State-of-the-art for alcohol dehydrogenase development and the prospect of its applications in bio-based furan compounds valorization

doi:10.12211/2096-8280.2023-059

Abstract

Abstract:

Alcohol dehydrogenase (ADH) is found widely in living cells, where it catalyzes the oxidation or reduction between hydroxyl group and carbonyl group. It also catalyzes redox reactions of a variety of organic compounds with high selectivity. In recent years, with the research progress of the catalytic mechanism, structural information, molecular modification, and reaction systems, ADH has shown great promise in the highly selective catalysis of various bio-based platform compounds. One example is the oxidation or reduction of furan derivatives such as furfural and 5-hydroxymethylfurfural, which are important sustainable building blocks for bio-jet fuels and biomaterials that are derived from tandem hydrolysis, isomerization, and dehydration of hemicelluloses and celluloses fractions in lignocellulose matrixes. This review focuses on the cutting-edge technologies in molecular design and directional engineering of ADH. In addition, the intensification of cofactor regeneration processes, including chemical-driven, enzyme-driven, and photo/electricity-driven pathways were also summarized. These methods could be the solutions to the negative aspects, such as expensive cost, poor stability, and the poor circulating efficiency of the nicotinamide cofactors that are indispensable assisted in typical ADH catalysis process. Moreover, the latest research progresses of ADH in catalysis of bio-based furans platform chemicals were also discussed. Apart from using ADH solely for the activation of the hydroxyl and carbonyl groups in the biobased furan derivates and the production of oxidative and reductive products, there is also of great promise in cascade ADH catalysis and other chemical or biological catalysis processes in one-pot under relatively mild conditions to valorize the furan derivates into valuable fine chemicals. Meanwhile, the whole-cell catalytic process that involves ADH and in vivo cofactors regeneration also possesses potentials in biobased furans valorization, with the advantages of low catalyst loading and processing costs. Overall, the researches of ADH in catalysis biological furans valorization has entered a new stage. With the further exploration of the potential applications of ADH, its role in the transformation of biomass resources will be increasingly important, particularly in the industrial process in the future.

Key words: alcohol dehydrogenase, furan compounds, oxidation-reduction reaction, cofactor regeneration, biomass valorization

摘要：

醇脱氢酶（alcohol dehydrogenase， ADH）广泛存在于生物体内，可应用于多种有机物选择性氧化还原。近年来，随着对酶的催化机理、结构认知、分子改造和反应系统构建及强化等方面研究的逐渐深入，ADH在生物基平台化合物的高选择性催化氧化还原方面展现出巨大潜力。本文综述了ADH分子设计和定向改造的前沿技术和进展，面向常见的辅因子依赖性ADH催化过程中的辅因子高成本及稳定性差等局限，聚焦酶反应过程中的辅因子再生强化技术，梳理了适用于ADH催化系统的化学驱动、酶驱动和光电驱动辅因子再生路径，并从单酶催化体系开发、多酶协同催化系统挖掘、全细胞催化技术发展与应用等多个角度概述了ADH在催化生物基呋喃化合物方向的最新研究进展。随着对ADH应用潜力的进一步挖掘，未来ADH有望成为生物基呋喃增值工业化进程中的重要组成部分，为能源生产的绿色发展提供助力。

关键词: 醇脱氢酶, 呋喃类化合物, 氧化还原反应, 辅因子再生, 生物质增值

CLC Number:

Q554+.1

Xiangshi LIU, Yilu WU, Peng ZHAN, Tianhao HUANG, Di CAI, Peiyong QIN. State-of-the-art for alcohol dehydrogenase development and the prospect of its applications in bio-based furan compounds valorization[J]. Synthetic Biology Journal, 2023, 4(6): 1122-1139.

刘庠诗, 吴奕禄, 詹鹏, 黄天灏, 蔡的, 秦培勇. 醇脱氢酶的研究进展及其催化增值生物基呋喃化合物前景展望[J]. 合成生物学, 2023, 4(6): 1122-1139.

