体外多酶分子机器产氢应用中的氢酶研究

doi:10.12211/2096-8280.2024-052

合成生物学 ›› 2024, Vol. 5 ›› Issue (6): 1461-1484.DOI: 10.12211/2096-8280.2024-052

体外多酶分子机器产氢应用中的氢酶研究

李怡霏¹^,²^,³, 陈艾¹^,²^,³, 孙俊松¹, 张以恒²^,³^,⁴

^1.中国科学院上海高等研究院低碳生物转化团队，上海 201210
^2.中国科学院天津工业生物技术研究所低碳合成工程生物学（全国）重点实验室，天津 300308
^3.中国科学院天津工业生物技术研究所体外合成生物学中心，天津 300308
^4.合成生物学海河实验室，天津 300308

收稿日期:2024-07-09 修回日期:2024-09-25 出版日期:2024-12-31 发布日期:2025-01-10
通讯作者: 孙俊松,张以恒
作者简介:李怡霏（2000—），女，硕士研究生。研究方向为氢酶参与的体外多酶分子机器构建。 E-mail：liyf@sari.ac.cn
孙俊松（1974—），男，博士，研究员。研究方向为氢酶表达、微生物代谢改造及生物合成。 E-mail：sunjs@sari.ac.cn
张以恒（1971—），男，博士，研究员，中国科学院天津工业生物技术研究所低碳合成工程生物学（全国）重点实验室主任，曾任美国弗吉尼亚理工大学终身正教授。研究方向为体外合成生物学、新质生物制造、生物炼制和淀粉储能。 E-mail：zhang_xw@tib.cas.cn
基金资助:
国家重点研发计划“合成生物学”重点专项“糖水氢电系统——体外多酶高效产氢及氢电装置的基础及工程研究”(2022YFA0912000)

Studies on hydrogenases for hydrogen production using in vitro synthetic enzymatic biosystems

Yifei LI¹^,²^,³, Ai CHEN¹^,²^,³, Junsong SUN¹, Yi-Heng P. Job ZHANG²^,³^,⁴

^1.Low Carbon Biotransformation Group，Shanghai Advanced Research Institute，Chinese Academy of Sciences，Shanghai 201210，China
^2.Key Laboratory of Engineering Biology for Low-Carbon Manufacturing，Tianjin Institute of Industrial Biotechnology，Chinese Academy of Sciences，Tianjin 300308，China
^3.In Vitro Synthetic Biology Center，Tianjin Institute of Industrial Biotechnology，Chinese Academy of Sciences，Tianjin 300308，China
^4.Haihe Laboratory of Synthetic Biology，Tianjin 300308，China

Received:2024-07-09 Revised:2024-09-25 Online:2024-12-31 Published:2025-01-10
Contact: Junsong SUN, Yi-Heng P. Job ZHANG

摘要/Abstract

摘要：

氢酶是生物制氢和氢能利用的最关键酶，它是一类广泛分布的对氧敏感的多亚基金属复合酶。体外多酶分子机器是体外生物转化技术中的高效酶生物催化系统，利用该分子机器生产氢气是一种新型高效的绿氢生产技术，它突破微生物产氢的Thauer极限，将葡萄糖产氢的转化率提高至接近化学理论值（1 mol葡萄糖裂解水生产12 mol氢气），代表着生物产氢的未来方向。氢酶的制备及催化性能是限制多酶分子机器产氢技术广泛应用的主要瓶颈；氧气不仅抑制氢酶的活性，也是氢酶转录翻译及翻译后加工的重要影响因素。体外多酶分子机器对氢酶的耐氧性能、热稳定性及高周转性能等参数提出高要求。本文结合氢酶在多酶分子机器制氢应用中的技术障碍，针对迫切的基础科学问题，分别从氢酶分类、结构功能、重组表达技术进展、（仿生）辅酶的适配等方面对其进行了相关的总结，并初步对氧的抑制机制、微生物重组表达氢酶以及产氢人工电子传递链的优化等难点问题的研究进行了跟踪，期待能够为氢酶在体外合成生物学的应用提供参考。

