真核微藻脂质代谢工程的研究进展和展望

doi:10.12211/2096-8280.2023-044

摘要/Abstract

摘要：

真核微藻作为一类重要的生物资源，脂质含量高，广泛应用于能源、化工和食品等领域。然而，真核微藻生物能源成本偏高，其产业化应用仍然面临着一系列挑战。通过代谢工程手段改造微藻，促进脂质的合成与积累，可提高微藻脂质生产的经济可行性。本文介绍真核微藻脂质代谢途径和关键酶基因，并总结了不同培养条件下代谢途径相关基因在转录水平上的变化。还探讨通过代谢工程调控脂质合成相关酶、转录因子和竞争途径等方法，以提高微藻脂质含量和调整脂肪酸组成。通过基因组学、转录组学和蛋白质组学数据的整合分析可揭示脂质代谢中的关键节点和主效调控因子，有助于确定代谢工程的潜在目标。此外，基因工具和基因编辑技术的开发和拓展可显著提高转化效率，实现对微藻底盘细胞的精准改造。通过重塑能量和碳代谢途径，可设计优化微藻脂质生物合成过程。在微藻遗传工具、基因编辑技术、代谢通路调控和产业化等方面的进一步研究和探索对于推动微藻脂质工程的研究和发展具有重要意义。

关键词: 真核微藻, 生物质精制, 脂质代谢, 能量代谢, 合成生物学

Abstract:

Microalgae represent a diverse group of photosynthetic organisms that are widely found in various ecosystems on the Earth. They play a crucial role in carbon dioxide bio-fixation. Apart from their efficient growth through photosynthesis, many microalgae can also grow robustly under heterotrophic and mixotrophic conditions for high biomass production. Due to their high lipid content and the presence of diverse fatty acid and lipid species, microalgae have a wide range of applications in industries of energy, chemicals, and food. However, the high production cost associated with microalgae-based bioenergy poses a significant challenge for large-scale implementation. To overcome this, there is a growing interest in engineering microalgae to enhance lipid biosynthesis and accumulation, which holds promise for improving the economic feasibility of microalgal lipid production. This requires a better understanding of lipid metabolism and regulation in microalgae. This article provides an overview of recent advances in the elucidation of lipid metabolic pathways, the roles of key enzyme genes involved in lipid metabolism, and the transcriptional regulation of lipid metabolic pathways under different cultivation conditions in eukaryotic microalgae. It also summarizes strategies for metabolic engineering aiming for manipulating lipid biosynthesis-related enzymes, transcription factors, and competing pathways to increase lipid content and/or modify fatty acid composition in microalgae. Integrated analysis of genomics, transcriptomics, and proteomics data can help identify crucial nodes and key regulators in lipid metabolism, facilitating the identification of potential targets for metabolic engineering. Furthermore, the rapid development of genetic tools and gene editing technologies has significantly improved transformation efficiency and enabled precise gene modification, providing a foundation for genetic engineering of microalgae. By reshaping energy and carbon metabolic pathways, it becomes possible to design and optimize lipid biosynthesis processes in microalgae for a better production. Further research and exploration in genetic tools, gene editing technologies, metabolic pathway regulation, and large-scale implementation are of utmost importance for driving the research and development of microalgal lipid engineering.

Key words: eukaryotic microalgae, biorefineries, lipid metabolism, energy metabolism, synthetic biology

中图分类号:

Q819

孙翰, 刘进. 真核微藻脂质代谢工程的研究进展和展望[J]. 合成生物学, 2023, 4(6): 1140-1160.

Han SUN, Jin LIU. Research progress and prospects in lipid metabolic engineering of eukaryotic microalgae[J]. Synthetic Biology Journal, 2023, 4(6): 1140-1160.

