面向高效光驱固碳产醇的蓝细菌合成生物技术研究进展

doi:10.12211/2096-8280.2023-051

合成生物学 ›› 2023, Vol. 4 ›› Issue (6): 1161-1177.DOI: 10.12211/2096-8280.2023-051

面向高效光驱固碳产醇的蓝细菌合成生物技术研究进展

孙绘梨¹^,²^,³, 崔金玉¹^,²^,³, 栾国栋¹^,²^,³, 吕雪峰¹^,²^,³

^1.中国科学院青岛生物能源与过程研究所，中国科学院生物燃料重点实验室，山东青岛 266101
^2.山东能源研究院，山东青岛 266101
^3.青岛新能源山东省实验室，山东青岛 266101

收稿日期:2023-07-17 修回日期:2023-08-24 出版日期:2023-12-31 发布日期:2024-01-19
通讯作者: 吕雪峰
作者简介:孙绘梨（1995—），女，博士后。研究方向为蓝细菌合成生物技术研究，包括光驱固碳细胞工厂的构建和底盘细胞生理功能认识与改造。E-mail：sunhl@qibebt.ac.cn
吕雪峰（1974—），男，研究员，博士生导师，中国科学院青岛生物能源与过程研究所所长。研究方向为合成生物学与绿色生物制造，在光驱固碳产能蓝细菌的人工设计与构建及真菌天然产物药物等。E-mail：lvxf@qibebt.ac.cn
基金资助:
国家重点研发计划(2021YFA0909700);国家自然科学基金(32270103);山东省博士后创新项目(SDCX-ZG-202202036);中国博士后科学基金第70 批面上项目(2021M703320)

Progress of cyanobacterial synthetic biotechnology for efficient light-driven carbon fixation and ethanol production

Huili SUN¹^,²^,³, Jinyu CUI¹^,²^,³, Guodong LUAN¹^,²^,³, Xuefeng LYU¹^,²^,³

^1.Key Laboratory of Biofuels，Qingdao Institute of Bioenergy and Bioprocess Technology，Chinese Academy of Sciences，Qingdao 266101，Shandong，China
^2.Shandong Energy Institute，Qingdao 266101，Shandong，China
^3.Qingdao New Energy Shandong Laboratory，Qingdao 266101，Shandong，China

Received:2023-07-17 Revised:2023-08-24 Online:2023-12-31 Published:2024-01-19
Contact: Xuefeng LYU

摘要/Abstract

摘要：

蓝细菌能够直接利用二氧化碳和太阳能通过光合作用生产乙醇，为提供绿色生物燃料提供了一条有前途的可持续路线。光驱固碳合成乙醇是最具代表性的蓝细菌光合生物制造技术。乙醇并不属于典型的蓝细菌天然代谢物，蓝细菌产醇细胞工厂的构建需要通过向基因组中导入异源的丙酮酸脱羧酶并结合异源/内源醇脱氢酶的过量表达来实现；在过去二十多年间，通过蛋白、途径、底盘、工艺层面的系统优化，蓝细菌产醇细胞工厂的效能得到有效提高，乙醇成为目前产量最高、产率最高、碳流分配率最高的蓝细菌代谢工程产物。本文总结并比较了“蓝细菌生物质炼制产醇”“蓝细菌固碳产糖-产醇”“蓝细菌固碳直接产醇”等三种光驱固碳产醇技术路线，并以构建光合细胞工厂驱动二氧化碳一站式转化为乙醇的技术路线为主，从乙醇合成途径优化与强化、蓝细菌光合碳代谢网络的调节与重塑、代谢网络模型与计算机辅助设计引导细胞工厂构建和优化三个方面对蓝细菌高效光驱固碳合成乙醇的技术发展历程和基本现状进行了介绍，特别是强调了计算生物学、系统代谢工程、生物材料嵌合等研究手段在本领域的应用进展；在此基础上，也从新型底盘开发、高通量筛选技术应用、细胞工厂的稳定性与鲁棒性优化等角度对蓝细菌固碳产醇的未来发展方向进行了展望。

关键词: 蓝细菌, 光合生物制造, 合成生物学, 代谢工程, 乙醇

Abstract:

The utilization of solar energy and carbon dioxide by cyanobacterial cell factories for photosynthetic ethanol production represents a promising and sustainable route towards green biofuels. Ethanol is one of the most representative products of the cyanobacterial photosynthetic biomanufacturing technology. Cyanobacterial ethanol production systems could serve as models for developing and optimizing advanced synthetic biology and metabolic engineering strategies. While most known cyanobacterial species lack the ability to synthesize and accumulate ethanol, the introduction and overexpression of heterologous pyruvate decarboxylase and alcohol dehydrogenase (heterologous or native) are required to enable ethanol synthesis in cyanobacteria. In the past decades, the performance of ethanol-producing cyanobacterial cell factories has been significantly improved through systematic optimization of proteins, pathways, chassis cells, and cultivation techniques. Cyanobacterial ethanol production technology has yielded the highest titer, productivity, and carbon partitioning ratio among all current cyanobacterial biomanufacturing systems. Recent advances have led to further improved efficiency of cyanobacterial ethanol photosynthetic production. Based on extensive systems biology data and rapidly developing computer modeling technologies, more accurate simulations of cyanobacterial physiological characteristics and metabolic networks have become possible. These simulations facilitate the identification of potential modification targets, thereby enhancing ethanol production capacity and guiding the design of next-generation alcohol-producing cell factories. With a more comprehensive understanding of cyanobacteria physiology and metabolism, systematic genome modifications and pathway optimizations have been performed, resulting in further improved ethanol productivity and final titers. Concurrently, efforts have been made to improve model strains and evaluate newly emerging non-conventional strains to establish more robust and efficient ethanol production processes. In conclusion, this review summarizes and compares three technological routes of light-driven carbon fixation and ethanol production in cyanobacteria, introduces the technological development trajectory and basic status of efficient light-driven carbon fixation for ethanol synthesis by cyanobacteria, provides valuable and up-to-date insights to facilitate the development of more promising cyanobacterial ethanol photosynthetic production technologies and explores future challenges and directions in this dynamic field.

Key words: cyanobacteria, photosynthetic biomanufacturing, synthetic biology, metabolic engineering, ethanol

中图分类号:

Q815

孙绘梨, 崔金玉, 栾国栋, 吕雪峰. 面向高效光驱固碳产醇的蓝细菌合成生物技术研究进展[J]. 合成生物学, 2023, 4(6): 1161-1177.

Huili SUN, Jinyu CUI, Guodong LUAN, Xuefeng LYU. Progress of cyanobacterial synthetic biotechnology for efficient light-driven carbon fixation and ethanol production[J]. Synthetic Biology Journal, 2023, 4(6): 1161-1177.

