Progress in biomanufacturing of lipids and single cell protein from one-carbon compounds

doi:10.12211/2096-8280.2024-013

Progress in biomanufacturing of lipids and single cell protein from one-carbon compounds

Liang ZHAO¹^,³, Zhenshuai LI¹^,², Liping FU¹^,³, Ming LU¹^,³, Shian WANG¹^,⁴, Quan ZHANG⁵, Licheng LIU², Fuli LI¹, Ziyong LIU¹^,³

^1.CAS Key Laboratory of Biofuel，Qingdao C1 refinery research engineering center，Qingdao Institute of Bioenergy and Bioprocess Technology，Chinese Academy of Sciences，Qingdao 266101，Shandong，China
^2.Ocean University of China，Qingdao 266100，Shandong，China
^3.Shandong Energy Institute，Qingdao 266101，Shandong，China
^4.Qingdao New Energy Shandong Laboratory，Qingdao 266101，Shandong，China
^5.Sinopec Dalian Research Institute of Petroleum and Petrochemicals，Dalian 116045，Liaoning，China

Received:2024-02-04 Revised:2024-05-08 Published:2024-05-27
Contact: Ziyong LIU

生物转化一碳化合物原料产油脂与单细胞蛋白研究进展

赵亮¹^,³, 李振帅¹^,², 付丽平¹^,³, 吕明¹^,³, 王士安¹^,⁴, 张全⁵, 刘立成², 李福利¹, 刘自勇¹^,³

^1.中国科学院生物燃料重点实验室，青岛市碳一炼制工程研究中心，中国科学院青岛生物能源与过程研究所，山东青岛 266101
^2.中国海洋大学，山东青岛 266100
^3.山东能源研究院，山东青岛 266101
^4.青岛新能源山东省实验室，山东青岛 266101
^5.中国石油化工股份有限公司大连研究院，辽宁大连 116045

通讯作者: 刘自勇
作者简介:赵亮（1996—），男，硕士，助理工程师。研究方向为解脂耶氏酵母油脂与蛋白发酵工艺优化。E-mail：zhaoliang@qibebt.ac.cn
刘自勇（1983—），男，副研究员，硕士生导师。研究方向为厌氧梭菌高效转化木质纤维素和合成气生产生物乙醇、丁醇和长链油脂等。E-mail：liuzy@qibebt.ac.cn
基金资助:
国家重点研发计划项目(2023YFA0914400);国家自然科学基金面上项目(32370039);中国科学院战略性先导科技专项（C类）(XDC0110302)

Abstract

Abstract:

One-carbon compounds are liquid or gaseous substances that can be naturally occurring or produced in industrial processes, offering the advantages of being abundant, cost-effective, and sustainable to produce. They are anticipated to serve as fundamental raw materials for the next phase of bio-manufacturing, encompassing easily transportable and storable liquid methanol, formic acid, and gaseous CO₂, CO, and CH₄. China is currently focusing on reducing carbon emissions and aims to progressively achieve the targets of carbon peak and carbon neutrality through diverse approaches. Amidst the flourishing landscape of bio-manufacturing, microorganisms are being genetically manipulated using synthetic biology techniques to efficiently harness one-carbon compounds for the creation of high-value products like lipids and single-cell protein. This initiative aims to reduce dependence on imported food and fossil resources, serving as a strategic measure to alleviate food and energy crises. This review presents a comprehensive overview of the most recent advancements in converting one-carbon compounds into valuable oils and single-cell proteins through the utilization of metabolic pathways, chassis genetic modification, and other methodologies involving methylotrophic microorganisms, acetogenic bacteria, yeast, and other microorganisms. It discusses pertinent studies on enhancing molecular engineering strains through the fermentation process using one-carbon compounds and includes research cases focusing on the production of ultra-long-chain fatty acids. Furthermore, it collates industrial instances related to the conversion of one-carbon compounds from research institutions or companies. Lastly, by addressing the constraints in metabolic pathway design and genetic tools for utilizing one-carbon compound strains, as well as the energy conversion challenges between acetogenic bacteria and lipids-producing microorganisms, it offers foresight into the future opportunities and obstacles encountered in the bio-manufacturing of lipids and single-cell proteins. It suggests advancing inter-disciplinary, efficient systematic integration for fermentation within complex systemic bio-manufacturing processes, driving exploration on the biological conversion of one-carbon compounds, proposing novel solutions to current theoretical and practical challenges, and providing guidance for practical applications and industrial advancements.

