Recent advances in metabolic engineering of clostridia for n-butanol production

doi:10.12211/2096-8280.2020-080

Abstract

Abstract:

n-Butanol is a bulk chemical and renewable and alternative vehicle fuel. It can be produced from biomass by some microorganisms, which has potential to replace the unsustainable and environment-hazardous petroleum refining methods. In this work, we review and compare various metabolic pathways and chassis cells for n-butanol production, and highlight that clostridia are natural cell factories for n-butanol production with obvious advantages in butanol titer and productivity. However, strains from this species are still unsatisfactory, which includes inefficient strain genetic modification, low n-butanol yield/ratio, rigid n-butanol synthesis pathway, and low substrate utilization spectrum.Fortunately, synthetic biotechnology has significantly accelerated the development of genetic manipulation tools, and many of them including TargeTron, allelic-exchange, CRISPR/Cas system mediated gene and base editing tools have been developed and applied in clostridia. Various genetic operations such as insertion, deletion, substitution, site mutation, and regulation of target gene expression can be efficiently implemented in clostridia, which laid a foundation for its metabolic engineering. As a result, significant progress has been made in increasing n-butanol titer/yield/ratio, reconstructing and refining the n-butanol synthesis pathway in clostridial chassis, as well as enhancing pentose utilization. The enhancement of the n-butanol pathway and the weakening or deletion of pathways for producing by-products such as acetone, acetic acid, butyric acid have increased butanol titer and ratio.In addition, unconventional clostridia including cellulolytic and gas-fermenting strains have been metabolically engineered for homo-butanol fermentation through decoupling with acetone production. Moreover, genetic manipulation tools also facilitate the reconstruction of the clostridial pentose (xylose and arabinose) transport/metabolism pathway and analysis of carbon catabolite repression (CCR) mechanism, which greatly improved the utilization of pentose. In this article, we review the above metabolic engineering strategies and important milestones of n-butanol production, and address the current bottlenecks and future trends. With the driving of synthetic biotechnology, the cost of n-butanol production by clostridia will be reduced, making it eventually competitive in the market.

Key words: genetic tools, butanol titer, butanol ratio, pathway reconstruction, pentose metabolism

摘要：

正丁醇是大宗化学品和可再生、替代性车用燃料，可由微生物发酵生产，以替代现有的高污染/不可持续的石油炼制方法。本文首先回顾和比较了各种正丁醇合成途径和底盘细胞，指出梭菌是天然的正丁醇细胞工厂，且在丁醇产量和生产强度上有明显优势，但仍受制于菌株性能不足，具体表现在菌株遗传改造困难，正丁醇产量不高，副产物较多，正丁醇合成途径刚性强，以及底物利用效率低等方面。幸运的是，合成生物学的发展加速了产正丁醇梭菌的遗传操作工具开发。很多遗传操作工具如TargeTron、CRISPR/Cas系统介导的基因和碱基编辑工具已经被开发出来。梭菌内已经可以高效实现靶标基因插入、删除、替换、点突变以及表达水平调控等各种操作，这为梭菌正丁醇代谢工程奠定了良好的基础。正丁醇合成途径的增强及副产物如丙酮、乙酸、丁酸等竞争途径的弱化或者删除，提升了丁醇的产量、比例；同时，一些非常规梭菌被代谢工程改造用于同型丁醇发酵，实现丁醇与丙酮生产的解耦；另外，遗传操作工具还为梭菌的戊糖转运/代谢途径以及碳源代谢抑制效应的调控机制的解析和重构提供了便利，极大改善了梭菌戊糖利用效率。相信在合成生物技术的驱动下，梭菌生产正丁醇的成本将大幅降低，最终走向市场。

关键词: 遗传操作工具, 正丁醇产量, 正丁醇比例, 途径重构, 戊糖代谢

CLC Number:

Q815

WEN Zhiqiang, SUN Xiaoman, WANG Qingzhuo, LI Yanan, LIU Wenzheng, JIANG Yu, YANG Sheng. Recent advances in metabolic engineering of clostridia for n-butanol production[J]. Synthetic Biology Journal, 2021, 2(2): 194-221.

