药学学报, 2022, 57(5): 1322-1335
林春草, 陈大伟, 戴均贵*. 黄酮类化合物合成生物学研究进展[J]. 药学学报, 2022, 57(5): 1322-1335.
LIN Chun-cao, CHEN Da-wei, DAI Jun-gui*. Advances of synthetic biology of flavonoids[J]. Acta Pharmaceutica Sinica, 2022, 57(5): 1322-1335.

林春草, 陈大伟, 戴均贵*
中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 中国医学科学院酶与天然药物生物催化重点实验室, 国家卫生健康委员会天然药物生物合成重点实验室, 北京 100050
黄酮类化合物是植物中广泛分布的一大类次级代谢产物,已报道的化合物超过10 000种,结构多样且部分具有抗癌、抗炎等重要的药理活性。黄酮类化合物生物合成相关的基因及酶、生物合成途径解析已有诸多报道;近年来,其合成生物学研究亦取得了较大进展。本文在概述黄酮类化合物生物合成的基础上,对近20年(2001~2021年)报道的黄酮类化合物合成生物学研究进展进行了综述,介绍了代表性黄酮类化合物的细胞工厂创建概况,并对相应的代谢瓶颈及优化策略进行了讨论和展望。
关键词:    黄酮类化合物      合成生物学      生物合成途径      细胞工厂      代谢瓶颈     
Advances of synthetic biology of flavonoids
LIN Chun-cao, CHEN Da-wei, DAI Jun-gui*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, CAMS Key Laboratory of Enzymes and Natural Drug Biocatalysis, NHC Key Laboratory of Natural Drug Biosynthesis, Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100050, China
Flavonoids is one of the biggest families of the plant-derived secondary metabolites with structural diversity. Until now, over 10 000 kinds of flavonoids with distinct structures have been purified and identified from plants, and some of them possess a range of important pharmacological effects, such as anticancer, anti-inflammatory and so on. So far, a number of genes and enzymes responsible for the biosynthesis of flavonoids have been reported, especially, a great of progress has been achieved in the synthetic biology of flavonoids in the recent years. Herein, based upon a brief introduction on the biosynthesis of flavonoids, this review summarizes the research advances in synthetic biology of flavonoids in the past two decades (2001-2021), highlighting the cell factories construction of the representative flavonoids. And, a brief discussion and prospects of the relevant metabolic bottlenecks and optimizing strategies are proposed.
Key words:    flavonoids    synthetic biology    biosynthetic pathway    cell factory    metabolic bottleneck   
收稿日期: 2022-01-04
DOI: 10.16438/j.0513-4870.2022-0008
基金项目: 国家重点研发计划资助项目(2020YFA0908000).
通讯作者: 戴均贵,Tel:86-10-63165195,E-mail:jgdai@imm.ac.cn
Email: jgdai@imm.ac.cn
PDF(765KB) Free
林春草  在本刊中的所有文章
陈大伟  在本刊中的所有文章
戴均贵*  在本刊中的所有文章

[1] Dixon RA, Pasinetti GM. Flavonoids and isoflavonoids:from plant biology to agriculture and neuroscience[J]. Plant Physiol, 2010, 154:453-457.
[2] Walsh CT, Tang Y. Natural Product Biosynthesis:Chemical Logic and Enzymatic Machinery[M]. London:The Royal Society of Chemistry, 2020.
[3] Novák K, Lisá L, Skrdleta V. Rhizobial nod gene-inducing activity in pea nodulation mutants:dissociation of nodulation and flavonoid response[J]. Physiol Plant, 2004, 120:546-555.
[4] Harborne JB, Williams CA. Advances in flavonoid research since 1992[J]. Phytochemistry, 2000, 55:481-504.
[5] Salehi B, Fokou PVT, Sharifi-Rad M, et al. The therapeutic potential of naringenin:a review of clinical trials[J]. Pharmaceuticals, 2019, 12:11.