图/表 5

参考文献 165

1	KATIYAR R, GURJAR B R, BISWAS S, et al. Microalgae: an emerging source of energy based bio-products and a solution for environmental issues[J]. Renewable and Sustainable Energy Reviews, 2017, 72: 1083-1093.
2	CARNEIRO M L N M, PRADELLE F, BRAGA S L, et al. Potential of biofuels from algae: comparison with fossil fuels, ethanol and biodiesel in Europe and Brazil through life cycle assessment (LCA)[J]. Renewable and Sustainable Energy Reviews, 2017, 73: 632-653.
3	HEPBURN C, ADLEN E, BEDDINGTON J, et al. The technological and economic prospects for CO₂ utilization and removal[J]. Nature, 2019, 575(7781): 87-97.
4	TZACHOR A, RICHARDS C E, HOLT L. Future foods for risk-resilient diets[J]. Nature Food, 2021, 2(5): 326-329.
5	ADESANYA V O, CADENA E, SCOTT S A, et al. Life cycle assessment on microalgal biodiesel production using a hybrid cultivation system[J]. Bioresource Technology, 2014, 163: 343-355.
6	KUMAR K, MISHRA S K, SHRIVASTAV A, et al. Recent trends in the mass cultivation of algae in raceway ponds[J]. Renewable and Sustainable Energy Reviews, 2015, 51: 875-885.
7	TIBOCHA-BONILLA J D, ZUÑIGA C, GODOY-SILVA R D, et al. Advances in metabolic modeling of oleaginous microalgae[J].Biotechnology for Biofuels, 2018, 11(1): 1241.
8	LI J, ZHU D L, NIU J F, et al. An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis [J]. Biotechnology Advances, 2011, 29(6): 568-574.
9	RUIZ J, OLIVIERI G, DE VREE J, et al. Towards industrial products from microalgae[J]. Energy & Environmental Science, 2016, 9(10): 3036-3043.
10	MASRI M A, GARBE D, MEHLMER N, et al. A sustainable, high-performance process for the economic production of waste-free microbial oils that can replace plant-based equivalents[J]. Energy & Environmental Science, 2019, 12(9): 2717-2732.
11	SUN H, MAO X M, WU T, et al. Novel insight of carotenoid and lipid biosynthesis and their roles in storage carbon metabolism in Chlamydomonas reinhardtii [J]. Bioresource Technology, 2018, 263: 450-457.
12	LAURENS L M L, MARKHAM J, TEMPLETON D W, et al. Development of algae biorefinery concepts for biofuels and bioproducts; a perspective on process-compatible products and their impact on cost-reduction[J]. Energy & Environmental Science, 2017, 10(8): 1716-1738.
13	ROTH M S, COKUS S J, GALLAHER S D, et al. Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(21): E4296-E4305.
14	BLANC-MATHIEU R, SANCHEZ-FERANDIN S, EYRE-WALKER A, et al. Organellar inheritance in the green lineage: insights from Ostreococcus tauri [J]. Genome Biology and Evolution, 2013, 5(8): 1503-1511.
15	WEISS T L, JOHNSTON J S, FUJISAWA K, et al. Genome size and phylogenetic analysis of the A and L races of Botryococcus braunii [J]. Journal of Applied Phycology, 2011, 23(5): 833-839.
16	MERCHANT S S, PROCHNIK S E, VALLON O, et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions[J]. Science, 2007, 318(5848): 245-250.
17	FAN J H, NING K, ZENG X W, et al. Genomic foundation of starch-to-lipid switch in oleaginous Chlorella spp[J]. Plant Physiology, 2015, 169(4): 2444-2461.
18	GUARNIERI M T, BOORE J L, HENARD C A, et al. LDKB00000000.1 Chlorella vulgaris [DB/OL]. NCBI, 2015[2023-06-01]. .
19	BROUARD J S, OTIS C, LEMIEUX C, et al. The exceptionally large chloroplast genome of the green alga Floydiella terrestris illuminates the evolutionary history of the Chlorophyceae[J]. Genome Biology and Evolution, 2010, 2: 240-256.
20	PALENIK B, GRIMWOOD J, AERTS A, et al. The tiny eukaryote Ostreococcus provides genomic insights into the paradox of plankton speciation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(18): 7705-7710.
21	WORDEN A Z, LEE J H, MOCK T, et al. Green evolution and dynamic adaptations revealed by genomes of the marine picoeukaryotes Micromonas [J]. Science, 2009, 324(5924): 268-272.
22	PROCHNIK S E, UMEN J, NEDELCU A M, et al. Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri [J]. Science, 2010, 329(5988): 223-226.
23	BLANC-MATHIEU R, VERHELST B, DERELLE E, et al. An improved genome of the model marine alga Ostreococcus tauri unfolds by assessing Illumina de novo assemblies[J].BMC Genomics, 2014, 15: 1103.
24	GAO C F, WANG Y, SHEN Y, et al. Oil accumulation mechanisms of the oleaginous microalga Chlorella protothecoides revealed through its genome, transcriptomes, and proteomes[J].BMC Genomics, 2014, 15: 582.
25	BOWLER C, ALLEN A E, BADGER J H, et al. The Phaeodactylum genome reveals the evolutionary history of diatom genomes[J]. Nature, 2008, 456(7219): 239-244.
26	ARMBRUST E V, BERGES J A, BOWLER C, et al. The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[J]. Science, 2004, 306(5693): 79-86.
27	NOZAKI H, TAKANO H, MISUMI O, et al. A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae [J].BMC Biology, 2007, 5: 28.
28	OHTA N, MATSUZAKI M, MISUMI O, et al. Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae [J]. DNA Research, 2003, 10(2): 67-77.
29	TAJIMA N, SATO S, MARUYAMA F, et al. Analysis of the complete plastid genome of the unicellular red alga Porphyridium purpureum [J]. Journal of Plant Research, 2014, 127(3): 389-397.
30	WANG D M, NING K, LI J, et al. Nannochloropsis genomes reveal evolution of microalgal oleaginous traits[J]. PLoS Genetics, 2014, 10(1): e1004094.
31	RADAKOVITS R, JINKERSON R E, FUERSTENBERG S I, et al. Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana [J]. Nature Communications, 2012, 3: 686.
32	MUÑOZ C F, SÜDFELD C, NADUTHODI M I S, et al. Genetic engineering of microalgae for enhanced lipid production[J]. Biotechnology Advances, 2021, 52: 107836.
33	JIA J, HAN D X, GERKEN H G, et al. Molecular mechanisms for photosynthetic carbon partitioning into storage neutral lipids in Nannochloropsis oceanica under nitrogen-depletion conditions[J]. Algal Research, 2015, 7: 66-77.
34	LIU J, SUN Z, MAO X M, et al. Multiomics analysis reveals a distinct mechanism of oleaginousness in the emerging model alga Chromochloris zofingiensis [J]. The Plant Journal, 2019, 98(6): 1060-1077.
35	KAMALANATHAN M, PIERANGELINI M, SHEARMAN L A, et al. Impacts of nitrogen and phosphorus starvation on the physiology of Chlamydomonas reinhardtii [J]. Journal of Applied Phycology, 2016, 28(3): 1509-1520.
36	LIANG J B, IQBAL S, WEN F, et al. Phosphorus-induced lipid class alteration revealed by lipidomic and transcriptomic profiling in oleaginous microalga Nannochloropsis sp. PJ12[J]. Marine Drugs, 2019, 17(9): 519.
37	JIN X J, GONG S Q, YANG B J, et al. Transcriptomic analysis for phosphorus limitation-induced β-glucans accumulation in Chlorella sorokiniana SCSIO 46784 during the early phase of growth[J]. Algal Research, 2021, 54: 102208.
38	HULATT C J, SMOLINA I, DOWLE A, et al. Proteomic and transcriptomic patterns during lipid remodeling in Nannochloropsis gaditana [J]. International Journal of Molecular Sciences, 2020, 21(18): 6946.
39	MANDOTRA S K, KUMAR P, SUSEELA M R, et al. Evaluation of fatty acid profile and biodiesel properties of microalga Scenedesmus abundans under the influence of phosphorus, pH and light intensities[J]. Bioresource Technology, 2016, 201: 222-229.
40	HE Q N, YANG H J, WU L, et al. Effect of light intensity on physiological changes, carbon allocation and neutral lipid accumulation in oleaginous microalgae[J]. Bioresource Technology, 2015, 191: 219-228.
41	LI Y L, SUN H, WANG Y N, et al. Integrated metabolic tools reveal carbon alternative in Isochrysis zhangjiangensis for fucoxanthin improvement[J]. Bioresource Technology, 2022, 347: 126401.
42	ATTA M, IDRIS A, BUKHARI A, et al. Intensity of blue LED light: a potential stimulus for biomass and lipid content in fresh water microalgae Chlorella vulgaris [J]. Bioresource Technology, 2013, 148: 373-378.
43	GAYTÁN-LUNA D E, OCHOA-ALFARO A E, ROCHA-URIBE A, et al. Effect of green and red light in lipid accumulation and transcriptional profile of genes implicated in lipid biosynthesis in Chlamydomonas reinhardtii [J]. Biotechnology Progress, 2016, 32(6): 1404-1411.
44	VIELER A, WU G X, TSAI C H, et al. Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779[J]. PLoS Genetics, 2012, 8(11): e1003064.
45	POLINER E, BUSCH A W U, NEWTON L, et al. Aureochromes maintain polyunsaturated fatty acid content in Nannochloropsis oceanica [J]. Plant Physiology, 2022, 189(2): 906-921.
46	PATELOU M, INFANTE C, DARDELLE F, et al. Transcriptomic and metabolomic adaptation of Nannochloropsis gaditana grown under different light regimes[J]. Algal Research, 2020, 45: 101735.
47	WU L F, CHEN P C, LEE C M. The effects of nitrogen sources and temperature on cell growth and lipid accumulation of microalgae[J]. International Biodeterioration & Biodegradation, 2013, 85: 506-510.
48	CALHOUN S, BELL T A S, DAHLIN L R, et al. A multi-omic characterization of temperature stress in a halotolerant Scenedesmus strain for algal biotechnology[J]. Communications Biology, 2021, 4: 333.
49	XING G L, YUAN H L, YANG J S, et al. Integrated analyses of transcriptome, proteome and fatty acid profilings of the oleaginous microalga Auxenochlorella protothecoides UTEX 2341 reveal differential reprogramming of fatty acid metabolism in response to low and high temperatures[J]. Algal Research, 2018, 33: 16-27.
50	SU H, FENG J, LV J P, et al. Molecular mechanism of lipid accumulation and metabolism of oleaginous Chlorococcum sphacosum GD from soil under salt stress[J]. International Journal of Molecular Sciences, 2021, 22(3): 1304.
51	QU D H, MIAO X L. Carbon flow conversion induces alkali resistance and lipid accumulation under alkaline conditions based on transcriptome analysis in Chlorella sp. BLD[J]. Chemosphere, 2021, 265: 129046.
52	SINGH R D, SETHY S, GHOSH S, et al. UV and γ-radiation induced molecular changes for rapid lipid accumulation in Chlorella sorokiniana [J]. Biomass and Bioenergy, 2022, 163: 106493.
53	NAJAFABADI H A, MALEKZADEH M, JALILIAN F, et al. Effect of various carbon sources on biomass and lipid production of Chlorella vulgaris during nutrient sufficient and nitrogen starvation conditions[J]. Bioresource Technology, 2015, 180: 311-317.
54	UDAYAN A, ARUMUGAM M. Selective enrichment of eicosapentaenoic acid (20:5n-3) in N. oceanica CASA CC201 by natural auxin supplementation[J]. Bioresource Technology, 2017, 242: 329-333.
55	XUE L L, JIANG J G. Cultivation of Dunaliella tertiolecta intervened by triethylamine enhances the lipid content[J]. Algal Research, 2017, 25: 136-141.
56	ROESSLER P G. Changes in the activities of various lipid and carbohydrate biosynthetic enzymes in the diatom Cyclotella cryptica in response to silicon deficiency[J]. Archives of Biochemistry and Biophysics, 1988, 267(2): 521-528.