闻志强, 孙小曼, 汪庆卓, 李亚楠, 刘文正, 蒋宇, 杨晟. 梭菌正丁醇代谢工程研究进展[J]. 合成生物学, 2021, 2(2): 194-221.

Figures/Tables 11

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107	MERMELSTEIN L D, PAPOUTSAKIS E T, PETERSEN D J, et al. Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon [J]. Biotechnology and Bioengineering, 1993, 42(9): 1053-1060.
108	JIANG Y, XU C, DONG F, et al. Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicum increases the butanol ratio [J]. Metabolic Engineering, 2009, 11(4-5): 284-291.
109	LEHMANN D, HOENICKE D, EHRENREICH A, et al. Modifying the product pattern of Clostridium acetobutylicum [J]. Applied Microbiology and Biotechnology, 2012, 94(3): 743-754.
110	BAO T, FENG J, JIANG W, et al. Recent advances in n-butanol and butyrate production using engineered Clostridium tyrobutyricum [J]. World Journal of Microbiology & Biotechnology, 2020, 36(9): 138.
111	YU L, ZHAO J, XU M, et al. Metabolic engineering of Clostridium tyrobutyricum for n-butanol production: effects of CoA transferase [J]. Applied Microbiology and Biotechnology, 2015, 99(11): 4917-4930.
112	ZHANG J, YU L, XU M, et al. Metabolic engineering of Clostridium tyrobutyricum for n-butanol production from sugarcane juice [J]. Applied Microbiology and Biotechnology, 2017, 101(10): 4327-4337.
113	YU L, XU M, TANG I C, et al. Metabolic engineering of Clostridium tyrobutyricum for n-butanol production from maltose and soluble starch by overexpressing α-glucosidase [J]. Applied Microbiology and Biotechnology, 2015, 99(14): 6155-6165.
114	DU Y, JIANG W, YU M, et al. Metabolic process engineering of Clostridium tyrobutyricum delta ack-adhE2 for enhanced n-butanol production from glucose: effects of methyl viologen on NADH availability, flux distribution, and fermentation kinetics [J]. Biotechnology and Bioengineering, 2015, 112(4): 705-715.
115	WEN Z, LEDESMA-AMARO R, LIN J, et al. Improved n-butanol production from Clostridium cellulovorans by integrated metabolic and evolutionary engineering [J]. Applied and Environmental Microbiology, 2019, 85(7): e02560-18.
116	BAO T, ZHAO J, LI J, et al. n-Butanol and ethanol production from cellulose by Clostridium cellulovorans overexpressing heterologous aldehyde/alcohol dehydrogenases [J]. Bioresource Technology, 2019, 285: 121316.
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119	JIA D, JIANG W, GU Y. Research progresses in gas-fermenting clostridia [J]. Microbiology China, 2019, 46(2): 374-387.
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121	LEMGRUBER R D S P, VALGEPEA K, TAPPEL R, et al. Systems-level engineering and characterisation of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB) [J]. Metabolic Engineering, 2019, 53: 14-23.
122	VALGEPEA K, LOI K Q, BEHRENDORFF J B, et al. Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum [J]. Metabolic Engineering, 2017, 41: 202-211.
123	W-C KAO, LIN D-S, CHENG C-L, et al. Enhancing butanol production with Clostridium pasteurianum CH₄ using sequential glucose–glycerol addition and simultaneous dual-substrate cultivation strategies [J]. Bioresource Technology, 2013, 135: 324-330.
124	PYNE M E, SOKOLENKO S, LIU X, et al. Disruption of the reductive 1,3-propanediol pathway triggers production of 1,2-propanediol for sustained glycerol fermentation by Clostridium pasteurianum [J]. Applied and Environmental Microbiology, 2016, 82(17): 5375-5388.
125	SCHWARZ K M, GROSSE-HONEBRINK A, DERECKA K, et al. Towards improved butanol production through targeted genetic modification of Clostridium pasteurianum [J]. Metabolic Engineering, 2017, 40: 124-137.
126	CORNILLOT E, NAIR R V, PAPOUTSAKIS E T, et al. The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain [J]. Journal of Bacteriology, 1997, 179(17): 5442-5447.
127	NAIR R V, PAPOUTSAKIS E T. Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 restores vigorous butanol production [J]. Journal of Bacteriology, 1994, 176(18): 5843-5846.
128	SILLERS R, CHOW A, TRACY B, et al. Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance [J]. Metabolic Engineering, 2008, 10(6): 321-332.
129	LEE J Y, Y-S JANG, LEE J, et al. Metabolic engineering of Clostridium acetobutylicum M5 for highly selective butanol production [J]. Biotechnology Journal, 2009, 4(10): 1432-1440.
