无细胞多酶分子机器赋能二氧化碳的高值利用及其挑战

doi:10.12211/2096-8280.2022-033

摘要/Abstract

摘要：

二氧化碳等温室气体排放导致的全球气候变暖及相应的气候灾难日益严重，开发高效二氧化碳捕获和利用技术迫在眉睫。利用生物制造技术固定二氧化碳是合成生物学工程科学的一个重要攻关方向，其中挖掘和组装无细胞多酶分子机器赋能二氧化碳高值利用，由于背景清晰、调控相对简单、副反应少和产率高等优点，受到越来越多的关注。近期，James C. Liao教授团队人工设计和开发了新型的多酶复合分子机器，建立了用于二氧化碳固定的闭合循环反应-还原型乙醛酸-丙酮酸合成路径（reductive glyoxylate-pyruvate synthesis cycle），可以理论上实现2分子二氧化碳（碳酸氢盐）到1分子乙醛酸的合成反应，并设计和尝试了反应过程中监控辅因子浓度以提高酶稳定性的策略。本文基于设计和组装体外多酶分子机器，聚焦辅因子工程以及利用多酶分子机器固定二氧化碳所面临的挑战等角度讨论这篇工作并简述作者的相关思考。

关键词: 二氧化碳固定, 多酶分子机器, 无细胞催化, 辅因子工程, 酶稳定性

Abstract:

Global warming, mainly caused by the emission of carbon dioxide, is becoming a serious problem, and it is urgent to develop efficient carbon dioxide capture and utilization technologies. The use of biomanufacturing technology to fix carbon dioxide is an important research direction in synthetic biology. The mining and in vitro assembly of cell-free multi-enzyme machines have the great potential to facilitate the conversion of carbon dioxide into high-value products. The advantages associated with cell-free biosynthesis, such as clear background, relatively simple metabolic regulation, and high-yield production, make it ideal for biomanufacturing. Recently, the team of James C. Liao designed and developed a novel multi-enzyme molecular machine, and established a reductive glyoxylate-pyruvate synthesis cycle, which can theoretically realize the conversion of 2 molecules of carbon dioxide (bicarbonate) to 1 molecule of glyoxylic acid. They also designed and developed a strategy to control the concentration of cofactors during the reaction to improve the enzyme stabilities. In this comment, we discuss this work from the perspectives of in vitro multi-enzyme assembly and cofactor engineering, and point to associated challenges of carbon dioxide utilization by multi-enzyme machines.

Key words: CO₂ fixation, in vitro muti-enzyme machines, cell-free synthetic biology, cofactor engineering, enzyme stability

中图分类号:

Q816

刘建明, 曾安平. 无细胞多酶分子机器赋能二氧化碳的高值利用及其挑战[J]. 合成生物学, 2022, 3(5): 825-832.

Jianming LIU, Anping ZENG. Cell-free multi-enzyme machines for CO₂ capture, utilization and its associated challenges[J]. Synthetic Biology Journal, 2022, 3(5): 825-832.

图/表 4

图1 人工设计的二氧化碳固定多酶分子机器rGPS-MCG路径和CETCH路径（其中rGPS-MCG路径由体外19个酶组成，需要NADPH、NADH、ATP、FAD和CoA等多种辅因子；而CETCH路径由体外17个酶组成，需要NADPH、ATP、FAD和CoA等多种辅因子）TS—羟基丙二酸半醛；2PG—2-磷酸甘油酸；PEP—磷酸烯醇式丙酮酸

Fig. 1 The artificial CO2-fixation pathways rGPS-MCG and CETCH cycle(The rGPS-MCG pathway consists of 19 enzymes and its function requires different cofactors, including NADPH, NADH, ATP, FAD and CoA; The CETCH cycle consists of 17 enzymes and it requires NADPH, ATP, FAD and CoA as cofactors.) TS—Tartronate semialdehyde; 2PG—2-phospho-D-glycerate; PEP—phosphoenolpyruvate