[6] Chen AY, Chen YC. A review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention[J]. Food Chem, 2013, 138:2099-2107.
[7] Ko KP. Isoflavones:chemistry, analysis, functions and effects on health and cancer[J]. Asian Pac J Cancer Prev, 2014, 15:7001-7010.
[8] Lee MH, Lin CC. Comparison of techniques for extraction of isoflavones from the root of Radix Puerariae:ultrasonic and pressurized solvent extractions[J]. Food Chem, 2007, 105:223-228.
[9] Lee DYW, Zhang WY, Karnati VVR. Total synthesis of puerarin, an isoflavone C-glycoside[J]. Tetrahedron Lett, 2003, 44:6857-6859.
[10] Wang YC, Chen S, Yu O. Metabolic engineering of flavonoids in plants and microorganisms[J]. Appl Microbiol Biotechnol, 2011, 91:949-956.
[11] Liu XY, Luo LL, Ma Y, et al. Biopathway construction of plant natural products[J]. Acta Pharm Sin (药学学报), 2021, 56:3285-3299.
[12] Ko YS, Kim JW, Lee JA, et al. Tools and strategies of systems metabolic engineering for the development of microbial cell factories for chemical production[J]. Chem Soc Rev, 2020, 49:4615-4636.
[13] Lim CG, Fowler ZL, Hueller T, et al. High-yield resveratrol production in engineered Escherichia coli[J]. Appl Environ Microbiol, 2011, 77:3451-3460.
[14] Paddon CJ, Westfall PJ, Pitera DJ, et al. High-level semi-synthetic production of the potent antimalarial artemisinin[J]. Nature, 2013, 496:528-532.
[15] Wang PP, Wei W, Ye W, et al. Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency[J]. Cell Discov, 2019, 5:5.
[16] Gao S, Zhou HR, Zhou JW, et al. Promoter-library-based pathway optimization for efficient (2S)-naringenin production from p-coumaric acid in Saccharomyces cerevisiae[J]. J Agric Food Chem, 2020, 68:6884-6891.
[17] Wu JJ, Du GC, Zhou JW, et al. Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy[J]. Metab Eng, 2013, 16:48-55.
[18] Zhu QL, Xie XR, Lin HX, et al. Isolation and functional characterization of a phenylalanine ammonia-lyase gene (SsPAL1) from Coleus (Solenostemon scutellarioides (L.) Codd)[J]. Molecules, 2015, 20:16833-16851.
[19] Wang ZW, Jian XY, Zhao YC, et al. Functional characterization of cinnamate 4-hydroxylase from Helianthus annuus Linn using a fusion protein method[J]. Gene, 2020, 758:144950.
[20] Jendresen CB, Stahlhut SG, Li MJ, et al. Highly active and specific tyrosine ammonia-lyases from diverse origins enable enhanced production of aromatic compounds in bacteria and Saccharomyces cerevisiae[J]. Appl Environ Microbiol, 2015, 81:4458-4476.
[21] Li ZB, Li CF, Li J, et al. Molecular cloning and functional characterization of two divergent 4-coumarate: coenzyme A ligases from kudzu (Pueraria lobata)[J]. Biol Pharm Bull, 2014, 37:113-122.
[22] Trantas E, Panopoulos N, Ververidis F. Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae[J]. Metab Eng, 2009, 11:355-366.
[23] Li MJ, Kildegaard KR, Chen Y, et al. De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae[J]. Metab Eng, 2015, 32:1-11.
[24] Nakajima O, Akiyama T, Hakamatsuka T, et al. Isolation, sequence and bacterial expression of a cDNA for chalcone synthase from the cultured cells of Pueraria lobata[J]. Chem Pharm Bull, 1991, 39:1911-1913.
[25] Leonard E, Lim KH, Saw PN, et al. Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli[J]. Appl Environ Microbiol, 2007, 73:3877-3886.
