Progress in construction and applications of methanotrophic cell factory for chemicals biosynthesis

doi:10.12211/2096-8280.2021-011

Abstract

Abstract:

Methane has been considered as a potential carbon source in industrial biotechnology because of its abundance, sustainability, high reducibility, and microbial availability. The biological conversion of methane into chemicals or fuels does not only reduce greenhouse gas emissions, but also substitute food-based substrates used in bio-manufacturing. Methanotrophs are gram-negative bacteria, and most are isolated from methane-plentiful environments. Owing to the presence of the methane monooxygenase, methanotrophs constitute a unique group of microbes. As an important industrially-promising microorganism with the characteristics of robust and anti-contamination ability, methanotrophs capable of growing with methane as the sole energy and carbon source play a significant role in carbon-neutral society by replacing petroleum-based products with biosynthesized products. Therefore, studies on methanotrophs for the biological conversion of methane have attracted extensive attention in recent years. With the rapid development of genetic manipulations tools and strategies for metabolically-engineered methanotrophs construction, including gene editing methods, regulation of metabolic pathways, and bio-elements mining, methanotrophic cell factories have been employed to efficiently convert methane into a variety of bulk chemicals and biofuels. In this review, biosynthetic technologies related to bioconversion of under-utilized methane ranging from fundamental understanding, systematic analysis, metabolic engineering to bio-product production are introduced. The genetic manipulations tools of methanotrophs, the approaches of methanotrophic cell factory construction, and the enhancement of methane assimilation efficiency are summarized from the aspects including the research progress of genetic engineering of methanotrophs, the regulation of methane carbon flux, the overexpression of heterologous pathway genes, and the accumulation of metabolic intermediates. Besides, the applications of genomics, transcriptomics, metabolomics, and metabolic modeling have been also deployed to facilitate the methane metabolism in methanotrophs chassis. Finally, given the strategy of 'waste-to-value' production, the challenges and opportunities for methane bioconversion by methanotrophs are also discussed and prospected based on industrial applications in terms of the research progress in the biosynthesis of methane-based acids, terpenes, alcohols, and other chemicals.

Key words: methane, methanotrophs, cell factory, construction strategy, chemical biosynthesis

摘要：

由于来源广泛且储量丰富，甲烷被认为是极具应用潜力的下一代生物碳源。嗜甲烷菌是一种分离自富含甲烷环境中的革兰氏阴性细菌，其体内含有独特的甲烷单加氧酶能够让这类微生物以甲烷为唯一碳源和能源进行生长、代谢与产物合成。作为一种重要的工业微生物，嗜甲烷菌在甲烷生物转化利用、温室气体减排和“碳中和”策略开发方面具有重要意义。近年来，随着嗜甲烷菌基因编辑方法、代谢路径调控、生物元件挖掘等菌种构建工具和策略的不断开发，嗜甲烷菌人工细胞可高效转化甲烷生物合成多种大宗化学品和生物燃料。本文围绕遗传改造工具、甲烷碳流调控、异源途径表达和代谢节点累积等方面的研究进展，概述了构建嗜甲烷菌人工细胞的方法和提高甲烷同化效率的策略。同时介绍了基因组学、转录组学、代谢组学等组学研究方法在调控嗜甲烷菌底盘碳代谢流向和通量中的应用。最后，结合生物转化甲烷合成酸类、萜类、醇类等化学品的研究，分析并展望了嗜甲烷菌工业化应用所面临的挑战和机遇。

关键词: 甲烷, 嗜甲烷菌, 细胞工厂, 构建策略, 化学品生物合成

CLC Number:

Q819

Shuqi GUO, Ziyue JIAO, Qiang FEI. Progress in construction and applications of methanotrophic cell factory for chemicals biosynthesis[J]. Synthetic Biology Journal, 2021, 2(6): 1017-1029.

郭树奇, 焦子悦, 费强. 基于化学品生物合成的嗜甲烷菌人工细胞构建及应用进展[J]. 合成生物学, 2021, 2(6): 1017-1029.

Figures/Tables 4

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功能	质粒	筛选标记	菌株	参考文献
基因复制	pBHR1	Km, Cm	Methylomonas sp. 16a	[36]
基因复制	pAWP78	Km	M. buryatense 5G	[34]
基因表达	pAWP89	Km	M. buryatense 5G	[34]
基因表达	pCAH01:emGFP	Km	M. buryatense 5G	[37]
基因敲除	pK18mobsacB	Km, SacB	Methylococcus capsulatus (Bath)	[38]
基因敲除	pCM184	Gm, SacB	Methylomicrobium alcaliphilum 20Z	[39]
基因转移技术
接合转移	pRK2013	Km	Methylomonas sp. 16a	[40]
接合转移	pBHR1	Km	Methylomonas sp. 16a	[40]
电转化	pAWP89 （线性化）	Km	M. buryatense 5G	[41]
电转化	pheS^AG （线性化DNA片段）	Km	M. buryatense 5G	[10]

功能	质粒	筛选标记	菌株	参考文献
基因复制	pBHR1	Km, Cm	Methylomonas sp. 16a	[36]
基因复制	pAWP78	Km	M. buryatense 5G	[34]
基因表达	pAWP89	Km	M. buryatense 5G	[34]
基因表达	pCAH01:emGFP	Km	M. buryatense 5G	[37]
基因敲除	pK18mobsacB	Km, SacB	Methylococcus capsulatus (Bath)	[38]
基因敲除	pCM184	Gm, SacB	Methylomicrobium alcaliphilum 20Z	[39]
基因转移技术
接合转移	pRK2013	Km	Methylomonas sp. 16a	[40]
接合转移	pBHR1	Km	Methylomonas sp. 16a	[40]
电转化	pAWP89 （线性化）	Km	M. buryatense 5G	[41]
电转化	pheS^AG （线性化DNA片段）	Km	M. buryatense 5G	[10]

