RESUMO
Threonine dehydratase and acetohydroxy acid synthase are critical enzymes in the L-isoleucine biosynthesis pathway of Corynebacterium glutamicum, but their activities are usually feedback-inhibited. In this study, we characterized a feedback-resistant threonine dehydratase and an acetohydroxy acid synthase from an L-isoleucine producing strain C. glutamicum JHI3-156. Sequence analysis showed that there was only a single amino acid substitution (Phe383Val) in the feedback-resistant threonine dehydratase, and there were three mutated amino acids (Pro176Ser, Asp426Glu, and Leu575Trp) in the big subunit of feedback-resistant acetohydroxy acid synthase. The mutated threonine dehydratase over-expressed in E. coli not only showed completely resistance to L-isoleucine inhibition, but also showed enhanced activity. The mutated acetohydroxy acid synthase over-expressed in E. coli showed more resistance to L-isoleucine inhibition than the wild type. Over-expression of the feedback-resistant threonine dehydratase or acetohydroxy acid synthase in C. glutamicum JHI3-156 led to increase of L-isoleucine production; co-expression of them in C. glutamicum JHI3-156 led to 131.7% increase in flask cultivation, and could produce 30.7g/L L-isoleucine in 72-h fed-batch fermentation. These results would be useful to enhance L-isoleucine production in C. glutamicum.
Assuntos
Acetolactato Sintase , Proteínas de Bactérias , Corynebacterium glutamicum , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Isoleucina , Treonina Desidratase , Substituição de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Escherichia coli/enzimologia , Escherichia coli/genética , Isoleucina/biossíntese , Isoleucina/genética , Mutação de Sentido Incorreto , Treonina Desidratase/biossíntese , Treonina Desidratase/genéticaRESUMO
NADPH is the key cofactor in L-isoleucine (Ile) biosynthetic pathway. To increase the Ile biosynthesis in Corynebacterium glutamicum ssp. lactofermentum JHI3-156, NADPH supply needs to be enhanced. Here NAD kinase, the key enzyme for the de novo biosynthesis of NADP(+) and NADPH, were cloned and expressed in JHI3-156, and their influences on Ile production were analysed. Meanwhile, enzyme properties of NAD kinase from JHI3-156 (CljPpnK) were compared with that from C. glutamicum ssp. lactofermentum ATCC 13869 (ClPpnK). Four variations existed between CljPpnK and ClPpnK. Both PpnKs were poly(P)/ATP-dependent NAD kinases that used ATP as the preferred phosphoryl donor and NAD(+) as the preferred acceptor. CljPpnK exhibited a higher activity and stability than ClPpnK and less sensitivity towards the effectors NADPH, NADP(+), and NADH, partly due to the variations between them. The S57P variation decreased their activity. Expression of CljppnK and ClppnK in JHI3-156 increased the ATP-NAD(+) kinase activity by 69- and 47-fold, respectively, the intracellular NADP(+) concentration by 36% and 101%, respectively, the NADPH concentration by 95% and 42%, respectively, and Ile production by 37% and 24%, respectively. These results suggest that overexpressing NAD kinase is a useful metabolic engineering strategy to improve NADPH supply and isoleucine biosynthesis.
Assuntos
Corynebacterium glutamicum/metabolismo , Isoleucina/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Corynebacterium glutamicum/genética , DNA Bacteriano/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Genes Bacterianos , Engenharia Metabólica/métodos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NADP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Regulação para CimaRESUMO
The effect of both dissolved oxygen (DO) and pH on L: -isoleucine production by batch culture of Brevibacterium lactofermentum was investigated. A two-stage agitation speed control strategy was developed, and the isoleucine production reached 23.3 g L(-1) in a relative short time (52 h), increased by 11.6% compared to the results obtained in the single agitation speed control process. In order to make sure whether the combination of DO and pH control can boost the production by a mutual effect, different control modes were conducted, based on the data obtained from the two-stage agitation speed control strategy and the analysis of kinetics parameters at different pH values. The results showed that the mode of combining two-stage DO with two-stage pH control strategy was the optimal for isoleucine production. The isoleucine production can reach 26.6 g L(-1) at 56 h, increased by 14.3% comparing to that obtained by the single two-stage DO control strategy.
