合成生物技术驱动酪丁酸梭菌细胞工厂开发的研究进展

doi:10.12211/2096-8280.2022-022

合成生物学 ›› 2022, Vol. 3 ›› Issue (6): 1174-1200.DOI: 10.12211/2096-8280.2022-022

合成生物技术驱动酪丁酸梭菌细胞工厂开发的研究进展

刘家宇¹, 杨智晗², 杨蕾², 朱丽英³, 朱政明¹, 江凌¹^,⁴

^1.南京工业大学食品与轻工学院，江苏南京 211816
^2.南京工业大学生物与制药工程学院，江苏南京 211816
^3.南京工业大学化学与分子工程学院，江苏南京 211816
^4.南京工业大学材料化学工程国家重点实验室，江苏南京 211816

收稿日期:2022-04-14 修回日期:2022-05-25 出版日期:2022-12-31 发布日期:2023-01-17
通讯作者: 朱政明,江凌
作者简介:刘家宇（1998—），男，硕士研究生。研究方向为微生物系统与合成生物学。E-mail：liujoy@njtech.edu.cn
朱政明（1989—），男，博士，副教授。研究方向为微生物系统与合成生物学。E-mail：zhuzm@njtech.edu.cn
江凌（1982—），男，博士，教授。研究方向为发酵工程与酶工程。E-mail：jiangling@njtech.edu.cn
基金资助:
国家自然科学基金优秀青年科学基金项目(31922070);国家自然科学基金青年科学基金项目(22008114);国家自然科学基金山东联合基金重点项目(U2106228);江苏省自然科学基金青年基金项目(BK20200684);江苏省先进生物制造创新中心资助(XTC2205)

Advances in the development of Clostridium tyrobutyricum cell factories driven by synthetic biotechnology

Jiayu LIU¹, Zhihan YANG², Lei YANG², Liying ZHU³, Zhengming ZHU¹, Ling JIANG¹^,⁴

^1.School of Food and Light Industry，Nanjing Tech University，Nanjing 211816，Jiangsu，China
^2.School of Biological and Pharmaceutical Engineering，Nanjing Tech University，Nanjing 211816，Jiangsu，China
^3.School of Chemical and Molecular Engineering，Nanjing Tech University，Nanjing 211816，Jiangsu，China
^4.State Key Laboratory of Chemical Engineering of Materials，Nanjing Tech University，Nanjing 211816，Jiangsu，China

Received:2022-04-14 Revised:2022-05-25 Online:2022-12-31 Published:2023-01-17
Contact: Zhengming ZHU, Ling JIANG

摘要/Abstract

摘要：

作为一种重要的工业微生物和新型益生菌，酪丁酸梭菌是厌氧条件下代谢多种底物产生丁酸的优势菌株，在其他精细化学品生产和大健康领域亦具有广泛应用。然而，获取生产效率高、鲁棒性强的高版本酪丁酸梭菌细胞工厂，仍然面临着遗传转化效率低、遗传操作工具有限、调控手段单一等诸多挑战。近年来，随着合成生物学的不断发展和酪丁酸梭菌生物信息数据的逐步完善，多种研究策略和技术，包括基因编辑系统等，被用于设计和构筑酪丁酸梭菌底盘细胞高效合成各种精细化学品。本文首先对酪丁酸梭菌独特的生理特性进行了概述。然后，对酪丁酸梭菌底盘细胞改造过程中涉及的系统生物学方法以及遗传操作工具的构建方法与技术进行了总结。同时，探讨了酪丁酸梭菌中多类型代谢调控策略以及群体感应系统的开发及其在合成精细化学品中的应用。最后，从遗传转化效率提升、基因编辑工具拓展、基因回路设计与重构高通量筛选平台建立、一碳气体利用等方面对酪丁酸梭菌底盘细胞的创制进行了展望。

关键词: 酪丁酸梭菌, 底盘细胞, 合成生物技术, 系统生物学, 代谢工程

Abstract:

As an important industrial microorganism and a novel probiotic, Clostridium tyrobutyricum is a superior strain for metabolizing various substrates to produce butyric acid under anaerobic condition, presenting a great potential for the valorization of agricultural wastes. Consequently, this bacterium with high yield of butyric acid has also been widely used in other fields, such as fine chemical production and human health. However, there are still many challenges in the construction of highly productive and robust C. tyrobutyricum cell factories. For example, the genetic transformation efficiency is rather low, due to the presence of restriction-modification systems. Gene editing tools are less developed and strain construction suffers from tedious processes and low efficiency. Moreover, genetic modification of C. tyrobutyricum is limited to a single mode of metabolic regulation, either knockout or overexpression, which is far behind the conventional model hosts. In recent years, with the continuous rapid development of synthetic biology and the collection of increasing amounts of C. tyrobutyricum bioinformatics data, a variety of research strategies and techniques, particularly the gene editing systems, have been employed to design and construct C. tyrobutyricum cell factories for efficient production of various fine chemicals. In this paper, we firstly provide an overview of the unique physiological properties of C. tyrobutyricum, including substrate range, environmental adaptability, butyric acid synthesis pathway, as well as the one-carbon gas fixation and energy metabolism pathways. Subsequently, we summarize the systems biology methods as well as the genetic manipulation tools for the modification of C. tyrobutyricum chassis cell, such as biological elements, the conjugation system, and the CRISPR/Cas system. Meanwhile, we discuss the static and dynamic metabolic regulation strategies and two types of quorum sensing systems (agr-type andRRNPP-type) as well as their applications in the synthesis of fine chemicals in C. tyrobutyricum. Finally, we prospect the trends for the creation of C. tyrobutyricum chassis cell, in terms of enhancing genetic transformation efficiency, expanding gene editing tools, designing and reconstructing gene circuits, establishing high-throughput screening platforms, and utilizing one-carbon gas.

Key words: Clostridium tyrobutyricum, chassis cells, synthetic biotechnology, systems biology, metabolic engineering

中图分类号:

刘家宇, 杨智晗, 杨蕾, 朱丽英, 朱政明, 江凌. 合成生物技术驱动酪丁酸梭菌细胞工厂开发的研究进展[J]. 合成生物学, 2022, 3(6): 1174-1200.

Jiayu LIU, Zhihan YANG, Lei YANG, Liying ZHU, Zhengming ZHU, Ling JIANG. Advances in the development of Clostridium tyrobutyricum cell factories driven by synthetic biotechnology[J]. Synthetic Biology Journal, 2022, 3(6): 1174-1200.