57	DUNAHAY T G, JARVIS E E, DAIS S S, et al. Manipulation of microalgal lipid production using genetic engineering[J]. Applied Biochemistry and Biotechnology, 1996, 57: 223-231.
58	GOMMA A E, LEE S K, SUN S M, et al. Improvement in oil production by increasing malonyl-CoA and glycerol-3-phosphate pools in Scenedesmus quadricauda [J]. Indian Journal of Microbiology, 2015, 55(4): 447-455.
59	CHEN J W, LIU W J, HU D X, et al. Identification of a malonyl CoA-acyl carrier protein transacylase and its regulatory role in fatty acid biosynthesis in oleaginous microalga Nannochloropsis oceanica [J]. Biotechnology and Applied Biochemistry, 2017, 64(5): 620-626.
60	RENGEL R, SMITH R T, HASLAM R P, et al. Overexpression of acetyl-CoA synthetase (ACS) enhances the biosynthesis of neutral lipids and starch in the green microalga Chlamydomonas reinhardtii [J]. Algal Research, 2018, 31: 183-193.
61	YAN J F, CHENG R B, LIN X Z, et al. Overexpression of acetyl-CoA synthetase increased the biomass and fatty acid proportion in microalga Schizochytrium [J]. Applied Microbiology and Biotechnology, 2013, 97(5): 1933-1939.
62	OSADA K, MAEDA Y, YOSHINO T, et al. Enhanced NADPH production in the pentose phosphate pathway accelerates lipid accumulation in the oleaginous diatom Fistulifera solaris [J]. Algal Research, 2017, 23: 126-134.
63	NIU Y F, WANG X, HU D X, et al. Molecular characterization of a glycerol-3-phosphate acyltransferase reveals key features essential for triacylglycerol production in Phaeodactylum tricornutum [J].Biotechnology for Biofuels, 2016, 9: 60.
64	MUÑOZ C F, WEUSTHUIS R A, D'ADAMO S, et al. Effect of single and combined expression of lysophosphatidic acid acyltransferase, glycerol-3-phosphate acyltransferase, and diacylglycerol acyltransferase on lipid accumulation and composition in Neochloris oleoabundans [J]. Frontiers in Plant Science, 2019, 10: 1573.
65	MARAVI D K, KUMAR S, SHARMA P K, et al. Ectopic expression of AtDGAT1, encoding diacylglycerol O-acyltransferase exclusively committed to TAG biosynthesis, enhances oil accumulation in seeds and leaves of Jatropha [J]. Biotechnology for Biofuels, 2016, 9: 226.
66	WANG Z K, HUANG W J, CHANG J M, et al. Overexpression of SiDGAT1, a gene encoding acyl-CoA: diacylglycerol acyltransferase from Sesamum indicum L. increases oil content in transgenic Arabidopsis and soybean[J]. Plant Cell, Tissue and Organ Culture (PCTOC), 2014, 119(2): 399-410.
67	NIU Y F, ZHANG M H, LI D W, et al. Improvement of neutral lipid and polyunsaturated fatty acid biosynthesis by overexpressing a type 2 diacylglycerol acyltransferase in marine diatom Phaeodactylum tricornutum [J]. Marine Drugs, 2013, 11(11): 4558-4569.
68	LI D W, CEN S Y, LIU Y H, et al. A type 2 diacylglycerol acyltransferase accelerates the triacylglycerol biosynthesis in heterokont oleaginous microalga Nannochloropsis oceanica [J]. Journal of Biotechnology, 2016, 229: 65-71.
69	DENG X D, GU B, LI Y J, et al. The roles of acyl-CoA: diacylglycerol acyltransferase 2 genes in the biosynthesis of triacylglycerols by the green algae Chlamydomonas reinhardtii [J]. Molecular Plant, 2012, 5(4): 945-947.
70	COUSO I, PÉREZ-PÉREZ M E, FORD M M, et al. Phosphorus availability regulates TORC1 signaling via LST8 in Chlamydomonas [J]. The Plant Cell, 2020, 32(1): 69-80.
71	NGAN C Y, WONG C H, CHOI C, et al. Lineage-specific chromatin signatures reveal a regulator of lipid metabolism in microalgae[J]. Nature Plants, 2015, 1: 15107.
72	BAJHAIYA A K, DEAN A P, ZEEF L A H, et al. PSR1 is a global transcriptional regulator of phosphorus deficiency responses and carbon storage metabolism in Chlamydomonas reinhardtii [J]. Plant Physiology, 2016, 170(3): 1216-1234.
73	JIA B, XIE X F, WU M, et al. Understanding the functions of endogenous DOF transcript factor in Chlamydomonas reinhardtii [J]. Biotechnology for Biofuels, 2019, 12: 67.
74	ZHANG J H, HAO Q, BAI L L, et al. Overexpression of the soybean transcription factor GmDof4 significantly enhances the lipid content of Chlorella ellipsoidea [J]. Biotechnology for Biofuels, 2014, 7: 128.
75	LI D W, BALAMURUGAN S, YANG Y F, et al. Transcriptional regulation of microalgae for concurrent lipid overproduction and secretion[J]. Science Advances, 2019, 5(1): eaau3795.
76	YAMAOKA Y, SHIN S, CHOI B Y, et al. The bZIP1 transcription factor regulates lipid remodeling and contributes to ER stress management in Chlamydomonas reinhardtii [J]. The Plant Cell, 2019, 31(5): 1127-1140.
77	AJJAWI I, VERRUTO J, AQUI M, et al. Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator[J]. Nature Biotechnology, 2017, 35(7): 647-652.
78	RADAKOVITS R, EDUAFO P M, POSEWITZ M C. Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum [J]. Metabolic Engineering, 2011, 13: 89-95.
79	GONG Y M, GUO X J, WAN X, et al. Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum [J]. Journal of Basic Microbiology, 2011, 51(6): 666-672.
80	PENG K T, ZHENG C N, XUE J, et al. Delta 5 fatty acid desaturase upregulates the synthesis of polyunsaturated fatty acids in the marine diatom Phaeodactylum tricornutum [J]. Journal of Agricultural and Food Chemistry, 2014, 62(35): 8773-8776.
81	HAMILTON M L, HASLAM R P, NAPIER J A, et al. Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids[J]. Metabolic Engineering, 2014, 22: 3-9.
82	SHI H S, LUO X, WU R N, et al. Production of eicosapentaenoic acid by application of a delta-6 desaturase with the highest ALA catalytic activity in algae[J]. Microbial Cell Factories, 2018, 17: 7.
83	POLINER E, PULMAN J A, ZIENKIEWICZ K, et al. A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production[J]. Plant Biotechnology Journal, 2018, 16: 298-309.
84	NORASHIKIN M N, LOH S H, AZIZ A, et al. Metabolic engineering of fatty acid biosynthesis in Chlorella vulgaris using an endogenous omega-3 fatty acid desaturase gene with its promoter[J]. Algal Research, 2018, 31: 262-275.
85	WANG X, DONG H P, WEI W, et al. Dual expression of plastidial GPAT1 and LPAT1 regulates triacylglycerol production and the fatty acid profile in Phaeodactylum tricornutum [J].Biotechnology for Biofuels, 2018, 11: 318.
86	LIU J, LIU M J, PAN Y F, et al. Metabolic engineering of the oleaginous alga Nannochloropsis for enriching eicosapentaenoic acid in triacylglycerol by combined pulling and pushing strategies[J]. Metabolic Engineering, 2022, 69: 163-174.
87	XIN Y, LU Y D, LEE Y Y, et al. Producing designer oils in industrial microalgae by rational modulation of Co-evolving type-2 diacylglycerol acyltransferases[J]. Molecular Plant, 2017, 10(12): 1523-1539.
88	LU N, WEI D, JIANG X L, et al. Regulation of lipid metabolism in the snow alga Chlamydomonas nivalis in response to NaCl stress: an integrated analysis by cytomic and lipidomic approaches[J]. Process Biochemistry, 2012, 47(7): 1163-1170.
89	SUN H, REN Y Y, MAO X M, et al. Harnessing C/N balance of Chromochloris zofingiensis to overcome the potential conflict in microalgal production[J]. Communications Biology, 2020, 3: 186.
90	SUN H, WU T, CHEN S H Y, et al. Powerful tools for productivity improvements in microalgal production[J]. Renewable and Sustainable Energy Reviews, 2021, 152: 111609.
91	KONG F T, LIANG Y X, LÉGERET B, et al. Chlamydomonas carries out fatty acid β-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase[J]. The Plant Journal, 2017, 90(2): 358-371.
92	CRAIG W, GARGANO D, SCOTTI N, et al. Direct gene transfer in potato: a comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts[J]. Plant Cell Reports, 2005, 24(10): 603-611.
93	NG S L, HARIKRISHNA J A, BAKAR F ABU, et al. Heterologous expression of the Streptococcus pneumoniae yoeB and pezT toxin genes is lethal in Chlorella vulgaris [J]. Algal Research, 2016, 19: 21-29.
94	YANG B, LIU J, LIU B, et al. Development of a stable genetic system for Chlorella vulgaris—a promising green alga for CO₂ biomitigation[J]. Algal Research, 2015, 12: 134-141.
95	TALEBI A F, TOHIDFAR M, TABATABAEI M, et al. Genetic manipulation, a feasible tool to enhance unique characteristic of Chlorella vulgaris as a feedstock for biodiesel production[J]. Molecular Biology Reports, 2013, 40(7): 4421-4428.
96	LIU L L, WANG Y Q, ZHANG Y C, et al. Development of a new method for genetic transformation of the green alga Chlorella ellipsoidea [J]. Molecular Biotechnology, 2013, 54(2): 211-219.
97	BAI L L, YIN W B, CHEN Y H, et al. A new strategy to produce a defensin: stable production of mutated NP-1 in nitrate reductase-deficient Chlorella ellipsoidea [J]. PLoS One, 2013, 8(1): e54966.
98	HUANG C C, CHEN M W, HSIEH J L, et al. Expression of mercuric reductase from Bacillus megaterium MB1 in eukaryotic microalga Chlorella sp. DT: an approach for mercury phytoremediation[J]. Applied Microbiology and Biotechnology, 2006, 72(1): 197-205.
99	LIU J, SUN Z, GERKEN H, et al. Genetic engineering of the green alga Chlorella zofingiensis: a modified norflurazon-resistant phytoene desaturase gene as a dominant selectable marker[J]. Applied Microbiology and Biotechnology, 2014, 98(11): 5069-5079.
100	LI F J, GAO D W, HU H H. High-efficiency nuclear transformation of the oleaginous marine Nannochloropsis species using PCR product[J]. Bioscience, Biotechnology, and Biochemistry, 2014, 78(5): 812-817.
101	KILIAN O, BENEMANN C S E, NIYOGI K K, et al. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.[J]. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(52): 21265-21269.
102	KANG N K, JEON S, KWON S, et al. Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina [J].Biotechnology for Biofuels, 2015, 8: 200.
103	WEI L, XIN Y, WANG Q T, et al. RNAi-based targeted gene knockdown in the model oleaginous microalgae Nannochloropsis oceanica [J]. The Plant Journal, 2017, 89(6): 1236-1250.
104	SHIH C H, CHEN H Y, LEE H C, et al. Purple chromoprotein gene serves as a new selection marker for transgenesis of the microalga Nannochloropsis oculata [J]. PLoS One, 2015, 10(3): e0120780.
105	SIMON D P, ANILA N, GAYATHRI K, et al. Heterologous expression of β-carotene hydroxylase in Dunaliella salina by Agrobacterium-mediated genetic transformation[J]. Algal Research, 2016, 18: 257-265.
106	FENG S Y, FENG W P, ZHAO L, et al. Preparation of transgenic Dunaliella salina for immunization against white spot syndrome virus in crayfish[J]. Archives of Virology, 2014, 159(3): 519-525.
107	姜国忠, 吕玉民, 牛向丽, 等. 盐藻肌动蛋白基因启动子驱动的bar基因表达作为核转化筛选标记[J]. 遗传学报, 2005, 32(4): 424-433.
	JIANG G Z, LÜ Y M, NIU X L, et al. The actin gene promoter-driven bar as a dominant selectable marker for nuclear transformation of Dunaliella salina [J]. Acta Genetica Sinica, 2005, 32(4): 424-433.