图/表 3

参考文献 92

1	KENNES D, ABUBACKAR H N, DIAZ M, et al. Bioethanol production from biomass: carbohydrate vs syngas fermentation[J]. Journal of Chemical Technology & Biotechnology, 2016, 91(2): 304-317.
2	KEASLING J D, CHOU H. Metabolic engineering delivers next-generation biofuels[J]. Nature Biotechnology, 2008, 26(3): 298-299.
3	THANGAVELU S K, AHMED A S, ANI F N. Review on bioethanol as alternative fuel for spark ignition engines[J]. Renewable and Sustainable Energy Reviews, 2016, 56: 820-835.
4	TABAH B, PULIDINDI I N, CHITTURI V R, et al. Utilization of solar energy for continuous bioethanol production for energy applications[J]. RSC Advances, 2016, 6(29): 24203-24209.
5	AGARWAL A K. Biofuels (alcohols and biodiesel) applications as fuels for internal combustion engines[J]. Progress in Energy and Combustion Science, 2007, 33(3): 233-271.
6	WEBER C, FARWICK A, BENISCH F, et al. Trends and challenges in the microbial production of lignocellulosic bioalcohol fuels[J]. Applied Microbiology and Biotechnology, 2010, 87(4): 1303-1315.
7	RUDE M A, SCHIRMER A. New microbial fuels: a biotech perspective[J]. Current Opinion in Microbiology, 2009, 12(3): 274-281.
8	HIMMEL M E, DING S Y, JOHNSON D K, et al. Biomass recalcitrance: engineering plants and enzymes for biofuels production[J]. Science, 2007, 315(5813): 804-807.
9	JI S Q, WANG B, LU M, et al. Direct bioconversion of brown algae into ethanol by thermophilic bacterium Defluviitalea phaphyphila [J]. Biotechnology for Biofuels, 2016, 9: 81.
10	LI H F, LIU T F, WEI P F, et al. High-rate CO₂ electroreduction to C₂₊ products over a copper-copper iodide catalyst[J]. Angewandte Chemie International Edition, 2021, 60(26): 14329-14333.
11	WEI J A, YAO R W, HAN Y, et al. Towards the development of the emerging process of CO₂ heterogenous hydrogenation into high-value unsaturated heavy hydrocarbons[J]. Chemical Society Reviews, 2021, 50(19): 10764-10805.
12	CHEN J E, ZHA Y J, LIU B, et al. Rationally designed water enriched nano reactor for stable CO₂ hydrogenation with near 100% ethanol selectivity over diatomic palladium active sites[J]. ACS Catalysis, 2023, 13(10): 7110-7121.
13	LIU Z H, WANG K, CHEN Y, et al. Third-generation biorefineries as the means to produce fuels and chemicals from CO₂ [J]. Nature Catalysis, 2020, 3(3): 274-288.
14	WATERBURY J B, WATSON S W, GUILLARD R R L, et al. Widespread occurrence of a unicellular, marine, planktonic, cyanobacterium[J]. Nature, 1979, 277(5694): 293-294.
15	ROUSSEAUX C, GREGG W. Interannual variation in phytoplankton primary production at a global scale[J]. Remote Sensing, 2014, 6(1): 1-19.
16	FLOMBAUM P, GALLEGOS J L, GORDILLO R A, et al. Present and future global distributions of the marine cyanobacteria Prochlorococcus and Synechococcus [J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(24): 9824-9829.
17	ATSUMI S, HIGASHIDE W, LIAO J C. Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde[J]. Nature Biotechnology, 2009, 27(12): 1177-1180.
18	ANGERMAYR S A, HELLINGWERF K J, LINDBLAD P, et al. Energy biotechnology with cyanobacteria[J]. Current Opinion in Biotechnology, 2009, 20(3): 257-263.
19	LU X F. A perspective: photosynthetic production of fatty acid-based biofuels in genetically engineered cyanobacteria[J]. Biotechnology Advances, 2010, 28(6): 742-746.
20	OLIVER J W K, ATSUMI S. Metabolic design for cyanobacterial chemical synthesis[J]. Photosynthesis Research, 2014, 120(3): 249-261.
21	DENG M D, COLEMAN J R. Ethanol synthesis by genetic engineering in cyanobacteria[J]. Applied and Environmental Microbiology, 1999, 65(2): 523-528.
22	DEXTER J, ARMSHAW P, SHEAHAN C, et al. The state of autotrophic ethanol production in cyanobacteria[J]. Journal of Applied Microbiology, 2015, 119(1): 11-24.
23	齐允晶, 王家林, 栾国栋, 等. 蓝细菌乙醇光合细胞工厂的发展与展望[J]. 生物工程学报, 2017, 33(6): 891-909.
	QI Y J, WANG J L, LUAN G D, et al. Cyanobacteria cell factories for ethanol photosynthetic production: development and prospect[J]. Chinese Journal of Biotechnology, 2017, 33(6): 891-909.
24	LUAN G D, LU X F. Engineering cyanobacteria for efficient photosynthetic production: ethanol case study [M/OL]//Cyanobacteria biotechnology. 2021: 237-266 [2023-07-01]. .
25	FERNÁNDEZ F G A, REIS A, WIJFFELS R H, et al. The role of microalgae in the bioeconomy[J]. New Biotechnology, 2021, 61: 99-107.