130	OU J, XU N, ERNST P, et al. Process engineering of cellulosic n-butanol production from corn-based biomass using Clostridium cellulovorans [J]. Process Biochemistry, 2017, 62: 144-150.
131	YU L, XU M, TANG I C, et al. Metabolic engineering of Clostridium tyrobutyricum for n-butanol production through co-utilization of glucose and xylose [J]. Biotechnology and Bioengineering, 2015, 112(10): 2134-2141.
132	GU Y, JIANG Y, YANG S, et al. Utilization of economical substrate-derived carbohydrates by solventogenic clostridia: pathway dissection, regulation and engineering [J]. Current Opinion in Biotechnology, 2014, 29: 124-131.
133	GU Y, LI J, ZHANG L, et al. Improvement of xylose utilization in Clostridium acetobutylicum via expression of the talA gene encoding transaldolase from Escherichia coli [J]. Journal of Biotechnology, 2009, 143(4): 284-287.
134	JIN L, ZHANG H, CHEN L, et al. Combined overexpression of genes involved in pentose phosphate pathway enables enhanced D-xylose utilization by Clostridium acetobutylicum [J]. Journal of Biotechnology, 2014, 173: 7-9.
135	XIAO H, GU Y, NING Y, et al. Confirmation and elimination of xylose metabolism bottlenecks in glucose phosphoenolpyruvate-dependent phosphotransferase system-deficient Clostridium acetobutylicum for simultaneous utilization of glucose, xylose, and arabinose [J]. Applied and Environmental Microbiology, 2011, 77(22): 7886-7895.
136	LI Z, XIAO H, JIANG W, et al. Improvement of solvent production from xylose mother liquor by engineering the xylose metabolic pathway in Clostridium acetobutylicum EA 2018 [J]. Applied Biochemistry and Biotechnology, 2013, 171(3): 555-568.
137	REN C, GU Y, HU S, et al. Identification and inactivation of pleiotropic regulator CcpA to eliminate glucose repression of xylose utilization in Clostridium acetobutylicum [J]. Metabolic Engineering, 2010, 12(5): 446-454.
138	BRUDER M, MOO-YOUNG M, CHUNG D A, et al. Elimination of carbon catabolite repression in Clostridium acetobutylicum-a journey toward simultaneous use of xylose and glucose [J]. Applied Microbiology and Biotechnology, 2015, 99(18): 7579-7588.
139	XIAO H, LI Z L, JIANG Y, et al. Metabolic engineering of D-xylose pathway in Clostridium beijerinckii to optimize solvent production from xylose mother liquid [J]. Metabolic Engineering, 2012, 14(5): 569-578.
140	GU Y, DING Y, REN C, et al. Reconstruction of xylose utilization pathway and regulons in Firmicutes [J]. BMC Genomics, 2010, 11:255.
141	LIU L, ZHANG L, TANG W, et al. Phosphoketolase pathway for xylose catabolism in Clostridium acetobutylicum Revealed by C-13 metabolic flux analysis [J]. Journal of Bacteriology, 2012, 194(19): 5413-5422.
142	SERVINSKY M D, GERMANE K L, LIU S, et al. Arabinose is metabolized via a phosphoketolase pathway in Clostridium acetobutylicum ATCC 824 [J]. Journal of Industrial Microbiology & Biotechnology, 2012, 39(12): 1859-1867.
143	ZHANG L, LEYN S A, GU Y, et al. Ribulokinase and transcriptional regulation of arabinose metabolism in Clostridium acetobutylicum [J]. Journal of Bacteriology, 2012, 194(5): 1055-1064.
144	SUN Z, CHEN Y, YANG C, et al. A novel three-component system-based regulatory model for D-xylose sensing and transport in Clostridium beijerinckii [J]. Molecular Microbiology, 2015, 95(4): 576-589.
145	LI J X, WANG C Y, YANG G H, et al. Molecular mechanism of environmental D-xylose perception by a XylFII-LytS complex in bacteria [J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(31): 8235-8240.
146	NESSLER S, FIEULAINE S, PONCET S, et al. HPr kinase/phosphorylase, the sensor enzyme of catabolite repression in Gram-positive bacteria: structural aspects of the enzyme and the complex with its protein substrate [J]. Journal of Bacteriology, 2003, 185(14): 4003-4010.
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153	WILLSON B J, KOVACS K, WILDING-STEELE T, et al. Production of a functional cell wall-anchored minicellulosome by recombinant Clostridium acetobutylicum ATCC 824 [J]. Biotechnol Biofuels, 2016, 9:109.
154	WEN Z, LEDESMA-AMARO R, LU M, et al. Combined evolutionary engineering and genetic manipulation improve low pH tolerance and butanol production in a synthetic microbial Clostridium community [J]. Biotechnology and Bioengineering, 2020, 117(7): 2008-2022.