108	ZHANG J Y, JEON H, SIM S J, et al. Homologous sense and antisense expression of a gene in Dunaliella tertiolecta [J]. Planta, 2015, 242(4): 1051-1058.
109	GEORGIANNA D R, HANNON M J, MARCUSCHI M, et al. Production of recombinant enzymes in the marine alga Dunaliella tertiolecta [J]. Algal Research, 2013, 2(1): 2-9.
110	KADONO T, MIYAGAWA-YAMAGUCHI A, KIRA N, et al. Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum [J]. Scientific Reports, 2015, 5: 18708.
111	ZHANG C Y, HU H H. High-efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation[J]. Marine Genomics, 2014, 16: 63-66.
112	POULSEN N, CHESLEY P M, KRÖGER N. Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae)[J]. Journal of Phycology, 2006, 42(5): 1059-1065.
113	SHARON-GOJMAN R, MAIMON E, LEU S, et al. Advanced methods for genetic engineering of Haematococcus pluvialis (Chlorophyceae, Volvocales)[J]. Algal Research, 2015, 10: 8-15.
114	GUTIÉRREZ C L, GIMPEL J, ESCOBAR C, et al. Chloroplast genetic tool for the green microalgae Haematococcus pluvialis (Chlorophyceae, Volvocales)₁ [J]. Journal of Phycology, 2012, 48(4): 976-983.
115	STEINBRENNER J, SANDMANN G. Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis[J]. Applied and Environmental Microbiology, 2006, 72(12): 7477-7484.
116	KATHIRESAN S, CHANDRASHEKAR A, RAVISHANKAR G A, et al. Agrobacterium-mediated transformation in the green alga Haematococcus pluvialis(Chlorophyceae, Volvocales)[J]. Journal of Phycology, 2009, 45(3): 642-649.
117	MUTO M, FUKUDA Y, NEMOTO M, et al. Establishment of a genetic transformation system for the marine pennate diatom Fistulifera sp. strain JPCC DA0580—a high triglyceride producer[J]. Marine Biotechnology, 2013, 15(1): 48-55.
118	JAEGER D, HÜBNER W, HUSER T, et al. Nuclear transformation and functional gene expression in the oleaginous microalga Monoraphidium neglectum [J]. Journal of Biotechnology, 2017, 249: 10-15.
119	CHUNGJATUPORNCHAI W, KITRAKSA P, FA-AROONSAWAT S. Stable nuclear transformation of the oleaginous microalga Neochloris oleoabundans by electroporation[J].Journal of Applied Phycology, 2016, 28(1): 191-199.
120	BERRIOS H, ZAPATA M, RIVAS M. A method for genetic transformation of Botryococcus braunii using a cellulase pretreatment[J]. Journal of Applied Phycology, 2016, 28(1): 201-208.
121	ÚBEDA-MÍNGUEZ P, CHILEH T, DAUTOR Y, et al. Tools for microalgal biotechnology: development of an optimized transformation method for an industrially promising microalga—Tetraselmis chuii [J]. Journal of Applied Phycology, 2015, 27(1): 223-232.
122	ORTIZ-MATAMOROS M F, VILLANUEVA M A, ISLAS-FLORES T. Transient transformation of cultured photosynthetic dinoflagellates (Symbiodinium spp.) with plant-targeted vectors[J]. Ciencias Marinas, 2015, 41(1): 21-32.
123	ZORIN B, GRUNDMAN O, KHOZIN-GOLDBERG I, et al. Development of a nuclear transformation system for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and genetic complementation of a mutant strain, deficient in arachidonic acid biosynthesis[J]. PLoS One, 2014, 9(8): e105223.
124	PRASAD B, VADAKEDATH N, JEONG H J, et al. Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species)[J].Applied Microbiology and Biotechnology, 2014, 98(20): 8629-8639.
125	RATHOD J P, PRAKASH G, PANDIT R, et al. Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri [J].Photosynthesis Research, 2013, 118(1/2): 141-146.
126	HONG W K, HEO S Y, OH B R, et al. A transgene expression system for the marine microalgae Aurantiochytrium sp. KRS101 using a mutant allele of the gene encoding ribosomal protein L44 as a selectable transformation marker for cycloheximide resistance[J]. Bioprocess and Biosystems Engineering, 2013, 36(9): 1191-1197.
127	GUO S L, ZHAO X Q, TANG Y, et al. Establishment of an efficient genetic transformation system in Scenedesmus obliquus [J]. Journal of Biotechnology, 2013, 163(1): 61-68.
128	CHENG R B, MA R J, LI K, et al. Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium [J]. Microbiological Research, 2012, 167(3): 179-186.
129	CUI Y L, WANG J F, JIANG P, et al. Transformation of Platymonas (Tetraselmis) subcordiformis (Prasinophyceae, Chlorophyta) by agitation with glass beads[J]. World Journal of Microbiology and Biotechnology, 2010, 26(9): 1653-1657.
130	LAPIDOT M, RAVEH D, SIVAN A, et al. Stable chloroplast transformation of the unicellular red alga Porphyridium species[J]. Plant Physiology, 2002, 129: 7-12.
131	KAWATA Y, YANO S, KOJIMA H, et al. Transformation of Spirulina platensis strain C1 (Arthrospira sp. PCC9438) with Tn5 transposase-transposon DNA-cation liposome complex[J]. Marine Biotechnology, 2004, 6(4): 355-363.
132	JAENICKE L, KUHNE W, SPESSERT R, et al. Cell-wall lytic enzymes (autolysins) of Chlamydomonas reinhardtii are (hydroxy)proline-specific proteases[J]. European Journal of Biochemistry, 1987, 170(1/2): 485-491.
133	ZHU Z, SUN J, FA Y, et al. Enhancing microalgal lipid accumulation for biofuel production[J]. Frontiers in Microbiology, 2022, 13: 1024441.
134	WU X G, GIRIDHAR BABU A, KIM B L, et al. Ultrasound-mediated intracellular delivery of fluorescent dyes and DNA into microalgal cells[J]. Algal Research, 2016, 15: 210-216.
135	KINDLE K L, SCHNELL R A, FERNÁNDEZ E, et al. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase[J]. The Journal of Cell Biology, 1989, 109(6): 2589-2601.
136	SØRENSEN I, FEI Z J, ANDREAS A, et al. Stable transformation and reverse genetic analysis of Penium margaritaceum: a platform for studies of charophyte green algae, the immediate ancestors of land plants[J]. The Plant Journal, 2014, 77(3): 339-351.
137	JIA Y L, XUE L X, LIU H T, et al. Characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from the halotolerant alga Dunaliella salina and inhibition of its expression by RNAi[J]. Current Microbiology, 2009, 58(5): 426-431.
138	RANDHAWA S, SENGAR S. The evolution and history of gene editing technologies[M/OL]//Progress in molecular biology and translational science. Amsterdam: Elsevier, 2021, 178: 1-62 [2023-07-01]. .
139	SIZOVA I, GREINER A, AWASTHI M, et al. Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases[J]. The Plant Journal, 2013, 73(5): 873-882.
140	DABOUSSI F, LEDUC S, MARÉCHAL A, et al. Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology[J]. Nature Communications, 2014, 5: 3831.
141	WEYMAN P D, BEERI K, LEFEBVRE S C, et al. Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis[J]. Plant Biotechnology Journal, 2015, 13(4): 460-470.
142	NGUYEN T H T, PARK S, JEONG J, et al. Enhancing lipid productivity by modulating lipid catabolism using the CRISPR-Cas9 system in Chlamydomonas [J]. Journal of Applied Phycology, 2020, 32(5): 2829-2840.
143	FENG S Y, HU L N, ZHANG Q H, et al. CRISPR/Cas technology promotes the various application of Dunaliella salina system[J].Applied Microbiology and Biotechnology, 2020, 104(20): 8621-8630.
144	LIN W R, NG I S. Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation[J]. Enzyme and Microbial Technology, 2020, 133: 109458.
145	VERRUTO J, FRANCIS K, WANG Y J, et al. Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies[J]. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(30): E7015-E7022.
146	WANG Q T, GONG Y H, HE Y H, et al. Genome engineering of Nannochloropsis with hundred-kilobase fragment deletions by Cas9 cleavages[J]. The Plant Journal, 2021, 106(4): 1148-1162.
147	BAEK K, KIM D H, JEONG J, et al. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins[J]. Scientific Reports, 2016, 6: 30620.
148	SCHRODA M, BLÖCKER D, BECK C F. The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas [J]. The Plant Journal, 2000, 21(2): 121-131.
149	FRANKLIN S, NGO B, EFUET E, et al. Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast[J]. The Plant Journal, 2002, 30(6): 733-744.
150	JIA Y L, LI S K, ALLEN G, et al. A novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter for expressing transgenes in the halotolerant alga Dunaliella salina [J]. Current Microbiology, 2012, 64(5): 506-513.
151	HOU Q T, QIU S, LIU Q, et al. Selenoprotein-transgenic Chlamydomonas reinhardtii [J]. Nutrients, 2013, 5(3): 624-636.
152	CROZET P, NAVARRO F J, WILLMUND F, et al. Birth of a photosynthetic chassis: a MoClo toolkit enabling synthetic biology in the microalga Chlamydomonas reinhardtii [J]. ACS Synthetic Biology, 2018, 7(9): 2074-2086.
153	SUN H, REN Y Y, FAN Y W, et al. Systematic metabolic tools reveal underlying mechanism of product biosynthesis in Chromochloris zofingiensis [J]. Bioresource Technology, 2021, 337: 125406.
154	SUN H, WANG Y X, HE Y J, et al. Microalgae-derived pigments for the food industry[J]. Marine Drugs, 2023, 21(2): 82.
155	SCHUHMANN H, LIM D K, SCHENK P M. Perspectives on metabolic engineering for increased lipid contents in microalgae[J]. Biofuels, 2012, 3(1): 71-86.
156	YAMORI W, SHIKANAI T. Physiological functions of cyclic electron transport around photosystemⅠin sustaining photosynthesis and plant growth[J]. Annual Review of Plant Biology, 2016, 67: 81-106.
157	XUE J, BALAMURUGAN S, LI D W, et al. Glucose-6-phosphate dehydrogenase as a target for highly efficient fatty acid biosynthesis in microalgae by enhancing NADPH supply[J]. Metabolic Engineering, 2017, 41: 212-221.
158	SCHIEVANO A, SCIARRIA T P, VANBROEKHOVEN K, et al. Electro-fermentation-merging electrochemistry with fermentation in industrial applications[J]. Trends in Biotechnology, 2016, 34(11): 866-878.
159	CHOI O, KIM T, WOO H M, et al. Electricity-driven metabolic shift through direct electron uptake by electroactive heterotroph Clostridium pasteurianum [J]. Scientific Reports, 2014, 4: 6961.
160	MICHELET L, ZAFFAGNINI M, MORISSE S, et al. Redox regulation of the Calvin-Benson cycle: something old, something new[J]. Frontiers in Plant Science, 2013, 4: 470.
161	KUREK I, CHANG T K, BERTAIN S M, et al. Enhanced thermostability of Arabidopsis Rubisco activase improves photosynthesis and growth rates under moderate heat stress[J]. The Plant Cell, 2007, 19(10): 3230-3241.
162	YAMORI W, MASUMOTO C, FUKAYAMA H, et al. Rubisco activase is a key regulator of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature[J]. The Plant Journal, 2012, 71(6): 871-880.
163	VUORISTO K S, MARS A E, SANDERS J P M, et al. Metabolic engineering of TCA cycle for production of chemicals[J]. Trends in Biotechnology, 2016, 34(3): 191-197.
164	SAINI R, KAPOOR R, KUMAR R, et al. CO₂ utilizing microbes—a comprehensive review[J]. Biotechnology Advances, 2011, 29(6): 949-960.
165	XIONG W, MORGAN J A, UNGERER J, et al. The plasticity of cyanobacterial metabolism supports direct CO₂ conversion to ethylene[J]. Nature Plants, 2015, 1: 15053.

