梭菌分子遗传改造工具研究进展

doi:10.12211/2096-8280.2022-041

合成生物学 ›› 2022, Vol. 3 ›› Issue (6): 1201-1217.DOI: 10.12211/2096-8280.2022-041

梭菌分子遗传改造工具研究进展

刘佳昕¹, 程驰¹^,², 李欣启¹, 汪超俊², 张颖², 薛闯¹^,²

^1.大连理工大学生物工程学院，大连市合成生物学应用转化工程技术研究中心，辽宁大连 116024
^2.大连理工大学宁波研究院，浙江宁波 315016

收稿日期:2022-07-18 修回日期:2022-08-30 出版日期:2022-12-31 发布日期:2023-01-17
通讯作者: 程驰,薛闯
作者简介:刘佳昕（1998—），女，硕士研究生。主要进行生物学研究。E-mail：ljx123456@mail.dlut.edu.cn
程驰（1990—），女，副教授，研究生导师。主要研究领域为：①能源微生物遗传操作工具开发及代谢改造；②化学-生物耦合的CO2固定。E-mail：cheng.chi@dlut.edu.cn
薛闯（1982—），男，教授，博士生导师。主要从事生物质新能源的生产、分离纯化以及高效代谢菌株构建的研究：①生物法生产燃料乙醇及酵母菌的代谢网络研究；②微生物发酵法生产丁醇；③生物基化学品的高效分离；④有机膜的制备及分离技术；⑤先进能源生产菌株的构建及代谢途径信号转导；⑥激酶的生物信息分析。E-mail：xue.1@dlut.edu.cn
基金资助:
国家重点研发计划(2021YFC2102500);国家自然科学基金(21878035);大连市杰出青年科技人才支持计划(2021RJ03);大连理工大学基本科研业务费（DUT21RC（3）003, DUT22ZD102）;辽宁省“百千万人才工程”经费

Recent progress in the molecular genetic modification tools of Clostridium

Jiaxin LIU¹, Chi CHENG¹^,², Xinqi LI¹, Chaojun WANG², Ying ZHANG², Chuang XUE¹^,²

^1.Engineering Research Center of Application and Transformation for Synthetic Biology Dalian，School of Bioengineering，Dalian University of Technology，Dalian 116024，Liaoning，China
^2.NingBo Institute of Dalian University of Technology，Ningbo 315016，Zhejiang，China

Received:2022-07-18 Revised:2022-08-30 Online:2022-12-31 Published:2023-01-17
Contact: Chi CHENG, Chuang XUE

摘要/Abstract

摘要：

梭状芽孢杆菌是一类革兰氏阳性、可内生孢子的严格厌氧型细菌，可产生多种化学物质，包括现如今极具潜力的新型生物燃料丁醇。通过分子改造以提高梭菌发酵的浓度及产率一直是一项亟需突破的重要课题，但该方向的研究长期受限于梭菌不完善的遗传操作工具。近年来，随着分子生物学的快速发展，适用于梭菌的基因编辑工具不断发展，梭菌中已有反义RNA技术、TargeTron、基于同源重组或CRISPR/Cas系统介导的基因编辑技术等多种遗传操作工具，可以基本实现靶标基因插入、删除、替换、点突变以及表达水平调控等各种操作。文中对上述遗传操作工具研究进展进行了总结，并着重讨论了以重组酶为代表的新型遗传操作技术及其在梭菌中的应用潜力。今后应进一步优化现有的梭菌分子遗传改造工具，重点突破梭菌自身同源重组效率低下等技术难点，同时应大力发展新的基因编辑技术，如以CRISPR技术为核心的多位点共编辑系统、噬菌体重组酶介导的多拷贝定点和随机整合技术等。

关键词: 梭状芽孢杆菌, 基因编辑工具, 遗传改造, 重组酶, CRISPR

Abstract:

Clostridium are Gram-positive, strictly anaerobic, endospore-forming bacteria that produce a variety of chemicals, including butanol, which is now a promising new biofuel. Improving the fermentation titer and yield of Clostridium by genetic modification has always been an important challenge that needs to be broken through, but it has long been hindered by the limitation of genetic manipulation tools of Clostridium. In recent years, with the continuous development of molecular biology, gene editing tools for Clostridium have been continuously developed. Many genetic manipulation tools such as plasmid-based gene overexpression, antisense RNA technology, transposon-based mutagenesis, group Ⅱ intron-mediated gene inactivation, and homologous recombination-based or CRISPR/Cas-mediated gene editing technology have been developed. Various operations such as target gene insertion, deletion, substitution, point mutation, and gene expression level regulation have been accomplished in Clostridium. In this review, we summarize the research progress in the molecular genetic modification tools of Clostridium, and especially discuss the potential application of new technologies, such as recombinase-based gene editing technology. Although the application of the recombinase system in Clostridium is rarely reported and discussed, the future application value and significance of this technology should be paid attention to. In the future, optimization of the existing molecular genetic modification technologies in Clostridium is still imperative, such as overcoming the low efficiency of homogeneous recombination in Clostridium, improving the stability and transformation efficiency of plasmids, solving the off-target problem of antisense RNA technology and type Ⅱ intron technology, reducing the toxicity of Cas9 protein, and so on. At the same time, new gene editing technologies should be developed, focusing on emerging technologies including CRISPR/Cas-mediated multi-locus editing systems, phage recombinase-mediated multiplex genome editing, targeted or random multi-copy gene integration, and so on. It is believed that with the development and improvement of genetic modification tools, Clostridium will be able to fully each its potential biorefinery capacity and make an important contribution to the green biosynthesis of bioenergy and bio-based chemicals.

Key words: Clostridium, gene editing tools, genetic modification, recombinase, CRISPR

刘佳昕, 程驰, 李欣启, 汪超俊, 张颖, 薛闯. 梭菌分子遗传改造工具研究进展[J]. 合成生物学, 2022, 3(6): 1201-1217.

Jiaxin LIU, Chi CHENG, Xinqi LI, Chaojun WANG, Ying ZHANG, Chuang XUE. Recent progress in the molecular genetic modification tools of Clostridium[J]. Synthetic Biology Journal, 2022, 3(6): 1201-1217.

图/表 4

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遗传操作工具	优点	缺点	梭菌菌株	基因	文献
基于游离质粒的基因过表达	设计简单，易于操作	质粒不稳定，需靠抗生素维持	C. acetobutylicum ATCC 824 C. acetobutylicum DSM 792 C. paraputrificum M-21 C. tyrobutyricum JM1 C. tyrobutyricum ATCC 25755 C. perfringens SM101 C. cellulolyticum H10	spo0A, hydA, adhE2, adC, groESL, txeR, tcdC, pdc-adhII	[28-38]
反义RNA技术	致死率低，易于筛选	仅在转录水平调控	C. acetobutylicum ATCC 824	ptb, buk, CoAT	[39-41]
转座子突变技术	可用于建立突变体库	难以控制插入位点	C. difficile CD37 C. perfringens Strain 13	random	[42-43]
二型内含子技术	操作简单，几乎适用于所有梭菌	脱靶概率较大，存在极性效应	C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052	glcG, cbei2385, xylR, bdhA, bdhB, ptb, ack, adc	[44-47]
同源重组	可精确进行基因编辑	同源重组效率很低	C. acetobutylicum NCIMB 8052	gutD, spo0A	[48]
反筛标记介导的等位基因替换	相比纯粹的等位基因替换，效率有所提升	受制于第一次单交换效率	C. acetobutylicum ATCC 824	adh	[49]
Ⅰ-SceⅠ归巢内切酶介导的等位基因替换	适用范围广，在革兰氏阳性和阴性菌中均可使用	设计复杂，操作烦琐，周期长	C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052	adc, glcG, xylR	[50]
噬菌体丝氨酸重组酶介导的位点特异性基因编辑技术	适用于大片段DNA快速整合到宿主染色体上	受限于附着位点attb/p，应用范围小	C. ljungdahlii DSM 13528	thl, crt, bcd, etfB, etfA, hbd, ptb, buk	[51]
Red/ET重组酶介导的同源重组	所需同源臂较短、不受限制性酶切位点限制	尚不完善	C. acetobutylicum SMB009	ermC	[52]
CRISPR/Cas9系统介导的基因编辑	提高同源重组效率，可实现精确编辑	毒性大，很难获得转化子	C. acetobutylicum DSM792 C. saccharoperbutylacetonicum N1-4 C. cellulovorans C. autoethanogenum DSM10061	hprK, cac1502, pta, buk, clocel2243, adhE1, ctf,pyrE, 2,3-bdh	[53-57]
CRISPR/Cas9n系统介导的基因编辑	相比于CRISPR/Cas9毒性有所降低，转化子数目增加	仍有毒性，转化子依然偏少	C. cellulolyticum H10 C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052	pyrF, spo0A	[58-59]
CRISPR/dCas9系统介导的基因表达下调	相比于CRISPR/Cas9毒性大大降低，易于得到转化子	依赖于sgRNA和基因；仅在转录水平调控	C. pasteurianum ATCC 6013 C. acetobutylicum DSM792	hprK, glpX	[53]