Figures/Tables 8

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酶的来源	底盘菌株	改造方法	改造结果	参考文献
Clostridium beijerinckii	Escherichia coli	利用PROSS对来源于Clostridium beijerinckii的乙醇脱氢酶基因中Ser24Pro、Gly182Ala、Gly196Ala、His222Asp、Ser250Glu和Ser254Arg六个位点进行定点诱变	催化活性增大到野生型9倍	[28]
Thermoanaerobacter ethanolicus	Escherichia coli	使用两步PCR法对来源于Thermoanaerobacter ethanolicus的乙醇脱氢酶基因中Ile86Ala等底物结合位点附近残基进行定点诱变	底物结合活性位点增大	[29]
Thermoanaerobacter ethanolicus	Escherichia coli	通过PCR技术对来源于Thermoanaerobacter ethanolicus 39E的乙醇脱氢酶基因的Trp110Ala残基发生定点突变，并在Escherichia coli中过表达	ADH的活性位点增大提升对映体选择性热稳定性提升	[30]
Bacillus stearothermophilus	Escherichia coli	定点诱变来源于Bacillus stearothermophilus LLD-R的乙醇脱氢酶基因的Glu11Lys/Pro242Ala残基，并在Escherichia coli中过表达	重组菌株ADH表达量增加该ADH耐热性进一步提升	[31]
Thermoanaerobacter brockii	Escherichia coli	定点诱变来源于Thermoanaerobacter brockii的乙醇脱氢酶基因的Asp275Pro残基，并在Escherichia coli过表达	提高ADH分子的热稳定性	[32]
Escherichia coli BL21(ED3)	Escherichia coli	使Escherichia coli共表达GDH与EcYjgB	重组菌株HMF耐受性提升 HMF到BHMF催化效率提升	[33]
Thermoanaerobacter ethanolicus	—	定点诱变来源于Thermoanaerobacter ethanolicus的乙醇脱氢酶基因的Ser39Tyr和Cys295Ala两个残基	对映体选择性提高	[34]
Pyrococcus furiosus	Escherichia coli	定点诱变来源于Pyrococcus furiosus的乙醇脱氢酶基因的Lys249Gly/His255Arg两个残基，并在Escherichia coli中过表达	ADH由NADH依赖性变为多种辅因子依赖型	[35]
Deinococcus geothermalis	—	定点诱变来源于Deinococcus geothermalis的乙醇脱氢酶基因的Asp55Asn等位于辅因子结合位点的残基	ADH由NADH依赖型变成NADPH依赖型	[36]
Rana perezi	—	定点诱变来源于Rana perezi的乙醇脱氢酶基因的Gly223Asp/Thr224Ile/His225Asn三个连续残基	ADH由NADPH依赖型变成NADH依赖型	[37]

酶的来源	底盘菌株	改造方法	改造结果	参考文献
Clostridium beijerinckii	Escherichia coli	利用PROSS对来源于Clostridium beijerinckii的乙醇脱氢酶基因中Ser24Pro、Gly182Ala、Gly196Ala、His222Asp、Ser250Glu和Ser254Arg六个位点进行定点诱变	催化活性增大到野生型9倍	[28]
Thermoanaerobacter ethanolicus	Escherichia coli	使用两步PCR法对来源于Thermoanaerobacter ethanolicus的乙醇脱氢酶基因中Ile86Ala等底物结合位点附近残基进行定点诱变	底物结合活性位点增大	[29]
Thermoanaerobacter ethanolicus	Escherichia coli	通过PCR技术对来源于Thermoanaerobacter ethanolicus 39E的乙醇脱氢酶基因的Trp110Ala残基发生定点突变，并在Escherichia coli中过表达	ADH的活性位点增大提升对映体选择性热稳定性提升	[30]
Bacillus stearothermophilus	Escherichia coli	定点诱变来源于Bacillus stearothermophilus LLD-R的乙醇脱氢酶基因的Glu11Lys/Pro242Ala残基，并在Escherichia coli中过表达	重组菌株ADH表达量增加该ADH耐热性进一步提升	[31]
Thermoanaerobacter brockii	Escherichia coli	定点诱变来源于Thermoanaerobacter brockii的乙醇脱氢酶基因的Asp275Pro残基，并在Escherichia coli过表达	提高ADH分子的热稳定性	[32]
Escherichia coli BL21(ED3)	Escherichia coli	使Escherichia coli共表达GDH与EcYjgB	重组菌株HMF耐受性提升 HMF到BHMF催化效率提升	[33]
Thermoanaerobacter ethanolicus	—	定点诱变来源于Thermoanaerobacter ethanolicus的乙醇脱氢酶基因的Ser39Tyr和Cys295Ala两个残基	对映体选择性提高	[34]
Pyrococcus furiosus	Escherichia coli	定点诱变来源于Pyrococcus furiosus的乙醇脱氢酶基因的Lys249Gly/His255Arg两个残基，并在Escherichia coli中过表达	ADH由NADH依赖性变为多种辅因子依赖型	[35]
Deinococcus geothermalis	—	定点诱变来源于Deinococcus geothermalis的乙醇脱氢酶基因的Asp55Asn等位于辅因子结合位点的残基	ADH由NADH依赖型变成NADPH依赖型	[36]
Rana perezi	—	定点诱变来源于Rana perezi的乙醇脱氢酶基因的Gly223Asp/Thr224Ile/His225Asn三个连续残基	ADH由NADPH依赖型变成NADH依赖型	[37]