门（分类）	种	基因库			参考文献
门（分类）	种	细胞核	线粒体	叶绿体	参考文献
绿藻门	Chromochloris zofingiensis SAG UTEX32	SRR5310949-5310954	SRR5310949-5310954	SRR5310949-5310954	[13]
	Ostreococcus tauri RCC1115	NERT00000000	—	—	[14]
	Botryococcus braunii	MVGU00000000	KR057902.1	KM462884.1	[15]
	Chlamydomonas reinhardtii CC503 cw92 mt+	ABCN00000000	U03843	BK000554.2	[16]
	Auxenochlorella pyrenoidosa FACHB-9	ANZC00000000	—	—	[17]
	Chlorella vulgaris	LDKB00000000	—	AB001684.1	[18]
	Floydiella terrestris	ADIC01000000	—	—	[19]
	Ostreococcus lucimarinus CCE9901	GCA_000092065.1	—	—	[20]
	Micromonas pusilla CCMP1545	ACCP00000000	FJ858268.1	FJ858269	[21]
	Micromonas sp. RCC299	GCA_000090985.2	FJ859351.1	FJ858267.1	[21]
	Volvox carteri Eve	ACJH00000000	—	—	[22]
	Ostreococcus tauri RCC 4221	CAID00000000	CR954200.2	CR954199.2	[23]
	Auxenochlorella protothecoides UTEX 2341/0710	MUYL00000000 APJO00000000	—	/KC63163 4.1	[24]
硅藻门	Phaeodactylum tricornutum CCAP1055/1	ABQD00000000	HQ840789.1	EF067920.1	[25]
硅藻门	Thalassiosira pseudonana CCMP1335	AAFD00000000	DQ186202.1	EF067921.1	[26]
红藻门	Cyanidioschzon merolae 10D	AP006483	D89861.1	AB002583.1	[27-28]
红藻门	Porphyridium purpureum NIES 2140	AROW00000000	—	AP012987.1	[29]
真眼点藻门	Nannochloropsis oceanica IMET1	MPCS00000000.1	KC598090.1	KC568462.1	[30]
真眼点藻门	Nannochloropsis gaditana CCMP526	AGNI00000000	KC012945.1	KC012944.1	[31]

