一碳生物转化合成有机酸的研究进展

doi:10.12211/2096-8280.2024-023

摘要/Abstract

摘要：

有机酸在食品、医药、化工、农业等领域有着广泛的应用。目前有机酸的生产主要以微生物发酵法为主，采用糖类为原料，然而长此以往可能面临“与人争粮”的困境。CO、CO₂、甲烷、甲醇和甲酸等含有一个碳原子的物质被称为一碳（one carbon，C1）资源，其来源广泛且价格低廉，有望成为生物制造的替代原料，且C1原料生物转化有助于缓解温室效应、助力“碳中和”目标。本文总结了近年来CO₂、甲烷和甲醇生物合成3种重要有机酸（3-羟基丙酸、乳酸、琥珀酸）的研究进展，主要论述了C1生物利用途径、有机酸的生物合成途径以及代谢工程策略，也讨论了C1合成有机酸的挑战及应对措施，并展望了有机酸产业化新路线，尤其是化学催化与生物转化耦合以CO₂为原料合成有机酸。本综述对于C1生物炼制以及有机酸产业升级具有一定的参考意义。

关键词: C1生物炼制, 代谢工程, 3-羟基丙酸, 乳酸, 琥珀酸

Abstract:

Organic acids, as important platform chemicals, have been widely used in food, pharmaceutical, chemical industries and agriculture. Currently, microbial production of organic acids relies mainly on sugars as feedstocks, which may suffer from the competition with food and arable lands. One carbon (C1) molecules such as CO, CO₂, methane, methanol and formic acid are widespread and inexpensive, which are considered as ideal feedstocks for future bio-manufacturing. Bioconversion of C1 feedstocks toward the production of organic acids helps to mitigate greenhouse effect and realize carbon neutrality. Therefore C1 sources have been regarded as raw materials of third generation biorefinery, and natural C1 utilizing microbes attracted increasing attention. Although some microorganisms have native biosynthetic pathway of organic acids, the production efficiency is usually lower than expected. This review summarizes the recent progress on the biosynthesis of organic acids (3-hydroxypropionic acid, lactic acid and succinic acid) from C1 feedstocks using synthetic biology methods. First, the native C1 utilizing pathways are summarized, including CO₂, CO, methane, methanol and formic acid. Then the metabolic engineering strategies to improve organic acids production were systematically reviewed, including the optimization of rate-limiting enzymes expression, enhancement of the supply of precursor and cofactor, cofactor engineering, and inhibition of the product degradation. In addition, the challenges, solutions, and prospects of C1 bioconversion to organic acids are also discussed, and coupling chemical catalysis and biological transformation may provide a promising industrial route for organic acids production. In particular, methanol is an ideal C1 feedstock with many advantages like convenient storage and transportation, high liquid-to-liquid mass transfer efficiency, and it can be massively produced from CO₂ by "liquid sunshine" technology. Therefore constructing high efficient methanol cell factory may enable organic acids production from CO₂, a carbon neutral production manner. This review may provide a guidance for C1 biorefinery and industrial bioproduction of organic acids.

Key words: C1 biorefinery, metabolic engineering, 3-hydroxyproionic acid, lactic acid, succinic acid

中图分类号:

禹伟, 高教琪, 周雍进. 一碳生物转化合成有机酸的研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-023.

Wei YU, Jiaoqi GAO, Yongjin ZHOU. Bioconversion of one carbon feedstocks for producing organic acids[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-023.