26	RAHUL S M, SUNDARAMAHALINGAM M A, SHIVAMTHI C S, et al. Insights about sustainable biodiesel production from microalgae biomass: a review[J]. International Journal of Energy Research, 2021, 45(12): 17028-17056.
27	LUAN G D, ZHANG S S, WANG M, et al. Progress and perspective on cyanobacterial glycogen metabolism engineering[J]. Biotechnology Advances, 2019, 37(5): 771-786.
28	SONG K, TAN X M, LIANG Y J, et al. The potential of Synechococcus elongatus UTEX 2973 for sugar feedstock production[J]. Applied Microbiology and Biotechnology, 2016, 100(18): 7865-7875.
29	AIKAWA S, NISHIDA A, HO S H, et al. Glycogen production for biofuels by the euryhaline cyanobacteria Synechococcus sp. strain PCC 7002 from an oceanic environment[J]. Biotechnology for Biofuels, 2014, 7: 88.
30	COMER A D, ABRAHAM J P, STEINER A J, et al. Enhancing photosynthetic production of glycogen-rich biomass for use as a fermentation feedstock[J]. Frontiers in Energy Research, 2020, 8: 93.
31	HAGEMANN M. Molecular biology of cyanobacterial salt acclimation[J]. FEMS Microbiology Reviews, 2011, 35(1): 87-123.
32	DUCAT D C, AVELAR-RIVAS J A, WAY J C, et al. Rerouting carbon flux to enhance photosynthetic productivity[J]. Applied and Environmental Microbiology, 2012, 78(8): 2660-2668.
33	LIN P C, ZHANG F Z, PAKRASI H B. Enhanced production of sucrose in the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973[J]. Scientific Reports, 2020, 10: 390.
34	ZHANG S S, SUN J H, FENG D D, et al. Unlocking the potentials of cyanobacterial photosynthesis for directly converting carbon dioxide into glucose[J]. Nature Communications, 2023, 14: 3425.
35	DUAN Y K, LUO Q, LIANG F Y, et al. Sucrose secreted by the engineered cyanobacterium and its fermentability[J]. Journal of Ocean University of China, 2016, 15(5): 890-896.
36	HAYS S G, YAN L L W, SILVER P A, et al. Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction[J]. Journal of Biological Engineering, 2017, 11(1): 4.
37	SANTOS-MERINO M, YUN L S, DUCAT D C. Cyanobacteria as cell factories for the photosynthetic production of sucrose[J]. Frontiers in Microbiology, 2023, 14: 1126032.
38	郗欣彤, 张彬彬, 毛绍名, 等. 蓝细菌光驱固碳合成蔗糖技术的发展与展望[J]. 生物工程学报, 2019, 35(8): 1411-1423.
	CHI X T, ZHANG S S, MAO S M, et al. Cyanobacteria based photosynthetic production of sucrose: development and prospect[J]. Chinese Journal of Biotechnology, 2019, 35(8): 1411-1423.
39	LUO D X, HU Z S, CHOI D G, et al. Life cycle energy and greenhouse gas emissions for an ethanol production process based on blue-green algae[J]. Environmental Science & Technology, 2010, 44(22): 8670-8677.
40	ARORA P, CHANCE R R, FISHBECK T, et al. Lifecycle greenhouse gas emissions for an ethanol production process based on genetically modified cyanobacteria: CO₂ sourcing options[J]. Biofuels, Bioproducts and Biorefining, 2020, 14(6): 1324-1334.
41	GAO Z X, ZHAO H, LI Z M, et al. Photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria[J]. Energy & Environmental Science, 2012, 5(12): 9857-9865.
42	CHOI Y N, PARK J M. Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis sp. PCC 6803[J]. Bioresource Technology, 2016, 213: 54-57.
43	ASPLUND-SAMUELSSON J, JANASCH M, HUDSON E P. Thermodynamic analysis of computed pathways integrated into the metabolic networks of E. coli and Synechocystis reveals contrasting expansion potential[J]. Metabolic Engineering, 2018, 45: 223-236.
44	BARTASUN P, PRANDI N, STORCH M, et al. The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux in Synechocystis sp. PCC 6803[J]. PeerJ, 2019, 7: e7529.
45	LIANG F Y, ENGLUND E, LINDBERG P, et al. Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio[J]. Metabolic Engineering, 2018, 46: 51-59.
46	ROUSSOU S, ALBERGATI A, LIANG F Y, et al. Engineered cyanobacteria with additional overexpression of selected Calvin-Benson-Bassham enzymes show further increased ethanol production[J]. Metabolic Engineering Communications, 2021, 12: e00161.
47	CHOU H H, SU H Y, CHOW T J, et al. Engineering cyanobacteria with enhanced growth in simulated flue gases for high-yield bioethanol production[J]. Biochemical Engineering Journal, 2021, 165: 107823.
48	GAO E B, WU J H, YE P L, et al. Rewiring carbon flow in Synechocystis PCC 6803 for a high rate of CO₂-to-ethanol under an atmospheric environment[J]. Frontiers in Microbiology, 2023, 14: 1211004.
49	WANG M, LUAN G D, LU X F. Engineering ethanol production in a marine cyanobacterium Synechococcus sp. PCC7002 through simultaneously removing glycogen synthesis genes and introducing ethanolgenic cassettes[J]. Journal of Biotechnology, 2020, 317: 1-4.
