好氧性嗜甲烷菌生物能供给与调控的研究进展

doi:10.16085/j.issn.1000-6613.2022-1544

摘要/Abstract

摘要：

为应对温室气体过度排放所带来环境和能源挑战，甲烷生物转化成为一种新颖的、具有潜力的解决方案。由于好氧性嗜甲烷菌能够利用其天然甲烷代谢途径完成甲烷的氧化和同化，使其在全球碳循环中发挥着重要的作用。随着生物制造技术的不断发展，好氧性嗜甲烷菌已被开发为合成生物基化学品的必要平台。目前，利用基因尺度代谢模型、多组学分析和代谢工程改造已对好氧性嗜甲烷菌的能量代谢和还原力供给进行了系统的解析和优化。本文首先从底物水平磷酸化和氧化磷酸化两个关键途径，概述了好氧性嗜甲烷菌能量供给与代谢通量的互作关系，然后重点介绍和讨论了能量流和碳-氮代谢之间调控策略以及最新研究进展，最后展望了好氧性嗜甲烷菌在生物能供给强化、工具和策略的发展方向和面临的挑战，为构建高效的嗜甲烷菌细胞工厂提供了理论依据和实践指导。

关键词: 好氧性嗜甲烷菌, 甲烷, 氧化路径, 代谢调控, 能量供给

Abstract:

The biological conversion of methane is a novel and potential solution in dealing with the environmental and energy challenges caused by greenhouse gas emissions. Aerobic methanotrophs are capable of utilizing their native metabolic pathways to oxidize and assimilate methane, which plays an important role in the global carbon cycle. As the development of biomanufacturing, aerobic methanotrophs have been considered as an essential platform for the biosynthesis of chemicals and fuels. At present, the energy metabolism pathway and reducing power supply of methanotrophs have been systematically evaluated and optimized by utilizing genome-scale metabolic network model, multi-omics analysis and metabolic engineering transformation. In this paper, the energy supply and metabolism characteristics of aerobic methanotrophs in substrate-level phosphorylation and oxidative phosphorylation pathways were firstly introduced, and regulation strategies between energy flow and carbon-nitrogen metabolism as well as the latest research progress were emphatically summarized and discussed. Finally, the development direction and challenges of biological energy supply of methanotrophs were prospected in terms of enhancement, tools, and strategies, which will provide profound information for the construction of efficient methanotrophic-cell factory.

Key words: aerobic methanotrophs, methane, oxidation pathway, metabolic manipulation, energy supply

中图分类号:

Q819

侯千姿, 郭心怡, 焦子悦, 费强. 好氧性嗜甲烷菌生物能供给与调控的研究进展[J]. 化工进展, 2023, 42(1): 86-93.

HOU Qianzi, GUO Xinyi, JIAO Ziyue, FEI Qiang. Research progress on energy supply and regulation of aerobic methanotrophs[J]. Chemical Industry and Engineering Progress, 2023, 42(1): 86-93.

图/表 5

表1 好氧性嗜甲烷菌能量代谢途径的产能概况

途径	NAD(P)H/个	FADH₂/个	ATP/个	参考文献
底物水平磷酸化
EMP途径	2	—	3.67	[16, 22]
ED途径	2	—	2	[16, 22]
TCA途径	6	2	2(GTP)	[21, 23]
氧化磷酸化
电子传递链	—	—	33.67^①	[16, 21-23]
电子传递链	—	—	29.5^②	[16, 21-23]

图1 好氧性嗜甲烷菌的底物水平磷酸化途径实线表示生化反应；虚线表示多步生化反应；橙色代表EMP途径；绿色代表ED途径；蓝色代表TCA循环；灰色代表细胞生长RuMP循环—单磷酸核酮糖循环；F6P—果糖-6-磷酸；PPi—焦磷酸；Pi—无机磷酸盐；ED途径—2-酮-3-脱氧-6-磷酸葡糖酸途径；EMP途径—己糖二磷酸途径；FBP—果糖-1,6-二磷酸；DAP—磷酸二羟基丙酮；GAP—甘油醛-3-磷酸；3PG—3-磷酸甘油酸；PEP—磷酸烯醇式丙酮酸；PYR—丙酮酸；TCA循环—三羧酸循环；G6P—葡萄糖-6-磷酸；6PG—6-磷酸葡萄糖；KDPG—2-酮-3-脱氧-6-磷酸葡萄糖酸；Acetyl-CoA—乙酰辅酶A；Citrate—柠檬酸；α-ketoglutarate—α-酮戊二酸；Succinate—琥珀酸；Fumarate—富马酸；Malate—苹果酸；Oxaloacetate—草酰乙酸