图/表 11

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底物		成分	发酵方式	产物	滴度 /（g/L）	产率 /[g/（L·h）]	产量 /（g/g）	改造方法	参考文献
单糖	葡萄糖	—	补料分批	丁酸	48.2	0.50	0.38	过表达pfkA、pykA	[22]
			补料分批	丁醇	约45	—	0.30	敲除ack，过表达adhE2	[23]
			FBB反应器	丁酸	52.2	0.41	0.37	过表达groESL	[24]
			FBB反应器	丁醇	约54	—	0.33	敲除ack，过表达adhE2	[23]
			分批	丁酸	—	0.20	0.36	敲除ptb	[25]
			分批	丁醇	约10.5	约0.24	约0.24	敲除ack，过表达adhE2、ctfAB	[26]
			重复FBB反应器	丁酸	86.9	1.1	0.46	—^①	[27]
	木糖	—	FBB反应器	丁酸	13.6	—	0.44	敲除ack	[28]
	木糖	—	分批	丁酸	—	0.22	0.35	敲除ptb	[25]
	果糖	—	FBB反应器	丁酸	49.4	2.60	0.48	—	[20]
二糖	蔗糖	葡萄糖、果糖	FBB反应器	丁酸	45.8	—	0.43	—	[20]
	蔗糖	葡萄糖、果糖	分批	丁醇	18.8	0.20	0.21	敲除ack，过表达adhE2、ctfABK	[29]
	麦芽糖	葡萄糖	分批	丁醇	17.3	0.40	0.17	敲除ack，过表达adhE2、aglul	[30]
农业原料及农业废弃物	咖啡残渣水解物	木糖、阿拉伯糖、半乳糖	分批	丁酸	34.3	0.36	0.37	过表达galKETP	[21]
	甘蔗糖蜜	果糖、葡萄糖	补料分批	丁酸	45.71	—	0.39	过表达scrBAK	[31]
	甘蔗汁	果糖、葡萄糖	分批	丁醇	14.8	0.15	0.21	敲除ack，过表达adhE2、scrBAK	[29]
	甘蔗渣水解物	木糖、阿拉伯糖、半乳糖	补料分批	丁酸	20.9	0.51	0.48	—	[32]
	菊粉	果糖、葡萄糖	分批	氢气	—	620 ml/（L·h）	3.7 mol/mol	过表达外源菊粉酶	[33]
	菊粉	果糖、葡萄糖	补料分批FBB 反应器	丁酸	60.4	1.14	0.38	—	[15]
	玉米秸秆水解物	葡萄糖、木糖	分批	丁酸	16.1	0.38	0.35	过表达groESL	[14]
	玉米面粉水解物	葡萄糖、木糖	补料分批FBB 反应器	丁酸	46.0	6.8	0.45	—	[19]
	玉米纤维水解物	葡萄糖、木糖	补料分批FBB 反应器	丁酸	29.0	2.9	0.47	—	[17]
	玉米皮水解物	葡萄糖、木糖	重复FBB反应器	丁酸	20.8	0.42	0.39	—	[34]
	小麦秸秆水解物	葡萄糖、木糖	分批	丁酸	20.0	0.21	0.33	—	[35]
	稻草水解物	葡萄糖、木糖	分批	丁酸	16.9	0.40	0.35	过表达groESL	[14]
	高粱秸秆和甜菜秸秆水解物	葡萄糖、木糖	分批	丁酸	58.8	1.9	0.52	—	[36]

