微藻破囊壶菌产功能性脂肪酸DHA研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2024-0011

生物技术通报 ›› 2024, Vol. 40 ›› Issue (6): 81-94.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0011

微藻破囊壶菌产功能性脂肪酸DHA研究进展

张美玉¹^,²(), 赵玉斌³, 王灵云³, 宋元达¹^,², 赵新河¹^,²(), 任晓洁¹^,²()

1.山东理工大学农业工程与食品科学学院考林腊特列杰微生物脂质国际研究中心，淄博 255000
2.山东省海洋微生物细胞工厂国际合作联合实验室，淄博 255000
3.鲁洲生物科技有限公司，临沂 276400

收稿日期:2024-01-04 出版日期:2024-06-26 发布日期:2024-05-15
通讯作者: 任晓洁，女，博士，副教授，研究方向：微生物代谢工程；E-mail: renxiaojie@sdut.edu.cn；
赵新河，男，博士，副教授，研究方向：发酵工程；E-mail: zhaoxinhe@sdut.edu.cn
作者简介:张美玉，女，硕士研究生，研究方向：微生物代谢工程；E-mail: zmeiyu1118@163.com
基金资助:
山东省自然科学基金项目(ZR2023MC194);山东省自然科学基金项目(ZR2019BC099);促进与加拿、澳大利亚、新西兰及拉美地区科研合作与高层次人才培养项目(2022-1007)

Research Progress in the Production of Functional Fatty Acid DHA by Microalga Thraustochytrids

ZHANG Mei-yu¹^,²(), ZHAO Yu-bin³, WANG Ling-yun³, SONG Yuan-da¹^,², ZHAO Xin-he¹^,²(), REN Xiao-jie¹^,²()

1. Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and Food Science, Shandong University of Technology, Zibo 255000
2. International Cooperative Joint Laboratory for Marine Microbial Cell Factories, Shandong Province, Zibo 255000
3. Luzhou Bio-Chem Technology Co. Ltd., Linyi 276400

Received:2024-01-04 Published:2024-06-26 Online:2024-05-15

摘要/Abstract

摘要：

二十二碳六烯酸（DHA）作为人体必需的多不饱和脂肪酸，在维护心血管健康、抗癌、支持视觉和脑功能等方面至关重要。传统的深海鱼油提取DHA方法存在鱼腥味重、工艺繁琐等问题，迫使研究者寻求更为高效、环保的替代方案。破囊壶菌（Thraustochytrids）凭借其生长迅速、低重金属污染以及高DHA含量的特性，成为工业化生产DHA的潜力微生物之一。当前在破囊壶菌发酵生产DHA的过程中，依然需要解决一系列关键问题，包括提高发酵产量、降低成本等。本文旨在全面阐述破囊壶菌发酵生产DHA的研究现状，包括菌株筛选与改良、DHA生物合成途径、遗传转化及代谢工程、发酵控制策略等方面。首先，总结归纳了对野生型菌株的自然筛选和诱变改良等方法，不断提高破囊壶菌中DHA产油量。其次，详细介绍了破囊壶菌DHA合成途径的研究进展，着重分析了生物合成途径中关键辅助因子在DHA生产中的作用。此外，概述了外源DNA传递到破囊壶菌细胞的遗传转化技术的应用现状，为提高其遗传转化效率和稳定性提供重要参考。在DHA代谢调控方面，探讨了氮限制对DHA合成的促进作用以及温度和氧气供应对生产效率的影响。最后，对利用破囊壶菌生产DHA存在的主要瓶颈问题和未来发展趋势进行了总结，以推动其在医药、保健品和食品等领域的广泛应用，实现工业规模下的高效生产。

关键词: 破囊壶菌, 二十二碳六烯酸（DHA）, 代谢工程, 遗传转化, 菌种选育, 生物合成途径, 发酵策略

Abstract:

Docosahexaenoic acid（DHA）, an essential polyunsaturated fatty acid in human body, plays a crucial role in maintaining cardiovascular health, combating cancer, and supporting vision and brain function. Traditional methods of extracting DHA from deep-sea fish oil suffer from strong fish odor and complex processing, driving researchers to explore more efficient and environmentally friendly alternatives. Thraustochytrids, with their rapid growth, low heavy metal contamination, and high DHA content, have emerged as potential microorganisms for industrial DHA production. However, several key issues persist in DHA production through Thraustochytrids fermentation, including enhancing fermentation production and reducing costs. This paper aims to comprehensively review the research status of DHA production through Thraustochytrids fermentation, covering strain screening and improvement, DHA biosynthesis pathways, genetic transformation, metabolic engineering, and fermentation control strategies. The paper first summarizes methods for natural screening and mutagenic improvement of wild-type strains to enhance DHA oil production in Thraustochytrids. It then delves into the research progress of DHA biosynthesis pathways in Thraustochytrids, emphasizing the role of key cofactors in DHA production. Additionally, it outlines the current application status of genetic transformation technology for introducing exogenous DNA into Thraustochytrids cells, offering valuable insights for enhancing genetic transformation efficiency and stability. Regarding DHA metabolism regulation, the paper discusses the promoting effect of nitrogen limitation on DHA synthesis and the impact of temperature and oxygen supply on production efficiency. Finally, it summarizes the main challenges and future development trends in leveraging Thraustochytrids for DHA production, aiming to facilitate their widespread application in pharmaceuticals, nutraceuticals, and food industries, and achieve efficient large-scale production.

Key words: Thraustochytrids, docosahexaenoic acid（DHA）, metabolic engineering, genetic transformation, breeding of bacterial strains, biosynthetic pathway, fermentation strategy

张美玉, 赵玉斌, 王灵云, 宋元达, 赵新河, 任晓洁. 微藻破囊壶菌产功能性脂肪酸DHA研究进展[J]. 生物技术通报, 2024, 40(6): 81-94.

ZHANG Mei-yu, ZHAO Yu-bin, WANG Ling-yun, SONG Yuan-da, ZHAO Xin-he, REN Xiao-jie. Research Progress in the Production of Functional Fatty Acid DHA by Microalga Thraustochytrids[J]. Biotechnology Bulletin, 2024, 40(6): 81-94.

图/表 5

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菌株 Bacterial species	来源 Source	产油特性 Oil production characteristics	参考文献Reference
橙黄壶菌ZJWZ-7 Aurantiochytrium ZJWZ-7	中国沿海红树林 Chinese coastal mangroves	DHA和饱和脂肪酸分别达到1.66 g/L和1.68 g/L DHA and saturated fatty acids reach 1.66 g/L and 1.68 g/L respectively	[13]
裂殖壶菌LU310 Schizochytrium sp. LU310	温州红树林 Wenzhou angroves	DHA产量高达24.74 g/L DHA production reaches as high as 24.74 g/L	[14]
裂殖壶菌ABC101 Schizochytrium sp. ABC101	海水中 Seawater	DHA达到11.0 g/L DHA reaches 11.0 g/L	[15]
裂殖壶菌S31 Schizochytrium sp. S31	美国加利福尼亚河 California River, USA	DHA产率高达300 mg/（L·d） DHA productivity reaches 300 mg/（L·d）	[16]
橙黄壶菌TL18 Aurantiochytrium sp. TL18	英国水域 British waters	DHA产量占多不饱和脂肪酸的83% DHA production accounts for 83% of polyunsaturated fatty acids	[17]
橙黄壶菌YB-05 Aurantiochytrium sp. YB-05	沙特阿拉伯红海沿岸的腐叶中Decayed leaves from the Red Sea in Saudi Arabia	DHA含量为24.85% DHA content is 24.85%	[18]
橙黄壶菌SW1 Aurantiochytrium sp. SW1	马来西亚 Malaysia	DHA产量达到3.6 g/L DHA production reaches 3.6 g/L	[19]

