Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase l-isoleucine production in Corynebacterium glutamicum

Yin, Lianghong; Hu, Xiaoqing; Xu, De-Xiang; Ning, Jianfei; Chen, Jian; Wang, Xiaoyuan

doi:10.1016/j.ymben.2012.06.002

Cited by 72 publications

(49 citation statements)

References 45 publications

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“…For example, overexpressing the feedbackresistant TD and AHAS in C. glutamicum IWJ001 produced 30.7 g/L l-isoleucine after 72 H fermentation in a 3-L fermentor [8]. Overexpressing the global regulator Lrp, the export system BrnFE, and the ribosome elongation and recycling factors could also increase L-isoleucine production in C. glutamicum [9][10][11][12].…”

Section: Metabolic Pathways Associated With the L-isoleucine Biosynthmentioning

confidence: 99%

Enhancing pentose phosphate pathway in Corynebacterium glutamicum to improve l‐isoleucine production

Wang

et al. 2016

Biotech and App Biochem

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Three genes, gnd, pgl, and fbp, relevant to the pentose phosphate pathway (PPP) were overexpressed in Corynebacterium glutamicum IWJ001, leading to increase of l-isoleucine production. The transcriptional levels of gnd, pgl, and fbp significantly increased in IWJ001/pDXW-8-gnd-fbp-pgl. Compared with the control strain IWJ001/pDXW-8, intracellular NADPH/NADP ratios in IWJ001/pDXW-8-gnd and IWJ001/pDXW-8-gnd-fbp cells grown for 36 H increased threefold and fourfold, respectively, indicating that overexpression of gnd and fbp redirected the carbon flux to PPP. Intracellular NADPH/NADP ratio in IWJ001/pDXW-8-gnd-fbp-pgl grown for 36 H was similar to IWJ001/pDXW-8, suggesting that the NADPH produced by PPP could be quickly consumed for l-isoleucine production. 10.9 and 28.96 g/L of l-isoleucine was produced in IWJ001/pDXW-8-gnd-fbp-pgl in shake flask cultivation and fed-batch fermentation, respectively. In addition, IWJ001/pDXW-8-gnd-fbp-pgl grew fast, its dry cell weight reached 49 g/L after 48 H, whereas the start strain IWJ001/pDXW-8 reached only 40 g/L. After 96 H fermentation, l-isoleucine yield on glucose in IWJ001/pDXW-8-gnd-fbp-pgl reached 0.138 g/g. The results demonstrate that carbon flux redirection to PPP is an efficient approach to enhance l-isoleucine production in C. glutamicum.

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Section: Metabolic Pathways Associated With the L-isoleucine Biosynthmentioning

confidence: 99%

Enhancing pentose phosphate pathway in Corynebacterium glutamicum to improve l‐isoleucine production

Wang

et al. 2016

Biotech and App Biochem

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“…The pET28a vector endows TD protein with N-terminal His-Tag. Thus, TD protein was purified by Ni-NTA affinity column under buffer B (0.5 mM NaCl, 0.5 mM imidazole, and 20 mM HEPES, pH 8.0) (Yin et al 2012). Protein concentrations were determined by the method of Bradford (1976).…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…Protein concentrations were determined by the method of Bradford (1976). TD activity was determined according to the published method with slight modification (Yin et al 2012;Guillouet et al 1999). Enzyme assay was performed in 100 mM potassium phosphate buffer (pH 8.0) containing 1 mM pyridoxal phosphate and 40 mM threonine.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…On one hand, the introduction of point mutations into the genome of isoleucineproducing strain can largely increase isoleucine production (Vogt et al 2015). On the other hand, co-expression of TD and AHAS in an isoleucine-producing strain significantly increases isoleucine production (Yin et al 2012). From the aforementioned, TD is important in the development of a strain that exhibits high performance in isoleucine production.…”

Section: Introductionmentioning

confidence: 99%

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Generation of mutant threonine dehydratase and its effects on isoleucine synthesis in Corynebacterium glutamicum

Guo

Han

et al. 2015

World J Microbiol Biotechnol

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Isoleucine synthesis is strongly regulated by its end product (isoleucine) in Corynebacterium glutamicum, especially at threonine dehydratase (TD) node. Multiple alignments of TD sequences of C. glutamicum and other sources were performed. According to the structural analysis, three TD variants were constructed by site-directed mutagenesis. These TD variants improved the performance of the holoenzyme. The specific activity of V140M variant was 1.5-fold higher than that of the wild-type TD, whereas F383A variant showed complete resistance to feedback inhibition by isoleucine. V140M-F383A variant had all the advantages of V140M and F383A variants and displayed 1.5-fold specific activity and complete resistance to isoleucine. In C. glutamicum, overexpression of V140M, F383A, and V140M-F383A variants accumulated 0.55, 0.63, and 0.73 g/l isoleucine, and overexpression of wild-type TD produced 0.47 g/l isoleucine. Thus, these novel TD variants, particularly V140M-F383A, showed great potential in isoleucine synthesis.

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“…It would be better to use microorganism that can produce l-methionine for SAM production. Corynebacterium glutamicum is well known for its ability to produce various amino acids [19][20][21][22]. C. glutamicum strains that could produce high level of l-methionine have been reported [23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S‐adenosyl‐l‐methionine

Han

Wang

2015

Biotech and App Biochem

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Two genes encoding methionine adenosyltransferase, SAM2 from Saccharomyces cerevisiae and metK from Corynebacterium glutamicum, were individually cloned into pDXW-8, the shuttle vector between Escherichia coli and C. glutamicum, and overexpressed in E. coli DH5α and C. glutamicum ATCC13032. In DH5α, both genes were overexpressed and their protein products showed the activity of methionine adenosyltransferase. In ATCC13032, metK was overexpressed, its product MetK showed the enzyme activity and could convert l-methionine to S-adenosyl-l-methionine (SAM). However, when SAM2 was overexpressed in ATCC13032, neither the enzyme activity nor the conversion of SAM from l-methionine was observed. Reverse transcription PCR analysis and SDS-PAGE showed that SAM2 was transcribed but not translated in C. glutamicum. Therefore, SAM2-C, a mutant SAM2, was constructed by codon optimization, and overexpressed in ATCC13032; it was well transcribed and translated, and could convert l-methionine to SAM. Finally, SAM2-C and metK were individually overexpressed in E. coli BL21(DE3), and their products SAM2-C and MetK were purified and characterized. The optimum activity for both enzymes was found at pH 8.5 and 35 °C; SAM2-C and MetK have similar K for ATP, but quite different K for l-methionine. These results suggest that SAM2-C and MetK can be useful for developing C. glutamicum to produce SAM.

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Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase l-isoleucine production in Corynebacterium glutamicum

Cited by 72 publications

References 45 publications

Enhancing pentose phosphate pathway in Corynebacterium glutamicum to improve l‐isoleucine production

Enhancing pentose phosphate pathway in Corynebacterium glutamicum to improve l‐isoleucine production

Generation of mutant threonine dehydratase and its effects on isoleucine synthesis in Corynebacterium glutamicum

Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S‐adenosyl‐l‐methionine

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