图2 好氧性嗜甲烷菌的甲烷氧化过程的三种电子传递模式红色箭头表示氧化还原臂模式；蓝色箭头表示直接耦合模式；绿色箭头表示向上电子转移模式；灰色实线箭头表示生化反应；灰色虚线箭头表示电子传递链F0F1-ATPase—ATP合酶；Ⅰ—NADH脱氢酶，复合体Ⅰ；Ⅲ—细胞色素还原酶，复合体Ⅲ；Ⅳ—细胞色素氧化酶，复合体Ⅳ；Mxa—甲醇脱氢酶；Cytc—细胞色素c；UQH2—泛醌；pMMO—颗粒状的膜相关甲烷单加氧化酶

图3 好氧性嗜甲烷菌的碳代谢实线表示生化反应；虚线表示多步生化反应；黄色表示甲烷氧化途径；紫色表示RuMP循环；绿色表示H4F途径；粉色表示丝氨酸循环；灰色表示细胞生长；可溶性甲烷单加氧化酶pMMO—颗粒状的膜相关甲烷单加氧化酶；Cytc—细胞色素c；MeDH—甲醇脱氢酶；H4MPT—四氢甲烷喋呤途径；FDH—甲酸脱氢酶；5,10-Methylene-THF—5,10-亚甲基四氢叶酸；H4F途径—四氢叶酸途径；RuMP循环—单磷酸核酮糖循环；X5P—木酮糖5-磷酸；R5P—核糖-5-磷酸；GAP—甘油醛-3-磷酸；S7P—景天庚酮-7-磷酸；E4P—赤藓糖-4-磷酸；Ru5P—核酮糖-5-磷酸；H6P—己酮糖-6-磷酸

图4 好氧性嗜甲烷菌的氮代谢实线表示生化反应；虚线表示跨膜转运cNOR—细胞色素c依赖的一氧化氮还原酶；CytS—细胞色素C-β；HAO—羟胺脱氢酶；NiR—NO-形成亚硝酸盐还原酶；NirBD—亚硝酸盐还原酶；Amt—铵转运蛋白；NR—硝酸还原酶；HAR—羟胺还原酶；URE—脲酶；pMMO—颗粒状的膜相关甲烷单加氧化酶；NH2OH—羟胺；MFS—硝酸盐/亚硝酸盐转运蛋白