[26] Mameda R, Waki T, Kawai Y, et al. Involvement of chalcone reductase in the soybean isoflavone metabolon:identification of GmCHR5, which interacts with 2-hydroxyisoflavanone synthase[J]. Plant J, 2018, 96:56-74.
[27] Ayabe S, Udagawa A, Furuya T. NAD(P)H-dependent 6'-deoxychalcone synthase activity in Glycyrrhiza echinata cells induced by yeast extract[J]. Arch Biochem Biophys, 1988, 261:458-462.
[28] Ralston L, Subramanian S, Matsuno M, et al. Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases[J]. Plant Physiol, 2005, 137:1375-1388.
[29] Zhao Q, Zhang Y, Wang G, et al. A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis[J]. Sci Adv, 2016, 2:e1501780.
[30] Prescott AG, Stamford NPJ, Wheeler G, et al. In vitro properties of a recombinant flavonol synthase from Arabidopsis thaliana[J]. Phytochemistry, 2002, 60:589-593.
[31] Salanoubat M, Lemcke K, Rieger M, et al. Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana[J]. Nature, 2000, 408:820-822.
[32] Jung W, Yu O, Lau SM, et al. Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes[J]. Nat Biotechnol, 2000, 18:208-212.
[33] Akashi T, Aoki T, Ayabe S. Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis[J]. Plant Physiol, 2005, 137:882-891.
[34] Kim BG, Kim H, Hur HG, et al. Regioselectivity of 7-O-methyltransferase of poplar to flavones[J]. J Biotechnol, 2006, 126:241-247.
[35] Zhao Q, Cui MY, Levsh O, et al. Two CYP82D enzymes function as flavone hydroxylases in the biosynthesis of root-specific 4'-deoxyflavones in Scutellaria baicalensis[J]. Mol Plant, 2018, 11:135-148.
[36] Amor ILB, Hehn A, Guedone E, et al. Biotransformation of naringenin to eriodictyol by Saccharomyces cerevisiea functionally expressing flavonoid 3'-hydroxylase[J]. Nat Prod Commun, 2010, 5:1893-1898.
[37] Thill J, Miosic S, Gotame TP, et al. Differential expression of flavonoid 3'-hydroxylase during fruit development establishes the different B-ring hydroxylation patterns of flavonoids in Fragaria×ananassa and Fragaria vesca[J]. Plant Physiol Biochem, 2013, 72:72-78.
[38] Wang X, Li CF, Zhou C, et al. Molecular characterization of the C-glucosylation for puerarin biosynthesis in Pueraria lobata[J]. Plant J, 2017, 90:535-546.
[39] Noguchi A, Saito A, Homma Y, et al. A UDP-glucose:isoflavone 7-O-glucosyltransferase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis[J]. J Biol Chem, 2007, 282:23581-23590.
[40] Chen RD, Liu X, Zou JH, et al. Regio-and stereospecific prenylation of flavonoids by Sophora flavescens prenyltransferase[J]. Adv Synth Catal, 2013, 355:1817-1828.
[41] Li JH, Chen RD, Wang RS, et al. Biocatalytic access to diverse prenylflavonoids by combining a regiospecific C-prenyltransferase and a stereospecific chalcone isomerase[J]. Acta Pharm Sin B, 2018, 8:678-686.
[42] Li JH, Chen RD, Wang RS, et al. GuA6DT, a regiospecific prenyltransferase from Glycyrrhiza uralensis, catalyzes the 6-prenylation of flavones[J]. Chembiochem, 2014, 15:1673-1681.
[43] Wang RS, Chen RD, Li JH, et al. Molecular characterization and phylogenetic analysis of two novel regio-specific flavonoid prenyltransferases from Morus alba and Cudrania tricuspidate[J]. J Biol Chem, 2014, 289:35815-35825.
[44] Feng KP, Chen RD, Xie KB, et al. A regiospecific rhamnosyltransferase from Epimedium pseudowushanense catalyzes the 3-O-rhamnosylation of prenylflavonols[J]. Org Biomol Chem, 2018, 16:452-458.
