强化细胞外排提高工程菌类黄酮产量的策略

doi:10.13560/j.cnki.biotech.bull.1985.2022-1006

摘要/Abstract

摘要：

类黄酮的抑菌谱广、抗氧化活性高，在食品和生物医药领域应用广泛，市场需求量大，依赖于从植物中提取的现有生产技术存在诸多弊端，微生物发酵法生产类黄酮成为国际竞相发展的前沿技术。通过代谢途径改造和基因优化构建高产菌株是现有研究的主要策略，较少考虑类黄酮对生产菌的抑菌作用。考虑到生产菌生长受到抑制会限制发酵中类黄酮积累，本文在总结分析动物、植物、微生物中类黄酮外排机制与关键基因的基础上，结合相关基因在工程菌中成功表达的案例，提出将类黄酮外排机制和关键基因引入工程菌构建的策略，以期为类黄酮高产菌株的构建提供借鉴。

关键词: 类黄酮, 类黄酮外排, 衍生化, ABC转运蛋白, 转基因工程菌

Abstract:

Flavonoid has a broad spectrum of antibacterial activity and high antioxidant activity, showing extensive usage and large market in food and biomedical fields，but there are many disadvantages in the currently used methods to extract flavonoid from plants. Thus microbial fermentation has become the most potential technique to produce flavonoid around the world. The main current strategy to solve this issue is to construct high-yield strains through metabolic pathway modification and gene optimization, but the antibacterial effect of flavonoid on the producing strain was little considered. Considering the antibacterial activity of flavonoid may inhibit the bacterial growth and thus limit the flavonoid production of microbial fermentation, we proposed that introducing or strengthening the flavonoid efflux may be helpful to enhancing the flavonoid production of microbial fermentation, based on the flavonoid efflux mechanism and key genes in animals, plants and microorganisms, as well as combined with cases of the relevant genes that have been successfully expressed in genetically modified bacteria, aiming to provide useful information for the construction of high-flavonoid-producing microorganisms.

Key words: flavonoids, efflux of flavonoid, derivatives, ATP-binding cassette transporter, genetically engineered bacteria

马芳芳, 刘冠闻, 庞冰, 蒋春美, 师俊玲. 强化细胞外排提高工程菌类黄酮产量的策略[J]. 生物技术通报, 2023, 39(5): 63-76.

MA Fang-fang, LIU Guan-wen, PANG Bing, JIANG Chun-mei, SHI Jun-ling. Strategies of Increasing Flavonoid Production in Engineered Bacteria by Intensifying the Efflux of Flavonoid in Cells[J]. Biotechnology Bulletin, 2023, 39(5): 63-76.

图/表 7

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基因 Gene	转运蛋白Transporter	位置/转运方向 Location/Transport direction	类黄酮 Flavonoid	基因来源 Source of gene	工程菌 Engineering bacterium	参考文献 Reference
Abcc1 （NC_000082）	MRP	小肠基底外侧膜/到血液	柚皮素Naringin 橙皮素Hesperidin （葡萄糖醛酸化和甲基化Glucuronidation and methylation）	小家鼠 Mus musculus（House mouse）	大肠杆菌Escherichia coli 金黄色葡萄糖球菌Staphylococcus aureus	[29-30]
Abcc2 （NC_000085）	MRP	小肠细胞顶点/到小肠	柚皮素Naringin 橙皮素Hesperidin （葡萄糖醛酸化和甲基化Glucuronidation and methylation）	小家鼠 Mus musculus（House mouse）	苏云金芽孢杆菌Bacillus thuringiensis	[29,31]
Abcc3 （NC_000077）	MRP	小肠基底外侧膜/到血液	柚皮素Naringin （葡萄糖醛酸化和甲基化Glucuronidation and methylation）	小家鼠 Mus musculus（House mouse）	大肠杆菌Escherichia coli 金黄色葡萄糖球菌Staphylococcus aureus	[29-30]
MRP2 （NC_000017）	MRP	小肠基底外侧膜/到血液	表没食子儿茶素没食子酸酯Epigallocatechin gallate（硫酸化和甲基化Sulfation and methylation）	人类 Homo sapiens（Human）	酿酒酵母Saccharomyces cerevisiae	[32]
ABCB1 （NC_000007）	P-gp	肠道管腔细胞/参与外排	4'-羟-6-甲基黄酮 4'-hydroxy-6- Methylflavone 4'-羟-6-甲氧基黄酮 4'-hydroxy-6-methoxyflavone 4'-羟-β-萘基黄酮 4'-hydroxy-β- Naphthoflavone	人类 Homo sapiens（Human）	金黄色葡萄糖球菌Staphylococcus aureus MRSA6	[33-34]
ABCG2 （NC_000072）	BCRP	肠道管腔细胞/参与外排	染料木素Genistein （硫酸化和葡萄糖醛酸化Sulfation and glucuronidation）	小家鼠 Mus musculus（House mouse）	大肠杆菌Escherichia coli	[35-36]