底物		成分	发酵方式	产物	滴度 /（g/L）	产率 /[g/（L·h）]	产量 /（g/g）	改造方法	参考文献
单糖	葡萄糖	—	补料分批	丁酸	48.2	0.50	0.38	过表达pfkA、pykA	[22]
			补料分批	丁醇	约45	—	0.30	敲除ack，过表达adhE2	[23]
			FBB反应器	丁酸	52.2	0.41	0.37	过表达groESL	[24]
			FBB反应器	丁醇	约54	—	0.33	敲除ack，过表达adhE2	[23]
			分批	丁酸	—	0.20	0.36	敲除ptb	[25]
			分批	丁醇	约10.5	约0.24	约0.24	敲除ack，过表达adhE2、ctfAB	[26]
			重复FBB反应器	丁酸	86.9	1.1	0.46	—^①	[27]
	木糖	—	FBB反应器	丁酸	13.6	—	0.44	敲除ack	[28]
	木糖	—	分批	丁酸	—	0.22	0.35	敲除ptb	[25]
	果糖	—	FBB反应器	丁酸	49.4	2.60	0.48	—	[20]
二糖	蔗糖	葡萄糖、果糖	FBB反应器	丁酸	45.8	—	0.43	—	[20]
	蔗糖	葡萄糖、果糖	分批	丁醇	18.8	0.20	0.21	敲除ack，过表达adhE2、ctfABK	[29]
	麦芽糖	葡萄糖	分批	丁醇	17.3	0.40	0.17	敲除ack，过表达adhE2、aglul	[30]
农业原料及农业废弃物	咖啡残渣水解物	木糖、阿拉伯糖、半乳糖	分批	丁酸	34.3	0.36	0.37	过表达galKETP	[21]
	甘蔗糖蜜	果糖、葡萄糖	补料分批	丁酸	45.71	—	0.39	过表达scrBAK	[31]
	甘蔗汁	果糖、葡萄糖	分批	丁醇	14.8	0.15	0.21	敲除ack，过表达adhE2、scrBAK	[29]
	甘蔗渣水解物	木糖、阿拉伯糖、半乳糖	补料分批	丁酸	20.9	0.51	0.48	—	[32]
	菊粉	果糖、葡萄糖	分批	氢气	—	620 ml/（L·h）	3.7 mol/mol	过表达外源菊粉酶	[33]
	菊粉	果糖、葡萄糖	补料分批FBB 反应器	丁酸	60.4	1.14	0.38	—	[15]
	玉米秸秆水解物	葡萄糖、木糖	分批	丁酸	16.1	0.38	0.35	过表达groESL	[14]
	玉米面粉水解物	葡萄糖、木糖	补料分批FBB 反应器	丁酸	46.0	6.8	0.45	—	[19]
	玉米纤维水解物	葡萄糖、木糖	补料分批FBB 反应器	丁酸	29.0	2.9	0.47	—	[17]
	玉米皮水解物	葡萄糖、木糖	重复FBB反应器	丁酸	20.8	0.42	0.39	—	[34]
	小麦秸秆水解物	葡萄糖、木糖	分批	丁酸	20.0	0.21	0.33	—	[35]
	稻草水解物	葡萄糖、木糖	分批	丁酸	16.9	0.40	0.35	过表达groESL	[14]
	高粱秸秆和甜菜秸秆水解物	葡萄糖、木糖	分批	丁酸	58.8	1.9	0.52	—	[36]

固碳途径	固碳种类	酶总数	关键酶	酶比活力^①/[µmol/(min·mg)]	能量来源	产物	ATP 消耗
WL途径^[47-48]	CO₂	8	甲酸脱氢酶	439	氢气	乙酰辅酶A	<1
WL途径^[47-48]	CO₂	8	CO脱氢酶	14 000	氢气	乙酰辅酶A	<1
还原性甘氨酸途径^[49]	CO₂	5	还原性甘氨酸裂解复合物	—	—	乙酰辅酶A	2
卡尔文循环^[50]	CO₂	11	RuBisCO	304.3	光	3-磷酸甘油醛	9
还原性TCA循环^[50]	CO₂	8	2-酮戊二酸合酶	35.2	光和硫	乙酰辅酶A	2
还原性TCA循环^[50]	CO₂	8	ATP-柠檬酸裂合酶	26.7	光和硫	乙酰辅酶A	2
DC/HB循环^[50]	CO₂/HCO₃^-	14	4-羟基丁酰辅酶A脱水酶	—	氢和硫	乙酰辅酶A	5
HP/HB循环^[50]	HCO₃^-	15	4-羟基丁酰辅酶A脱水酶	—	氢和氧	乙酰辅酶A	6
3-HP双循环^[51-52]	HCO₃^-	18	丙二酰辅酶A还原酶	80	光和硫	丙酮酸	7
3-HP双循环^[51-52]	HCO₃^-	18	丙酰辅酶A合酶	22	光和硫	丙酮酸	7