门（分类）	种	基因库			参考文献
门（分类）	种	细胞核	线粒体	叶绿体	参考文献
绿藻门	Chromochloris zofingiensis SAG UTEX32	SRR5310949-5310954	SRR5310949-5310954	SRR5310949-5310954	[13]
	Ostreococcus tauri RCC1115	NERT00000000	—	—	[14]
	Botryococcus braunii	MVGU00000000	KR057902.1	KM462884.1	[15]
	Chlamydomonas reinhardtii CC503 cw92 mt+	ABCN00000000	U03843	BK000554.2	[16]
	Auxenochlorella pyrenoidosa FACHB-9	ANZC00000000	—	—	[17]
	Chlorella vulgaris	LDKB00000000	—	AB001684.1	[18]
	Floydiella terrestris	ADIC01000000	—	—	[19]
	Ostreococcus lucimarinus CCE9901	GCA_000092065.1	—	—	[20]
	Micromonas pusilla CCMP1545	ACCP00000000	FJ858268.1	FJ858269	[21]
	Micromonas sp. RCC299	GCA_000090985.2	FJ859351.1	FJ858267.1	[21]
	Volvox carteri Eve	ACJH00000000	—	—	[22]
	Ostreococcus tauri RCC 4221	CAID00000000	CR954200.2	CR954199.2	[23]
	Auxenochlorella protothecoides UTEX 2341/0710	MUYL00000000 APJO00000000	—	/KC63163 4.1	[24]
硅藻门	Phaeodactylum tricornutum CCAP1055/1	ABQD00000000	HQ840789.1	EF067920.1	[25]
硅藻门	Thalassiosira pseudonana CCMP1335	AAFD00000000	DQ186202.1	EF067921.1	[26]
红藻门	Cyanidioschzon merolae 10D	AP006483	D89861.1	AB002583.1	[27-28]
红藻门	Porphyridium purpureum NIES 2140	AROW00000000	—	AP012987.1	[29]
真眼点藻门	Nannochloropsis oceanica IMET1	MPCS00000000.1	KC598090.1	KC568462.1	[30]
真眼点藻门	Nannochloropsis gaditana CCMP526	AGNI00000000	KC012945.1	KC012944.1	[31]