菌株	底物	代谢途径	正丁醇/g·L^-1	文献
E. coli	甘油	CoA依赖的途径	0.552	[10]
E. coli	葡萄糖	CoA依赖的途径	15	[23]
E. coli	葡萄糖	反式β氧化途径	14	[24]
E. coli	葡萄糖	ACP依赖的途径	0.3	[25]
E. coli	葡萄糖	2-酮酸依赖的途径	1	[26]
E. coli	葡萄糖+丁酸	(截短的)CoA依赖的途径	6.2	[27]
E. coli	葡萄糖+甘油	CoA依赖的途径	18.3	[28]
E. coli	葡萄糖	CoA依赖的途径	20	[29]
S. cerevisiae	半乳糖	CoA依赖的途径	0.00 025	[11]
S. cerevisiae	葡萄糖	CoA依赖的途径	0.120	[21]
S. cerevisiae	葡萄糖+甘氨酸	2-酮酸依赖的途径	0.092	[22]
S. cerevisiae	葡萄糖	2-酮酸依赖的途径	0.2428	[20]
S. cerevisiae	葡萄糖	2-酮酸依赖的途径	0.835	[30]
Y. lipolytica	葡萄糖	CoA依赖的途径	0.123	[15]
B. subtilis	甘油	CoA依赖的途径	0.024	[13]
L. brevis	葡萄糖	CoA依赖的途径	0.3	[14]
Synechococcus 7942	CO₂	Malonyl-CoA (CoA)依赖的途径	0.404	[19]
S.7942	CO₂	CoA依赖的途径	0.030	[19]
S.7942	CO₂	CoA依赖的途径	0.404	[18]
S.7942	CO₂	CoA依赖的途径	0.4187	[31]
Synechocystis PCC 6803	CO₂	CoA依赖的途径	4.8	[32]
K. pneumonia	甘油	CoA依赖的途径	0.0150	[17]
K. pneumonia	甘油	2-酮酸依赖的途径	0.0287	[17]
K. pneumonia	甘油	2-酮酸依赖的途径	0.100	[16]
P. putida	甘油	CoA依赖的途径	0.122	[13]
Thermoanaerobacterium saccharolyticum	木糖	CoA依赖的途径	1.05	[33]
C. tyrobutyricum	葡萄糖	CoA依赖的途径	26.2	[34]
C. saccharoperbutylacetonicum N1-4	葡萄糖+木糖	CoA依赖的途径	16	[35]
C. pasteurianum	甘油+葡萄糖	CoA依赖的途径	21.1	[36]
C. acetobutylicum ATCC 824	葡萄糖	CoA依赖的途径	11.27	[37]
C. beijerinckii BA101	葡萄糖	CoA依赖的途径	18.6	[38]
C. cellulovorans	碱处理玉米棒芯	CoA依赖的途径	4.97	[39]
C. ljungdahlii	CO₂	CoA依赖的途径	0.148	[40]
C. carboxidivorans P7	CO₂	CoA依赖的途径	1.67	[41]
C. autoethanogenum	CO	CoA依赖的途径	1.54	[42]
C. cellulovorans/ C. beijerinckii	碱处理玉米棒芯	CoA依赖的途径	8.3	[43]
C. cellulovorans/ C. beijerinckii	碱处理玉米棒芯	CoA依赖的途径	11.5	[44]

