Advances in methanol bio-transformation

doi:10.12211/2096-8280.2020-017

Abstract

Abstract:

Methanol represents a promising feedstock due to its specific characteristics for storage and transport, which has been widely applied in chemical industry as raw material, intermediate and fuel. Methanol bio-transformation by microbes may further expand the methanol-based production of high quality fuels and various chemicals, which will drive clean utilization of inferior coal and natural gas. We here review recent advances in methanol-based bio-refinery. We first summarize current progress in methanol production through chemical and biological processes, which shows that the chemical process is now the main route for methanol supply. However, emerging technologies like methane oxidation (chemical or biological) and CO₂ hydrogenation may achieve a renewable, sustainable and clean route for methanol production. We then discuss the engineering of methylotrophs (bacterial and yeast) for producing a variety of chemicals from methanol (Top-Down approach), which indicates that improved genetic tools may further optimize these cell factories for industrial applications. We also summarize recent advances on engineering artificial methylotrophs in numerous model organisms such as Escherichia coli, Corynebacterium glutamicum and Saccharomyces cerevisiae (Bottom-Up approach). The advances in synthetic biology, metabolic engineering and adaptive laboratory evolution will facilitate the construction of robust microbial cell factories for methanol biotransformation, which will expand substrate resource for bio-refinery and the product portfolio of methanol.

Key words: methanol, bio-refinery, methylotrophic yeast, synthetic biology, metabolic engineering

摘要：

甲醇以其易储存和易运输等特性，成为极具应用潜力的原料。除了传统的基于合成气（CO，H₂，CO₂）和化学催化的甲醇合成，甲烷氧化（化学法和生物法）和CO₂加氢技术逐步成熟，特别是CO₂加氢技术将有望实现甲醇可持续洁净合成。甲醇生物转化有望进一步拓展现有甲醇转化路线，推动我国煤炭资源洁净利用以及CO₂利用。本文详细综述了国内外甲醇生物炼制研究进展：①以甲基营养型微生物，包括细菌和甲醇酵母，为宿主构建细胞工厂实现氨基酸以及平台化合物等合成；②在模式微生物中构建甲醇代谢途径实现甲醇利用与转化。通过天然/人工甲基营养型代谢途径相互借鉴，采用代谢工程与合成生物学策略，提高甲醇利用效率、底物与产物耐受能力，将推动甲醇生物转化、拓展生物炼制原料供应路线。

关键词: 甲醇, 生物炼制, 甲醇酵母, 合成生物学, 代谢工程

CLC Number:

Q815

GAO Jiaoqi, ZHOU Yongjin. Advances in methanol bio-transformation[J]. Synthetic Biology Journal, 2020, 1(2): 158-173.

高教琪, 周雍进. 甲醇生物转化的机遇与挑战[J]. 合成生物学, 2020, 1(2): 158-173.

Figures/Tables 6

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宿主	培养基	产物	产量	备注	参考文献
甲醇芽孢杆菌	甲醇，酵母粉，基础盐培养基	L-谷氨酸	69.0g/L	批式补料	[39]
甲醇芽孢杆菌	甲醇，酵母粉，基础盐培养基	L-赖氨酸	11.0g/L	批式补料	[39]
甲醇芽孢杆菌	甲醇，酵母粉，基础盐培养基	γ-氨基丁酸	9.0g/L	批式补料	[40]
甲醇芽孢杆菌	甲醇，酵母粉，基础盐培养基	尸胺	10.2g/L	批式补料	[41]
扭脱甲基杆菌	甲醇，基础盐培养基	甲羟戊酸	2.3g/L	ALE &批式补料	[42]
扭脱甲基杆菌	甲醇，琥珀酸，基础盐培养基	3-羟基丙酸	91.0mg/L	摇瓶	[43]
扭脱甲基杆菌	甲醇，基础盐培养基	2-羟基丁酸	2.1g/L	批式补料	[44]
扭脱甲基杆菌	甲醇，基础盐培养基	1-丁醇	25.5mg/L	ALE & 摇瓶	[45]
扭脱甲基杆菌	甲醇，基础盐培养基	α-葎草烯	1.7g/L	摇瓶两相发酵	[46]
巴斯德毕赤酵母	甲醇，基础盐培养基	莫那克林J、洛伐他汀	60.0mg/L 14.4mg/L	摇瓶	[47]
巴斯德毕赤酵母	甲醇， YPD培养基	D-乳酸	3.5g/L	试管	[48]
多形汉逊酵母	甲醇基础盐培养基	谷胱甘肽	约1.3g/L	批式补料	[49]

