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Enhanced Low Molecular Weight Poly-γ-Glutamic Acid Production in Recombinant Bacillus subtilis 1A751 with Zinc Ion

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Abstract

Poly-γ-glutamic acid (γ-PGA) is a novel biodegradable polyamide material. Microbial fermentation is the only way to produce γ-PGA, but the molecular weight of γ-PGA varied depending on different strains and culture conditions used. The molecular weight of γ-PGA is a main factor affecting the utilization of γ-PGA. It is urgent to find an efficient way to prepare γ-PGA with specific molecular weight, especially low molecular weight. Bacillus subtilis ECUST is a glutamate-dependent strain that produces γ-PGA. In this study, a recombinant B. subtilis harboring the γ-PGA synthase gene cluster pgsBCAE of our preciously identified γ-PGA–producing B. subtilis ECUST was constructed. Assay of γ-PGA contents and properties showed that recombinant B. subtilis 1A751-pBNS2-pgsBCAE obtained the ability to synthesize γ-PGA with low molecular weight (about 10 kDa). The excessive addition of glutamate inhibited the γ-PGA synthesis, while the addition of Zn2+ could promote the synthesis of γ-PGA by increasing the transcription of pgsB but had no effect on the molecular weight of synthesized γ-PGA. Under optimized conditions, γ-PGA produced by recombinant B. subtilis 1A751-pBNS2-pgsBCAE increased from initial 0.54 g/L to 3.9 g/L, and the glutamate conversion rate reached 78%. Recombinant B. subtilis 1A751-pBNS2-pgsBCAE has the potential for efficient preparation of low molecular weight γ-PGA.

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Abbreviations

CTAB:

cetyltrimethylammonium bromide

γ-PGA:

poly-γ-glutamic acid

PCR:

polymerase chain reaction

TLC:

thin-layer chromatography

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Funding

This work is financially supported by the National Key Research and Development Program of China (2017YFB0309302), the National High Technology Research and Development Program of China (863 Program) (No. SS2014AA021202), and “Shu Guang” project of Shanghai Municipal Education Commission and Shanghai Education Development Foundation (15SG28).

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Authors and Affiliations

  1. School of Biotechnology, State Key Laboratory of Bioreactor Engineering, R&D Center of Separation and Extraction Technology in Fermentation Industry, East China University of Science and Technology, Shanghai, 200237, China

    Sijing Jiang, Liqiang Fan, Mengyao Zhao, Yongjun Qiu & Liming Zhao

  2. Shanghai Collaborative Innovation Center for Biomanufacturing Technology (SCICBT), Shanghai, 200237, China

    Yongjun Qiu & Liming Zhao

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  1. Sijing Jiang
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  2. Liqiang Fan
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  5. Liming Zhao
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Correspondence to Liqiang Fan or Liming Zhao.

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Jiang, S., Fan, L., Zhao, M. et al. Enhanced Low Molecular Weight Poly-γ-Glutamic Acid Production in Recombinant Bacillus subtilis 1A751 with Zinc Ion. Appl Biochem Biotechnol 189, 411–423 (2019). https://doi.org/10.1007/s12010-019-03004-2

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