类型	酶/菌种名称	底物	产物	特点	参考文献
单酶催化	HLADH	FAL	FOL	使用肌红蛋白作为催化剂使辅因子再生证明人造辅因子BNAH可用于替代呋喃合成过程中的NAD(P)H等 48 h FOL产率高达93%	[63]
	HLADH	FAL	FCA	使用血红蛋白作为催化剂使辅因子再生 48 h FAL转化率100%，FCA产率达到98%	[64]
		HMF	HMFCA	使用血红蛋白作为催化剂使辅因子再生 60 h HMF转化率100%，HMFCA产率81%
		FFCA	FDCA	使用血红蛋白作为催化剂使辅因子再生 108 h FFCA转化率79%，FDCA产率54%
		DFF	FDCA	使用血红蛋白作为催化剂使辅因子再生 60 h DFF转化率100%，FDCA产率96%
	ADH	FAL	FOL	通过构筑Rh电子介体-固定化ADH复合电极，电化学方式辅助辅因子再生 FAL还原为FOL的选择性达96.4%，产率90.0%	[65] [93]
	ADH	FAL	FOL	使用附着有CdSe/ZnS纳米颗粒的C₃N₄作为光催化剂驱动辅因子再生达到近100%的FAL转化率，产物FOL浓度为0.6 mmol/L	[73]
	BovADH	FAL	FCA	对羰基氧化成羧基有高反应活性 FAL、HMF转化率达99% 在pH8.5情况下活性温度在40 ℃以上	[102]
	BovADH	HMF	HMFCA	对羰基氧化成羧基有高反应活性 FAL、HMF转化率达99% 在pH8.5情况下活性温度在40 ℃以上
	EcADH	FAL	FCA	对羰基氧化成羧基有高反应活性 FAL、HMF转化率达99% 在pH8.5情况下活性温度在40 ℃以上
	EcADH	HMF	HMFCA	对羰基氧化成羧基有高反应活性 FAL、HMF转化率达99% 在pH8.5情况下活性温度在40 ℃以上
	PpADH	FAL	FCA	在pH8.5下活性温度在40 ℃以上对羰基氧化成羧基有高反应活性，FAL、HMF转化率达99%
	PpADH	HMF	HMFCA	在pH8.5下活性温度在40 ℃以上对羰基氧化成羧基有高反应活性，FAL、HMF转化率达99%
多酶级联催化	GDH + ADH + AcTs	HMF	BHMF	设计了一种共表达GDH与EcYjgB的大肠杆菌重组菌株，实现了HMF的还原与辅因子的再生，BHMF产率高达15 g/(L·h)，HMF收率大于99% 使用AcTs催化BHMF酯化反应，HMF生产BHMF双酯总产率达到88%	[33]
	AlaDH + ADH + ω-TA	BHMF	呋喃氨基醇	可通过控制级联中助溶剂的种类和比例控制氨基醇和二胺的选择性辅因子与氨供体在级联体系中可循环使用在10% DME，20 ℃下BHMF的转化率和二胺的产率均达到了99%	[103]
	ADH + L-AlaDH + ω-TA	HMF	呋喃二甲胺	设计了一种用于固定化酶的多孔载体，使固定化酶活性下降不超过10%， ADH回收利用率达84% HMF转化率达80%	[104]
	GOase + ADH	HMF	FDCA	通过控制不同的底物浓度、反应时间、CaCO₃的添加量、两种酶的浓度和比例，实现FDCA、FFCA可控合成 10 mmol/L HMF，1.6 µmol/L GOase与36 µmol/L HLADH反应60h时的HMF转化率达到99%，FDCA产率95%	[105]
	GOase + ADH	HMF	FFCA	通过控制不同的底物浓度、反应时间、CaCO₃的添加量、两种酶的浓度和比例，实现FDCA、FFCA可控合成 100 mmol/L HMF，3.2 µmol/L GOase与66 µmol/L SADH反应48h时的HMF转化率达到99%，FFCA产率97%	[105]
全细胞催化	Comamonas testosteroni SC1588	HMF	HMFCA	对HMF耐受性好，低浓度FOL可提高菌体活性，组氨酸可进一步提高菌体耐受性并控制pH HMF转化率达100%，且对HMFCA的选择性为87%～88%	[33]
	Saccharomyces cerevisiae NL22	FAL	FOL	对FAL耐受性好，在100 mmol/L FAL中仍保持高催化活性 8 h FAL转化率达到98%，还原为FOL选择性高达87.9%	[106]
	Escherichia coli TS	FAL	FA	优化含HLADH的Escherichia coli TS菌体催化活性 72 h将25 mmol/L FAL完全转化为FA	[107]