菌株 Bacterial species	来源 Source	产油特性 Oil production characteristics	参考文献Reference
橙黄壶菌ZJWZ-7 Aurantiochytrium ZJWZ-7	中国沿海红树林 Chinese coastal mangroves	DHA和饱和脂肪酸分别达到1.66 g/L和1.68 g/L DHA and saturated fatty acids reach 1.66 g/L and 1.68 g/L respectively	[13]
裂殖壶菌LU310 Schizochytrium sp. LU310	温州红树林 Wenzhou angroves	DHA产量高达24.74 g/L DHA production reaches as high as 24.74 g/L	[14]
裂殖壶菌ABC101 Schizochytrium sp. ABC101	海水中 Seawater	DHA达到11.0 g/L DHA reaches 11.0 g/L	[15]
裂殖壶菌S31 Schizochytrium sp. S31	美国加利福尼亚河 California River, USA	DHA产率高达300 mg/（L·d） DHA productivity reaches 300 mg/（L·d）	[16]
橙黄壶菌TL18 Aurantiochytrium sp. TL18	英国水域 British waters	DHA产量占多不饱和脂肪酸的83% DHA production accounts for 83% of polyunsaturated fatty acids	[17]
橙黄壶菌YB-05 Aurantiochytrium sp. YB-05	沙特阿拉伯红海沿岸的腐叶中Decayed leaves from the Red Sea in Saudi Arabia	DHA含量为24.85% DHA content is 24.85%	[18]
橙黄壶菌SW1 Aurantiochytrium sp. SW1	马来西亚 Malaysia	DHA产量达到3.6 g/L DHA production reaches 3.6 g/L	[19]

菌株 Bacterial species	突变方法 Mutation method	结果 Result	参考文献Reference
橙黄壶菌CG Aurantiochytrium sp.CGMCC 6208	重离子诱变 Heavy ion mutagenesis	DHA产量由21 g/L提高到27 g/L DHA production increased from 21 g/L to 27 g/L	[21]
橙黄壶菌SW1 Aurantiochytrium sp. SW1	ARTP	生物量、油脂和DHA产量分别提高30%、65%和80% Biomass, lipid content, and DHA production increased by 30%, 65%, and 80%, respectively	[22]
裂殖壶菌B4D1 Schizochytrium sp. B4D1	紫外诱变 UV mutagenesis	DHA含量达到总油脂的41.24%，提高了11.70% The DHA content reached 41.24% of total lipids, increasing by 11.70%	[23]
裂殖壶菌SR21 Schizochytrium limacinum SR21	ARTP	DHA产量达到6. 59 g/L，提高了46.12% The DHA production reached 6.59 g/L, representing a 46.12% increase	[24]
裂殖壶菌ATCC20888 Schizochytrium sp. ATCC20888	低能离子诱变 Low-energy ion mutagenesis	生物量为48.01 g/L，DHA产量最高为6.52 g/L Biomass was 48.01 g/L, with a maximum DHA production of 6.52 g/L	[25]
橙黄壶菌PKU#SW7 Aurantiochytrium sp. PKU#SW7	紫外诱变 UV mutagenesis	生物量为6.62 g/L，DHA在脂肪酸中所占比例由29.97%增加到33.43% Biomass was 6.62 g/L, and the proportion of DHA in fatty acids increased from 29.97% to 33.43%	[26]
裂殖壶菌M209059 Schizochytrium sp. M209059	NTG结合紫外诱变 NTG combined with UV mutagenesis	DHA占总脂肪酸的比例达到56.22%，提高了38.88% The proportion of DHA in total fatty acids reached 56.22%, representing an increase of 38.88%	[27]
裂殖壶菌 Schizochytrium sp.	甲烷磺酸乙酯（EMS）结合紫外诱变 EMS combined with UV mutagenesis	脂质含量和DHA产量分别提高了8.53%和21.58% Lipid content and DHA production increased by 8.53% and 21.58%, respectively	[28]
裂殖壶菌31 Schizochytrium sp. 31	EMS和ARTP复合诱变 EMS and ARTP combined mutagenesis	DHA产量达到7.2 g/L，比亲本菌株提高35.6% The DHA production reached 7.2 g/L, representing a 35.6% increase compared to the parental strain	[29]
裂殖壶菌ALE70 Schizochytrium sp. ALE70	耐热性ALE Heat-resistant ALE	油脂产量和DHA产量分别提高了4.31倍和4.33倍 The lipid production and DHA production increased by 4.31-fold and 4.33-fold, respectively	[30]
裂殖壶菌HX-308 Schizochytrium sp. HX-308	高氧ALE High-oxygen ALE	细胞干重达到84.34 g/L，DHA产量为26.40 g/L Cell dry weight reached 84.34 g/L, and DHA production was 26.40 g/L	[31]
裂殖壶菌HX-308 Schizochytrium sp. HX-308	低温与高盐度双因子协同ALE Synergistic ALE of Low Temperature and High Salinity	DHA产量为38.12 g/L，比亲本菌株高57.52% The DHA production is 38.12 g/L, which is 57.52% higher than the parental strain	[32]
裂殖壶菌 Schizochytrium limacinum B4D1	低温ALE Low-temperature ALE	DHA含量提高了11.22% DHA content increased by 11.22%	[33]