关键词: 氢酶, 生物产氢, 体外多酶分子机器, 仿生辅酶, 人工电子传递链

Abstract:

Hydrogenases are the most important enzymes in biological hydrogen production and hydrogen energy utilization. They are widely distributed, oxygen-sensitive, multiunit complexed metal enzymes. In vitro synthetic enzymatic biosystems (ivSEB) is a type of in vitro biotransformation (ivBT) technology, which is an emerging biomanufacturing powerhouse that combines microbial fermentation with enzymatic biocatalysis, allowing for novel and efficient hydrogen production, also breaking the Thauer limit and achieving a yield of hydrogen close to the theoretical value of chemistry (1 mole of glucose to produce 12 moles of hydrogen in maximum). It represents the future direction of biological hydrogen production. However, the recombinant expression of hydrogenase is the main bottleneck limiting the wide application of ivSEB for hydrogen production technology. Hydrogenases are widely distributed in all life domains, but are oxygen-sensitive and mostly consist of metalloproteins with multi-subunits, bearing [Fe] only, [NiFe] or [FeFe] dinuclear core in their catalytic center. Oxygen not only inhibits the activity of hydrogenase, but also affects the transcription of the enzyme-encoding gene and post-translational process of the enzymes. As a result, the levels of recombinant hydrogenase are usually low and the enzymatic activities are also incomparable to the native enzymes, often leading to high production costs due to the strict anaerobic purification procedures. In order to meet the requirements of industrial hydrogen production, hydrogenases must possess excellent catalytic properties, such as a high catalytic turnover number, great thermal stability, and the ability to tolerate trace amounts of oxygen. This review summarizes the studies on the structural and catalytic characterizations of hydrogenases, including their classification, oxygen resistance mechanisms, and progress in recombinant expression. Additionally, the evolution of natural electron transfer chains and the design of artificial routes, which can improve hydrogen production efficiency and reduce costs, are briefly discussed. The review also discussed the progress in the studies on the mechanisms of hydrogenases’ tolerance toward oxygen, the strategies for microbial expression of recombinant hydrogenases as well as the optimization of the artificial electron transfer chains adapted for the production of hydrogen using ivSEB, in expectations of promoting the applications of hydrogenases involved ivSEB, from renewable energy storage, anaerobic artificial respiration, to clean hydrogenation or dehydrogenation in biocatalysis.

Key words: hydrogenase, bioproduction of hydrogen, in vitro synthetic enzymatic biosystems, biomimetic coenzyme, artificial electron transport chain

中图分类号:

Q819

李怡霏, 陈艾, 孙俊松, 张以恒. 体外多酶分子机器产氢应用中的氢酶研究[J]. 合成生物学, 2024, 5(6): 1461-1484.

Yifei LI, Ai CHEN, Junsong SUN, Yi-Heng P. Job ZHANG. Studies on hydrogenases for hydrogen production using in vitro synthetic enzymatic biosystems[J]. Synthetic Biology Journal, 2024, 5(6): 1461-1484.