Key words: One-carbon compound, lipids, single cell protein, biomanufacturing, metabolic engineering, fermentation process

摘要：

一碳化合物是一类产生于自然界或工业过程中的液态或气态物质，其具有来源广泛、价格低廉、可持续生产的优势，有望于成为新一代生物制造关键原料，包括液态的甲醇、甲酸，以及气态的CO₂、CO、CH₄等。在生物制造蓬勃发展的背景下，通过合成生物学手段改造微生物，使之利用一碳化合物高效生产油脂与单细胞蛋白等高附加值产品，降低对粮食、化石资源进口的依赖，成为缓解粮食能源危机的有效战略举措。本文综述了甲基营养型微生物、产乙酸菌以及酵母等微生物通过代谢途径、底盘遗传改造等方法，将一碳化合物转化为高附加值油脂与单细胞蛋白的最新研究进展；介绍了一些通过发酵工艺控制优化分子工程菌株利用一碳化合物的相关研究；同时收集了部分一碳化合物转化相关研究机构或企业的产业化案例。最后，针对一碳化合物利用菌株的代谢通路设计与遗传工具存在的限制问题，以及产乙酸菌与产油微生物之间的能量转化矛盾，展望了未来生物制造油脂与单细胞蛋白的前景和面临的挑战，提出在复杂系统性的生物制造过程中，发展多学科交叉的高效系统集成发酵，以期对一碳化合物的生物转化研究产生推动作用，为攻克目前存在的理论与实践难题提供新思路，并对实际应用与产业化发展提供参考。

关键词: 一碳化合物, 油脂, 单细胞蛋白, 生物制造, 代谢工程, 发酵工艺

CLC Number:

Q816

Liang ZHAO, Zhenshuai LI, Liping FU, Ming LU, Shian WANG, Quan ZHANG, Licheng LIU, Fuli LI, Ziyong LIU. Progress in biomanufacturing of lipids and single cell protein from one-carbon compounds[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-013.

赵亮, 李振帅, 付丽平, 吕明, 王士安, 张全, 刘立成, 李福利, 刘自勇. 生物转化一碳化合物原料产油脂与单细胞蛋白研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-013.

