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

Dong-Feng Liu,Shuang-Jiang Liu,Bo Zhang,Yi-Ming Liu,Cheng-Ying Jiang,Li-Xia Liu,Chen Yang,Guo-Min Ai,Qing-Xiang Zheng,Chang Liu

doi:10.1186/1475-2859-13-40

Abstract

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.

Highlights

Shikimic acid (SA) is a key chiral starting molecule for the synthesis of the neuramidase inhibitor GS4104 against viral influenza
B. subtilis strain 1A474 had been used for genome mapping and its genome had a mutation on the SA kinase gene
We observed that SA production increased by 8% in B. subtilis, from 3.2 g/L in the control strain BSSA/pSAAroA/pDGSAAroD to 3.46 g/L in strain BSSA/Ωpyk::pSApyk

Summary

Introduction

Shikimic acid (SA) is a key chiral starting molecule for the synthesis of the neuramidase inhibitor GS4104 against viral influenza. Shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid, SA), is a key chiral starting material for the synthesis of the antiviral neuraminidase inhibitor GS4104 [1,2]. It is a key metabolic intermediate of the shikimate pathway for biosynthesis of aromatic amino acids (L-Phe, L-Trp, and L-Tyr) and many alkaloids in plants and microorganisms [3,4,5]. Beginning with phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), SA is synthesized via first 4 reactions of the shikimate pathway (Figure 1) This shikimate pathway is essential to bacterial growth and there has been no report on excessive accumulation of SA by microorganisms. Knowledge of the metabolic fluxes of those genetically engineered strains of E. coli and B. subtilis is still missing

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