Journal
FRONTIERS IN MICROBIOLOGY
Volume 12, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2021.714449
Keywords
multiplex genome engineering; natural genetic transformation; all-in-one vector; competency factor; recombinase; Bacillus subtilis
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Funding
- Strategic Priority Research Program of the Chinese Academy of Sciences [XDA17010503]
- Innovation Academy for Green Manufacture, Chinese Academy of Sciences [IAGM-2019-A02]
- National Natural Science Foundation of China [31800073, 31170103]
- National Science and Technology Major Project [2018ZX10101004003003]
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SMGE is a rapid and efficient method for simultaneous multiplex genome engineering in Bacillus subtilis through one-step natural transformation. By adjusting the transformed DNA, competence factors and recombinases, co-editing frequencies are improved by over 27-fold, successfully generating single to octuplet variants with genetic diversity.
Multiplex engineering at the scale of whole genomes has become increasingly important for synthetic biology and biotechnology applications. Although several methods have been reported for engineering microbe genomes, their use is limited by their complex procedures using multi-cycle transformations. Natural transformation, involving in species evolution by horizontal gene transfer in many organisms, indicates its potential as a genetic tool. Here, we aimed to develop simultaneous multiplex genome engineering (SMGE) for the simple, rapid, and efficient design of bacterial genomes via one-step of natural transformation in Bacillus subtilis. The transformed DNA, competency factors, and recombinases were adapted to improved co-editing frequencies above 27-fold. Single to octuplet variants with genetic diversity were simultaneously generated using all-in-one vectors harboring multi-gene cassettes. To demonstrate its potential application, the tyrosine biosynthesis pathway was further optimized for producing commercially important resveratrol by high-throughput screening of variant pool in B. subtilis. SMGE represents an accelerated evolution platform that generates diverse multiplex mutations for large-scale genetic engineering and synthetic biology in B. subtilis.
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