Figures/Tables 6

References 112

1	国家统计局. 中华人民共和国2023年国民经济和社会发展统计公报[R/OL]. (2024-02-29)[2024-03-01]. .
	National Bureau of Statistics. Statistical Communiqué of the People's Republic of China on the 2023 National Economic and Social Development[R/OL]. (2024-02-29)[2024-03-01]. .
2	中国人民共和国海关总署. 海关统计:2023年1至12月部分进口商品主要贸易方式量值表(人民币值)[EB/OL]. [2024-03-01]. .
	General Administration of Customs of the People's Republic of China. Customs Statistics: Table of Quantities and Values of Main Trade Modes of Selected Imported Commodities from January to December 2023 (in RMB)[EB/OL]. [2024-03-01]. .
3	中华人民共和国自然资源部. 全国石油天然气资源勘查开采通报(2020年度)[R/OL]. (2021-09-17)[2024-01-01]. .
	Ministry of Natural Resources of the People's Republic of China. National Bulletin on Oil and Gas Resources Exploration and Exploitation (2020) [R/OL]. (2021-09-17)[2024-01-01]. .
4	AGENCY INTERNATIONAL ENERGY. CO₂ Emissions in 2022[R/OL]. IEA. [2024-01-01]. .
5	肖俊夫, 陈德敏, 高艳红. "双碳"目标下再生资源利用的碳减排效应及作用机制研究[J]. 重庆大学学报(社会科学版), 2023, 46(10): 2.
	XIAO J F, CHEN D M, GAO Y H. Research on the effect and mechanism of renewable resources utilization on carbon emission reduction under "double-carbon" target[J]. Journal of Chongqing University(Social Science Edition), 2023, 46(10): 2.
6	JIANG W, LI C, LI Y J, et al. Metabolic engineering strategies for improved lipid production and cellular physiological responses in yeast Saccharomyces cerevisiae [J]. Journal of Fungi, 2022, 8(5): 427.
7	YAN D, YE S Y, HE Y, et al. Fatty acids and lipid mediators in inflammatory bowel disease: from mechanism to treatment[J]. Frontiers in Immunology, 2023, 14: 1286667.
8	CHEUNG P C K. Chemical evaluation of some lesser known edible mushroom mycelia produced in submerged culture from soy milk waste[J]. Food Chemistry, 1997, 60(1): 61-65.
9	ATHANASIADIS I, BOSKOU D, KANELLAKI M, et al. Effect of carbohydrate substrate on fermentation by kefir yeast supported on delignified cellulosic materials[J]. Journal of Agricultural and Food Chemistry, 2001, 49(2): 658-663.
10	CHOI M H, PARK Y H. Growth of Pichia guilliermondii A9, an osmotolerant yeast, in waste brine generated from kimchi production[J]. Bioresource Technology, 1999, 70(3): 231-236.
11	ANUPAMA, RAVINDRA P. Value-added food: single cell protein[J]. Biotechnology Advances, 2000, 18(6): 459-479.
12	CAI P, WU X Y, DENG J, et al. Methanol biotransformation toward high-level production of fatty acid derivatives by engineering the industrial yeast Pichia pastoris [J]. Proceedings of the National Academy of Sciences of the United States of America, 2022, 119(29): e2201711119.
13	GAO L, MENG J, DAI W L, et al. Deciphering cell wall sensors enabling the construction of robust P. pastoris for single-cell protein production[J]. Biotechnology for Biofuels and Bioproducts, 2023, 16(1): 178.
14	高琳惠, 蔡鹏, 周雍进. 甲醇酵母代谢工程研究进展[J]. 生物工程学报, 2021, 37(3): 966-979.
	GAO L H, CAI P, ZHOU Y J. Advances in metabolic engineering of methylotrophic yeasts[J]. Chinese Journal of Biotechnology, 2021, 37(3): 966-979.
15	GAO J Q, LI Y X, YU W, et al. Rescuing yeast from cell death enables overproduction of fatty acids from sole methanol[J]. Nature Metabolism, 2022, 4(7): 932-943.
16	ZHOU H L, WANG F, MAO H L, et al. Efficient expression of heterologous protein by engineered Komagataella phaffii by harnessing a bioelectrical CO₂ reduction system[J]. Biochemical Engineering Journal, 2023, 191: 108762.
17	VOGL T, FISCHER J E, HYDEN P, et al. Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris [J]. AMB Express, 2020, 10(1): 38.
18	SAKAI Y, AKIYAMA M, KONDOH H, et al. High-level secretion of fungal glucoamylase using the Candida boidinii gene expression system[J]. Biochimica et Biophysica Acta, 1996, 1308(1): 81-87.
19	SCHNEIDER K, PEYRAUD R, KIEFER P, et al. The ethylmalonyl-CoA pathway is used in place of the glyoxylate cycle by Methylobacterium extorquens AM1 during growth on acetate[J]. Journal of Biological Chemistry, 2012, 287(1): 757-766.
20	MO X H, ZHANG H, WANG T M, et al. Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis[J]. Applied Microbiology and Biotechnology, 2020, 104(10): 4515-4532.
21	BÉLANGER L, FIGUEIRA M M, BOURQUE D, et al. Production of heterologous protein by Methylobacterium extorquens in high cell density fermentation[J]. FEMS Microbiology Letters, 2004, 231(2): 197-204.
22	KLEIN V J, BRITO L F, PEREZ-GARCIA F, et al. Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol[J]. Microbial Biotechnology, 2023, 16(5): 1011-1026.
23	WENDISCH V F, KOSEC G, HEUX S, et al. Aerobic utilization of methanol for microbial growth and production[J]. Advances in Biochemical Engineering/Biotechnology, 2022, 180: 169-212.
24	SCHRADER J, SCHILLING M, HOLTMANN D, et al. Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria[J]. Trends in Biotechnology, 2009, 27(2): 107-115.
25	ZHAN C J, LI X W, LAN G X, et al. Reprogramming methanol utilization pathways to convert Saccharomyces cerevisiae to a synthetic methylotroph[J]. Nature Catalysis, 2023, 6(5): 435-450.
26	WANG G K, OLOFSSON-DOLK M, HANSSON F G, et al. Engineering yeast Yarrowia lipolytica for methanol assimilation[J]. ACS Synthetic Biology, 2021, 10(12): 3537-3550.
27	GLEIZER S, BEN-NISSAN R, BAR-ON Y M, et al. Conversion of Escherichia coli to generate all biomass carbon from CO₂ [J]. Cell, 2019, 179(6): 1255-1263.e12.
28	KIM S, LINDNER S N, ASLAN S, et al. Growth of E. coli on formate and methanol via the reductive glycine pathway[J]. Nature Chemical Biology, 2020, 16(5): 538-545.
29	TONG S, ZHAO L Z, ZHU D L, et al. From formic acid to single-cell protein: genome-scale revealing the metabolic network of Paracoccus communis MA5[J]. Bioresources and Bioprocessing, 2022, 9(1): 55.
30	赵立枝, 朱大玲, 李德茂. 利用甲酸产单细胞蛋白菌株的筛选及发酵工艺[J]. 饲料研究, 2021, 44(9): 88-93.