菌株 Bacterial species	突变方法 Mutation method	结果 Result	参考文献Reference
橙黄壶菌CG Aurantiochytrium sp.CGMCC 6208	重离子诱变 Heavy ion mutagenesis	DHA产量由21 g/L提高到27 g/L DHA production increased from 21 g/L to 27 g/L	[21]
橙黄壶菌SW1 Aurantiochytrium sp. SW1	ARTP	生物量、油脂和DHA产量分别提高30%、65%和80% Biomass, lipid content, and DHA production increased by 30%, 65%, and 80%, respectively	[22]
裂殖壶菌B4D1 Schizochytrium sp. B4D1	紫外诱变 UV mutagenesis	DHA含量达到总油脂的41.24%，提高了11.70% The DHA content reached 41.24% of total lipids, increasing by 11.70%	[23]
裂殖壶菌SR21 Schizochytrium limacinum SR21	ARTP	DHA产量达到6. 59 g/L，提高了46.12% The DHA production reached 6.59 g/L, representing a 46.12% increase	[24]
裂殖壶菌ATCC20888 Schizochytrium sp. ATCC20888	低能离子诱变 Low-energy ion mutagenesis	生物量为48.01 g/L，DHA产量最高为6.52 g/L Biomass was 48.01 g/L, with a maximum DHA production of 6.52 g/L	[25]
橙黄壶菌PKU#SW7 Aurantiochytrium sp. PKU#SW7	紫外诱变 UV mutagenesis	生物量为6.62 g/L，DHA在脂肪酸中所占比例由29.97%增加到33.43% Biomass was 6.62 g/L, and the proportion of DHA in fatty acids increased from 29.97% to 33.43%	[26]
裂殖壶菌M209059 Schizochytrium sp. M209059	NTG结合紫外诱变 NTG combined with UV mutagenesis	DHA占总脂肪酸的比例达到56.22%，提高了38.88% The proportion of DHA in total fatty acids reached 56.22%, representing an increase of 38.88%	[27]
裂殖壶菌 Schizochytrium sp.	甲烷磺酸乙酯（EMS）结合紫外诱变 EMS combined with UV mutagenesis	脂质含量和DHA产量分别提高了8.53%和21.58% Lipid content and DHA production increased by 8.53% and 21.58%, respectively	[28]
裂殖壶菌31 Schizochytrium sp. 31	EMS和ARTP复合诱变 EMS and ARTP combined mutagenesis	DHA产量达到7.2 g/L，比亲本菌株提高35.6% The DHA production reached 7.2 g/L, representing a 35.6% increase compared to the parental strain	[29]
裂殖壶菌ALE70 Schizochytrium sp. ALE70	耐热性ALE Heat-resistant ALE	油脂产量和DHA产量分别提高了4.31倍和4.33倍 The lipid production and DHA production increased by 4.31-fold and 4.33-fold, respectively	[30]
裂殖壶菌HX-308 Schizochytrium sp. HX-308	高氧ALE High-oxygen ALE	细胞干重达到84.34 g/L，DHA产量为26.40 g/L Cell dry weight reached 84.34 g/L, and DHA production was 26.40 g/L	[31]
裂殖壶菌HX-308 Schizochytrium sp. HX-308	低温与高盐度双因子协同ALE Synergistic ALE of Low Temperature and High Salinity	DHA产量为38.12 g/L，比亲本菌株高57.52% The DHA production is 38.12 g/L, which is 57.52% higher than the parental strain	[32]
裂殖壶菌 Schizochytrium limacinum B4D1	低温ALE Low-temperature ALE	DHA含量提高了11.22% DHA content increased by 11.22%	[33]