基因 Gene	转运蛋白Transporter	位置/转运方向 Location/Transport direction	类黄酮 Flavonoid	基因来源 Source of gene	工程菌 Engineering bacterium	参考文献 Reference
Abcc1 （NC_000082）	MRP	小肠基底外侧膜/到血液	柚皮素Naringin 橙皮素Hesperidin （葡萄糖醛酸化和甲基化Glucuronidation and methylation）	小家鼠 Mus musculus（House mouse）	大肠杆菌Escherichia coli 金黄色葡萄糖球菌Staphylococcus aureus	[29-30]
Abcc2 （NC_000085）	MRP	小肠细胞顶点/到小肠	柚皮素Naringin 橙皮素Hesperidin （葡萄糖醛酸化和甲基化Glucuronidation and methylation）	小家鼠 Mus musculus（House mouse）	苏云金芽孢杆菌Bacillus thuringiensis	[29,31]
Abcc3 （NC_000077）	MRP	小肠基底外侧膜/到血液	柚皮素Naringin （葡萄糖醛酸化和甲基化Glucuronidation and methylation）	小家鼠 Mus musculus（House mouse）	大肠杆菌Escherichia coli 金黄色葡萄糖球菌Staphylococcus aureus	[29-30]
MRP2 （NC_000017）	MRP	小肠基底外侧膜/到血液	表没食子儿茶素没食子酸酯Epigallocatechin gallate（硫酸化和甲基化Sulfation and methylation）	人类 Homo sapiens（Human）	酿酒酵母Saccharomyces cerevisiae	[32]
ABCB1 （NC_000007）	P-gp	肠道管腔细胞/参与外排	4'-羟-6-甲基黄酮 4'-hydroxy-6- Methylflavone 4'-羟-6-甲氧基黄酮 4'-hydroxy-6-methoxyflavone 4'-羟-β-萘基黄酮 4'-hydroxy-β- Naphthoflavone	人类 Homo sapiens（Human）	金黄色葡萄糖球菌Staphylococcus aureus MRSA6	[33-34]
ABCG2 （NC_000072）	BCRP	肠道管腔细胞/参与外排	染料木素Genistein （硫酸化和葡萄糖醛酸化Sulfation and glucuronidation）	小家鼠 Mus musculus（House mouse）	大肠杆菌Escherichia coli	[35-36]

基因 Gene	转运蛋白 Transporter	转运底物 Substrate	分布位置 Location	基因来源 Gene source	工程菌Engineering microorganism	参考文献 Reference
TRANSPARENT TESTA12（tt12）（AJ294464）	MATE	花青素-3-葡萄糖Cyanidin-3-O-glucoside	花青素苷液泡Anthocyanin vacuole	拟南芥Arabidopsis thaliana	大肠杆菌Escherichia coli酿酒酵母Saccharomyces cerevisiae	[51-52]
花青素-3-葡萄糖转移酶 Cyanidin 3-O-Glucosyltransferase（3GT）（NC_003076）	UDP-d-葡萄糖转运蛋白 UDP-d-glucose transporter proteins GLF	花青素-3-葡萄糖Cyanidin-3-O-glucoside	细胞质Cytoplasm	拟南芥Arabidopsis thaliana	大肠杆菌Escherichia coli BL21	[53]
花青素-3-葡萄糖转移酶 Cyanidin 3-O-Glucosyltransferase（3GT）（NC_003076）	UDP-d-葡萄糖转运蛋白 UDP-d-glucose transporter proteins GALU1	花青素-3-葡萄糖Cyanidin-3-O-glucoside	细胞质Cytoplasm	拟南芥Arabidopsis thaliana	谷氨酸棒状杆菌Corynebacterium glutamicum	[54]
ZmMrp3 （AY609318）	MRP	花青素-3-葡萄糖Cyanidin-3-O-glucoside	花青素苷液泡Anthocyanin vacuole	玉米 Zea mays	大肠杆菌Escherichia coli	[55]
Bronze-2（Bz2）（NC_050096）	GST	花青素-3-葡萄糖Cyanidin-3-O-glucoside	细胞质Cytoplasm	玉米 Zea mays	大肠杆菌Escherichia coli	[56]
葡萄原花青素O-甲基转移酶Vitis vinifera Anthocyanin O-methyltransferase（VvAOMT）（NC_012007）	花色素苷O-甲基转移酶Anthocyanin O-methyltransferase（AOMT）	花青素-3-葡萄糖Cyanidin 3-O-glucoside，甲基花青素3-O-葡萄糖 Peonidin 3-O- glucoside	细胞质Cytoplasm	葡萄 Vitis vinifera	大肠杆菌Escherichia coli DH5α and BL21	[57]

