title: Metabolic flux responses to genetic modification for shikimic acid production by Bacillus subtilis strains.
abstract: BACKGROUND
  Shikimic 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.
  
  
  RESULTS
  B. 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.
  
  
  CONCLUSIONS
  Of 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.
authors.forename: Dong-Feng
authors.initials: DF
authors.lastname: Liu
authors.forename: Guo-Min
authors.initials: GM
authors.lastname: Ai
authors.forename: Qing-Xiang
authors.initials: QX
authors.lastname: Zheng
authors.forename: Chang
authors.initials: C
authors.lastname: Liu
authors.forename: Cheng-Ying
authors.initials: CY
authors.lastname: Jiang
authors.forename: Li-Xia
authors.initials: LX
authors.lastname: Liu
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: Yi-Ming
authors.initials: YM
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. chenyang@sibs.ac.cn.
authors.forename: Chen
authors.initials: C
authors.lastname: Yang
authors.forename: Shuang-Jiang
authors.initials: SJ
authors.lastname: Liu
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D002952:Citric Acid Cycle; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis; D053858:Metabolic Networks and Pathways; D010427:Pentose Phosphate Pathway; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012765:Shikimic Acid / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012765:Shikimic Acid; D002244:Carbon; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase; D005947:Glucose