菌株 Bacterial species	基因 Gene	电击参数 Electric shock parameters	结果 Result	参考文献 Reference
裂殖壶菌S31 Schizochytrium sp. S31	过表达来自隐甲藻的苹果酸酶和高山被孢霉的ELO3基因 Overexpression of malate synthase from Thalassiosira pseudonana and ELO3 gene from Mortierella alpina	2 000 V, 50 μF, 200 Ω	DHA产量为3.54 g/L，占细胞干重的26.70% DHA production reached 3.54 g/L, accounting for 26.70% of cell dry weight	[52]
裂殖壶菌ATCC 20888 Schizochytrium sp. ATCC 20888	过表达硫氧还蛋白还原酶、乙醛脱氢酶、谷胱甘肽过氧化物酶和葡萄糖-6 -磷酸脱氢酶的基因 Overexpression of genes encoding thioredoxin reductase, aldehyde dehydrogenase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase	1 500 V, 200 Ω, 50 μF	DHA产量达到13.3 g/L DHA production reached 13.3 g/L	[53]
橙黄壶菌SZU445 Aurantiochytrium sp. SZU445	敲除2,4 -二烯基辅酶A还原酶基因 Knockout of the gene encoding 2,4-dienoyl-CoA reductase	1 500 V, 50 μF, 200 Ω	DHA产量占总脂肪酸的53.97% DHA production accounted for 53.97% of total fatty acids	[54]
裂殖壶菌H016 Schizochytrium sp. H016	过表达内源葡萄糖- 6 -磷酸脱氢酶基因 Overexpression of endogenous glucose-6-phosphate dehydrogenase gene	1 400 V, 200 Ω, 50 μF	DHA产量达到17.01 g/L，提高了31.47% The DHA production reached 17.01 g/L, representing a 31.47% increase	[55]
裂殖壶菌TIO110 Schizochytrium sp. TIO1101	过表达大肠杆菌乙酰辅酶A合成酶基因 Overexpression of Escherichia coli acetyl-CoA synthase gene	2 100 V, 4.5 ms	生物量和脂肪酸比例分别增加了29.9%和11.3% Biomass and fatty acid proportion increased by 29.9% and 11.3%, respectively	[56]
裂殖壶菌ATCC1381 Schizochytrium sp. ATCC1381	过表达丙二酰辅酶A转移酶 Overexpression of acyltransferase	7.5 kV/cm, 50 μF, 50 Ω	总脂产量提高到110.5 g/L，比野生菌株提高39.6%，DHA产量为47.39 g/L Total lipid production increased to 110.5 g/L, a 39.6% increase compared to the wild-type strain, with DHA production at 47.39 g/L	[57]
裂殖壶菌PKU#Mn42 Schizochytrium sp. PKU#Mn42	抗氧化基因超氧化物歧化酶 Antioxidant gene superoxide dismutase	1 000 V, 50 μF, 500 Ω	多不饱和脂肪酸（PUFA）产量增加1.37 倍 Polyunsaturated fatty acids（PUFA）production increased by 1.37 times	[58]

微藻破囊壶菌产功能性脂肪酸DHA研究进展

Research Progress in the Production of Functional Fatty Acid DHA by Microalga Thraustochytrids

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