Abstract
Objective
To improve the production of short branched-chain acyl-CoAs for avermectin biosynthesis, the functional expression of the branched chain α-keto acid dehydrogenase complex (BKDH) from Streptomyces avermitilis was systematically optimized by selectively regulating individual subunit expression in Escherichia coli.
Results
Functional expression of the BKDH complex was achieved by independent and selective optimization of individual subunit genes of the complex. Codon optimization significantly improved the expression of complex component proteins BkdH and LpdA1 but expression of BkdF and BkdG depended on coexpression of the bkdH gene. The optimized BKDH complex supplied sufficient short branched-chain acyl-CoA to synthesize phlorisovalerophenone, a key intermediate in bitter acid (humulone) synthesis. We also developed a novel p15A origin-derived high-copy-number vector system for expression; the yield of PIVP was 350 ng/mOD.
Conclusion
Through optimization strategies, we obtained stable, functional expression of the BKDH complex in E. coli, which could be applied in the heterologous production of numerous high-value-added chemicals, especially polyketides.
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Acknowledegments
This work is supported by the National Basic Research Program of China (973 Program) (No. 2013CB734003); the Key Research Program of the Chinese Academy of Sciences (No. ZDRW-ZS-2016-3-1); and the Sichuan Science and Technology Support Program (No. 2016JZ0006). We are greatly thankful for the support of Metabolomics Facility at Technology Center for Protein Sciences of Tsinghua University.
Supporting information
Supplementary Table 1—Strains, genes and plasmids.
Supplementary Table 2—Primers used in this study.
Supplementary Fig. 1—SDS-PAGE analysis of each subunit of the BKDH complex after codon optimization.
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Cui, Q., Zhou, F., Liu, W. et al. Avermectin biosynthesis: stable functional expression of branched chain α-keto acid dehydrogenase complex from Streptomyces avermitilis in Escherichia coli by selectively regulating individual subunit gene expression. Biotechnol Lett 39, 1567–1574 (2017). https://doi.org/10.1007/s10529-017-2389-z
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DOI: https://doi.org/10.1007/s10529-017-2389-z