菌株	底物	代谢途径	正丁醇/g·L^-1	文献
E. coli	甘油	CoA依赖的途径	0.552	[10]
E. coli	葡萄糖	CoA依赖的途径	15	[23]
E. coli	葡萄糖	反式β氧化途径	14	[24]
E. coli	葡萄糖	ACP依赖的途径	0.3	[25]
E. coli	葡萄糖	2-酮酸依赖的途径	1	[26]
E. coli	葡萄糖+丁酸	(截短的)CoA依赖的途径	6.2	[27]
E. coli	葡萄糖+甘油	CoA依赖的途径	18.3	[28]
E. coli	葡萄糖	CoA依赖的途径	20	[29]
S. cerevisiae	半乳糖	CoA依赖的途径	0.00 025	[11]
S. cerevisiae	葡萄糖	CoA依赖的途径	0.120	[21]
S. cerevisiae	葡萄糖+甘氨酸	2-酮酸依赖的途径	0.092	[22]
S. cerevisiae	葡萄糖	2-酮酸依赖的途径	0.2428	[20]
S. cerevisiae	葡萄糖	2-酮酸依赖的途径	0.835	[30]
Y. lipolytica	葡萄糖	CoA依赖的途径	0.123	[15]
B. subtilis	甘油	CoA依赖的途径	0.024	[13]
L. brevis	葡萄糖	CoA依赖的途径	0.3	[14]
Synechococcus 7942	CO₂	Malonyl-CoA (CoA)依赖的途径	0.404	[19]
S.7942	CO₂	CoA依赖的途径	0.030	[19]
S.7942	CO₂	CoA依赖的途径	0.404	[18]
S.7942	CO₂	CoA依赖的途径	0.4187	[31]
Synechocystis PCC 6803	CO₂	CoA依赖的途径	4.8	[32]
K. pneumonia	甘油	CoA依赖的途径	0.0150	[17]
K. pneumonia	甘油	2-酮酸依赖的途径	0.0287	[17]
K. pneumonia	甘油	2-酮酸依赖的途径	0.100	[16]
P. putida	甘油	CoA依赖的途径	0.122	[13]
Thermoanaerobacterium saccharolyticum	木糖	CoA依赖的途径	1.05	[33]
C. tyrobutyricum	葡萄糖	CoA依赖的途径	26.2	[34]
C. saccharoperbutylacetonicum N1-4	葡萄糖+木糖	CoA依赖的途径	16	[35]
C. pasteurianum	甘油+葡萄糖	CoA依赖的途径	21.1	[36]
C. acetobutylicum ATCC 824	葡萄糖	CoA依赖的途径	11.27	[37]
C. beijerinckii BA101	葡萄糖	CoA依赖的途径	18.6	[38]
C. cellulovorans	碱处理玉米棒芯	CoA依赖的途径	4.97	[39]
C. ljungdahlii	CO₂	CoA依赖的途径	0.148	[40]
C. carboxidivorans P7	CO₂	CoA依赖的途径	1.67	[41]
C. autoethanogenum	CO	CoA依赖的途径	1.54	[42]
C. cellulovorans/ C. beijerinckii	碱处理玉米棒芯	CoA依赖的途径	8.3	[43]
C. cellulovorans/ C. beijerinckii	碱处理玉米棒芯	CoA依赖的途径	11.5	[44]