[45] Feng KP, Chen RD, Xie KB, et al. Ep7GT, a glycosyltransferase with sugar donor flexibility from Epimedium pseudowushanense, catalyzes the 7-O-glycosylation of baohuoside[J]. Org Biomol Chem, 2019, 17:8106-8114.
[46] Wang PP, Li CJ, Li XD, et al. Complete biosynthesis of the potential medicine icaritin by engineered Saccharomyces cerevisiae and Escherichia coli[J]. Sci Bull, 2021, 66:1906-1916.
[47] Muhammad A, Feng XD, Rasool A, et al. Production of plant natural products through engineered Yarrowia lipolytica[J]. Biotechnol Adv, 2020, 43:107555.
[48] Chen TT, Zhou YY, Lu YH, et al. Advances in heterologous biosynthesis of plant and fungal natural products by modular co-culture engineering[J]. Biotechnol Lett, 2019, 41:27-34.
[49] Rodriguez A, Kildegaard KR, Li MJ, et al. Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis[J]. Metab Eng, 2015, 31:181-188.
[50] Liu QL, Yu T, Li XW, et al. Rewiring carbon metabolism in yeast for high level production of aromatic chemicals[J]. Nat Commun, 2019, 10:4976.
[51] Gao S, Lyu YB, Zeng WZ, et al. Efficient biosynthesis of (2S)-naringenin from p-coumaric acid in Saccharomyces cerevisiae[J]. J Agric Food Chem, 2020, 68:1015-1021.
[52] Zhou SH, Hao TT, Zhou JW. Fermentation and metabolic pathway optimization to de novo synthesize (2S)-naringenin in Escherichia coli[J]. J Microbiol Biotechnol, 2020, 30:1574-1582.
[53] Koopman F, Beekwilder J, Crimi B, et al. De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae[J]. Microb Cell Fact, 2012, 11:155.
[54] Lyu XM, Zhao GL, Ng KR, et al. Metabolic engineering of Saccharomyces cerevisiae for de novo production of kaempferol[J]. J Agric Food Chem, 2019, 67:5596-5606.
[55] Ganesan V, Li ZH, Wang XN, et al. Heterologous biosynthesis of natural product naringenin by co-culture engineering[J]. Synth Syst Biotechnol, 2017, 2:236-242.
[56] Zhang W, Liu H, Li X, et al. Production of naringenin from D-xylose with co-culture of E. coli and S. cerevisiae[J]. Eng Life Sci, 2017, 17:1021-1029.
[57] Lv YK, Marsafari M, Koffas M, et al. Optimizing oleaginous yeast cell factories for flavonoids and hydroxylated flavonoids biosynthesis[J]. ACS Synth Biol, 2019, 8:2514-2523.
[58] Palmer CM, Miller KK, Nguyen A, et al. Engineering 4-coumaroyl-CoA derived polyketide production in Yarrowia lipolytica through a β-oxidation mediated strategy[J]. Metab Eng, 2020, 57:174-181.
[59] Eichenberger M, Hansson A, Fischer D, et al. De novo biosynthesis of anthocyanins in Saccharomyces cerevisiae[J]. FEMS Yeast Res, 2018.DOI:10.1093/femsyr/foy046.
[60] Leonard E, Lim KH, Saw PN, et al. Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli[J]. Appl Environ Microbiol, 2007, 73:3877-3886.
[61] Leonard E, Yan YJ, Fowler ZL, et al. Strain improvement of recombinant Escherichia coli for efficient production of plant flavonoids[J]. Mol Pharm, 2008, 5:257-265.
[62] Wu JJ, Zhang X, Zhou JW, et al. Efficient biosynthesis of (2S)-pinocembrin from D-glucose by integrating engineering central metabolic pathways with a pH-shift control strategy[J]. Bioresour Technol, 2016, 218:999-1007.
[63] Yan YJ, Huang LX, Koffas MAG. Biosynthesis of 5-deoxyflavanones in microorganisms[J]. Biotechnol J, 2007, 2:1250-1262.