50	NAMAKOSHI K, NAKAJIMA T, YOSHIKAWA K, et al. Combinatorial deletions of glgC and phaCE enhance ethanol production in Synechocystis sp. PCC 6803[J]. Journal of Biotechnology, 2016, 239: 13-19.
51	VELMURUGAN R, INCHAROENSAKDI A. Co-cultivation of two engineered strains of Synechocystis sp. PCC 6803 results in improved bioethanol production[J]. Renewable Energy, 2020, 146: 1124-1133.
52	VELMURUGAN R, INCHAROENSAKDI A. Metal oxide mediated extracellular NADPH regeneration improves ethanol production by engineered Synechocystis sp. PCC 6803[J]. Frontiers in Bioengineering and Biotechnology, 2019, 7: 148.
53	EHIRA S, TAKEUCHI T, HIGO A. Spatial separation of photosynthesis and ethanol production by cell type-specific metabolic engineering of filamentous cyanobacteria[J]. Applied Microbiology and Biotechnology, 2018, 102(3): 1523-1531.
54	HIGO A, EHIRA S. Spatiotemporal gene repression system in the heterocyst-forming multicellular cyanobacterium Anabaena sp. PCC 7120 [J]. ACS Synthetic Biology, 2019, 8(4): 641-646.
55	LASRY TESTA R, DELPINO C, ESTRADA V, et al. In silico strategies to couple production of bioethanol with growth in cyanobacteria[J]. Biotechnology and Bioengineering, 2019, 116(8): 2061-2073.
56	LUAN G D, QI Y J, WANG M, et al. Combinatory strategy for characterizing and understanding the ethanol synthesis pathway in cyanobacteria cell factories[J]. Biotechnology for Biofuels, 2015, 8: 184.
57	RAJ K C, TALARICO L A, INGRAM L O, et al. Cloning and characterization of the Zymobacter palmae pyruvate decarboxylase gene (pdc) and comparison to bacterial homologues[J]. Applied and Environmental Microbiology, 2002, 68(6): 2869-2876.
58	QUINN L, ARMSHAW P, SOULIMANE T, et al. Zymobacter palmae pyruvate decarboxylase is less effective than that of Zymomonas mobilis for ethanol production in metabolically engineered Synechocystis sp. PCC6803[J]. Microorganisms, 2019, 7(11): 494.
59	LEVIN-KARP A, BARENHOLZ U, BAREIA T, et al. Quantifying translational coupling in E. coli synthetic operons using RBS modulation and fluorescent reporters[J]. ACS Synthetic Biology, 2013, 2(6): 327-336.
60	ZHOU J, ZHU T C, CAI Z, et al. From cyanochemicals to cyanofactories: a review and perspective[J]. Microbial Cell Factories, 2016, 15: 2.
61	LIANG F Y, LINDBLAD P. Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803[J]. Metabolic Engineering, 2016, 38: 56-64.
62	KAMENNAYA N A, AHN S, PARK H, et al. Installing extra bicarbonate transporters in the cyanobacterium Synechocystis sp. PCC 6803 enhances biomass production[J]. Metabolic Engineering, 2015, 29: 76-85.
63	PRICE G D, BADGER M R, WOODGER F J, et al. Advances in understanding the cyanobacterial CO₂-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants[J]. Journal of Experimental Botany, 2008, 59(7): 1441-1461.
64	KUPRIYANOVA E V, SINETOVA M A, BEDBENOV V S, et al. Putative extracellular α-class carbonic anhydrase, EcaA, of Synechococcus elongatus PCC 7942 is an active enzyme: a sequel to an old story[J]. Microbiology, 2018, 164(4): 576-586.
65	ZINGARO K A, TERRY PAPOUTSAKIS E. GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1,2,4-butanetriol and ethanol with complex and unpredictable patterns[J]. Metabolic Engineering, 2013, 15: 196-205.
66	YOSHIKAWA K, HIRASAWA T, SHIMIZU H. Effect of malic enzyme on ethanol production by Synechocystis sp. PCC 6803[J]. Journal of Bioscience and Bioengineering, 2015, 119(1): 82-84.
67	TAKAHASHI H, UCHIMIYA H, HIHARA Y. Difference in metabolite levels between photoautotrophic and photomixotrophic cultures of Synechocystis sp. PCC 6803 examined by capillary electrophoresis electrospray ionization mass spectrometry[J]. Journal of Experimental Botany, 2008, 59(11): 3009-3018.
68	VIDAL R, LÓPEZ-MAURY L, GUERRERO M G, et al. Characterization of an alcohol dehydrogenase from the cyanobacterium Synechocystis sp. strain PCC 6803 that responds to environmental stress conditions via the Hik34-Rre1 two-component system[J]. Journal of Bacteriology, 2009, 191(13): 4383-4391.
69	JIANG Z Y, LÜ C Q, WU H. Photoregeneration of NADH using carbon-containing TiO₂ [J]. Industrial & Engineering Chemistry Research, 2005, 44(12): 4165-4170.
70	SHI Q, YANG D, JIANG Z Y, et al. Visible-light photocatalytic regeneration of NADH using P-doped TiO₂ nanoparticles[J]. Journal of Molecular Catalysis B: Enzymatic, 2006, 43(1/2/3/4): 44-48.