参考文献 52

1	FELDMAN D R, COLLINS W D, BIRAUD S C, et al. Observationally derived rise in methane surface forcing mediated by water vapour trends[J]. Nature Geoscience, 2018, 11(4): 238-243.
2	鞠鑫鑫, 郭建斌, 杨守军, 等. 成年奶牛温室气体排放分析[J]. 中国沼气, 2022, 40(3): 9-17.
	JU Xinxin, GUO Jianbin, YANG Shoujun, et al. Greenhouse gas emissions of a mature dairy cattle[J]. China Biogas, 2022, 40(3): 9-17.
3	许志杰, 孙浩捷. 全球甲烷浓度不断升高[J]. 生态经济, 2022, 38(6): 5-8.
	XU Zhijie, SUN Haojie. Global methane concentration continues to rise[J]. Ecological Economy, 2022, 38(6): 5-8.
4	WEN Huijiao, CHEN Qing, ZHENG Guojun. Enantioselective synthesis of (1S,4R)-N-(benzylcarbamoyl)-4-aminocyclopent-2-en-1-ol by Candida antarctica lipase B [J]. Chinese Chemical Letters, 2015,26(11): 1431-1434.
5	马延和. 生物制造产业是生物经济重点发展方向[J]. 中国生物工程杂志, 2022, 42(5): 4-5.
	MA Yanhe. Bio-manufacturing industry is the key development direction of bio-economy[J]. China Biotechnology, 2022, 42(5): 4-5.
6	HU Lizhen, GUO Shuqi, WANG Bo, et al. Bio-valorization of C₁ gaseous substrates into bioalcohols: potentials and challenges in reducing carbon emissions[J]. Biotechnology Advances, 2022, 59:107954.
7	MERUVU Haritha, WU Hui, JIAO Ziyue, et al. From nature to nurture: essence and methods to isolate robust methanotrophic bacteria[J]. Synthetic and Systems Biotechnology, 2020, 5(3): 173-178.
8	胡礼珍, 王佳, 袁波, 等. 碳一气体生物利用进展[J]. 生物加工过程, 2017, 15(6):17-25.
	HU Lizhen, WANG Jia, YUAN Bo, et al. Production of biofuels and chemicals from C₁ gases by microorganisms: status and prospects[J]. Chinese Journal of Bioprocess Engineering, 2017, 15(6):17-25.
9	费强, 高子熹, 傅容湛. 一种利用嗜甲烷菌生产单细胞蛋白和可发酵糖的方法：CN114045235A[P]. 2022-02-15.
	FEI Qiang, GAO Zixi, FU Rongzhan. A method of producing single cell protein and glycogen by methanotrophic bacteria: CN114045235A[P]. 2022-02-15.
10	HU Lizhen, GUO Shuqi, YAN Xin, et al. Exploration of an efficient electroporation system for heterologous gene expression in the genome of Methanotroph [J]. Frontiers in Microbiology, 2021, 12:717033.
11	PURI Aaron W, OWEN Sarah, CHU Frances, et al. Genetic tools for the industrially promising methanotroph Methylomicrobium buryatense [J]. Applied and Environmental Microbiology, 2015, 81(5):1775-1781.
12	郭树奇, 焦子悦, 费强. 基于化学品生物合成的嗜甲烷菌人工细胞构建及应用进展[J]. 合成生物学, 2021, 2(6): 1017-1029.
	GUO Shuqi, JIAO Ziyue, FEI Qiang. Progress in construction and applications of methanotrophic cell factory for chemicals biosynthesis[J]. Synthetic Biology Journal, 2021, 2(6): 1017-1029.
13	郭树奇, 费强. 甲烷生物利用及嗜甲烷菌的工程改造[J]. 生物工程学报, 2021, 37(3): 816-830.
	GUO Shuqi, FEI Qiang. Bioconversion of methane by metabolically engineered methanotrophs[J]. Chinese Journal of Biotechnology, 2021, 37(3): 816-830.
14	NGUYEN Anh Duc, CHAU Tin Hoang Trung, LEE EunYeol. Methanotrophic microbial cell factory platform for simultaneous conversion of methane and xylose to value-added chemicals[J]. Chemical Engineering Journal, 2020, 420(2): 127632.
15	HENARD Calvin A, AKBERDIN Ilya R, KALYUZHNAYA Marina G, et al. Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts [J]. Green Chemistry, 2019, 21(24):6731-6737.
16	HE Lian, GROOM Joseph D, LIDSTROM Mary E. The Entner-Doudoroff pathway is an essential metabolic route for Methylotuvimicrobium buryatense 5GB1C[J]. Applied and Environmental Microbiology, 2021, 87(3): e02481-20.
17	WARD Naomi, Øivind LARSEN, SAKWA James, et al. Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath)[J]. PLoS Biology, 2004, 2(10): e303.
18	MATSEN Janet B, YANG Song, STEIN Lisa Y, et al. Global molecular analyses of methane metabolism in methanotrophic alphaproteobacterium, Methylosinus trichosporium OB3b. Part I: Transcriptomic study[J]. Frontiers in Microbiology, 2013, 4: 40.
