Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis.
Appl Microbiol Biotechnol
; 104(1): 391-403, 2020 Jan.
Article
em En
| MEDLINE
| ID: mdl-31745574
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 DW9iâ³leuA (1.47 g/L and 1.79 g/L), the L-valine titers of combinatorial strain DW9iâ³leuA/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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MEDLINE
Assunto principal:
Inativação Gênica
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Engenharia Metabólica
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Sistemas CRISPR-Cas
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Bacillus licheniformis
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article