Abstract
Bacillus licheniformis has been regarded as an outstanding microbial cell factory for the production of biochemicals and enzymes. Due to lack of genetic tools to repress gene expression, metabolic engineering and gene function elucidation are limited in this microbe. In this study, an integrated CRISPR interference (CRISPRi) system was constructed in B. licheniformis. Several endogenous genes, including yvmC, cypX, alsD, pta, ldh, and essential gene rpsC, were severed as the targets to test this CRISPRi system, and the repression efficiencies were ranged from 45.02 to 94.00%. Moreover, the multiple genes were simultaneously repressed with high efficiency using this CRISPRi system. As a case study, the genes involved in by-product synthetic and l-valine degradation pathways were selected as the silence targets to redivert metabolic flux toward l-valine synthesis. Repression of acetolactate decarboxylase (alsD) and leucine dehydrogenase (bcd) led to 90.48% and 80.09 % increases in l-valine titer, respectively. Compared with the control strain DW9i△leuA (1.47 g/L and 1.79 g/L), the l-valine titers of combinatorial strain DW9i△leuA/pHYi-alsD-bcd were increased by 1.27-fold and 2.89-fold, respectively, in flask and bioreactor. Collectively, this work provides a feasible approach for multiplex metabolic engineering and functional genome studies of B. licheniformis.
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Funding
This study was funded by the National Key Research and Development Program of China (No. 2018YFA0900303), the National Natural Science Foundation of China (31972849), the Technical Innovation Special Fund of Hubei Province (No. 2018ACA149), and the Natural Science Foundation of Hubei Province of China (2019CFB319).
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S Chen and Y Zhan designed and supervised the study. Y Zhan, P Zheng, M He, and S Sun performed the experiments. Y Zhan and Y Xu analyzed the data and wrote the manuscript. D Wang, D Cai, X Ma, and S Chen revised the manuscript. All authors read and approved the final manuscript.
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Zhan, Y., Xu, Y., Zheng, P. et al. Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis. Appl Microbiol Biotechnol 104, 391–403 (2020). https://doi.org/10.1007/s00253-019-10230-5
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DOI: https://doi.org/10.1007/s00253-019-10230-5