西红花苷生源途径解析及其合成生物学研究进展

Research progress on biosynthesis and synthetic biology of crocin

天然产物是新药研发的重要资源,具有显著的抗疟疾、抗肿瘤、抗病毒感染等作用.珍稀名贵中药西红花的活性成分为西红花苷,属于脱辅基类胡萝卜素类化合物,具有保护中枢神经系统及预防心脑血管疾病的药理活性.西红花苷资源匮乏、价格高昂,极大限制其药物开发及临床应用.本文从西红花苷的化学表征、生物合成途径解析及合成生物学研究进行综述,展示了西红花苷的光谱学和质谱学特征,阐述其完整生物合成途径的解析,介绍了利用微生物细胞工厂生产5种西红花苷的最新突破.据此,进一步提出开展不同物种西红花苷生物合成途径关键酶的进化机制、合成生物学关键技术探索和条件优化等研究,以推动西红花苷工业化生产及新药研发,为西红花苷来源的中药资源可持续发展提供理论基础和方法指导.

Natural products are important resources for drug discovery and have marvelous roles in the treatment of various diseases,such as malaria,cancers,and viral infections.Crocins are the colored apocarotenoids derived from the stigmas of Crocus sativus and often used as a spice/colorant in the food industry.Pharmacologically,crocins confer protection to the central nervous system(especially in the treatment of Alzheimer’s disease)and prevent cardiovascular/cerebrovascular diseases.Unfortunately,the demand for crocins based on their pharmacological benefits combined with a complex harvesting process of C.sativus has driven the retail price of C.sativus stigmas up to 2000–7000€/kg.Thus,their expense and limited supply greatly restrict the development and clinical application of crocin-based pharmaceuticals.It is therefore urgent to develop an alternative,sustainable way to bolster the supply of crocins.In recent years,synthetic biology has been widely used to overcome shortages of plant-based natural products,including artemisinic acid,etoposide aglycone,opioids,cannabinoids,breviscapine,and ginsenosides.As such,the genetic tools required for crocin biosynthesis have attracted a great deal of attention.The crocin biosynthetic pathway includes three stages:Stage(I),the precursor geranylgeranyl pyrophosphate(GGPP)is generated by the methylerythritol-4-phosphate(MEP)biosynthetic pathway;stage(II),C40-carotenoids are produced via the carotenoid biosynthetic pathway;stage(III),crocins are synthesized by three key catalytic steps,namely,carotenoid cleavage,aldehyde oxidation,and glucosyl group transfer.A series of studies have successfully decoded the crocin biosynthetic pathway and led to its reconstitution in Saccharomyces cerevisiae and Escherichia coli.Hereby,we summarized the chemical characterization of crocins using spectroscopy and mass spectrometry,and elucidated stage(III)of the crocin biosynthetic pathway.Carotenoid cleavage dioxygenases(CCDs)degrade carotenoids.Cs CCD2,CaCCD2(Crocus ancyrensis),BdCCD4.1(Buddleja davidii),BdCCD4.3 and GjCCD4 a(Gardenia jasminoides)can cleave the 7,8(7′,8′)double bond of zeaxanthin to yield crocetin dialdehyde.Subsequently,crocetin dialdehyde was oxidized to crocetin by the catalysis of aldehyde dehydrogenases(ALDHs),including CsALDH54788,CsALDH3898,CsALDH20158,CsALDH11367,CsALDH3 I1,CsALDH3,and GjALDH2 C3.Finally,the biosynthesis of crocins from crocetin was catalyzed by UDP-glycosyltransferases(UGTs),including CsUGT74 AD1,GjUGT74 F8,GjUGT75 L6,GjUGT94 E5,GjUGT94 E13,Bs-GT(Bacillus subtilis 168),and Bc-GTA(Bacillus cereus WQ9-2).And then,we displayed the research progress on the synthetic biology of crocins.With the rapid updating of genetic tools,we have the potential to generate more types of crocins.The in vivo production of crocins is highlighted in this review.The engineered E.coli with GjUGT74 F8 and GjUGT94 E13 can catalyze crocetin into five types of crocins.Furthermore,we put forward some suggestions that might help to improve the bioproduction of crocins,such as the exploration of evolutionary mechanisms of the key enzymes and the optimization of reaction conditions,etc.Taken together,this review will facilitate the industrial production of crocins and the development of crocin-based pharmaceuticals,providing a theoretical basis and method guidance for the sustainable development of crocin-based traditional Chinese medicine.