	ZHAO L Z, ZHU D L, LI D M. Screening and fermentation process of single cell protein-producing strains by formic acid[J]. Feed Research, 2021, 44(9): 88-93.
31	TIAN J Z, DENG W S Y, ZHANG Z W, et al. Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery[J]. Nature Communications, 2023, 14(1): 7758.
32	WAN S, LAI M C, GAO X Y, et al. Recent progress in engineering Clostridium autoethanogenum to synthesize the biochemicals and biocommodities[J]. Synthetic and Systems Biotechnology, 2024, 9(1): 19-25.
33	HEFFERNAN J K, MAHAMKALI V, VALGEPEA K, et al. Analytical tools for unravelling the metabolism of gas-fermenting Clostridia[J]. Current Opinion in Biotechnology, 2022, 75: 102700.
34	王永伟, 施晶晶, 段涛, 等. 生物技术在粮油饲料资源增值转化中的应用研究进展[J]. 粮油食品科技, 2023, 31(5): 152-159.
	WANG Y W, SHI J J, DUAN T, et al. Research progress on the application of biotechnology in value-added conversion of grain, oil and feed resources[J]. Science and Technology of Cereals, Oils and Foods, 2023, 31(5): 152-159.
35	YI J H, HUANG H Y, LIANG J Y, et al. A heterodimeric reduced-ferredoxin-dependent methylenetetrahydrofolate reductase from syngas-fermenting Clostridium ljungdahlii [J]. Microbiology Spectrum, 2021, 9(2): e0095821.
36	ZHU H F, LIU Z Y, ZHOU X, et al. Energy conservation and carbon flux distribution during fermentation of CO or H₂/CO₂ by Clostridium ljungdahlii [J]. Frontiers in Microbiology, 2020, 11: 416.
37	路伟源. 甲烷氧化菌与氢氧化细菌共培养生产微生物蛋白工艺优化及机理研究[D]. 北京:北京化工大学, 2023.
	LU W Y. Process optimization and mechanism of microbial protein production by co-culture of methane oxidizing bacteria and hydrogen oxidizing bacteria[D]. Beijing: Beijing University of Chemical Technology, 2023.
38	严程, 梅娟, 赵由才. 好氧甲烷氧化菌及其工程应用进展[J]. 生物工程学报, 2022, 38(4): 1322-1338.
	YAN C, MEI J, ZHAO Y C. Engineering application of aerobic methane oxidizing bacteria(methanotrophs): a review[J]. Chinese Journal of Biotechnology, 2022, 38(4): 1322-1338.
39	RITALA A, HÄKKINEN S T, TOIVARI M, et al. Single cell protein-state-of-the-art, industrial landscape and patents 2001-2016[J]. Frontiers in Microbiology, 2017, 8: 2009.
40	傅晓莹, 乔玮博, 史硕博. 微生物利用一碳底物生产单细胞蛋白研究进展[J]. 食品科学, 2023, 44(3): 1-11.
	FU X Y, QIAO W B, SHI S B. Microbial production of single cell proteins from single carbon substrates: a review[J]. Food Science, 2023, 44(3): 1-11.
41	蔡朝阳, 何崭飞, 胡宝兰. 甲烷氧化菌分类及代谢途径研究进展[J]. 浙江大学学报(农业与生命科学版), 2016, 42(3): 273-281.
	CAI C Y, HE Z F, HU B L. Progresses in the classification and mechanism of methane-oxidizing bacteria[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2016, 42(3): 273-281.
42	WANG X, QIN J L, MA C, et al. Methanol assimilation with CO₂ reduction in Butyribacterium methylotrophicum and development of genetic toolkits for its engineering[J]. ACS Sustainable Chemistry & Engineering, 2021, 9(36): 12079-12090.
43	冯叨, 高教琪, 龚志伟, 等. 多形汉逊酵母代谢改造生产脂肪酸及发酵条件优化[J]. 生物工程学报, 2022, 38(2): 760-771.
	FENG D, GAO J Q, GONG Z W, et al. Production of fatty acids by engineered Ogataea polymorpha [J]. Chinese Journal of Biotechnology, 2022, 38(2): 760-771.
44	WU X Y, CAI P, GAO L H, et al. Efficient bioproduction of 3-hydroxypropionic acid from methanol by a synthetic yeast cell factory[J]. ACS Sustainable Chemistry & Engineering, 2023, 11(16): 6445-6453.
45	MENG J, LIU S F, GAO L, et al. Economical production of Pichia pastoris single cell protein from methanol at industrial pilot scale[J]. Microbial Cell Factories, 2023, 22(1): 198.
46	NATTERMANN M, WENK S, PFISTER P, et al. Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo [J]. Nature Communications, 2023, 14(1): 2682.
47	SINGH H B, KANG M K, KWON M, et al. Developing methylotrophic microbial platforms for a methanol-based bioindustry[J]. Frontiers in Bioengineering and Biotechnology, 2022, 10: 1050740.
48	BANG J, HWANG C H, AHN J H, et al. Escherichia coli is engineered to grow on CO₂ and formic acid[J]. Nature Microbiology, 2020, 5(12): 1459-1463.
49	ZHANG S Q, ZHANG L J, XU G, et al. A review on biodiesel production from microalgae: Influencing parameters and recent advanced technologies[J]. Frontiers in Microbiology, 2022, 13: 970028.
50	WEUSTER-BOTZ D. Process engineering aspects for the microbial conversion of C1 gases[M/OL]//One-carbon feedstocks for sustainable bioproduction. Cham: Springer International Publishing, 2021, 180: 33-56. (2021-07-22)[2024-01-01]. .
51	万赛, 王皓明, 马小清, 等. 碳一气体生物转化中的产乙酸菌改造与发酵工艺优化[J]. 生物工程学报, 2023, 39(6): 2410-2429.
	WAN S, WANG H M, MA X Q, et al. Genetic modification of acetogens and optimization of fermentation process in C1-gas bioconversion[J]. Chinese Journal of Biotechnology, 2023, 39(6): 2410-2429.
52	JIN T Z, WANG Y J, YAO S, et al. Bioconversion of carbon dioxide to succinate by Citrobacter [J]. Chemical Engineering Journal, 2023, 452: 139668.
53	ALBERTO R, WELTER C, KUBISCH C, et al. Co-fermenting pyrolysis aqueous condensate and pyrolysis syngas with anaerobic microbial communities enables L-malate production in a secondary fermentative stage[J]. Fermentation, 2022, 8(10): 512.
54	OH H J, GONG G T, AHN J H, et al. Effective hexanol production from carbon monoxide using extractive fermentation with Clostridium carboxidivorans P7[J]. Bioresource Technology, 2023, 367: 128201.
55	BAE J, SONG Y, LEE H, et al. Valorization of C1 gases to value-added chemicals using acetogenic biocatalysts[J]. Chemical Engineering Journal, 2022, 428: 131325.
56	DAI Z X, GU H L, ZHANG S J, et al. Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae [J]. Bioresource Technology, 2017, 245(Pt B): 1407-1412.
57	姚伦, 周雍进. 一碳化合物生物利用和转化研究进展[J]. 化工进展, 2023, 42(1): 16-29.
	YAO L, ZHOU Y J. Progress in microbial utilization of one-carbon feedstocks for biomanufacturing[J]. Chemical Industry and Engineering Progress, 2023, 42(1): 16-29.
