Simultaneous Multiplex Genome Engineering via Accelerated Natural Transformation in Bacillus subtilis.
Simultaneous Multiplex Genome Engineering via Accelerated Natural Transformation in Bacillus subtilis.
复制标题
通过枯草芽孢杆菌加速自然转化进行同步多重基因组工程
DOI：
10.3389/fmicb.2021.714449
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发表时间：
2021
影响因子：
5.2
通讯作者：
Wen T
中科院分区：
文献类型：
--
作者：
Deng A;Sun Z;Wang T;Cui D;Li L;Liu S;Huang F;Wen T
全基因组规模的多重工程对于合成生物学和生物技术应用变得越来越重要。尽管已经报道了几种用于工程化微生物基因组的方法，但它们的使用受到使用多循环转化的复杂程序的限制。在许多生物中，通过水平基因转移参与物种进化的自然转化显示了其作为遗传工具的潜力。本研究旨在通过枯草芽孢杆菌的一步自然转化，发展一种简单、快速、高效的细菌基因组设计的同步多重基因组工程（SMGE）。转化的DNA、感受态因子和重组酶适于提高共编辑频率超过27倍。使用携带多基因盒的一体化载体同时产生具有遗传多样性的单到八联体变体。为了证明其潜在的应用，通过高通量筛选B中的变体库，进一步优化酪氨酸生物合成途径以生产商业上重要的白藜芦醇。枯草芽孢杆菌SMGE代表了一个加速进化的平台，可以为B中的大规模基因工程和合成生物学产生多样化的多重突变。枯草杆菌。
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.