71	LUAN G D, LU X F. Tailoring cyanobacterial cell factory for improved industrial properties[J]. Biotechnology Advances, 2018, 36(2): 430-442.
72	ANFELT J, KACZMARZYK D, SHABESTARY K, et al. Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production[J]. Microbial Cell Factories, 2015, 14: 167.
73	VAN DER WOUDE A D, ANGERMAYR S A, PUTHAN VEETIL V, et al. Carbon sink removal: Increased photosynthetic production of lactic acid by Synechocystis sp. PCC6803 in a glycogen storage mutant[J]. Journal of Biotechnology, 2014, 184: 100-102.
74	KOCABAŞ P, ÇALIK P, ÇALIK G, et al. Analyses of extracellular protein production in Bacillus subtilis-Ⅰ: genome-scale metabolic model reconstruction based on updated gene-enzyme-reaction data[J]. Biochemical Engineering Journal, 2017, 127: 229-241.
75	SENGUPTA T, BHUSHAN M, WANGIKAR P P. Metabolic modeling for multi-objective optimization of ethanol production in a Synechocystis mutant[J]. Photosynthesis Research, 2013, 118(1/2): 155-165.
76	REPPAS N B. Metabolic switch: 20120164705[P/OL].2012-06-28[2023-07-01]. .
77	ERDRICH P, KNOOP H, STEUER R, et al. Cyanobacterial biofuels: new insights and strain design strategies revealed by computational modeling[J]. Microbial Cell Factories, 2014, 13(1): 128.
78	KNOOP H, STEUER R. A computational analysis of stoichiometric constraints and trade-offs in cyanobacterial biofuel production[J]. Frontiers in Bioengineering and Biotechnology, 2015, 3: 47.
79	KNOOP H, GRÜNDEL M, ZILLIGES Y, et al. Flux balance analysis of cyanobacterial metabolism: the metabolic network of Synechocystis sp. PCC 6803[J]. PLoS Computational Biology, 2013, 9(6): e1003081.
80	YU J J, LIBERTON M, CLIFTEN P F, et al. Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO₂ [J]. Scientific Reports, 2015, 5: 8132.
81	JAISWAL D, SENGUPTA A, SOHONI S, et al. Genome features and biochemical characteristics of a robust, fast growing and naturally transformable cyanobacterium Synechococcus elongatus PCC 11801 isolated from India[J]. Scientific Reports, 2018, 8: 16632.
82	WŁODARCZYK A, SELÃO T T, NORLING B, et al. Newly discovered Synechococcus sp. PCC 11901 is a robust cyanobacterial strain for high biomass production[J]. Communications Biology, 2020, 3(1): 215.
83	SENGUPTA S, JAISWAL D, SENGUPTA A, et al. Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid[J]. Biotechnology for Biofuels, 2020, 13: 89.
84	SENGUPTA A, PRITAM P, JAISWAL D, et al. Photosynthetic co-production of succinate and ethylene in a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801[J]. Metabolites, 2020, 10(6): 250.
85	SRIVASTAVA V, AMANNA R, ROWDEN S J L, et al. Adaptive laboratory evolution of the fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for improved solvent tolerance[J]. Journal of Bioscience and Bioengineering, 2021, 131(5): 491-500.
86	ABALDE-CELA S, GOULD A, LIU X, et al. High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform[J]. Journal of the Royal Society Interface, 2015, 12(106): 20150216.
87	CHEN D D, XU S M, LI S L, et al. Directly evolved AlkS-based biosensor platform for monitoring and high-throughput screening of alkane production[J]. ACS Synthetic Biology, 2023, 12(3): 832-841.
88	DU W, BURBANO P C, HELLINGWERF K J, et al. Challenges in the application of synthetic biology toward synthesis of commodity products by cyanobacteria via "direct conversion"[M/OL]//ZHANG W W, SONG X Y. Synthetic biology of cyanobacteria. Singapore: Springer, 2018: 3-26 [2023-07-01]. .
89	PIENKOS P T. Pilot scale integrated biorefinery for producing ethanol from hybrid algae: cooperative research and development final report, CRADA number CRD-10-389[R/OL]. 2013[2023-07-01]. .
90	SCHULZE K, LANG I, ENKE H K, et al. The use of fluorescence microscopy and image analysis for rapid detection of non-producing revertant cells of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002[J]. BMC Research Notes, 2015, 8: 160.
91	MOTOMURA K, SANO K, WATANABE S, et al. Synthetic phosphorus metabolic pathway for biosafety and contamination management of cyanobacterial cultivation[J]. ACS Synthetic Biology, 2018, 7(9): 2189-2198.
92	SELÃO T T, WŁODARCZYK A, NIXON P J, et al. Growth and selection of the cyanobacterium Synechococcus sp. PCC 7002 using alternative nitrogen and phosphorus sources[J]. Metabolic Engineering, 2019, 54: 255-263.