[64] Rodriguez A, Strucko T, Stahlhut SG, et al. Metabolic engineering of yeast for fermentative production of flavonoids[J]. Bioresour Technol, 2017, 245:1645-1654.
[65] Stahlhut SG, Siedler S, Malla S, et al. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli[J]. Metab Eng, 2015, 31:84-93.
[66] Akram M, Rasool A, An T, et al. Metabolic engineering of Yarrowia lipolytica for liquiritigenin production[J]. Chem Eng Sci, 2021, 230:116117.
[67] Wang X, Song ZJ, He X, et al. Antitumor and immunomodulatory activity of genkwanin on colorectal cancer in the APC(Min/+) mice[J]. Int Immunopharmacol, 2015, 29:701-707.
[68] Lee H, Kim BG, Kim M, et al. Biosynthesis of two flavones, apigenin and genkwanin, in Escherichia coli[J]. J Microbiol Biotechnol, 2015, 25:1442-1448.
[69] Li JH, Tian CF, Xia YH, et al. Production of plant-specific flavones baicalein and scutellarein in an engineered E. coli from available phenylalanine and tyrosine[J]. Metab Eng, 2019, 52:124-133.
[70] Ji DN, Li JH, Xu FL, et al. Improve the biosynthesis of baicalein and scutellarein via manufacturing self-assembly enzyme reactor in vivo[J]. ACS Synth Biol, 2021, 10:1087-1094.
[71] Dueber JE, Wu GC, Malmirchegini GR, et al. Synthetic protein scaffolds provide modular control over metabolic flux[J]. Nat Biotechnol, 2009, 27:753-759.
[72] Liu XN, Cheng J, Zhang GH, et al. Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches[J]. Nat Commun, 2018, 9:448.
[73] Duan LJ, Ding WT, Liu XN, et al. Biosynthesis and engineering of kaempferol in Saccharomyces cerevisiae[J]. Microb Cell Fact, 2017, 16:165.
[74] Du Y, Yang BR, Yi ZQ, et al. Engineering Saccharomyces cerevisiae coculture platform for the production of flavonoids[J]. J Agric Food Chem, 2020, 68:2146-2154.
[75] Dixon RA, Ferreira D. Genistein[J]. Phytochemistry, 2002, 60:205-211.
[76] Kim DH, Kim BG, Jung NR, et al. Production of genistein from naringenin using Escherichia coli containing isoflavone synthase-cytochrome P450 reductase fusion protein[J]. J Microbiol Biotechnol, 2009, 19:1612-1616.
[77] Kim BG. Biological synthesis of genistein in Escherichia coli[J]. J Microbiol Biotechnol, 2019, 30:770-776.
[78] Horinouchi S. Combinatorial biosynthesis of plant medicinal polyketides by microorganisms[J]. Curr Opin Chem Biol, 2009, 13:197-204.
[79] Leonard E, Koffas MAG. Engineering of artificial plant cytochrome P450 enzymes for synthesis of isoflavones by Escherichia coli[J]. Appl Environ Microbiol, 2007, 73:7246-7251.
[80] Liu QL, Liu Y, Li G, et al. De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories[J]. Nat Commun, 2021, 12:6085.
1.刘秀玉, 罗凌龙, 马莹, 卜俊玲, 胡志敏, 孙术富, 崔光红, 唐金富, 郭娟, 黄璐琦.植物天然产物途径创建[J]. 药学学报, 2021,56(12): 3285-3299
2.王冬, 刘怡, 许骄阳, 王金鹤, 戴住波, 张学礼, 黄璐琦.创建酿酒酵母细胞工厂高效生产人参皂苷前体达玛烯二醇II[J]. 药学学报, 2018,53(8): 1233-1241
3.何云飞 高伟 刘塔斯 李文渊 黄璐琦.二萜合酶的研究进展[J]. 药学学报, 2011,46(9): 1019-1025