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Genome-wide screening identifies promiscuous phosphatases impairing terpenoid biosynthesis in Escherichia coli

  • Applied microbial and cell physiology
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Abstract

Terpenoids are a large family of natural compounds that are important for both biotechnological applications and basic microorganism physiology. Inspired by the current literature, we hypothesized that recently deciphered phosphatase promiscuity may be an unexplored factor that negatively affects terpenoid biosynthesis by redirecting carbon flux away from the pathway via unrecognized catalytic activities on the phosphorylated intermediates. We used lycopene as a proof-of-concept to test this hypothesis. Based on an extensive bioinformatics analysis, we selected 56 phosphatase-encoding genes in Escherichia coli and constructed a knockdown library for these genes in a lycopene overproducer via CRISPR interference (CRISPRi). We screened this phosphatase knockdown library and observed enrichment (28 of 56) for genes that impair lycopene biosynthesis. Further scaled-up cultivation, combinatorial knockdown, and knockout assays in strains that overproduce lycopene or another terpenoid (β-carotene) confirmed the proposed relationship between promiscuous phosphatases and impaired terpenoid biosynthesis. This study hence suggests the necessity of reconsidering the interactions of promiscuous phosphatases with ubiquitous phosphorylated components of metabolic networks with respect to engineering metabolism.

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Funding

This study was supported by the National Key Research and Development Program of China (2016YFF0202303), the National Key Scientific Instrument and Equipment Project of NSFC (21627812), the General Program of NSFC (21676156), and the Tsinghua University Initiative Scientific Research Program (20161080108).

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Author notes

  1. Tianmin Wang, Jiahui Guo and Yangyang Liu contributed equally to this work.

Authors and Affiliations

  1. MOE Key Laboratory for Industrial Biocatalysis, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China

    Tianmin Wang, Jiahui Guo, Chong Zhang & Xin-Hui Xing

  2. Department of Biological and Chemical Engineering, Anhui Polytechnic University, Wuhu, 241000, China

    Yangyang Liu & Zhenglian Xue

  3. Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China

    Chong Zhang & Xin-Hui Xing

Authors
  1. Tianmin Wang
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  2. Jiahui Guo
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  3. Yangyang Liu
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  4. Zhenglian Xue
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  5. Chong Zhang
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  6. Xin-Hui Xing
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Contributions

T.W. proposed the idea of this work. T.W. and J.G. performed the preliminary strain construction, CRISPRi method establishment, and bioinformatics analysis. T.W., J.G., and Y.L. performed the screening experiments. T.W., Y.L. J.G., Z.X., C.Z., and X.X. analyzed the results. T.W., J.G., and C.Z. wrote the manuscript based on discussion among all authors. C.Z., Z.X., and X.X. supervised the project.

Corresponding authors

Correspondence to Zhenglian Xue or Chong Zhang.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Wang, T., Guo, J., Liu, Y. et al. Genome-wide screening identifies promiscuous phosphatases impairing terpenoid biosynthesis in Escherichia coli. Appl Microbiol Biotechnol 102, 9771–9780 (2018). https://doi.org/10.1007/s00253-018-9330-9

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  • DOI: https://doi.org/10.1007/s00253-018-9330-9

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