藻种	株	细胞器	转化方法	启动子	报告基因（标记）	参考文献
Chlamydomonas reinhardtii	dw15.1	细胞核	电穿孔	Hsp70A-RBCS2	ble	[91]
	137c	原生质体	农杆菌转化	ChlL	nptⅡ (GUS)	[92]
Chlorella vulgaris	UMT-M1	细胞核	农杆菌转化	CaMV35S/OlexA-TATA	hpt (GFP)	[93]
	CBS 15-2075	叶绿体	PEG介导	CaMV35S	aphⅧ (EGFP)	[94]
	211/11B	细胞核	基因枪	CaMV35S	nptⅡ (GUS)	[95]
Chlorella ellipsoidea	SD0801	细胞核	PEG介导	Ubi-1 (Polyubiquitin-1)	ble (GUS)	[96]
Chlorella ellipsoidea	Nrm-4	细胞核	电穿孔	Ubi	nptⅡ	[97]
Chlorella sp.	DT	细胞核	PEG介导	Actin1/CaMV35S	hpt (GUS)	[98]
Chromochloris zofingiensis	ATCC 30412	细胞核	电穿孔/基因枪	PDS/NIT/RBCS2	pds	[99]
Nannochloropsis sp.	PP983	细胞核	电穿孔	TUB	ble (GUS)	[100]
	W2J3B	细胞核	电穿孔	VCP1/2	ble	[101]
Nannochloropsis salina	CCMP1776	细胞核	基因枪	TUB	ble	[102]
Nannochloropsis oceanica	IMET1	细胞核	电穿孔	β-tubulin	ble (GUS)	[103]
Nannochloropsis oculata	NIES-2146	细胞核	纤维素酶预处理和电穿孔	Hsp70A-RBCS2	CP (purple chromoprotein)	[104]
Nannochloropsis gaditana	CCMP526	细胞核	电穿孔	TUB/UEP/Hsp70A	ble	[31]
Dunaliella salina	—	细胞核	农杆菌转化	RBCS2/CaMV35S	hptⅡ (GFP)	[105]
	UTEX-1644	细胞核	玻璃珠法	Ubi-1	pat	[106]
	L1644	细胞核	基因枪	Actin	bar	[107]
Dunaliella tertiolecta	—	细胞核	玻璃珠法	CaMV35S	bar (EGFP)	[108]
	SE0045	叶绿体	基因枪	PsbD	ereB	[109]
Phaeodactylum tricornutum	NRIA-0065	细胞核	基因枪	DIV/FCP	ble (EGFP)	[110]
	CCMP2561	细胞核	电穿孔	FCPB	ble (GFP/GUS)	[111]
Thalassiosira pseudonana	CCMP1335	细胞核	基因枪	FCP2	ble (EGFP)	[112]
Haematococcus pluvialis	1844	细胞核	基因枪	PsaD	ble/pds	[113]
	CCAP 34/7	叶绿体	PEG介导	RBCL	aadA	[114]
	NIES-144	细胞核	基因枪	PDS	pds	[115]
	—	细胞核	农杆菌转化	CaMV35S	hpt (GFP)	[116]
Fistulifera solaris	JPCC DA0580	细胞核	基因枪	GAPDH	AphⅧ (G418) (GFP)	[62]
	JPCC DA0580	叶绿体	基因枪	CaMV35S/FCPB/RSV	nptⅡ (GFP)	[117]
Monoraphidium neglectum	SAG 48.87	细胞核	电穿孔	CAB2 (Chlorophyll a/b-binding protein)	AphⅧ (HygromycinB)	[118]
Neochloris oleoabundans	UTEX 1185	细胞核	电穿孔	β2-tubulin	Hyg3 (HygromycinB)	[119]
Botryococcus braunii	UTEX572	细胞核	纤维素酶预处理和电穿孔	CaMV35S	aphⅧ	[120]
Tetraselmis chuii	CCAP 66/21B	细胞核	农杆菌转化	CaMV35S	ble	[121]
Symbiodinium spp.	Mf11.5b.1	细胞核	玻璃珠法	CaMV35S	nptⅡ	[122]
Lobosphaera incisa	SAG 2468	细胞核	电穿孔	RBCS	ble	[123]
Isochrysis sp.	H-13	细胞核	农杆菌转化	LAT	pds	[124]
Parachlorella kessleri	—	细胞核	农杆菌转化	CaMV35S	hpt	[125]
Fistulifera sp.	JPCC DA0580	细胞核	基因枪	FCPB	nptⅡ	[117]
Aurantiochytrium sp.	KRS101	细胞核	电穿孔	GAP	CYH	[126]
Scenedesmus obliquus	FSP-3	细胞核	电穿孔	CaMV35S	CAT	[127]
Schizochytrium sp.	TIO1101	细胞核	农杆菌转化	TEF1/CaMV35S	nptⅡ	[128]
Platymonas subcordiformis	—	细胞核	玻璃珠法	CaMV35S	—	[129]
Porphyridium sp.	—	叶绿体	基因枪	AHAS	AHAS	[130]
Arthrospira sp.	PCC9438	细胞核	电穿孔	CAT	CAT	[131]