遗传操作工具	原理及效果	优点	缺点	文献
RNA干扰	利用反义RNA干扰靶标基因转录	周期短，见效快	设计和操作烦琐，仅在转录水平控制	[62-63]
二型内含子插入失活	二型内含子可重编程靶向插入目的基因的特定位置	操作简单，几乎适用于所有梭菌	有一定的脱靶概率，可能造成极性效应	[83-85]
转座	利用Mariner转座子在梭菌染色体上随机插入使基因失活	随机插入失活，适合建库	不可用于靶向敲除	[83-85]
小RNA介导基因下调	小RNA形成的特定结构影响目的mRNA的转录水平	周期短，见效快	设计和操作烦琐，仅在转录水平上能调控	[82]
整合酶介导的染色体整合	噬菌体丝氨酸整合酶将目标基因整合到染色体预先设计的整合位点上	可以很方便进行大片段整合	需先将整合位点插入染色体靶标位置	[89]
等位基因替换	依赖同源重组单交换及之后的二次交换	可精确进行基因编辑	梭菌同源重效率低，过程耗时长	[70,88]
反筛标记介导的等位基因替换	在等位基因替换基础上，在同源臂内设计pyrE、mazF等反筛标记，方便筛选二次交换突变株	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	[71-72]
I-SceI归巢内切酶介导的等位基因替换	在等位基因替换基础上，在同源臂内设计I-SceI酶切位点，表达I-SceI切割双键，促进二次交换或帮助筛选二次交换突变株	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	[73]
单链寡核苷酸介导的点突变	重组蛋白RecT介导单链寡核苷酸更强的侵入和重组	可在较短同源臂时实现点突变	同源重组效率仍然较低	[90]
CRISPR/Cas9系统介导的基因编辑	Cas9在sgRNA引导下切割靶标DNA，造成双键断裂（平末端），依赖同源重组修复	增加第一次单交换效率，可实现多功能编辑	双键断裂的毒性大，转化子很难获得	[74-75]
CRISPR/nCas9系统介导的基因编辑	失活Cas9蛋白的其中一个切割蛋白结构域，使其仅能单链切割靶标DNA，造成缺刻，然后依赖同源重组修复	相比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	[74-75,77,81]
CRISPR/Cpf1系统介导的基因编辑	Cpf1在sgRNA引导下切割靶标DNA，造成双键断裂(有悬端)，依赖同源重组修复	悬端的存在有利于DNA修复；脱靶概率比CRISPR/Cas9低	仍有毒性，转化子依然偏少	[78]
CRISPR/dCas9系统介导的基因下调	失活Cas9蛋白的全部两个切割蛋白结构域，使其仅能与靶标DNA结合，利用位阻效应降低mRNA转录水平	对靶标基因转型转录调控，尤其适用于必需基因的操作	依赖于sgRNA和基因	[44,75,81]
CRISPR/Cas系统辅助的碱基编辑	将胞嘧啶脱氨酶和尿嘧啶DNA糖苷酶与Cas蛋白融合表达，在sgRNA引导下对目标碱基实现C-G到T-A的替换	理论上可对任何碱基进行编辑，不依赖于同源重组	碱基转换能力待拓展，编辑效率有待提升	[80]

