Simultaneous Multiplex Genome Engineering via Accelerated Natural Transformation in Bacillus subtilis.

Aihua Deng,Shuwen Liu,Fei Huang,Zhaopeng Sun,Di Cui,Tiantian Wang,Lai Li,Tingyi Wen

doi:10.3389/fmicb.2021.714449

Abstract

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.

Highlights

Multiplex engineering at the scale of whole genomes has become increasingly important for biological research and biotechnological applications
Recent advancements in genetic manipulation have led to the development of several molecular biology techniques for complete multiplex genome editing, such as multiplexed automated genome engineering (MAGE; Wang et al, 2009), conjugative assembly genome engineering (CAGE; Isaacs et al, 2011), “coselection” MAGE (CoS-MAGE; Wang et al, 2012a), multiplex genome editing by natural transformation (MuGENT; Dalia et al, 2014), and RNA-guided nuclease technology based on the microbial clustered regularly interspaced short palindromic repeats adaptive immune system (CRISPR-Cas9/ CRISPR-Cas12; Jiang et al, 2013; Mussolino and Cathomen, 2013; Wang et al, 2016; Westbrook et al, 2016; Qin et al, 2018; Liu et al, 2019)
We aimed to develop a simultaneous multiplex genome engineering (SMGE) method to randomly modify multiple chromosomal loci in B. subtilis via one-step natural transformation

Summary

Introduction

Multiplex engineering at the scale of whole genomes has become increasingly important for biological research and biotechnological applications. Utilizing highly efficient recombination with ssDNA oligonucleotides and multiple cycles of transformation, MAGE, CoS-MAGE, and CAGE have been used to introduce point mutations or small insertions/deletions into the genome of Escherichia coli for large-scale programming and evolution of cells (Wang et al, 2009; Isaacs et al, 2011; Wang et al, 2012a). These methods cannot be adapted for most microorganisms owing to low recombination efficiency and complex transformation procedure using successive multi-cycle transformations. A simple, rapid, and high-efficiency transformation system has to be developed for use in a wide range of microorganisms

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Conclusion