摘要
芽孢杆菌是一类重要的工业菌种,具有出色的产物分泌能力,在微生物发酵领域具有巨大的应用潜力,但在发酵过程中仍面临着发酵泡沫多、副产物多、产量受限等诸多问题,阻碍了菌种在工业化生产中的应用.CRISPR/Cas9技术在实现菌株多基因敲除、基因敲入和转录抑制等方面具有简便、高效的特点,可从基因表达水平层面调控菌株生理及代谢,提升菌株性能和目的产物发酵产量,以满足菌株工业化生产的需要.首先对CRISPR/Cas9的系统原理及其在芽孢杆菌中的构建策略、影响因素和效应蛋白元件研究现状进行概述,之后总结近年来应用CRISPR/Cas9技术改造芽孢杆菌发酵性能、调控产物分子量、异源表达产物基因、提升代谢性能4个方面的相关研究策略,最后总结CRISPR/Cas9在野生型芽孢杆菌基因编辑应用中目前存在的回复突变、部分野生型菌株编辑系统难建立以及外源DNA转化效率低等技术难题,并提出可能的解决思路:(1)开发运用普适性强的Cas9变种酶和sgRNA递送机制;(2)结合菌株生理开发载体转化方法;(3)挖掘菌株内源DNA组件;(4)深入基因功能表征鉴定,开发多元化编辑策略.本综述旨在为建立工业芽孢杆菌的高精度基因编辑调控系统和拓宽CRISPR/Cas9技术的改造应用范围以实现更高工业价值提供参考.
Strains of Bacillus with excellent secretory capacities are widely used in various industrial applications and have great application potential in the field of microbial fermentation and synthesis.However,many problems,such as excessive foam,abundant byproducts,and limited yield during Bacillus fermentation,hinder the application of Bacillus in industrial production.CRISPR/Cas9 technology is a simple and efficient way to achieve multigene knockout,gene knock-in,and transcription inhibition,resulting in regulating the physiology and metabolism of a strain at the gene level.These manipulations can improve the strain performance and yield of the target product to meet the requirements of industrial production.This study provides an overview of the systematic principles of CRISPR/Cas9,its construction strategies in Bacillus,and the status of the influencing factors and effector protein elements associated with CRISPR/Cas9.The application of CRISPR/Cas9 in the development of Bacillus fermentation strains is summarized,including modification of fermentation performance,regulation of molecular weight of products,heterologous expression of product genes,and enhancement of metabolic capacity.Finally,we conclude with the current technical difficulties in applying CRISPR/Cas9 in wild-type Bacillus genome editing,such as reverse mutation,unstable editing systems in some wild-type strains,and low efficiency of exogenous DNA transformation.However,feasible solutions have been proposed:(1)development and application of universal Cas9 variant enzymes and sgRNA delivery mechanisms;(2)improved vector transformation in combination with strain physiology;(3)exploration of endogenous DNA components;and(4)further functional characterization of genes and the use of diverse editing strategies.This review aims to provide a reference for establishing a highprecision genome editing and regulatory system for fermentation Bacillus strains and expand the spectrum of CRISPR/Cas9 technology retrofit applications to achieve higher industrial value.
作者
袁梦婷
闫志英
吕青阳
刘杨
杜亚玲
宾石玉
YUAN Mengting;YAN Zhiying;LYU Qingyang;LIU Yang;DU Yaling;BIN Shiyu(College of Life Sciences,Guangxi Normal University,Guiling 541006,China;Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China)
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2023年第5期1279-1288,共10页
Chinese Journal of Applied and Environmental Biology
基金
四川省自然科学基金项目(2023NSFSC1227)
成都市科技项目(2022-YF05-00690-SN)
中国科学院生物资源库项目(KFJ-BRP-009-004)
西部之光项目(2019XBZG_XBQNXZ_A_001)
四川省委组织部人才专项资助