类型	酶/菌种名称	底物	产物	特点	参考文献
单酶催化	HLADH	FAL	FOL	使用肌红蛋白作为催化剂使辅因子再生证明人造辅因子BNAH可用于替代呋喃合成过程中的NAD(P)H等 48 h FOL产率高达93%	[63]
	HLADH	FAL	FCA	使用血红蛋白作为催化剂使辅因子再生 48 h FAL转化率100%，FCA产率达到98%	[64]
		HMF	HMFCA	使用血红蛋白作为催化剂使辅因子再生 60 h HMF转化率100%，HMFCA产率81%
		FFCA	FDCA	使用血红蛋白作为催化剂使辅因子再生 108 h FFCA转化率79%，FDCA产率54%
		DFF	FDCA	使用血红蛋白作为催化剂使辅因子再生 60 h DFF转化率100%，FDCA产率96%
	ADH	FAL	FOL	通过构筑Rh电子介体-固定化ADH复合电极，电化学方式辅助辅因子再生 FAL还原为FOL的选择性达96.4%，产率90.0%	[65] [93]
	ADH	FAL	FOL	使用附着有CdSe/ZnS纳米颗粒的C₃N₄作为光催化剂驱动辅因子再生达到近100%的FAL转化率，产物FOL浓度为0.6 mmol/L	[73]
	BovADH	FAL	FCA	对羰基氧化成羧基有高反应活性 FAL、HMF转化率达99% 在pH8.5情况下活性温度在40 ℃以上	[102]
	BovADH	HMF	HMFCA	对羰基氧化成羧基有高反应活性 FAL、HMF转化率达99% 在pH8.5情况下活性温度在40 ℃以上
	EcADH	FAL	FCA	对羰基氧化成羧基有高反应活性 FAL、HMF转化率达99% 在pH8.5情况下活性温度在40 ℃以上
	EcADH	HMF	HMFCA	对羰基氧化成羧基有高反应活性 FAL、HMF转化率达99% 在pH8.5情况下活性温度在40 ℃以上
	PpADH	FAL	FCA	在pH8.5下活性温度在40 ℃以上对羰基氧化成羧基有高反应活性，FAL、HMF转化率达99%
	PpADH	HMF	HMFCA	在pH8.5下活性温度在40 ℃以上对羰基氧化成羧基有高反应活性，FAL、HMF转化率达99%
多酶级联催化	GDH + ADH + AcTs	HMF	BHMF	设计了一种共表达GDH与EcYjgB的大肠杆菌重组菌株，实现了HMF的还原与辅因子的再生，BHMF产率高达15 g/(L·h)，HMF收率大于99% 使用AcTs催化BHMF酯化反应，HMF生产BHMF双酯总产率达到88%	[33]
	AlaDH + ADH + ω-TA	BHMF	呋喃氨基醇	可通过控制级联中助溶剂的种类和比例控制氨基醇和二胺的选择性辅因子与氨供体在级联体系中可循环使用在10% DME，20 ℃下BHMF的转化率和二胺的产率均达到了99%	[103]
	ADH + L-AlaDH + ω-TA	HMF	呋喃二甲胺	设计了一种用于固定化酶的多孔载体，使固定化酶活性下降不超过10%， ADH回收利用率达84% HMF转化率达80%	[104]
	GOase + ADH	HMF	FDCA	通过控制不同的底物浓度、反应时间、CaCO₃的添加量、两种酶的浓度和比例，实现FDCA、FFCA可控合成 10 mmol/L HMF，1.6 µmol/L GOase与36 µmol/L HLADH反应60h时的HMF转化率达到99%，FDCA产率95%	[105]
	GOase + ADH	HMF	FFCA	通过控制不同的底物浓度、反应时间、CaCO₃的添加量、两种酶的浓度和比例，实现FDCA、FFCA可控合成 100 mmol/L HMF，3.2 µmol/L GOase与66 µmol/L SADH反应48h时的HMF转化率达到99%，FFCA产率97%	[105]
全细胞催化	Comamonas testosteroni SC1588	HMF	HMFCA	对HMF耐受性好，低浓度FOL可提高菌体活性，组氨酸可进一步提高菌体耐受性并控制pH HMF转化率达100%，且对HMFCA的选择性为87%～88%	[33]
	Saccharomyces cerevisiae NL22	FAL	FOL	对FAL耐受性好，在100 mmol/L FAL中仍保持高催化活性 8 h FAL转化率达到98%，还原为FOL选择性高达87.9%	[106]
	Escherichia coli TS	FAL	FA	优化含HLADH的Escherichia coli TS菌体催化活性 72 h将25 mmol/L FAL完全转化为FA	[107]