固碳途径	固碳种类	酶总数	关键酶	酶比活力^①/[µmol/(min·mg)]	能量来源	产物	ATP 消耗
WL途径^[47-48]	CO₂	8	甲酸脱氢酶	439	氢气	乙酰辅酶A	<1
WL途径^[47-48]	CO₂	8	CO脱氢酶	14 000	氢气	乙酰辅酶A	<1
还原性甘氨酸途径^[49]	CO₂	5	还原性甘氨酸裂解复合物	—	—	乙酰辅酶A	2
卡尔文循环^[50]	CO₂	11	RuBisCO	304.3	光	3-磷酸甘油醛	9
还原性TCA循环^[50]	CO₂	8	2-酮戊二酸合酶	35.2	光和硫	乙酰辅酶A	2
还原性TCA循环^[50]	CO₂	8	ATP-柠檬酸裂合酶	26.7	光和硫	乙酰辅酶A	2
DC/HB循环^[50]	CO₂/HCO₃^-	14	4-羟基丁酰辅酶A脱水酶	—	氢和硫	乙酰辅酶A	5
HP/HB循环^[50]	HCO₃^-	15	4-羟基丁酰辅酶A脱水酶	—	氢和氧	乙酰辅酶A	6
3-HP双循环^[51-52]	HCO₃^-	18	丙二酰辅酶A还原酶	80	光和硫	丙酮酸	7
3-HP双循环^[51-52]	HCO₃^-	18	丙酰辅酶A合酶	22	光和硫	丙酮酸	7

菌株名称	测序程度	基因组大小/Mb	G+C/%	蛋白数量	rRNA	tRNA	其他RNA
KCTC 5387	Complete	3.13	31.0	3015	19	63	5
L319	Complete	3.09	31.0	2922	19	63	5
W428	Complete	3.07	30.9	2945	16	51	5
Cirm BIA 2237	Chromosome	3.16	30.8	3069	3	52	5
FAM22553	Scaffold	3.09	31.0	2992	18	63	5
FAM22552	Scaffold	3.05	30.9	2923	18	68	5
DSM 663	Scaffold	3.15	30.7	3091	3	35	5
1001713B170207_170306_A1	Scaffold	3.02	31.0	2831	4	57	5
1001713B170207_170306_A10	Scaffold	3.05	30.5	2881	4	60	5
1001283B150210_160208_G3	Scaffold	3.07	30.5	2869	5	52	5
1001283B150210_160208_D6	Scaffold	3.00	30.5	2791	4	58	5
Cl_188	Scaffold	3.01	30.5	2876	3	40	5
Cl_171	Scaffold	3.04	30.5	2938	3	36	5
Cl_239	Scaffold	3.33	30.5	3219	4	48	5
Cl_117	Scaffold	3.11	31.0	3034	4	46	5
Cl_238	Scaffold	3.23	30.5	3115	4	30	5
Cl_84	Scaffold	3.05	30.5	2954	3	52	5
Cl_82	Scaffold	3.01	30.5	2916	4	45	5
Cl_64	Scaffold	3.17	31.0	3087	4	46	5
Cl_52	Scaffold	3.09	30.5	2940	2	42	5
Cl_80	Scaffold	3.09	30.5	3049	3	34	5
Cl_29	Scaffold	3.09	30.5	2988	4	37	5
MGYG-HGUT-00125	Scaffold	3.26	30.5	3101	9	63	5
24853	Scaffold	3.00	30.5	2664	4	59	5
ATCC 25755	Contig	3.01	30.5	2908	4	46	5
DIVETGP	Contig	3.02	30.5	2907	4	46	5
UC7086	Contig	3.06	30.5	2943	4	48	5
IFP923	Contig	3.19	30.5	3090	10	60	5

合成生物技术驱动酪丁酸梭菌细胞工厂开发的研究进展

Advances in the development of Clostridium tyrobutyricum cell factories driven by synthetic biotechnology

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启动子	类型	性质	参考文献
P _ptb	组成型	控制ptb的表达	[82]
P _thl	组成型	控制thl的表达	[33]
P _hydA	组成型	电子代谢关键酶基因hydA的启动子	[83]
P _ptk	诱导型	阿拉伯糖诱导	[84]
P _xylA	诱导型	受木糖诱导	[85]
P _bgaL	诱导型	受乳糖诱导	[86]
P _celC	诱导型	受纤维素诱导	[87]
P _Pcm-2tetO1	诱导型	受四环素诱导	[88]

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