58	CAI T, SUN H B, QIAO J, et al. Cell-free chemoenzymatic starch synthesis from carbon dioxide[J]. Science, 2021, 373(6562): 1523-1527.
59	YANG J G, SONG W, CAI T, et al. De novo artificial synthesis of hexoses from carbon dioxide[J]. Science Bulletin, 2023, 68(20): 2370-2381.
60	GAO F, LIU G Y, CHEN A B, et al. Artificial photosynthetic cells with biotic-abiotic hybrid energy modules for customized CO₂ conversion[J]. Nature Communications, 2023, 14(1): 6783.
61	MOON J, WASCHINGER L M, MÜLLER V. Lactate formation from fructose or C1 compounds in the acetogen Acetobacterium woodii by metabolic engineering[J]. Applied Microbiology and Biotechnology, 2023, 107(17): 5491-5502.
62	BENITO-VAQUERIZO S, NOUSE N, SCHAAP P J, et al. Model-driven approach for the production of butyrate from CO₂/H₂ by a novel co-culture of C. autoethanogenum and C. beijerinckii [J]. Frontiers in Microbiology, 2022, 13: 1064013.
63	POULALIER-DELAVELLE M, BAKER J P, MILLARD J, et al. Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum [J]. Frontiers in Bioengineering and Biotechnology, 2023, 11: 1213236.
64	SAMRANRIT T, TEEKA J, NGERNSOMBAT K, et al. Modulation of yeast oil production by Pseudozyma parantarctica CHC28 using xylose and organic acids and its conversion feasibility to bio-polyurethane foam[J]. Biochemical Engineering Journal, 2023, 198: 109025.
65	HUANG C C, CHEN Y R, CHENG S, et al. Enhanced acetate utilization for value-added chemicals production in Yarrowia lipolytica by integration of metabolic engineering and microbial electrosynthesis[J]. Biotechnology and Bioengineering, 2023, 120(10): 3013-3024.
66	ZHENG T T, ZHANG M L, WU L H, et al. Upcycling CO₂ into energy-rich long-chain compounds via electrochemical and metabolic engineering[J]. Nature Catalysis, 2022, 5: 388-396.
67	GAO Q, YANG J L, ZHAO X R, et al. Yarrowia lipolytica as a metabolic engineering platform for the production of very-long-chain wax esters[J]. Journal of Agricultural and Food Chemistry, 2020, 68(39): 10730-10740.
68	ZHAO X R, CHEN X L, YANG J L, et al. De novo synthesis of nervonic acid and optimization of metabolic regulation by Yarrowia lipolytica[J]. Bioresources and Bioprocessing, 2023, 10(1): 70.
69	LIU F X, LU Z W, LU T T, et al. Metabolic engineering of oleaginous yeast in the lipogenic phase enhances production of nervonic acid[J]. Metabolic Engineering, 2023, 80: 193-206.
70	WANG K F, LIN L, WEI P, et al. Combining orthogonal plant and non-plant fatty acid biosynthesis pathways for efficient production of microbial oil enriched in nervonic acid in Yarrowia lipolytica [J]. Bioresource Technology, 2023, 378: 129012.
71	SU H, SHI P H, SHEN Z S, et al. High-level production of nervonic acid in the oleaginous yeast Yarrowia lipolytica by systematic metabolic engineering[J]. Communications Biology, 2023, 6(1): 1125.
72	RAVI R K, NEERAJ A, YADAV R H. Assessment of microbial biomass for production of ecofriendly single-cell protein, bioenergy, and other useful products[M/OL]//Microbes in land use change management. Amsterdam: Elsevier, 2021: 267-284. (2021-08-27)[2024-01-01]. .
73	JONES S W, KARPOL A, FRIEDMAN S, et al. Recent advances in single cell protein use as a feed ingredient in aquaculture[J]. Current Opinion in Biotechnology, 2020, 61: 189-197.
74	MOLITOR B, MISHRA A, ANGENENT L T. Power-to-protein: converting renewable electric power and carbon dioxide into single cell protein with a two-stage bioprocess[J]. Energy & Environmental Science, 2019, 12(12): 3515-3521.
75	FU Y F, LI Y, LIDSTROM M. The oxidative TCA cycle operates during methanotrophic growth of the Type I methanotroph Methylomicrobium buryatense 5GB1[J]. Metabolic Engineering, 2017, 42: 43-51.
76	KHOSHNEVISAN B, TSAPEKOS P, ZHANG Y F, et al. Urban biowaste valorization by coupling anaerobic digestion and single cell protein production[J]. Bioresource Technology, 2019, 290: 121743.
77	NWAOKORIE U J, REINMETS K, DE LIMA L A, et al. Deletion of genes linked to the C₁-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum [J]. Frontiers in Bioengineering and Biotechnology, 2023, 11: 1167892.
78	舒芹, 李凯凯, 全拓, 等. 微生物蛋白作为优质替代蛋白资源的应用研究[J]. 未来食品科学, 2022(2): 96-106.
	SHU Q, LI K K, QUAN T, et al. Application and development of microbial proteins as high-quality alternative protein resources-a view[J]. Future Food Science, 2022(2): 96-106.
79	LIU Y Q, ZHANG Z W, JIANG W H, et al. Protein acetylation-mediated cross regulation of acetic acid and ethanol synthesis in the gas-fermenting Clostridium ljungdahlii [J]. Journal of Biological Chemistry, 2022, 298(2): 101538.
80	VALGEPEA K, TALBO G, TAKEMORI N, et al. Absolute proteome quantification in the gas-fermenting acetogen Clostridium autoethanogenum [J]. mSystems, 2022, 7(2): e00026-22.
81	DETSIOS N, MARAGOUDAKI L, ATSONIOS K, et al. Design considerations of an integrated thermochemical/biochemical route for aviation and maritime biofuel production[J/OL]. Biomass Conversion and Biorefinery. (2023-01-13)[2024-01-04]. .
82	ROBLES-IGLESIAS R, NICAUD J M, VEIGA M C, et al. Integrated fermentative process for lipid and β-carotene production from acetogenic syngas fermentation using an engineered oleaginous Yarrowia lipolytica yeast[J]. Bioresource Technology, 2023, 389: 129815.
83	ROBLES-IGLESIAS R, VEIGA M C, KENNES C. Sequential bioconversion of C₁-gases (CO, CO₂, syngas) into lipids, through the carboxylic acid platform, with Clostridium aceticum and Rhodosporidium toruloides [J]. Journal of Environmental Management, 2023, 347: 119097.
84	ODUOR W W, WANDERA S M, MURUNGA S I, et al. Enhancement of anaerobic digestion by co-digesting food waste and water hyacinth in improving treatment of organic waste and bio-methane recovery[J]. Heliyon, 2022, 8(9): e10580.
