Microbial Production Of Salidroside,Rosmarinic Acid And Its Analogues By Extending Shikimate Pathway

Plant natural products(PNPs)derived from the shikimate pathway are widely distributed in nature,and serve many functions in an organism's survivability due to the various biological and pharmacological activites.Alternative methods have been developed to produce PNPs including direct extraction from plants,chemical synthesis.However,direct extraction method has been limited due to inadequate natural resources and low content of plant tissues,and chemical synthesis has disadvantages of employing toxic chemicals and complex procedures.Biosynthesis of fine and specialty chemicals via the use of engineered microbes has arisen as a competitive alternative to traditional chemistry-based routes due to its fast growth,low cost and easier purification.Hence,microbial production remains a promising way to meet the commercial demand of PNPs.In this work,we are dedicated to designing and constructing new biosynthetic pathways to synthesize salidroside(SD),rosmarinic acid(RA)and its analogous derived from shikimate pathway in microorganisms.Rhodiola is a traditional Chinese medicinal herb,which is well-known as "Tibetan Ginseng" with a long history for folk use.Rhodiola is commonly used as a health food to reinforce immunity,memory,and learning,as well as relieve altitude sickness.SD,a Tyrosol glucoside,is an important active ingredient in plant Rhodiola and displays diverse biological properties such as anti-aging,anti-inflammation,cardiovascular disease prevention,nerve cell protection etc.Shas attracted much attention as potential nutraceutical supplements or drugs in multiple fields.In this work,we systematically engineered high yield production of SD in Saccharomyces cerevisiae.The yeast is well known for producing Tyrosol through Ehrlich pathway.The Tyrosol production was increased from 170 mg/L to 401 mg/L by overexpressing aromatic aldehyde synthase from Petroselinum crispum converting Tyrosine to the corresponding aldehyde.Glucosyltransferases from different resources were screened for better function for glucosylating Tyrosol in yeast via biotransformation experiments.Salidroside production from glucose reached 79 mg/L in the engineered yeast harboring the best glucosyltransferase screened.the metabolic flux was enhanced toward Tyrosine biosynthesis by overexpressing shikimate kinase from Escherichia coli and alleviation of feedback inhibition,and the SD production was further increased to 107 mg/L.In order to acquire a genetically stable strain for potential industrial production,the genes of SD biosynthesis were integrated into yeast chromosome,resulting in improvement of SD production to 239 mg/L.Finally,the production of SD was improved to 732 mg/L through fed-batch fermentation in a 5-L bioreactor.The work may provide an alternative way for large-scale production of SD instead of the traditional methods.RA and its analogues are important phenolic acids widely found in plant kingdom and display diverse biological activities such as antiviral,antioxidant,anti-inflammatory,anti-allergic,neuroprotective,which led to the extensive application in food,cosmetic and pharmaceutical industries.In this work,3,4-dihydroxyphenyllactic acid(DHPL)pathway was constructed and co-expressed with a RA synthase from Coleus blumei(CbRAS)and a 4-coumarate:Co A ligase from Arabidopsis thaliana(At4CL)in a Tyrosine overproducing E.coli strain.We obtained high yield production of RA(?130 mg/L)by co-incubation of the engineered strains with caffeic acid.Additionally,a new compound caffeoyl-phenyllactate(CP)was also produced by the engineered E.coli strain beyond the known compounds RA and isorinic acid(IA)that has been isolated from plants.The titers of RA,IA,CP are 127 mg/L,252 mg/L,55 mg/L,respectively.This work also sheds new light on the construction of cell factory of RA in E.coli.Additionally,we are dedicated to production of RA analogues in E.coli by precursor-directed biosynthesis.The process included donor substrate synthesis for CoA esters from carboxylic acids by 4-coumarate:CoA ligase and a conjugation step by RAS to form RA and its analogues with acceptor substrates.Incubation of the recombinant E.coli harboring 4-coumarate:CoA ligase from Arabidopsis thaliana(At4CL)and a RAS from Coleus blumei(CbRAS)with exogenously supplied phenyllactic acid(PL)and analogues as acceptor substrates and coumaric acid and analogues led to production of 21 compounds,including 14 unnatural RA analogues.This work presented here not only provides a new example of successful generation of plant derived natural products and unnatural analogues by precursor-directed biosynthesis,but also sheds new light on the substrate specificity of CbRAS.3-(4-Hydroxyphenyl)propionic acid(HPPA)is an aromatic compound used as primary intermediate of plant derived natural products,such as myricanol and phloretin,with important application in food industries.Additionally,it also serves as a key intermediate of pharmaceuticals such as cetraxate hydrochloride,esmolol hydrochloride.The traditional method for production of HPPA is chemical synthesis,which requires harsh reaction conditions and toxic chemicals.In this work,we aim to achieve the De novo synthesis of HPPA in S.cerevisiae,and it was conducted via overexpressing a Tyrosine ammonia-lyase from Flavobacterium johnsoniaeu(FjTAL)and a p-coumaroyl-CoA ligase from A.thaliana(At4CL),as well as using super long-chain enoyl-CoA reductase(ScTSC13)and acyl-CoA thioesterases from S.cerevisiae BY4742.The titer of HPPA reached to 70 mg/L.