valine, L-alanine and organic acids in the presence of low pantothenate concentrations. These findings can be used to redirect the carbon flux from glycolysis via pyruvate to the TCA cycle towards the desired product L-valine.