Abstract
Bacillus licheniformis WX-02 is a well-studied strain to produce poly-γ-glutamic acid (γ-PGA) with numerous applications. This study is to improve WX-02 strain’s capability of assimilating glycerol, a major byproduct of biofuels industries, through metabolic manipulation. Through gene knockout, the GlpK pathway was identified as the sole functional glycerol catabolism pathway, while the DhaK pathway was inactive for this strain under either aerobic or anaerobic conditions. The enhancement of glycerol utilization was attempted by substituting the native glpFK promoter with the constitutive promoter (P43), ytzE promoter (PytzE), and bacABC operon promoter (PbacA), respectively. The glycerol consumptions of the corresponding mutant strains WX02-P43glpFK, WX02-PytzEglpFK, and WX02-PbacAglpFK were 30.9, 26.42, and 18.8% higher than that of the WX-02 strain, respectively. The γ-PGA concentrations produced by the three mutant strains were 33.71, 23.39, and 30.05% higher than that of WX-02 strain, respectively. When biodiesel-derived crude glycerol was used as the carbon source, the mutant WX02-P43glpFK produced 16.63 g L−1 of γ-PGA, with a productivity of 0.35 g L−1 h−1. Collectively, this study demonstrated that glycerol can be used as an effective substrate for producing γ-PGA by metabolic engineering B. licheniformis strains.
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Acknowledgements
This study was funded by the National Program on Key Basic Research Project (973 Program, No. 2015CB150505), and the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (No.2013AA102801-52).
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C Zhu and S Chen designed and supervised the study. Y Zhan and B Sheng performed the experiments. Y Zhan, C Zhu, D Cai, Q Wang, Z Wen, and S Chen analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.
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All the strains, plasmids and primers used in this study were listed as the Supplementary Materials (Tables S1-S3). The result of mRNA relative transcription levels of TCA cycle, codY, ccpC, and gltT in WX02-P43glpFK was listed in the Supplementary Materials (Fig. S1).. This information is available free of charge via the Internet http://link.springer.com/journal/253
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Zhan, Y., Zhu, C., Sheng, B. et al. Improvement of glycerol catabolism in Bacillus licheniformis for production of poly-γ-glutamic acid. Appl Microbiol Biotechnol 101, 7155–7164 (2017). https://doi.org/10.1007/s00253-017-8459-2
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DOI: https://doi.org/10.1007/s00253-017-8459-2