表1 固碳途径中关键酶-羧化酶性质比较［5，9］

Tab. 1 Enzymatic properties of different carboxylases

酶	EC编号	催化的碳种类	比酶活速率 /[μmol/(min·mg)]	K_m/(mmol/L)	辅因子	氧气敏感性
Rubisco核酮糖-1,5-二磷酸羧化酶	4.1.1.39	二氧化碳	3.5	0.01～0.02	—	—
Ppc磷酸烯醇式丙酮酸羧化酶	4.1.1.31	$H C O 3 -$	35	0.06～0.2	—	—
Ccr巴豆酰辅酶A羧化酶		二氧化碳	130	14	NADPH	—
Pyc丙酮酸羧化酶	6.4.1.1	$H C O 3 -$	32	0.25～2.5	ATP	—
acetyl-CoA carboxylase乙酰辅酶A羧化酶	6.4.1.2	$H C O 3 -$	18	0.7～1.7	ATP	—
propionyl-CoA carboxylase丙酰辅酶A羧化酶	6.4.1.3	$H C O 3 -$	30	0.7～1.7	ATP	—
methylcrotonyl-CoA carboxylase甲基巴豆酰辅酶A羧化酶	6.4.1.4	$H C O 3 -$	6	0.8～0.9	ATP	—
acetone carboxylase丙酮羧化酶	6.4.1.6	二氧化碳	0.4	3×10^-6	ATP	—
2-oxoglutarate carboxylase 2-氧代戊二酸羧化酶	6.4.1.7	$H C O 3 -$	15		ATP	—
PFor丙酮酸:铁氧还原蛋白还原酶	1.2.7.1	二氧化碳	<1		TPP，Ferredoxin	+
2-ketoglutarate synthase酮戊二酸:铁氧还蛋白还原酶	1.2.7.3	二氧化碳	<1		TPP，Ferredoxin	+

表1 固碳途径中关键酶-羧化酶性质比较［5，9］

Tab. 1 Enzymatic properties of different carboxylases

酶	EC编号	催化的碳种类	比酶活速率 /[μmol/(min·mg)]	K_m/(mmol/L)	辅因子	氧气敏感性
Rubisco核酮糖-1,5-二磷酸羧化酶	4.1.1.39	二氧化碳	3.5	0.01～0.02	—	—
Ppc磷酸烯醇式丙酮酸羧化酶	4.1.1.31	$H C O 3 -$	35	0.06～0.2	—	—
Ccr巴豆酰辅酶A羧化酶		二氧化碳	130	14	NADPH	—
Pyc丙酮酸羧化酶	6.4.1.1	$H C O 3 -$	32	0.25～2.5	ATP	—
acetyl-CoA carboxylase乙酰辅酶A羧化酶	6.4.1.2	$H C O 3 -$	18	0.7～1.7	ATP	—
propionyl-CoA carboxylase丙酰辅酶A羧化酶	6.4.1.3	$H C O 3 -$	30	0.7～1.7	ATP	—
methylcrotonyl-CoA carboxylase甲基巴豆酰辅酶A羧化酶	6.4.1.4	$H C O 3 -$	6	0.8～0.9	ATP	—
acetone carboxylase丙酮羧化酶	6.4.1.6	二氧化碳	0.4	3×10^-6	ATP	—
2-oxoglutarate carboxylase 2-氧代戊二酸羧化酶	6.4.1.7	$H C O 3 -$	15		ATP	—
PFor丙酮酸:铁氧还原蛋白还原酶	1.2.7.1	二氧化碳	<1		TPP，Ferredoxin	+
2-ketoglutarate synthase酮戊二酸:铁氧还蛋白还原酶	1.2.7.3	二氧化碳	<1		TPP，Ferredoxin	+

表2 体外人工合成固碳途径性能比较

Tab. 2 Properties of in vitro carbon-fixation pathways

多酶途径	主要底物及浓度	主要产物及浓度	ATP/CO₂/(mol/mol)	多酶数量	反应条件	固碳速率 /[nmol/(min·mg)]
rGPS-MCG	0.4 mmol/L巴豆酰辅酶A+ 0.4 mmol/L磷酸烯醇式丙酮酸+ 100 mmol/L碳酸氢钠	0.7 mmol/L乙酰辅酶A+ 0.4 mmol/L甘油酸+ 1 mmol/L苹果酸	2.5	19	20 mL，6 h， pH 7.2	28.5
CETCH	0.2 mmol/L丙酰辅酶A+ 50 mmol/L碳酸氢钠	0.54 mmol/L乙醛酸	1	17	0.52 mL，1.5 h，pH 7.5	5.0
POAP	20 mmol/L乙酸钠+ 100 mmol/L碳酸氢钠	0.023 mmol/L草酸	1	4	50 ℃，厌氧， 20 min，pH 7.8	8.0
ASAP	100 mmol/L甲醇（二氧化碳还原）	1280 mg/L淀粉	0.5	11	4 h，30 ℃， pH 7.5	22.0

图2 辅因子浓度监测和调控（rGPS-MCG路径中辅因子ATP，NAD（P）H，FAD浓度的实时监测。ATP调控模块：肌酸激酶Cpk负责；NAD（P）H调控模块：甲酸脱氢酶FDH或6磷酸葡萄糖酸脱氢酶G6PDH负责；FAD调控模块：辣根过氧化物酶Hrp或电催化模块负责）

Fig. 2 Cofactor monitoring and regulation(The cofactors in rGPS-MCG pathway, including ATP, NAD(P)H and FAD, are monitored. ATP regulation module, creatine phosphate kinase Cpk is responsible for ATP regeneration; NAD(P)H regulation module, formate dehydrogenase or glucose-6-phosphate dehydrogenase G6PDH is responsible for NAD(P)H regeneration; FAD regulation module, horseradish peroxidase Hrp or electrochemical approach is used for FAD regeneration)

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