宿主	培养基	产物	产量	备注	参考文献
甲醇芽孢杆菌	甲醇，酵母粉，基础盐培养基	L-谷氨酸	69.0g/L	批式补料	[39]
甲醇芽孢杆菌	甲醇，酵母粉，基础盐培养基	L-赖氨酸	11.0g/L	批式补料	[39]
甲醇芽孢杆菌	甲醇，酵母粉，基础盐培养基	γ-氨基丁酸	9.0g/L	批式补料	[40]
甲醇芽孢杆菌	甲醇，酵母粉，基础盐培养基	尸胺	10.2g/L	批式补料	[41]
扭脱甲基杆菌	甲醇，基础盐培养基	甲羟戊酸	2.3g/L	ALE &批式补料	[42]
扭脱甲基杆菌	甲醇，琥珀酸，基础盐培养基	3-羟基丙酸	91.0mg/L	摇瓶	[43]
扭脱甲基杆菌	甲醇，基础盐培养基	2-羟基丁酸	2.1g/L	批式补料	[44]
扭脱甲基杆菌	甲醇，基础盐培养基	1-丁醇	25.5mg/L	ALE & 摇瓶	[45]
扭脱甲基杆菌	甲醇，基础盐培养基	α-葎草烯	1.7g/L	摇瓶两相发酵	[46]
巴斯德毕赤酵母	甲醇，基础盐培养基	莫那克林J、洛伐他汀	60.0mg/L 14.4mg/L	摇瓶	[47]
巴斯德毕赤酵母	甲醇， YPD培养基	D-乳酸	3.5g/L	试管	[48]
多形汉逊酵母	甲醇基础盐培养基	谷胱甘肽	约1.3g/L	批式补料	[49]

宿主	途径	基因	培养基	表征	文献
大肠杆菌	RuMP	BmMdh2, BmHps, BmPhi	¹³C甲醇+核糖	40%标记C进入中心碳代谢	[54]
	RuMP	BmMdh2, MlHps, MlPhi	¹³C甲醇+葡糖酸盐	24%甲醇进入中心代谢物，甲醇代谢速率加快	[74]
	RuMP	BsMdh, BmHps, BmPhi	1g/L酵母粉+甲醇	1g甲醇转化生物量0.29g（DCW），39% 甲醇C进入TCA循环，实现柚皮素合成	[73]
	RuMP	BmMdh, BmHps, BmPhi	葡萄糖，酵母粉，甲醇	琥珀酸得率提高7.7%，甲醇消耗 0.38～0.45 g/L	[72]
	丝氨酸	Mdh, Fdh, Fthfl, Mthfs, Mtk/Mcl, Agx1, SdaA, GlyA	¹³C甲醇+木糖	25%丙酮酸标记，甲醇消耗0.70mmol/（L·h·OD）	[53]
	SACA	BsMDH, GALS, ACPS, PTA	LB+甲醇	甲醇转化生物量0.03gDCW/g甲醇	[64]
	MCC	BmMDH, McHps, MlPhi, BaFpk, BsPta	—	¹³C标记证明循环发挥功能，无细胞体系合成乙醇和丁醇	[63]
谷氨酸棒状杆菌	RuMP	BmMdh, BshxlA, BshxlB	甲醇+核糖	比生长速率0.18h^-1，¹³C标记	[75]
谷氨酸棒状杆菌	RuMP	BsMdh, BmHps, BmPhi	甲醇+木糖	比生长速率0.03h^-1，63%标记¹³C进入代谢	[76]
酿酒酵母	XuMP	Aox, Cat, Das, Dak	甲醇+酵母粉	生物量增长12%，甲醇消耗2.4 g/L	[77]

宿主	途径	基因	培养基	表征	文献
大肠杆菌	RuMP	BmMdh2, BmHps, BmPhi	¹³C甲醇+核糖	40%标记C进入中心碳代谢	[54]
	RuMP	BmMdh2, MlHps, MlPhi	¹³C甲醇+葡糖酸盐	24%甲醇进入中心代谢物，甲醇代谢速率加快	[74]
	RuMP	BsMdh, BmHps, BmPhi	1g/L酵母粉+甲醇	1g甲醇转化生物量0.29g（DCW），39% 甲醇C进入TCA循环，实现柚皮素合成	[73]
	RuMP	BmMdh, BmHps, BmPhi	葡萄糖，酵母粉，甲醇	琥珀酸得率提高7.7%，甲醇消耗 0.38～0.45 g/L	[72]
	丝氨酸	Mdh, Fdh, Fthfl, Mthfs, Mtk/Mcl, Agx1, SdaA, GlyA	¹³C甲醇+木糖	25%丙酮酸标记，甲醇消耗0.70mmol/（L·h·OD）	[53]
	SACA	BsMDH, GALS, ACPS, PTA	LB+甲醇	甲醇转化生物量0.03gDCW/g甲醇	[64]
	MCC	BmMDH, McHps, MlPhi, BaFpk, BsPta	—	¹³C标记证明循环发挥功能，无细胞体系合成乙醇和丁醇	[63]
谷氨酸棒状杆菌	RuMP	BmMdh, BshxlA, BshxlB	甲醇+核糖	比生长速率0.18h^-1，¹³C标记	[75]
谷氨酸棒状杆菌	RuMP	BsMdh, BmHps, BmPhi	甲醇+木糖	比生长速率0.03h^-1，63%标记¹³C进入代谢	[76]
酿酒酵母	XuMP	Aox, Cat, Das, Dak	甲醇+酵母粉	生物量增长12%，甲醇消耗2.4 g/L	[77]

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