Metabolic flux responses to genetic modification for shikimic acid production by Bacillus subtilis strains

Microbial Cell Factories

Research

Qing-Xiang Zheng¹  Chen Yang¹  Li-Xia Liu¹  Bo Zhang²  Guo-Min Ai²  Yi-Ming Liu²  Chang Liu²  Cheng-Ying Jiang²  Shuang-Jiang Liu³  Dong-Feng Liu⁴ 

[1] Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China;State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China;State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China;Institute of Microbiology, Chinese Academy of Sciences, Beichen-Xilu, Beijing, 100101, Chaoyang District, China;State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China;School of Life Sciences, University of Science and Technology of China, 230026, Hefei, China;

关键词: Shikimic acid production;    Shikimate pathway;    Bacillus subtilis;    Metabolic flux assay (MFA);    aroA;    aroD;    tkt;    pyk;   

DOI  :  10.1186/1475-2859-13-40

 received in 2013-11-30, accepted in 2014-02-21,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundShikimic acid (SA) is a key chiral starting molecule for the synthesis of the neuramidase inhibitor GS4104 against viral influenza. Microbial production of SA has been extensively investigated in Escherichia coli, and to a less extent in Bacillus subtilis. However, metabolic flux of the high SA-producing strains has not been explored. In this study, we constructed with genetic manipulation and further determined metabolic flux with 13C-labeling test of high SA-producing B. subtilis strains.ResultsB. subtilis 1A474 had a mutation in SA kinase gene (aroI) and accumulated 1.5 g/L of SA. Overexpression of plasmid-encoded aroA, aroB, aroC or aroD in B. subtilis revealed that aroD had the most significantly positive effects on SA production. Simultaneous overexpression of genes for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroA) and SA dehydrogenase (aroD) in B. subtilis BSSA/pSAAroA/pDGSAAroD resulted in SA production of 3.2 g/L. 13C-Metabolic flux assay (MFA) on the two strains BSSA/pHCMC04/pDG148-stu and BSSA/pSAAroA/pDGSAAroD indicated the carbon flux from glucose to SA increased to 4.6% in BSSA/pSAAroA/pDGSAAroD from 1.9% in strain BSSA/pHCMC04/pDG148-stu. The carbon flux through tricarboxylic acid cycle significantly reduced, while responses of the pentose phosphate pathway and the glycolysis to high SA production were rather weak, in the strain BSSA/pSAAroA/pDGSAAroD. Based on the results from MFA, two potential targets for further optimization of SA production were identified. Experiments on genetic deletion of phosphoenoylpyruvate kinase gene confirmed its positive influence on SA production, while the overexpression of the transketolase gene did not lead to increase in SA production.ConclusionOf the genes involved in shikimate pathway in B. subtilis, aroD exerted most significant influence on SA accumulation. Overexpression of plasmid-encoded aroA and aroD doubled SA production than its parent strain. MFA revealed metabolic flux redistribution among phosphate pentose pathway, glycolysis, TCA cycle in the low and high SA-producing B. subtilis strains. The high SA producing strain BSSA/pSAAroA/pDGSAAroD had increased carbon flux into shikimate pathway and reduced flux into TCA cycle.

【 授权许可】

CC BY   
© Liu et al.; licensee BioMed Central Ltd. 2014