策略	菌株	操作	效果	参考文献
优化Adh2的活性	PCC 6803	采用集胞藻PCC 6803来源的偏好使用NADPH为辅因子的slr1192基因替代运动发酵单胞菌adh2基因	增强了乙醛还原催化步骤与蓝细菌代谢背景的适配性，将乙醇产率提高了50%	[41]
优化Pdc与Adh2的实际丰度和配比	PCC 6803	使用5种强度不同的RBS分别控制pdc与adh2的表达，并进行组合，评价其乙醇产量及酶活	发现乙醇合成通量更强烈地决定于pdc而非adh2的表达强度和丰度，在5种RBS控制的不同pdc表达强度的突变体中，pdc表达强度最高的藻株乙醇合成能力最强，培养7 d时的乙醇含量约为1 g/L	[44]
强化卡尔文循环以提高蓝细菌固碳效率并加强乙醇合成	PCC 6803	分别将RuBisCo、SBPase、FBA或TK基因与Pdc-Adh2途径共表达	分别将乙醇产量提高了55%、67%、37%和69%，同时，总生物量也分别增加7.7%、15.1%、8.8%和10.1%	[45]
		在表达Pdc-Adh2途径的基础上，共表达FBA与TK基因	相比于单独表达FBA藻株的乙醇产量提高9倍以上	[46]
		在表达Pdc-Adh2途径的基础上，共表达SBPase与FBA基因	相比FBA单独表达藻株的乙醇产量提高2.5倍	[46]
加强蓝细菌底盘藻株对无机碳源的吸收	PCC 7942	在表达Pdc-Adh2途径的基础上，过量表达ictB、ecaA以及groESL	提高了在模拟烟道气条件下藻株的生物质含量（提高约4倍，0.9 g/L，72 h）和乙醇含量（提高约20倍，0.2 g/L，72 h）	[47]
敲除丙酮酸消耗途径，并将TCA循环中的碳流重新导回丙酮酸节点以增强乙醇合成前体供应	PCC 6803	在表达Pdc-Adh2途径的基础上，敲除了PEP合成酶（PpsA）基因，催化糖原合成的关键酶（GlgC）基因，并过量表达大肠杆菌来源的苹果酸酶（MaeB）基因	乙醇产量提升至1.09 g/L（7 d）	[48]
阻断糖原合成途径	PCC 7002	将两个乙醇合成途径（Pdc-Adh2）拷贝整合至基因组中两个糖原合成酶（GlgA）编码基因的位点，从而同时加强乙醇合成通量并阻断糖原合成途径竞争	在实验室环境柱式反应器中乙醇产量达到2.2 g/L（10 d），在户外挂袋式培养中乙醇产量达到0.8 g/L（7 d）	[49]
阻断糖原和PHB的合成途径	PCC 6803	敲除糖原合成关键基因glgC	乙醇产量从0.212 g/L（3 d）提高至0.297 g/L（3 d）	[50]
		在以上基础上进一步敲除phaCE基因以阻断PHB合成路径	乙醇产量提高至0.332 g/L（3 d）	[50]
		对以上藻株进行缺氮处理	乙醇产量达到0.6 g/L（3 d）	[50]
共培养“碳汇”工程策略	PCC 6803	将敲除glgC和phaA基因以阻断糖原和PHB合成途径的藻株和整合了Pdc-Adh2途径的工程藻株进行共培养	双菌体系中的乙醇产量达到4.6 g/L（25 d），而单平台藻株（基因组同时进行乙醇合成途径整合和糖原、PHB合成途径阻断）的产量4.1 g/L（25 d）	[51]
补充还原力供应	PCC 6803	过量表达内源G6PDH编码基因zwf，并导入Pdc-Adh2途径	乙醇产量从0.44 g/L增加到0.59 g/L（14 d），同时生物量积累增加了50%	[42]
向蓝细菌培养体系中添加金属氧化物以介导NADPH再生	PCC 6803	在培养体系中添加MgO或Fe₂O₃	乙醇产量达到5.1 g/L或4.851 g/L（25 d）	[52]
区室化合成乙醇并靶向性模拟缺氮环境	PCC 7120	在异形胞中使用特异性启动的hupS启动子控制Pdc-Adh2的表达	乙醇产量达到1.68 g/L（23 d）	[53]
区室化合成乙醇并靶向性模拟缺氮环境	PCC 7120	在以上基础上，使用特异性靶向异形胞的CRISRPi基因表达系统抑制glnA的表达	乙醇产量提高了27%	[54]
通过优化的基因组尺度代谢网络模型，预测乙醇/生物量耦合突变体	PCC 6803	预测最优突变体为通过13个基因的敲除来达到耦联乙醇合成和细胞生长的效果	预测乙醇产量为3.498 g/L（4 d）	[55]