19	DE LA TORRE A, METIVIER A, CHU Frances, et al. Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1)[J]. Microbial Cell Factories, 2015, 14(1): 188.
20	VUILLEUMIER Stéphane, KHMELENINA Valentina N, BRINGEL Françoise, et al. Genome sequence of the haloalkaliphilic methanotrophic bacterium Methylomicrobium alcaliphilum 20Z[J]. Journal of Bacteriology, 2012, 194(2): 551-552.
21	朱圣庚, 徐长法. 生物化学(上、下册)[M]. 4版. 北京：高等教育出版社， 2017.
	ZHU Shenggeng, XU Changfa. Biochemistry (Volume 1 and 2)[M]. 4th ed. Beijing: High Education Press, 2017.
22	SCHOCKE Ludger, SCHINK Bernhard. Membrane-bound proton-translocating pyrophosphatase of syntrophus gentianae, a syntrophically benzoate-degrading fermenting bacterium[J]. European Journal of Biochemistry, 1998, 256(3): 589-594.
23	FU Yanfen, LI Yi, LIDSTROM Mary. The oxidative TCA cycle operates during methanotrophic growth of the Type Ⅰ methanotroph Methylomicrobium buryatense 5GB1[J]. Metabolic Engineering, 2017, 42: 43-51.
24	KALYUZHNAYA MG, YANG S, ROZOVA ON, et al. Highly efficient methane biocatalysis revealed in a methanotrophic bacterium[J]. Nature Communications, 2013, 4: 2785.
25	TROTSENKO Yuri A, MURRELL John Colin. Metabolic aspects of aerobic obligate methanotrophy[J]. Advances in Applied Microbiology, 2008, 63: 183-229.
26	HE Lian, FU Yanfen, LIDSTROM Mary E. Quantifying methane and methanol metabolism of “Methylotuvimicrobium buryatense”5GB1C under substrate limitation[J]. mSystems, 2019, 4(6): e00748-19.
27	STONE Kyle, HILLIARD Matthew, BADR Kiumars, et al. Comparative study of oxygen-limited and methane-limited growth phenotypes of Methylomicrobium buryatense 5GB1[J]. Biochemical Engineering Journal, 2020, 161: 107707.
28	GUPTA Ankit, AHMAD Ahmad, CHOTHWE Dipesh, et al. Genome-scale metabolic reconstruction and metabolic versatility of an obligate methanotroph Methylococcus capsulatus str. Bath[J]. Cold Spring Harbor Laboratory, 2018, 7: e6685.
29	KHMELENINA Valentina N, ROZOVA Olga N, TROTSENKO Yuri A. Characterization of the recombinant pyrophosphate-dependent 6-phosphofructokinases from Methylomicrobium alcaliphilum 20Z and Methylococcus capsulatus Bath[J]. Methods in Enzymology, 2011, 495: 1-14.
30	KHMELENINA Valentina N, Colin Murrell J, SMITH Thomas J, et al. Physiology and biochemistry of the aerobic methanotrophs[M]//Aerobic Utilization of Hydrocarbons, Oils, and Lipids. Switzerland: Springer Cham, 2018: 1-25.
31	FU Yanfen, HE Lian, REEVE Jennifer, BECK David A C, et al. Core metabolism shifts during growth on methanol versus methane in the methanotroph Methylomicrobium buryatense 5GB1[J]. Mbio, 2019, 10(2): e00406-19.
32	ROZOVA Olga N, KHMELENINA Valentina N, BOCHAROVA Ksenia A, et al. Role of NAD⁺-dependent malate dehydrogenase in the metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b[J]. Microorganisms, 2015, 3(1): 47-59.
33	DISPIRITO Alan A, KUNZ Ryan C, CHOI Don Won, et al. Chapter 7: Respiration in Methanotrophs[M]// Respiration in Archaea and Bacteria. Dordrecht: Springer, 2004: 149-168.
34	D-W CHOI, KUNZ Ryan C, BOYD Eric S, et al. The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH: quinone oxidoreductase complex from Methylococcus capsulatus bath[J]. Journal of Bacteriology, 2003, 185(19): 5755-5764.
35	KALYUZHNAYA Marina G, PURI Aaron W, LIDSTROM Mary E. Metabolic engineering in methanotrophic bacteria[J]. Metabolic Engineering, 2015, 29: 142-152.
36	NAIZABEKOV Sanzhar, LEE Eun Yeol. Genome-scale metabolic model reconstruction and in silico Investigations of methane metabolism in Methylosinus trichosporium OB3b[J]. Microorganisms, 2020, 8(3): 437.