菌株	产物种类	代谢途径/前体	产物	产量	参考文献
M. buryatense 5GB1C^[43]	有机酸	丙酮酸	L-乳酸	800 mg/L	[37]
	有机酸	丙酮酸	L-乳酸	600 mg/L	[33]
	有机酸	乙酰辅酶A	丁烯酸	70 mg/L	[44]
	有机酸	乙酰辅酶A	丁酸	40 mg/L	[44]
	有机酸	莽草酸途径	黏糠酸	12.4 mg/L	[45]
	有机酸	乙酰辅酶A	脂肪酸	111 mg/g DCW	[46]
M. alcaliphilum 20Z^[47]	生物醇	丙酮酸	2,3-丁二醇	86.2 mg/L	[14]
	生物醇	丙酮酸	2,3-丁二醇	361.3 mg/L	[39]
	有机酸	乙酰辅酶A	3-羟基丙酸	196.3 mg/L	[39]
	有机酸	莽草酸途径	黏糠酸	0.75 mg/L	[45]
	萜类	MEP 途径	α-蛇麻烯	0.75 mg/g DCW	[48]
	—	TCA循环	腐胺	98.08 mg/L	[49]
	有机酸	丙酮酸	乳酸	0.027 g/(g DCW·h)	[50]
	—	RuMP 循环	Shinorine	31 mg/L	[39]
M. capsulatus Bath^[51]	有机酸	莽草酸途径	黏糠酸	1.0 mg/L	[45]
	萜类	MEP 途径	异戊二烯	10 mg/L	[52]
	生物醇	TCA 循环	1,4-丁二醇	—	[53]
	生物醇	丙酮酸	异丙醇	220 mg/L^①	[54]
	生物醇	丙酮酸	异丙醇	1 mg/L	[54]
Methylomonas sp. DH-1^[55]	有机酸	TCA 循环	琥珀酸	195 mg/L	[56]
Methylomonas sp. DH-1^[55]	有机酸	丙酮酸	D-乳酸	1190 mg/L	[32]
Methylosinus trichosporium OB3b^[57]	有机酸	乙酰辅酶A	3-羟基丙酸	60.6 mg/L	[15]
Methylosinus trichosporium OB3b^[57]	—	TCA 循环	尸胺	283.6 mg/L	[58]
Methylomonas sp. 16a^[36]	萜类	MEP 途径	柠檬烯	0.5 mg/L	[59]
	萜类	MEP 途径	法尼烯	—	[20]
	萜类	MEP 途径	虾青素	2.4 mg/g DCW	[60]
	萜类	MEP 途径	虾青素	2.0 mg/g DCW	[36]

菌株	产物种类	代谢途径/前体	产物	产量	参考文献
M. buryatense 5GB1C^[43]	有机酸	丙酮酸	L-乳酸	800 mg/L	[37]
	有机酸	丙酮酸	L-乳酸	600 mg/L	[33]
	有机酸	乙酰辅酶A	丁烯酸	70 mg/L	[44]
	有机酸	乙酰辅酶A	丁酸	40 mg/L	[44]
	有机酸	莽草酸途径	黏糠酸	12.4 mg/L	[45]
	有机酸	乙酰辅酶A	脂肪酸	111 mg/g DCW	[46]
M. alcaliphilum 20Z^[47]	生物醇	丙酮酸	2,3-丁二醇	86.2 mg/L	[14]
	生物醇	丙酮酸	2,3-丁二醇	361.3 mg/L	[39]
	有机酸	乙酰辅酶A	3-羟基丙酸	196.3 mg/L	[39]
	有机酸	莽草酸途径	黏糠酸	0.75 mg/L	[45]
	萜类	MEP 途径	α-蛇麻烯	0.75 mg/g DCW	[48]
	—	TCA循环	腐胺	98.08 mg/L	[49]
	有机酸	丙酮酸	乳酸	0.027 g/(g DCW·h)	[50]
	—	RuMP 循环	Shinorine	31 mg/L	[39]
M. capsulatus Bath^[51]	有机酸	莽草酸途径	黏糠酸	1.0 mg/L	[45]
	萜类	MEP 途径	异戊二烯	10 mg/L	[52]
	生物醇	TCA 循环	1,4-丁二醇	—	[53]
	生物醇	丙酮酸	异丙醇	220 mg/L^①	[54]
	生物醇	丙酮酸	异丙醇	1 mg/L	[54]
Methylomonas sp. DH-1^[55]	有机酸	TCA 循环	琥珀酸	195 mg/L	[56]
Methylomonas sp. DH-1^[55]	有机酸	丙酮酸	D-乳酸	1190 mg/L	[32]
Methylosinus trichosporium OB3b^[57]	有机酸	乙酰辅酶A	3-羟基丙酸	60.6 mg/L	[15]
Methylosinus trichosporium OB3b^[57]	—	TCA 循环	尸胺	283.6 mg/L	[58]
Methylomonas sp. 16a^[36]	萜类	MEP 途径	柠檬烯	0.5 mg/L	[59]
	萜类	MEP 途径	法尼烯	—	[20]
	萜类	MEP 途径	虾青素	2.4 mg/g DCW	[60]
	萜类	MEP 途径	虾青素	2.0 mg/g DCW	[36]

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