图/表 6

图1 C1资源生物利用途径(a. CO2利用途径:CBB循环;b. CO2利用途径:还原TCA(rTCA)循环;c. CO2利用途径: 还原甘氨酸(rGly)途径;d. CO2、CO和甲酸利用途径:产乙酸菌的WL途径;e. CO2、CO和甲酸利用途径:产甲烷菌的WL途径;f. CO2利用途径:DC/HB循环;g. CO2利用途径:HP/HB循环;h. CO2利用途径: 3-HP循环;i. 甲醇利用途径:XuMP循环;j. 甲醇利用途径:RuMP循环;k. 甲醇和甲酸利用途径:丝氨酸循环;l. 甲烷利用途径:RuMP循环、CBB循环以及丝氨酸循环)

Fig. 1 The utilization pathways of C1 sources(a.CO2 utilizing pathway: CBB cycle; b. CO2 utilizing pathway: reductive TCA (rTCA) cycle; c. CO2 utilizing pathway: reductive Glycine (rGly) pathway; d. CO2, CO and formic acid utilizing pathway: WL pathway in acetogen; e. CO2, CO and formic acid utilizing pathway: WL pathway in methanogen; f. CO2 utilizing pathway: DC/HB cycle; g. CO2 utilizing pathway: HP/HB cycle; h. CO2 utilizing pathway: 3-HP cycle; i. methanol utilizing pathway: XuMP cycle; j. methanol utilizing pathway: RuMP cycle; k. methanol and formic acid utilizing pathway: serine cycle; l. methane utilizing pathway: RuMP cycle, CBB cycle and serine cycle.)

表1 C1原料合成有机酸

Table 1 Bio-production of organic acids from C1 feedstocks

产物	宿主	底物	培养条件	产量(g/L)	得率(%)	生产强度(g/L/d)	参考文献
3-HP	蓝细菌	CO₂	MM，摇瓶， 50 mL发酵体积	0.67	2.6	0.07	[50]
	蓝细菌	CO₂	MM，摇瓶， 20 mL发酵体积	0.84	8.0	0.14	[47]
	甲基弯菌	甲烷	MM，发酵罐， 50 mL发酵体积	0.06	2.4	0.03	[48]
	扭脱甲基杆菌	甲醇	MM，摇瓶， 50 mL发酵体积	0.07	2.0	0.04	[51]
	扭脱甲基杆菌	甲醇	MM，发酵罐， 1.8 L发酵体积	0.86	3.1	0.21	[52]
	多形汉逊酵母	甲醇	MM，摇瓶， 50 mL发酵体积	7.10	14.2	1.20	[53]
	巴斯德毕赤酵母	甲醇	MM，发酵罐， 300 mL发酵体积	48.2	23.0	3.7	[49]
L-乳酸	蓝细菌	CO₂	MM，摇瓶， 80 mL发酵体积	1.0	N.D.	0.03	[54]
	甲烷氧化菌	甲烷	MM，摇瓶， 2 mL发酵体积	0.6	N.D.	0.15	[55]
	多形汉逊酵母	甲醇	MM，摇瓶， 50 mL发酵体积	3.8	8.0	0.69	[56]
D-乳酸	蓝细菌	CO₂	MM，发酵罐， 100 mL发酵体积	1.3	N.D.	0.13	[57]
	甲基单胞菌	甲烷	MM，摇瓶， 12.5 mL发酵体积	1.2	24.5	0.20	[58]
	巴斯德毕赤酵母	甲醇	CM，摇瓶， 5 mL发酵体积	3.5	22.0	0.87	[59]
琥珀酸	蓝细菌	CO₂	MM，发酵罐，发酵体积未知	0.93	N.D.	0.19	[60]
	蓝细菌	CO₂	MM，摇瓶， 40 mL发酵体积	0.63	N.D.	0.32	[61]
	蓝细菌	CO₂	MM，摇瓶，发酵体积未知	1.8	N.D.	0.60	[62]
	蓝细菌	CO₂	MM，发酵罐， 1 L发酵体积	2.5	N.D.	0.23	[63]
	甲基单胞菌	甲烷	MM，发酵罐， 3.2 L发酵体积	0.20	7.9	0.04	[64]