图/表 8

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氢酶 Hydrogenase	表达宿主 Expression host	成熟蛋白 Maturation protein	全细胞活性 Whole cell activity	纯化酶活性 Purified enzyme activity	参考文献 References
CacHydA	C. acetobutylicum	Host	NR	162 ^②	[100]
CacHydA	E. coli	C. acetobutylicum	96 ^①	75 ^②	[101]
CacHydA	E. coli ∆iscR	C. acetobutylicum	1.3 ^①,*	96 ^②	[102]
CacHydA	S. elongatus	C. reinhardtii	NR	0.05 ^②	[103]
CacHydB	E. coli	C. acetobutylicum	NR	8.6 ^②	[101]
CbuHydA	E. coli	Host	500 ^①	NR	[104]
CpaHydA	C. pasteurianum	Host	1681 ^①	1236 ^②	[105]
CpaHydI	E. coli	S. oneidensis	NR	1087 ^②	[106]
CpaHydI	Synechococcus sp.	Host	NR	4.6 ^②	[98]
CreHydA1	C. reinhardtii	Host	13.8 ^②,*	741 ^②	[107]
CreHydA1	C. acetobutylicum	Host	NR	625～760 ^②	[108-109]
CreHydA1	E. coli	C. reinhardtii	NR	0.4 ^②	[110]
CreHydA1	E. coli	C. acetobutylicum	61 ^①	150 ^②	[101]
CreHydA1-Fd	E. coli	C. acetobutylicum	NR	1000 ^②	[111]
CreHydA1	E. coli	S. oneidensis	NR	641 ^②	[106]
CreHydA1	S. oneidensis	Host	NA	740 ^②	[112]
CreHydA1	Synechocystis sp.	Host	NR	0.1 ^②	[113]
CreHydA2	E. coli	C. acetobutylicum	108 ^①	116 ^②	[101]
CsuHydA	E. coli	S. oneidensis	NA	6.5 ^②	[114]
EhaHyd	E. coli	Host	NR	70 ^②	[115]
EhiHyd	E. coli	Host	NR	0.04 ^②	[116]
PgrHyd	E. coli	Host	NR	2131 ^②	[117]
SobHydA1	C. acetobutylicum	Host	NR	633 ^②	[108]
SonHydA	Anabaena sp.	S. oneidensis	NR	0.06 ^②	[118]

氢酶 Hydrogenase	表达宿主 Expression host	成熟蛋白 Maturation protein	全细胞活性 Whole cell activity	纯化酶活性 Purified enzyme activity	参考文献 References
CacHydA	C. acetobutylicum	Host	NR	162 ^②	[100]
CacHydA	E. coli	C. acetobutylicum	96 ^①	75 ^②	[101]
CacHydA	E. coli ∆iscR	C. acetobutylicum	1.3 ^①,*	96 ^②	[102]
CacHydA	S. elongatus	C. reinhardtii	NR	0.05 ^②	[103]
CacHydB	E. coli	C. acetobutylicum	NR	8.6 ^②	[101]
CbuHydA	E. coli	Host	500 ^①	NR	[104]
CpaHydA	C. pasteurianum	Host	1681 ^①	1236 ^②	[105]
CpaHydI	E. coli	S. oneidensis	NR	1087 ^②	[106]
CpaHydI	Synechococcus sp.	Host	NR	4.6 ^②	[98]
CreHydA1	C. reinhardtii	Host	13.8 ^②,*	741 ^②	[107]
CreHydA1	C. acetobutylicum	Host	NR	625～760 ^②	[108-109]
CreHydA1	E. coli	C. reinhardtii	NR	0.4 ^②	[110]
CreHydA1	E. coli	C. acetobutylicum	61 ^①	150 ^②	[101]
CreHydA1-Fd	E. coli	C. acetobutylicum	NR	1000 ^②	[111]
CreHydA1	E. coli	S. oneidensis	NR	641 ^②	[106]
CreHydA1	S. oneidensis	Host	NA	740 ^②	[112]
CreHydA1	Synechocystis sp.	Host	NR	0.1 ^②	[113]
CreHydA2	E. coli	C. acetobutylicum	108 ^①	116 ^②	[101]
CsuHydA	E. coli	S. oneidensis	NA	6.5 ^②	[114]
EhaHyd	E. coli	Host	NR	70 ^②	[115]
EhiHyd	E. coli	Host	NR	0.04 ^②	[116]
PgrHyd	E. coli	Host	NR	2131 ^②	[117]
SobHydA1	C. acetobutylicum	Host	NR	633 ^②	[108]
SonHydA	Anabaena sp.	S. oneidensis	NR	0.06 ^②	[118]