遗传操作工具	原理及效果	优点	缺点	文献
RNA干扰	利用反义RNA干扰靶标基因转录	周期短，见效快	设计和操作烦琐，仅在转录水平控制	[62-63]
二型内含子插入失活	二型内含子可重编程靶向插入目的基因的特定位置	操作简单，几乎适用于所有梭菌	有一定的脱靶概率，可能造成极性效应	[83-85]
转座	利用Mariner转座子在梭菌染色体上随机插入使基因失活	随机插入失活，适合建库	不可用于靶向敲除	[83-85]
小RNA介导基因下调	小RNA形成的特定结构影响目的mRNA的转录水平	周期短，见效快	设计和操作烦琐，仅在转录水平上能调控	[82]
整合酶介导的染色体整合	噬菌体丝氨酸整合酶将目标基因整合到染色体预先设计的整合位点上	可以很方便进行大片段整合	需先将整合位点插入染色体靶标位置	[89]
等位基因替换	依赖同源重组单交换及之后的二次交换	可精确进行基因编辑	梭菌同源重效率低，过程耗时长	[70,88]
反筛标记介导的等位基因替换	在等位基因替换基础上，在同源臂内设计pyrE、mazF等反筛标记，方便筛选二次交换突变株	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	[71-72]
I-SceI归巢内切酶介导的等位基因替换	在等位基因替换基础上，在同源臂内设计I-SceI酶切位点，表达I-SceI切割双键，促进二次交换或帮助筛选二次交换突变株	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	[73]
单链寡核苷酸介导的点突变	重组蛋白RecT介导单链寡核苷酸更强的侵入和重组	可在较短同源臂时实现点突变	同源重组效率仍然较低	[90]
CRISPR/Cas9系统介导的基因编辑	Cas9在sgRNA引导下切割靶标DNA，造成双键断裂（平末端），依赖同源重组修复	增加第一次单交换效率，可实现多功能编辑	双键断裂的毒性大，转化子很难获得	[74-75]
CRISPR/nCas9系统介导的基因编辑	失活Cas9蛋白的其中一个切割蛋白结构域，使其仅能单链切割靶标DNA，造成缺刻，然后依赖同源重组修复	相比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	[74-75,77,81]
CRISPR/Cpf1系统介导的基因编辑	Cpf1在sgRNA引导下切割靶标DNA，造成双键断裂(有悬端)，依赖同源重组修复	悬端的存在有利于DNA修复；脱靶概率比CRISPR/Cas9低	仍有毒性，转化子依然偏少	[78]
CRISPR/dCas9系统介导的基因下调	失活Cas9蛋白的全部两个切割蛋白结构域，使其仅能与靶标DNA结合，利用位阻效应降低mRNA转录水平	对靶标基因转型转录调控，尤其适用于必需基因的操作	依赖于sgRNA和基因	[44,75,81]
CRISPR/Cas系统辅助的碱基编辑	将胞嘧啶脱氨酶和尿嘧啶DNA糖苷酶与Cas蛋白融合表达，在sgRNA引导下对目标碱基实现C-G到T-A的替换	理论上可对任何碱基进行编辑，不依赖于同源重组	碱基转换能力待拓展，编辑效率有待提升	[80]