85	KÜÇÜKAĞA Y, FACCHIN A, KARA S, et al. Conversion of pyrolysis products into volatile fatty acids with a biochar-packed anaerobic bioreactor[J]. Industrial & Engineering Chemistry Research, 2022, 61(45): 16624-16634.
86	REGIS F, MONTEVERDE A H A, FINO D. A techno-economic assessment of bioethanol production from switchgrass through biomass gasification and syngas fermentation[J]. Energy, 2023, 274: 127318.
87	MA S, DONG C Q, HU X Y, et al. Techno-economic evaluation of a combined biomass gasification-solid oxide fuel cell system for ethanol production via syngas fermentation[J]. Fuel, 2022, 324: 124395.
88	PATI S, DE S, CHOWDHURY R. Exploring the hybrid route of bio-ethanol production via biomass co-gasification and syngas fermentation from wheat straw and sugarcane bagasse: model development and multi-objective optimization[J]. Journal of Cleaner Production, 2023, 395: 136441.
89	HEFFERNAN J K, LAI C Y, GONZALEZ-GARCIA R A, et al. Biogas upgrading using Clostridium autoethanogenum for value-added products[J]. Chemical Engineering Journal, 2023, 452: 138950.
90	PUIMAN L, ELISIÁRIO M P, CRASBORN L M L, et al. Gas mass transfer in syngas fermentation broths is enhanced by ethanol[J]. Biochemical Engineering Journal, 2022, 185: 108505.
91	PUIMAN L, ABRAHAMSON B, VAN DER LANS R G J M, et al. Alleviating mass transfer limitations in industrial external-loop syngas-to-ethanol fermentation[J]. Chemical Engineering Science, 2022, 259: 117770.
92	KÜÇÜKAĞA Y, FACCHIN A, STEFANELLI V, et al. Innovative char-sparger for improving volatile fatty acids (VFA) production in homoacetogenic fermentation of H₂/CO₂ with microbial mixed cultures (MMC)[J]. Chemical Engineering Journal, 2023, 471: 144165.
93	PERRET L, BOUKIS N, SAUER J. Influence of increased cell densities on product ratio and productivity in syngas fermentation[J]. Industrial & Engineering Chemistry Research, 2023, 62(35): 13799-13810.
94	VELVIZHI G, SARKAR O, ROVIRA-ALSINA L, et al. Conversion of carbon dioxide to value added products through anaerobic fermentation and electro fermentation: a comparative approach[J]. International Journal of Hydrogen Energy, 2022, 47(34): 15442-15455.
95	KIM J H, LEE M, JEONG H, et al. Recycling of minerals with acetate separation in biological syngas fermentation with an electrodialysis system[J]. Chemical Engineering Journal, 2023, 459: 141555.
96	MARIËN Q, REGUEIRA A, GANIGUÉ R. Steerable isobutyric and butyric acid production from CO₂ and H₂ by Clostridium luticellarii [J]. Microbial Biotechnology, 2024, 17(1): e14321.
97	KATAKOJWALA R, THARAK A, SARKAR O, et al. Design and evaluation of gas fermentation systems for CO₂ reduction to C2 and C4 fatty acids: Non-genetic metabolic regulation with pressure, pH and reaction time[J]. Bioresource Technology, 2022, 351: 126937.
98	LIU J, ZHOU W T, HE Q N, et al. Microbial lipid production from high concentration of volatile fatty acids via Trichosporon cutaneum for biodiesel preparation[J]. Applied Biochemistry and Biotechnology, 2022, 194(7): 2968-2979.
99	ZHOU W, WANG Y N, ZHANG J L, et al. A metabolic model of Lipomyces starkeyi for predicting lipogenesis potential from diverse low-cost substrates[J]. Biotechnology for Biofuels, 2021, 14(1): 148.
100	SUN S M, DING Y M, LIU M, et al. Comparison of glucose, acetate and ethanol as carbon resource for production of poly(3-hydroxybutyrate) and other acetyl-CoA derivatives[J]. Frontiers in Bioengineering and Biotechnology, 2020, 8: 833.
101	PEREIRA A S, LOPES M, MIRANDA S M, et al. Bio-oil production for biodiesel industry by Yarrowia lipolytica from volatile fatty acids in two-stage batch culture[J]. Applied Microbiology and Biotechnology, 2022, 106(8): 2869-2881.
102	REROP Z S, STELLNER N I, GRABAN P, et al. Bioconversion of a lignocellulosic hydrolysate to single cell oil for biofuel production in a cost-efficient fermentation process[J]. Fermentation, 2023, 9(2): 189.
103	BURGSTALLER L, OLIVER L, DIETRICH T, et al. Pilot scale production of single cell oil by Apiotrichum brassicae and Pichia kudriavzevii from acetic acid and propionic acid[J]. Applied Sciences, 2023, 13(8) : 4674.
104	MORENO J F, OULEGO P, COLLADO S, et al. Production of biolipids from volatile fatty acids of sewage sludge by Yarrowia lipolytica [J]. Fuel, 2023, 348: 128488.
105	NAVEIRA-PAZOS C, VEIGA M C, KENNES C. Accumulation of lipids by the oleaginous yeast Yarrowia lipolytica grown on carboxylic acids simulating syngas and carbon dioxide fermentation[J]. Bioresource Technology, 2022, 360: 127649.
106	MORALES-PALOMO S, GONZÁLEZ-FERNÁNDEZ C, TOMÁS-PEJÓ E. Prevailing acid determines the efficiency of oleaginous fermentation from volatile fatty acids[J]. Journal of Environmental Chemical Engineering, 2022, 10(2): 107354.
107	Kiverdi, Inc. [EB/OL]. [2023-12-29]. .
108	Air Protein Inc. [EB/OL]. [2024-01-02].
109	Newswire Contact PR. FGen AG, Ginkgo Bioworks Subsidiarya, Awarded Funding Through European Innovation Council's Pathfinder Programme For Sustainable Milk Protein Production Project[N/OL]. News from ginkgo bioworks. (2023-12-19)[2024-01-01]. .
110	中华人民共和国农业农村部. 中华人民共和国农业农村部公告第465号[EB/OL]. (2021-12-07)[2024.03.06]. .
	Ministy of Agnculure and Rural Afairs of the People's Republic of China. Announcement No. 465 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China[EB/OL]. (2021-12-07)[2024-03-06]. .
111	GORLA G, FERRER A, GIUSSANI B. Process understanding and monitoring: a glimpse into data strategies for miniaturized NIR spectrometers[J]. Analytica Chimica Acta, 2023, 1281: 341902.
112	SCHORN-GARCÍA D, EZENARRO J, ACEÑA L, et al. Spatially offset Raman spectroscopic (SORS) analysis of wine alcoholic fermentation: a preliminary study[J]. Fermentation, 2023, 9(2): 115.