氢酶 Hydrogenase	表达宿主 Expression host	成熟蛋白 Maturation protein	全细胞活性 Whole cell activity	纯化酶活性 Purified enzyme activity	参考文献 References
AmaHynSL	A. macleodi ∆HynSL	Host	0.03 ^①,*	0.1 ^③	[119]
AmaHynSL	E. coli	A. macleodii	3×10^-3～70×10^{-3 ①,}*	NR	[120-121]
AmaHyaAB	T. roseopersicina	Host, A. macleodii	5×10^{-3 ①}	NR	[122]
AflHydSL	E. coli	Host	NA	77 ^①	[123]
DgiHynAB	D. gigas ∆HynAB	Host	1.9 ^①,*	91 ^①	[124]
DgiHynAB	D. fructosovorans ∆HynAB	Host	0.2 ^②	NR	[125]
EcoHyd1	E. coli ∆Hyd1	Host	4×10^-2～7×10^{-2 ①,}*	1×10^-2～3×10^{-2 ①}	[126-127]
HmaMBH	E. coli	E. coli	0.07 ^①,*	0.03 ^①	[127]
NpuHupSL	E. coli	E. coli	208 ^①	NR	[128]
PfuSH	E. coli	P. furiosus	2.9 ^①	100 ^①	[43]
PfuSHI	T. kodakarensis	Host	23.6 ^④	880 ^④	[45]
ReuMBH	R. Eutropha H16	Host	1.0 ^③,*	170 ^③	[129]
ReuMBH	P. stutzeri	R. eutropha	17～19 ^③,*	NR	[130]
ReuRH	E. coli	R. eutropha	NR	0.8 ^②	[131]
ReuRH	E. coli	R. eutropha	1.2 ^②,*	230 ^②	[132]
RopSH	R. eutropha ∆SH ∆MBH	Host, R. opacus	5.9 ^①,*	NR	[133]
SynSH	E. coli	Synechocystis sp.	0.04 ^①,*	NR	[134]

氢酶 Hydrogenase	表达宿主 Expression host	成熟蛋白 Maturation protein	全细胞活性 Whole cell activity	纯化酶活性 Purified enzyme activity	参考文献 References
AmaHynSL	A. macleodi ∆HynSL	Host	0.03 ^①,*	0.1 ^③	[119]
AmaHynSL	E. coli	A. macleodii	3×10^-3～70×10^{-3 ①,}*	NR	[120-121]
AmaHyaAB	T. roseopersicina	Host, A. macleodii	5×10^{-3 ①}	NR	[122]
AflHydSL	E. coli	Host	NA	77 ^①	[123]
DgiHynAB	D. gigas ∆HynAB	Host	1.9 ^①,*	91 ^①	[124]
DgiHynAB	D. fructosovorans ∆HynAB	Host	0.2 ^②	NR	[125]
EcoHyd1	E. coli ∆Hyd1	Host	4×10^-2～7×10^{-2 ①,}*	1×10^-2～3×10^{-2 ①}	[126-127]
HmaMBH	E. coli	E. coli	0.07 ^①,*	0.03 ^①	[127]
NpuHupSL	E. coli	E. coli	208 ^①	NR	[128]
PfuSH	E. coli	P. furiosus	2.9 ^①	100 ^①	[43]
PfuSHI	T. kodakarensis	Host	23.6 ^④	880 ^④	[45]
ReuMBH	R. Eutropha H16	Host	1.0 ^③,*	170 ^③	[129]
ReuMBH	P. stutzeri	R. eutropha	17～19 ^③,*	NR	[130]
ReuRH	E. coli	R. eutropha	NR	0.8 ^②	[131]
ReuRH	E. coli	R. eutropha	1.2 ^②,*	230 ^②	[132]
RopSH	R. eutropha ∆SH ∆MBH	Host, R. opacus	5.9 ^①,*	NR	[133]
SynSH	E. coli	Synechocystis sp.	0.04 ^①,*	NR	[134]

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[3]	王俊婷, 郭潇佳, 李青, 万里, 赵宗保. 创制非天然辅酶偏好型甲醇脱氢酶[J]. 合成生物学, 2021, 2(4): 651-661.

体外多酶分子机器产氢应用中的氢酶研究

Studies on hydrogenases for hydrogen production using in vitro synthetic enzymatic biosystems

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