策略	菌株	操作	效果	参考文献
优化Adh2的活性	PCC 6803	采用集胞藻PCC 6803来源的偏好使用NADPH为辅因子的slr1192基因替代运动发酵单胞菌adh2基因	增强了乙醛还原催化步骤与蓝细菌代谢背景的适配性，将乙醇产率提高了50%	[41]
优化Pdc与Adh2的实际丰度和配比	PCC 6803	使用5种强度不同的RBS分别控制pdc与adh2的表达，并进行组合，评价其乙醇产量及酶活	发现乙醇合成通量更强烈地决定于pdc而非adh2的表达强度和丰度，在5种RBS控制的不同pdc表达强度的突变体中，pdc表达强度最高的藻株乙醇合成能力最强，培养7 d时的乙醇含量约为1 g/L	[44]
强化卡尔文循环以提高蓝细菌固碳效率并加强乙醇合成	PCC 6803	分别将RuBisCo、SBPase、FBA或TK基因与Pdc-Adh2途径共表达	分别将乙醇产量提高了55%、67%、37%和69%，同时，总生物量也分别增加7.7%、15.1%、8.8%和10.1%	[45]
		在表达Pdc-Adh2途径的基础上，共表达FBA与TK基因	相比于单独表达FBA藻株的乙醇产量提高9倍以上	[46]
		在表达Pdc-Adh2途径的基础上，共表达SBPase与FBA基因	相比FBA单独表达藻株的乙醇产量提高2.5倍	[46]
加强蓝细菌底盘藻株对无机碳源的吸收	PCC 7942	在表达Pdc-Adh2途径的基础上，过量表达ictB、ecaA以及groESL	提高了在模拟烟道气条件下藻株的生物质含量（提高约4倍，0.9 g/L，72 h）和乙醇含量（提高约20倍，0.2 g/L，72 h）	[47]
敲除丙酮酸消耗途径，并将TCA循环中的碳流重新导回丙酮酸节点以增强乙醇合成前体供应	PCC 6803	在表达Pdc-Adh2途径的基础上，敲除了PEP合成酶（PpsA）基因，催化糖原合成的关键酶（GlgC）基因，并过量表达大肠杆菌来源的苹果酸酶（MaeB）基因	乙醇产量提升至1.09 g/L（7 d）	[48]
阻断糖原合成途径	PCC 7002	将两个乙醇合成途径（Pdc-Adh2）拷贝整合至基因组中两个糖原合成酶（GlgA）编码基因的位点，从而同时加强乙醇合成通量并阻断糖原合成途径竞争	在实验室环境柱式反应器中乙醇产量达到2.2 g/L（10 d），在户外挂袋式培养中乙醇产量达到0.8 g/L（7 d）	[49]
阻断糖原和PHB的合成途径	PCC 6803	敲除糖原合成关键基因glgC	乙醇产量从0.212 g/L（3 d）提高至0.297 g/L（3 d）	[50]
		在以上基础上进一步敲除phaCE基因以阻断PHB合成路径	乙醇产量提高至0.332 g/L（3 d）	[50]
		对以上藻株进行缺氮处理	乙醇产量达到0.6 g/L（3 d）	[50]
共培养“碳汇”工程策略	PCC 6803	将敲除glgC和phaA基因以阻断糖原和PHB合成途径的藻株和整合了Pdc-Adh2途径的工程藻株进行共培养	双菌体系中的乙醇产量达到4.6 g/L（25 d），而单平台藻株（基因组同时进行乙醇合成途径整合和糖原、PHB合成途径阻断）的产量4.1 g/L（25 d）	[51]
补充还原力供应	PCC 6803	过量表达内源G6PDH编码基因zwf，并导入Pdc-Adh2途径	乙醇产量从0.44 g/L增加到0.59 g/L（14 d），同时生物量积累增加了50%	[42]
向蓝细菌培养体系中添加金属氧化物以介导NADPH再生	PCC 6803	在培养体系中添加MgO或Fe₂O₃	乙醇产量达到5.1 g/L或4.851 g/L（25 d）	[52]
区室化合成乙醇并靶向性模拟缺氮环境	PCC 7120	在异形胞中使用特异性启动的hupS启动子控制Pdc-Adh2的表达	乙醇产量达到1.68 g/L（23 d）	[53]
区室化合成乙醇并靶向性模拟缺氮环境	PCC 7120	在以上基础上，使用特异性靶向异形胞的CRISRPi基因表达系统抑制glnA的表达	乙醇产量提高了27%	[54]
通过优化的基因组尺度代谢网络模型，预测乙醇/生物量耦合突变体	PCC 6803	预测最优突变体为通过13个基因的敲除来达到耦联乙醇合成和细胞生长的效果	预测乙醇产量为3.498 g/L（4 d）	[55]

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面向高效光驱固碳产醇的蓝细菌合成生物技术研究进展

Progress of cyanobacterial synthetic biotechnology for efficient light-driven carbon fixation and ethanol production

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