遗传操作工具	优点	缺点	梭菌菌株	基因	文献
基于游离质粒的基因过表达	设计简单，易于操作	质粒不稳定，需靠抗生素维持	C. acetobutylicum ATCC 824 C. acetobutylicum DSM 792 C. paraputrificum M-21 C. tyrobutyricum JM1 C. tyrobutyricum ATCC 25755 C. perfringens SM101 C. cellulolyticum H10	spo0A, hydA, adhE2, adC, groESL, txeR, tcdC, pdc-adhII	[28-38]
反义RNA技术	致死率低，易于筛选	仅在转录水平调控	C. acetobutylicum ATCC 824	ptb, buk, CoAT	[39-41]
转座子突变技术	可用于建立突变体库	难以控制插入位点	C. difficile CD37 C. perfringens Strain 13	random	[42-43]
二型内含子技术	操作简单，几乎适用于所有梭菌	脱靶概率较大，存在极性效应	C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052	glcG, cbei2385, xylR, bdhA, bdhB, ptb, ack, adc	[44-47]
同源重组	可精确进行基因编辑	同源重组效率很低	C. acetobutylicum NCIMB 8052	gutD, spo0A	[48]
反筛标记介导的等位基因替换	相比纯粹的等位基因替换，效率有所提升	受制于第一次单交换效率	C. acetobutylicum ATCC 824	adh	[49]
Ⅰ-SceⅠ归巢内切酶介导的等位基因替换	适用范围广，在革兰氏阳性和阴性菌中均可使用	设计复杂，操作烦琐，周期长	C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052	adc, glcG, xylR	[50]
噬菌体丝氨酸重组酶介导的位点特异性基因编辑技术	适用于大片段DNA快速整合到宿主染色体上	受限于附着位点attb/p，应用范围小	C. ljungdahlii DSM 13528	thl, crt, bcd, etfB, etfA, hbd, ptb, buk	[51]
Red/ET重组酶介导的同源重组	所需同源臂较短、不受限制性酶切位点限制	尚不完善	C. acetobutylicum SMB009	ermC	[52]
CRISPR/Cas9系统介导的基因编辑	提高同源重组效率，可实现精确编辑	毒性大，很难获得转化子	C. acetobutylicum DSM792 C. saccharoperbutylacetonicum N1-4 C. cellulovorans C. autoethanogenum DSM10061	hprK, cac1502, pta, buk, clocel2243, adhE1, ctf,pyrE, 2,3-bdh	[53-57]
CRISPR/Cas9n系统介导的基因编辑	相比于CRISPR/Cas9毒性有所降低，转化子数目增加	仍有毒性，转化子依然偏少	C. cellulolyticum H10 C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052	pyrF, spo0A	[58-59]
CRISPR/dCas9系统介导的基因表达下调	相比于CRISPR/Cas9毒性大大降低，易于得到转化子	依赖于sgRNA和基因；仅在转录水平调控	C. pasteurianum ATCC 6013 C. acetobutylicum DSM792	hprK, glpX	[53]

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梭菌分子遗传改造工具研究进展

Recent progress in the molecular genetic modification tools of Clostridium

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