37	NGUYEN Anh Duc, LEE Eun Yeol. Engineered methanotrophy: a sustainable solution for methane-based industrial biomanufacturing[J]. Trends in Biotechnology, 2021, 39(4): 381-396.
38	AKBERDIN Ilya R, THOMPSON Merlin, HAMILTON Richard. Methane utilization in Methylomicrobium alcaliphilum 20Z^R: a systems approach[J]. Scientific Reports, 2018, 8(1): 2512.
39	LIEVEN Christian, PETERSEN Leander A H, JØRGENSEN Sten Bay, et al. A Genome-scale metabolic model for Methylococcus capsulatus (Bath) suggests reduced efficiency electron transfer to the particulate methane monooxygenase[J]. Frontiers in Microbiology, 2018, 9: 2947.
40	BORDEL Sergio, Yadira RODRÍGUEZ, HAKOBYAN Anna, et al. Genome scale metabolic modeling reveals the metabolic potential of three Type II methanotrophs of the genus Methylocystis[J]. Metabolic Engineering, 2019, 54: 191-199.
41	BORDEL Sergio, ROJAS Antonia, Raúl MUÑOZ. Reconstruction of a genome scale metabolic model of the polyhydroxybutyrate producing methanotroph Methylocystis parvus OBBP[J]. Microbial Cell Factories, 2013, 18(1): 104.
42	SUGDEN Scott, LAZIC M, SAUVAGEAU Dominic, et al. Transcriptomic and metabolomic responses to carbon and nitrogen sources in Methylomicrobium album BG8[J]. Applied Environmental Microbiology, 2021, 87(13): e0038521.
43	NGUYEN Anh Duc, PARK Joon Young, HWANG In Yeub, et al. Genome-scale evaluation of core one-carbon metabolism in gammaproteobacterial methanotrophs grown on methane and methanol[J]. Metabolic Engineering, 2020, 57: 1-12.
44	BUT S Y, EGOROVA S V, KHMELENINA V N, et al. Serine-glyoxylate aminotranferases from methanotrophs using different C₁-assimilation pathways[J]. Antonie Van Leeuwenhoek, 2019, 112(5): 741-751.
45	HU Lizhen, YANG Yongfu, YAN Xin, et al. Molecular mechanism associated with the impact of methane/oxygen gas supply ratios on cell growth of Methylomicrobium buryatense 5GB1 through RNA-seq[J]. Frontiers in Bioengineering and Biotechnology, 2020, 8: 263.
46	TENTORI Egidio F, FANG Shania, RICHARDSON Ruth E. RNA biomarker trends across Type Ⅰ and Type Ⅱ aerobic methanotrophs in response to methane oxidation rates and transcriptome response to short-term methane and oxygen limitation in Methylomicrobium album BG8[J]. Microbiology Spectrum, 2022, 10(3): e0000322.
47	GILMAN Alexey, FU Yanfen, HENDERSHOTT Melissa, et al. Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C[J]. PeerJ, 2017, 5: e3945.
48	KALUZHNAYA Marina, KHMELENINA Valentina, ESHINIMAEV Bulat, et al. Taxonomic characterization of new alkaliphilic and alkalitolerant methanotrophs from soda lakes of the southeastern transbaikal region and description of Methylomicrobium buryatense sp.nov[J]. Systematic and Applied Microbiology, 2001, 24(2): 166-176.
49	YU Woon Jong, LEE Jae Won, NGUYEN Ngoc Loi, et al. The characteristics and comparative analysis of methanotrophs reveal genomic insights into Methylomicrobium sp. enriched from marine sediments[J]. Systematic and Applied Microbiology, 2018, 41(5): 415-426.
50	CUI Jing, ZHANG Meng, CHEN Linxia, et al. Methanotrophs contribute to nitrogen fixation in emergent macrophytes[J]. Frontiers in Microbiology, 2022, 13: 851424.
51	刘国琴, 张曼夫. 生物化学[M]. 2版. 北京: 中国农业大学出版社, 2011.
	LIU Guoqin, ZHANG Manfu. Biochemistry[M]. 2th ed. Beijing: China Agricultural University Press, 2011.
52	GUO Shuqi, ZHANG Tianqing, CHEN Yunhao, et al. Transcriptomic profiling of nitrogen fixation and the role of NifA in Methylomicrobium buryatense 5GB1[J]. Applied Microbiology and Biotechnology, 2022, 106(8): 3191-3199.