基因 Gene	转运蛋白 Transporter	转运底物 Substrate	分布位置 Location	基因来源 Gene source	工程菌Engineering microorganism	参考文献 Reference
TRANSPARENT TESTA12（tt12）（AJ294464）	MATE	花青素-3-葡萄糖Cyanidin-3-O-glucoside	花青素苷液泡Anthocyanin vacuole	拟南芥Arabidopsis thaliana	大肠杆菌Escherichia coli酿酒酵母Saccharomyces cerevisiae	[51-52]
花青素-3-葡萄糖转移酶 Cyanidin 3-O-Glucosyltransferase（3GT）（NC_003076）	UDP-d-葡萄糖转运蛋白 UDP-d-glucose transporter proteins GLF	花青素-3-葡萄糖Cyanidin-3-O-glucoside	细胞质Cytoplasm	拟南芥Arabidopsis thaliana	大肠杆菌Escherichia coli BL21	[53]
花青素-3-葡萄糖转移酶 Cyanidin 3-O-Glucosyltransferase（3GT）（NC_003076）	UDP-d-葡萄糖转运蛋白 UDP-d-glucose transporter proteins GALU1	花青素-3-葡萄糖Cyanidin-3-O-glucoside	细胞质Cytoplasm	拟南芥Arabidopsis thaliana	谷氨酸棒状杆菌Corynebacterium glutamicum	[54]
ZmMrp3 （AY609318）	MRP	花青素-3-葡萄糖Cyanidin-3-O-glucoside	花青素苷液泡Anthocyanin vacuole	玉米 Zea mays	大肠杆菌Escherichia coli	[55]
Bronze-2（Bz2）（NC_050096）	GST	花青素-3-葡萄糖Cyanidin-3-O-glucoside	细胞质Cytoplasm	玉米 Zea mays	大肠杆菌Escherichia coli	[56]
葡萄原花青素O-甲基转移酶Vitis vinifera Anthocyanin O-methyltransferase（VvAOMT）（NC_012007）	花色素苷O-甲基转移酶Anthocyanin O-methyltransferase（AOMT）	花青素-3-葡萄糖Cyanidin 3-O-glucoside，甲基花青素3-O-葡萄糖 Peonidin 3-O- glucoside	细胞质Cytoplasm	葡萄 Vitis vinifera	大肠杆菌Escherichia coli DH5α and BL21	[57]

类黄酮 Flavonoid	外排基因 Efflux gene	微生物 Microorganism	转运蛋白家族 Transporter protein	参考文献 Reference
查尔酮Chalcone （2E）-1-（4'-氨苯基）-3-（4-甲氧苯基）- 2丙-烯-1-酮（2E）-1-（4'-aminophenyl）-3-（4-methoxyphenyl）-prop-2-en-1-one	MepA （NC_016941）	金黄色葡萄球菌Staphylococcus aureus	ABC转运蛋白 ABC transporters	[59-60]
木犀草素Luteolin 芹菜素Apigenin 山奈酚Kaempferol 鼠李素Rhamnetin	NorA （NC_007795）	金黄色葡萄球菌Staphylococcus aureus NCTC 8325	ABC转运蛋白 ABC transporters	[61⇓-63]
染料木素Genistein 芹菜素Apigenin 表儿茶素没食子酸酯Epicatechin gallate	NorA、TetK、MsrA（NC_007795）	金黄色葡萄球菌Staphylococcus aureus NCTC 8325	ABC转运蛋白 ABC transporters	[64-65]
二氢黄酮Flavonone	LmrA （NZ_CP059048）	乳酸乳球菌 Lactococcus lactis	ABC转运蛋白 ABC transporters	[66]
类黄酮Flavonoid	YvcC （AGE65090）	枯草芽孢杆菌 Bacillus subtilis	ABC转运蛋白 ABC transporters	[67]
类黄酮Flavonoid	DrrAB （AAC43341）	波赛链霉菌 Streptomyces peucetius	ABC转运蛋白 ABC transporters	[68]
原儿茶酸Protocatechuic-acid 龙胆酸Gentisic-acid 毛花甙丙Lanatoside-C 大豆甙元Diadzein	MexB （NC_002516）	铜绿假单胞菌Pseudomonas aeruginosa PAO1	RND转运蛋白 RND transporters	[69-70]
龙胆酸Gentisic-acid 毛花甙丙Lanatoside-C 大豆甙元Diadzein	AcrB （NC_000913）	大肠杆菌 Escherichia coli str. K-12 substr. MG1655	RND转运蛋白 RND transporters	[70-71]
表没食子儿茶素没食子酸酯Epigallocatechin gallate	CmeABC、CmeDEF （NC_002163）	空肠弯曲菌Campylobacter jejuni NCTC 11168	RND转运蛋白 RND transporters	[72]
槲皮素Quercetin	AcrAB-TolC （NC_000913）	大肠杆菌 Escherichia coli str. K-12 substr. MG1655	RND转运蛋白 RND transporters	[73-74]
硫酸原黄素Proflavine	Acel （QWB10945）	鲍曼不动杆菌Acinetobacter baumannii	PACE转运蛋白 PACE transporters	[75]