微生物类型	菌种	底物	代谢途径	代谢改造	产量/产率		产物	参考文献
天然甲醇酵母	P. pastoris	甲醇	XuMP途径	/	蛋白80.6 g/L	脂肪酸23.4 g/L	脂肪酸，单细胞蛋白	[12-14]
	O. polymorpha	甲醇		/	生物量26.6 g/L	脂肪酸15.9 g/L	脂肪酸，单细胞蛋白	[15]
	C. boidinii	甲醇		/	3.4 g/L		单细胞蛋白	[16-18]
天然甲醇细菌	M. extorquens	甲醇	RuMP途径，丝氨酸循环	/	干重561 g/L		单细胞蛋白	[19-21]
天然甲醇细菌	B. methanolicus	甲醇	RuMP途径，丝氨酸循环	/	干重30-144 g/L		单细胞蛋白	[22-24]
非天然甲醇酵母	S. cerevisiae	甲醇	XuMP途径	Mdh与XuMP途径共表达	/		单细胞蛋白	[25]
非天然甲醇酵母	Y. lipolytica	甲醇	RuMP、XuMP途径	表达杂合RuMP和XuMP途径基因，敲除内源甲醛脱氢酶	/		单细胞蛋白	[26]
大肠杆菌	E. coli	甲醇、甲酸、CO₂	还原性甘氨酸途径、CBB循环	利用还原性甘氨酸途径重新设计了中心碳代谢	2.8 ± 0.8 g 干重/mol 甲酸		单细胞蛋白	[27-28]
天然甲酸利用微生物	P. communis	甲酸	丝氨酸循环、四氢叶酸循环、糖酵解途径、TCA循环	/	1.7 g/L		单细胞蛋白	[29-30]
需钠弧菌	V. natriegens	甲酸	丝氨酸循环、TCA循环	重新连接丝氨酸循环和TCA循环	/		单细胞蛋白	[31]
产乙酸菌	Clostridium autoethanogenum	CO、CO₂	Wood-Ljungdahl途径	/	1×10⁸ t/a		单细胞蛋白	[32-34]
产乙酸菌	Clostridium ljungdahlii	CO、CO2	Wood-Ljungdahl途径	/	2 g/L		单细胞蛋白	[33,35-36]
氢氧化细菌	Hydrogenophaga	CO、CO2	反向三羧酸循环、CBB循环、Wood-Ljungdahl途径	/	0.9-1.7 g/L		单细胞蛋白	[37]
	Xanthobacter	CO、CO2		/			单细胞蛋白
	Aquamicrobium	CO、CO2		/			单细胞蛋白
	Defluviimonas	CO、CO2		/			单细胞蛋白
甲烷氧化菌	Proteobacteria-γ亚型	CH₄	RuMP途径	/	4 kg/m³/h		单细胞蛋白	[38-40]
	Proteobacteria-α亚型	CH₄	丝氨酸循环	/	/		单细胞蛋白
	Verrucomicrobia	CH₄	CBB循环	/	/		单细胞蛋白	[39,41]