藻种	株	细胞器	转化方法	启动子	报告基因（标记）	参考文献
Chlamydomonas reinhardtii	dw15.1	细胞核	电穿孔	Hsp70A-RBCS2	ble	[91]
	137c	原生质体	农杆菌转化	ChlL	nptⅡ (GUS)	[92]
Chlorella vulgaris	UMT-M1	细胞核	农杆菌转化	CaMV35S/OlexA-TATA	hpt (GFP)	[93]
	CBS 15-2075	叶绿体	PEG介导	CaMV35S	aphⅧ (EGFP)	[94]
	211/11B	细胞核	基因枪	CaMV35S	nptⅡ (GUS)	[95]
Chlorella ellipsoidea	SD0801	细胞核	PEG介导	Ubi-1 (Polyubiquitin-1)	ble (GUS)	[96]
Chlorella ellipsoidea	Nrm-4	细胞核	电穿孔	Ubi	nptⅡ	[97]
Chlorella sp.	DT	细胞核	PEG介导	Actin1/CaMV35S	hpt (GUS)	[98]
Chromochloris zofingiensis	ATCC 30412	细胞核	电穿孔/基因枪	PDS/NIT/RBCS2	pds	[99]
Nannochloropsis sp.	PP983	细胞核	电穿孔	TUB	ble (GUS)	[100]
	W2J3B	细胞核	电穿孔	VCP1/2	ble	[101]
Nannochloropsis salina	CCMP1776	细胞核	基因枪	TUB	ble	[102]
Nannochloropsis oceanica	IMET1	细胞核	电穿孔	β-tubulin	ble (GUS)	[103]
Nannochloropsis oculata	NIES-2146	细胞核	纤维素酶预处理和电穿孔	Hsp70A-RBCS2	CP (purple chromoprotein)	[104]
Nannochloropsis gaditana	CCMP526	细胞核	电穿孔	TUB/UEP/Hsp70A	ble	[31]
Dunaliella salina	—	细胞核	农杆菌转化	RBCS2/CaMV35S	hptⅡ (GFP)	[105]
	UTEX-1644	细胞核	玻璃珠法	Ubi-1	pat	[106]
	L1644	细胞核	基因枪	Actin	bar	[107]
Dunaliella tertiolecta	—	细胞核	玻璃珠法	CaMV35S	bar (EGFP)	[108]
	SE0045	叶绿体	基因枪	PsbD	ereB	[109]
Phaeodactylum tricornutum	NRIA-0065	细胞核	基因枪	DIV/FCP	ble (EGFP)	[110]
	CCMP2561	细胞核	电穿孔	FCPB	ble (GFP/GUS)	[111]
Thalassiosira pseudonana	CCMP1335	细胞核	基因枪	FCP2	ble (EGFP)	[112]
Haematococcus pluvialis	1844	细胞核	基因枪	PsaD	ble/pds	[113]
	CCAP 34/7	叶绿体	PEG介导	RBCL	aadA	[114]
	NIES-144	细胞核	基因枪	PDS	pds	[115]
	—	细胞核	农杆菌转化	CaMV35S	hpt (GFP)	[116]
Fistulifera solaris	JPCC DA0580	细胞核	基因枪	GAPDH	AphⅧ (G418) (GFP)	[62]
	JPCC DA0580	叶绿体	基因枪	CaMV35S/FCPB/RSV	nptⅡ (GFP)	[117]
Monoraphidium neglectum	SAG 48.87	细胞核	电穿孔	CAB2 (Chlorophyll a/b-binding protein)	AphⅧ (HygromycinB)	[118]
Neochloris oleoabundans	UTEX 1185	细胞核	电穿孔	β2-tubulin	Hyg3 (HygromycinB)	[119]
Botryococcus braunii	UTEX572	细胞核	纤维素酶预处理和电穿孔	CaMV35S	aphⅧ	[120]
Tetraselmis chuii	CCAP 66/21B	细胞核	农杆菌转化	CaMV35S	ble	[121]
Symbiodinium spp.	Mf11.5b.1	细胞核	玻璃珠法	CaMV35S	nptⅡ	[122]
Lobosphaera incisa	SAG 2468	细胞核	电穿孔	RBCS	ble	[123]
Isochrysis sp.	H-13	细胞核	农杆菌转化	LAT	pds	[124]
Parachlorella kessleri	—	细胞核	农杆菌转化	CaMV35S	hpt	[125]
Fistulifera sp.	JPCC DA0580	细胞核	基因枪	FCPB	nptⅡ	[117]
Aurantiochytrium sp.	KRS101	细胞核	电穿孔	GAP	CYH	[126]
Scenedesmus obliquus	FSP-3	细胞核	电穿孔	CaMV35S	CAT	[127]
Schizochytrium sp.	TIO1101	细胞核	农杆菌转化	TEF1/CaMV35S	nptⅡ	[128]
Platymonas subcordiformis	—	细胞核	玻璃珠法	CaMV35S	—	[129]
Porphyridium sp.	—	叶绿体	基因枪	AHAS	AHAS	[130]
Arthrospira sp.	PCC9438	细胞核	电穿孔	CAT	CAT	[131]

[1]	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.
[2]	晏雄鹰, 王振, 娄吉芸, 张皓瑜, 黄星宇, 王霞, 杨世辉. 生物燃料高效生产微生物细胞工厂构建研究进展[J]. 合成生物学, 2023, 4(6): 1082-1121.
[3]	刁志钿, 王喜先, 孙晴, 徐健, 马波. 单细胞拉曼光谱测试分选装备研制及应用进展[J]. 合成生物学, 2023, 4(5): 1020-1035.
[4]	卢挥, 张芳丽, 黄磊. 合成生物学自动化装置iBioFoundry的构建与应用[J]. 合成生物学, 2023, 4(5): 877-891.
[5]	白仲虎, 任和, 聂简琪, 孙杨. 高通量平行发酵技术的发展与应用[J]. 合成生物学, 2023, 4(5): 904-915.
[6]	吴玉洁, 刘欣欣, 刘健慧, 杨开广, 随志刚, 张丽华, 张玉奎. 基于高通量液相色谱质谱技术的菌株筛选与关键分子定量分析研究进展[J]. 合成生物学, 2023, 4(5): 1000-1019.
[7]	胡哲辉, 徐娟, 卞光凯. 自动化高通量技术在天然产物生物合成中的应用[J]. 合成生物学, 2023, 4(5): 932-946.
[8]	刘欢, 崔球. 原位电离质谱技术在微生物菌株筛选中的应用进展[J]. 合成生物学, 2023, 4(5): 980-999.
[9]	王雁南, 孙宇辉. 碱基编辑技术及其在微生物合成生物学中的应用[J]. 合成生物学, 2023, 4(4): 720-737.
[10]	刘晚秋, 季向阳, 许慧玲, 卢屹聪, 李健. 限制性内切酶的无细胞快速制备研究[J]. 合成生物学, 2023, 4(4): 840-851.
[11]	孙美莉, 王凯峰, 陆然, 纪晓俊. 解脂耶氏酵母底盘细胞的工程改造及应用[J]. 合成生物学, 2023, 4(4): 779-807.
[12]	孙智, 杨宁, 娄春波, 汤超, 杨晓静. 功能拓扑的理性设计及其在合成生物学中的应用[J]. 合成生物学, 2023, 4(3): 444-463.
[13]	赖奇龙, 姚帅, 查毓国, 白虹, 宁康. 微生物组生物合成基因簇发掘方法及应用前景[J]. 合成生物学, 2023, 4(3): 611-627.
[14]	孟巧珍, 郭菲. “可折叠性”在酶智能设计改造中的应用研究——以AlphaFold2为例[J]. 合成生物学, 2023, 4(3): 571-589.
[15]	王晟, 王泽琛, 陈威华, 陈珂, 彭向达, 欧发芬, 郑良振, 孙瑨原, 沈涛, 赵国屏. 基于人工智能和计算生物学的合成生物学元件设计[J]. 合成生物学, 2023, 4(3): 422-443.