出发菌株	基因型	增产策略及效果描述	正丁醇/g·L^-1	文献
C. acetobutylicum ATCC 824	+pykA+pfkA	过表达EMP途径的6-磷酸果糖激酶和丙酮酸激酶基因，增强碳源供应，正丁醇增加44.3%	19.12	[92]
C. acetobutylicum ATCC 824	+thl（R133G，H156N；G222V）	过表达点突变的thl（3处点突变减少辅酶A对硫酯酶的反馈抑制），增强主途径代谢，乙醇和正丁醇产量分别增加46%和18%，丙酮几乎没有变化	12.4	[93]
C. diolis DSM15140	+ter	过表达不可逆的反式烯酰辅酶A还原酶（来自齿垢密螺旋体），正丁醇产量提升50.5%	10.1	[94]
C. acetobutylicum ATCC 824	△adc,+gshAB+adhE+ctfAB,+thl+hbd+crt+bcd	正丁醇14.86g/L提升187%；乙醇产量3.25 g/L,提升278%;丙酮0.15 g/L，下降94.3%	14.86	[95]
C. acetobutylicum ATCC 824	△solR	敲除产溶剂操纵子的负调控因子，正丁醇产量增加	17.8	[96]
C. acetobutylicum ATCC 824	野生型	—	11.71	[97]
C. acetobutylicum ATCC 824	△solR	敲除产溶剂操纵子的负调控因子，正丁醇产量小幅提升	14.6	[97]
C. acetobutylicum ATCC 824	△solR+aad	敲除产溶剂操纵子的负调控因子后过表达醇醛脱氢酶基因（aad）	17.6	[97]
C. acetobutylicum ATCC 824	野生型	—	11.27	[37]
C. acetobutylicum ATCC 824	+aad+thl	过表达aad和thl增强主途径，但正丁醇产量和比例未见明显改变	11.34	[37]
C. acetobutylicum ATCC 824	+aad	过表达thl增强主途径，正丁醇产量和比例未见明显改变	11.86	[37]
C. acetobutylicum ATCC 824	+aad-ctfB	过表达aad及辅酶A转移酶基因ctfB的反义RNA。正丁醇产量小幅增加，但乙醇增加明显，导致正丁醇比例下降	13.19	[37]
C. acetobutylicum ATCC 824	+aad ^D458G	过表达点突变（485位天冬氨酸突变为甘氨酸）aad（对NADH和NADPH均有较好亲和力），正丁醇仅增加11.3%;乙醇大幅增加294%	14.8	[98]
C. acetobutylicum ATCC 824	+aad ^D458G +(Cb ald-Cl bdh)	过表达aad ^D458G，以及拜氏梭菌来源的醛脱氢酶基因(ald)，杨氏梭菌来源的正丁醇脱氢酶基因(bdh)，试图增强主途径，正丁醇产量增加27.1%,乙醇和丙酮均未大幅增加	16.9	[98]
C. acetobutylicum ATCC 824	△ack	TargeTron中断乙酸激酶基因（ack），试图阻断产乙酸途径，正丁醇产量提升22.9%,乙醇产量提升305%	8.6	[99]
C. acetobutylicum ATCC 824	△ack	TargeTron中断ack，试图阻断产乙酸途径，正丁醇产量几乎不变	11.3	[45]
C. acetobutylicum ATCC 824	△pta	TargeTron中断乙酰磷酸转移酶基因pta，试图阻断产乙酸途径，溶剂产量大幅增加，其中正丁醇增加45.8%	17.2	[45]
C. acetobutylicum ATCC 824	△ptb	TargeTron中断丁酰磷酸转移酶基因ptb，试图阻断产丁酸途径，正丁醇减少32.8%;乙醇产量12.1 g/L，增加505%；丙酮5.3 g/L	8	[100]
C. acetobutylicum ATCC 824	△ptb	TargeTron中断丁ptb，试图阻断产丁酸途径，正丁醇增加17.8%,乙醇0.9 g/L，几乎不变	13.9	[45]
C. acetobutylicum ATCC 824	△buk	TargeTron中断丁酸激酶基因buk，试图阻断产丁酸途径，正丁醇增加28.8%;乙醇1.9 g/L,增加111%；丙酮8 g/L,增加48.1%	15.2	[45]
C. acetobutylicum ATCC 824	△pta△buk	TargeTron连续中断pta和buk，正丁醇增加35.6%	16	[45]
C. acetobutylicum ATCC 824	△pta△buk +adhE1	TargeTron连续中断pta和buk后，过表达醇醛脱氢酶基因adhE1,正丁醇增加55.9%。乙醇3.0 g/L，增加233%；丙酮2.1 g/L，下降61.1%	18.4	[45]
C. acetobutylicum ATCC 824	△pta△buk +adhE1^D485G	TargeTron连续中断pta和buk后，过表达点突变的adhE1 ^D485G (对NADH和NADPH均有较好亲和力)，正丁醇增加60.2%；乙醇1.1 g/L，增加22.2%；丙酮1.5 g/L，降低72.2%	18.9	[45]
C. beijerinckii NCIMB 8052	△pta△buk	TargeTron连续中断pta和buk后意外发现可实现葡萄糖木糖同步发酵，消除了CCR效应，正丁醇增产36.4%	12.64	[101]
C. beijerinckiiCC101	+ctfAB+adhE2	过表达酸回用途径和正丁醇合成途径关键酶，正丁醇产量增加接近100%	12	[102]
C. acetobutylicum ATCC 824	△ldhA, △ctfAB, △ptb, △buk, +thl, hbd	删除乳酸脱氢酶基因ldhA、ctfAB、ptb、buk，过表达thl和3-羟基丁酰辅酶A脱氢酶基因（hbd），增强主途径，连续发酵且辅以气提移除正丁醇抑制	550^①	[103]