微生物类型	菌种	底物	代谢途径	代谢改造	产量/产率		产物	参考文献
天然甲醇酵母	P. pastoris	甲醇	XuMP途径	/	蛋白80.6 g/L	脂肪酸23.4 g/L	脂肪酸，单细胞蛋白	[12-14]
	O. polymorpha	甲醇		/	生物量26.6 g/L	脂肪酸15.9 g/L	脂肪酸，单细胞蛋白	[15]
	C. boidinii	甲醇		/	3.4 g/L		单细胞蛋白	[16-18]
天然甲醇细菌	M. extorquens	甲醇	RuMP途径，丝氨酸循环	/	干重561 g/L		单细胞蛋白	[19-21]
天然甲醇细菌	B. methanolicus	甲醇	RuMP途径，丝氨酸循环	/	干重30-144 g/L		单细胞蛋白	[22-24]
非天然甲醇酵母	S. cerevisiae	甲醇	XuMP途径	Mdh与XuMP途径共表达	/		单细胞蛋白	[25]
非天然甲醇酵母	Y. lipolytica	甲醇	RuMP、XuMP途径	表达杂合RuMP和XuMP途径基因，敲除内源甲醛脱氢酶	/		单细胞蛋白	[26]
大肠杆菌	E. coli	甲醇、甲酸、CO₂	还原性甘氨酸途径、CBB循环	利用还原性甘氨酸途径重新设计了中心碳代谢	2.8 ± 0.8 g 干重/mol 甲酸		单细胞蛋白	[27-28]
天然甲酸利用微生物	P. communis	甲酸	丝氨酸循环、四氢叶酸循环、糖酵解途径、TCA循环	/	1.7 g/L		单细胞蛋白	[29-30]
需钠弧菌	V. natriegens	甲酸	丝氨酸循环、TCA循环	重新连接丝氨酸循环和TCA循环	/		单细胞蛋白	[31]
产乙酸菌	Clostridium autoethanogenum	CO、CO₂	Wood-Ljungdahl途径	/	1×10⁸ t/a		单细胞蛋白	[32-34]
产乙酸菌	Clostridium ljungdahlii	CO、CO2	Wood-Ljungdahl途径	/	2 g/L		单细胞蛋白	[33,35-36]
氢氧化细菌	Hydrogenophaga	CO、CO2	反向三羧酸循环、CBB循环、Wood-Ljungdahl途径	/	0.9-1.7 g/L		单细胞蛋白	[37]
	Xanthobacter	CO、CO2		/			单细胞蛋白
	Aquamicrobium	CO、CO2		/			单细胞蛋白
	Defluviimonas	CO、CO2		/			单细胞蛋白
甲烷氧化菌	Proteobacteria-γ亚型	CH₄	RuMP途径	/	4 kg/m³/h		单细胞蛋白	[38-40]
	Proteobacteria-α亚型	CH₄	丝氨酸循环	/	/		单细胞蛋白
	Verrucomicrobia	CH₄	CBB循环	/	/		单细胞蛋白	[39,41]

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