[1]	赖诗妮, 江丽霞, 李军, 黄宏宇, 小林敬幸. 含碳掺氨燃料的研究进展[J]. 化工进展, 2023, 42(9): 4603-4615.
[2]	黄玉飞, 李子怡, 黄杨强, 金波, 罗潇, 梁志武. 光催化CO₂和CH₄重整催化剂研究进展[J]. 化工进展, 2023, 42(8): 4247-4263.
[3]	奚永兰, 王成成, 叶小梅, 刘洋, 贾昭炎, 曹春晖, 韩挺, 张应鹏, 田雨. 微纳米气泡在厌氧消化中的应用研究进展[J]. 化工进展, 2023, 42(8): 4414-4423.
[4]	刘洋, 叶小梅, 苗晓, 王成成, 贾昭炎, 曹春晖, 奚永兰. 农村有机生活垃圾干发酵氨胁迫下中试工艺[J]. 化工进展, 2023, 42(7): 3847-3854.
[5]	张凯, 吕秋楠, 李刚, 李小森, 莫家媚. 南海海泥中甲烷水合物的形貌及赋存特性[J]. 化工进展, 2023, 42(7): 3865-3874.
[6]	马源, 肖晴月, 岳君容, 崔彦斌, 刘姣, 许光文. CeO₂-Al₂O₃复合载体负载Ni基催化剂催化CO _x 共甲烷化性能[J]. 化工进展, 2023, 42(5): 2421-2428.
[7]	阮鹏, 杨润农, 林梓荣, 孙永明. 甲烷催化部分氧化制合成气催化剂的研究进展[J]. 化工进展, 2023, 42(4): 1832-1846.
[8]	何阳东, 常宏岗, 王丹, 陈昌介, 李雅欣. 熔融金属法甲烷裂解制氢和碳材料研究进展[J]. 化工进展, 2023, 42(3): 1270-1280.
[9]	张巍, 王锐, 缪平, 田戈. 全球可再生能源电转甲烷的应用[J]. 化工进展, 2023, 42(3): 1257-1269.
[10]	祝佳欣, 朱雯喆, 徐俊, 谢靖, 王文标, 谢丽. 基于导电材料强化抗生素胁迫厌氧消化的研究进展[J]. 化工进展, 2023, 42(2): 1008-1019.
[11]	赵同心, 赵磊, 张延平, 李寅. 甲酸微生物转化研究进展[J]. 化工进展, 2023, 42(1): 67-72.
[12]	赵锦波, 卞凤鸣. CO₂化学转化基础与应用研究进展[J]. 化工进展, 2022, 41(S1): 524-535.
[13]	闫鹏, 程易. 用于分布式制氢的甲烷蒸汽重整膜反应器的数值模拟[J]. 化工进展, 2022, 41(7): 3446-3454.
[14]	郑小梅, 林茹晶, 周文静, 徐泠, 张洪宁, 张昕颖, 谢丽. 微生物电解池辅助CO₂甲烷化阴极材料的研究进展[J]. 化工进展, 2022, 41(5): 2476-2486.
[15]	李楠, 贾帅, 孙振峰, 孙长宇, 陈光进, 李璟明. 注CO₂+N₂混合气改造-开采CH₄水合物储层[J]. 化工进展, 2022, 41(5): 2356-2363.