图2 C1原料合成3-HP的代谢途径及其改造策略ZWF1：葡萄糖-6-磷酸脱氢酶基因；GND1：葡萄糖酸-6-磷酸脱氢酶基因；EcPDH：大肠杆菌的丙酮酸脱氢酶系基因；ACC1：乙酰-CoA羧化酶基因；FAA1：脂酰-CoA合成酶基因；POX1：脂酰-CoA氧化酶基因；FAS：脂肪酸合成酶基因；MmACL：小鼠的ATP-柠檬酸裂解酶基因；ScIDP2：酿酒酵母的异柠檬酸脱氢酶基因；MCR-C：丙二酰-CoA还原酶C端基因；MCR-N：丙二酰-CoA还原酶N端基因

Fig. 2 Biosynthetic pathway and engineering strategies for 3-HP production from C1 feedstocksZWF1:Glucose-6-Phosphate Dehydrogenase Gene; GND1: 6-Phosphogluconate Dehydrogenase Gene; EcPDH: Escherichia coli Pyruvate Dehydrogenase Complex Gene; ACC1: Acetyl-CoA Carboxylase Gene; FAA1: Fatty Acyl-CoA Synthetase Gene; POX1: Fatty Acyl-CoA Oxidase Gene; FAS: Fatty Acid Synthase Gene; MmACL: Mouse ATP-Citrate Lyase Gene; ScIDP2: Saccharomyces cerevisiae Isocitrate Dehydrogenase Gene; MCR-C: Malonyl-CoA Reductase C-Terminal Gene; MCR-N: Malonyl-CoA Reductase N-Terminal Gene

图3 C1原料合成乳酸的代谢途径及其改造策略GPD1/2：甘油-3-磷酸脱氢酶1/2基因；ADH：乙醇脱氢酶基因；PDC1/5/6：丙酮酸脱羧酶1/5/6基因；ALDH：乙醛脱氢酶基因；L-LDH：L-乳酸脱氢酶基因；D-LDH：D-乳酸脱氢酶基因；CYB2：L-乳酸脱氢酶（细胞色素）基因；DLD1：D-乳酸脱氢酶基因；Ady2：乙酸转运蛋白；Jen1：单羧酸/H+同向转运蛋白；LldP：D-乳酸转运蛋白

Fig. 3 Biosynthetic pathway and engineering strategies for lactic acid production from C1 feedstocksGPD1/2: Glycerol-3-Phosphate Dehydrogenase 1/2 Genes; ADH: Alcohol Dehydrogenase Gene; PDC1/5/6: Pyruvate Decarboxylase 1/5/6 Genes; ALDH: Aldehyde Dehydrogenase Gene; L-LDH: L-Lactate Dehydrogenase Gene; D-LDH: D-Lactate Dehydrogenase Gene; CYB2: L-Lactate Dehydrogenase (Cytochrome) Gene; DLD1: D-Lactate Dehydrogenase Gene; Ady2: Acetate Transporter; Jen1: Monocarboxylate/H+ Symporter; LldP: D-Lactate Transporter

图4 C1原料合成琥珀酸的代谢途径及其改造策略PYC：丙酮酸羧化酶基因；PPC：磷酸烯醇式丙酮酸羧化酶基因；PCK：磷酸烯醇式丙酮酸羧激酶基因；MAE：苹果酸酶基因；MDH：苹果酸脱氢酶基因；FUM：延胡索酸酶基因；FRD：延胡索酸还原酶基因；SDH：琥珀酸脱氢酶基因

Fig. 4 Biosynthetic pathway and engineering strategies for succinic acid production from C1 feedstocksPYC: Pyruvate Carboxylase Gene; PPC: Phosphoenolpyruvate Carboxylase Gene; PCK: Phosphoenolpyruvate Carboxykinase Gene; MAE: Malic Enzyme Gene; MDH: Malate Dehydrogenase Gene; FUM: Fumarase Gene; FRD: Fumarate Reductase Gene; SDH: Succinate Dehydrogenase Gene

图5 可再生能源催化CO2还原制备甲醇结合甲醇生物转化合成有机酸是未来有机酸生产的潜力路径

Fig. 5 Combination of CO2 reduction and methanol bioconversion is a potential approach for industrial production of organic acids

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