Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum

Wang, Zhiwen; Chen, Tao; Ma, Xinbin; Shen, Zhuo; Zhao, Xiao‐Fan

doi:10.1016/j.biortech.2010.11.120

Cited by 81 publications

(61 citation statements)

References 34 publications

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“…NADPH/NADP + ratios in IWJ001/pDXW-8-gnd-fbp-pgl were close to that of the control strain IWJ001/pDXW-8; NADPH/NADP + ratios in IWJ001/pDXW-8-gnd and IWJ001/pDXW-8-gnd-fbp were higher than in IWJ001/pDXW-8-gnd-fbp-pgl. The decrease of intracellular NADPH/NADP + ratios in IWJ001/pDXW-8-gnd-fbp-pgl might be related to the quick usage of NADPH for l-isoleucine production and cell growth [18,19], consistent with the highest rates for both l-isoleucine production and cell growth of IWJ001/pDXW-8-gnd-fbp-pgl (Figs. 3A and 3B).…”

Section: Nadph Levels Changed In C Glutamicum Strains Overexpressingsupporting

confidence: 65%

“…1). Overexpression of G6PDH, 6PGDH, or PGL could enhance the production of poly-hydroxybutyrate [16], xylitol [17], hydrogen [18], riboflavin [19], and l-lysine [20]. Fructose 1,6-bisphosphatase (FBPase) encoded by fbp is part of the gluconeogenetic pathway and essential for C. glutamicum to grow on noncarbohydrates such as acetate.…”

Section: Metabolic Pathways Associated With the L-isoleucine Biosynthmentioning

confidence: 99%

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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: Nadph Levels Changed In C Glutamicum Strains Overexpressingsupporting

confidence: 65%

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 minimal medium and shake cultivation medium for physiological characterizations and fermentation procedure were described as previously reported by Wang et al [[35]]. Physiological characterizations were carried out in minimal medium and riboflavin production was carried out in shake cultivation medium.…”

Section: Methodsmentioning

confidence: 99%

Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis

Shi

Wang

et al. 2014

Microb Cell Fact

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BackgroundPurine nucleotides are essential metabolites for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and biosynthesis of several amino acids and riboflavin. Owing to the pivotal roles of purines in cell physiology, the pool of intracellular purine nucleotides must be maintained under strict control, and hence the de novo purine biosynthetic pathway is tightly regulated by transcription repression and inhibition mechanism. Deregulation of purine pathway is essential for this pathway engineering in Bacillus subtilis.ResultsDeregulation of purine pathway was attempted to improve purine nucleotides supply, based on a riboflavin producer B. subtilis strain with modification of its rib operon. To eliminate transcription repression, the pur operon repressor PurR and the 5’-UTR of pur operon containing a guanine-sensing riboswitch were disrupted. Quantitative RT-PCR analysis revealed that the relative transcription levels of purine genes were up-regulated about 380 times. Furthermore, site-directed mutagenesis was successfully introduced into PRPP amidotransferase (encoded by purF) to remove feedback inhibition by homologous alignment and analysis. Overexpression of the novel mutant PurF (D293V, K316Q and S400W) significantly increased PRPP amidotransferase activity and triggered a strong refractory effect on purine nucleotides mediated inhibition. Intracellular metabolite target analysis indicated that the purine nucleotides supply in engineered strains was facilitated by a stepwise gene-targeted deregulation. With these genetic manipulations, we managed to enhance the metabolic flow through purine pathway and consequently increased riboflavin production 3-fold (826.52 mg/L) in the purF-VQW mutant strain.ConclusionsA sequential optimization strategy was applied to deregulate the rib operon and purine pathway of B. subtilis to create genetic diversities and to improve riboflavin production. Based on the deregulation of purine pathway at transcription and metabolic levels, an extended application is recommended for the yield of products, like inosine, guanosine, adenosine and folate which are directly stemming from purine pathway in B. subtilis.

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“…However, human and eukaryotic microorganisms do not have ability to self-synthesized riboflavin and therefore its acquirement was made through daily diets. According to literature, it is estimated that more than 3000 metric tons are produced annually to cater demands for human supplement, animal husbandry and food colorant [2]. Current progress in biotechnology and poor economic return by conventional chemical synthesis route has turn biosynthesis fermentation as a popular choice in producing riboflavin [3].…”

Section: Introductionmentioning

confidence: 99%

Riboflavin adsorption onto multi-modal mesoporous carbon synthesized from polyethylene glycol 400

Din

Ahmad

Hameed

2013

Chemical Engineering Journal

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Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum

Cited by 81 publications

References 34 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

Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis

Riboflavin adsorption onto multi-modal mesoporous carbon synthesized from polyethylene glycol 400

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