De Novo Biosynthesis Taxifolin In Saccharomyces Cerevisiae Through Metabolic Engineering

(2S)-Taxifolin is a kind of flavonoid which belongs to plant nature product and mainly extracted from the wood of Pseudotsuga menziesii or Larix gmelinii.(2S)-taxifolin has many healthy care benefits and it plays an important precursor of many plant natural products like flavonolignan and anthocyanin in plant metabolic pathway.Most natural flavonoids ncluding (2S)-taxifolin,can be commercially obtained only via plant extraction,a process highly dependent on long breeding cycles and seasonal or regional availability,thereby restricting the extensive application of these useful compounds.Hence,microbial synthesis of flavonoids has received the attention of many researchers because of its clear advantages,such as a moderate reaction process rather than extreme reaction conditions,use of renewable feedstocks,and native chiral selectivity.With Saccharomyces cerevisiae was used as a platform strain,this project is focused on investigating and developing the general strategies which applied to biosynthesize high-value-added plant natural products,expressly (2S)-taxifolin,in microorganisms.Combined tools and strategies emerged in synthetic biology such as modular pathway engineering,promoter engineering,directed evolution,bioinformation analysis and high-throughput detection method,the bottlenecks in biosynthesis (2S)-taxifolin were verified and released.A high accumulation of (2S)-taxifolin and intermediate product–p-coumaric acid,(2S)-naringenin and (2S)-eriodcityol-was achieved.Tools and strategies applied and established in this project could be a valuable significance for other researchers.The major results in this project were highlighted below:(1)Constructed a de novo (2S)-naringenin production strain to verify the bottlenecks in (2S)-naringenin bioxynthesis pathway.(2S)-Naringenin is an important preco biosynthesis pathway in Saccharomyces cerevisiae is long and many intermediate products are involved including (2S)-naringenin.The (2S)-naringenin synthesis pathway genes,consisting of FjTAL(TyrosineammonialyasesfromFlavobacteriumjohnsoniae),Pc4CL(4-hydroxycinnamoyl-Co A ligase from Petroselinum crispum),PhCHS(Chalcone synthase from Petunia X hybrida),MsCHI(Chalcone isomerase from Medicago sativa),were integrated into the yeast genome to obtain a (2S)-naringenin production strain.After gene dosage experiments,the genes negatively regulating the shikimate pathway and inefficient chalcone synthase activity were verified as factors limiting (2S)-naringenin biosynthesis.With fed-batch process optimization of the engineered strain,the titer of (2S)-naringenin reached 648.63 mg·L^-1 from 2.50 g·L^-1 p-coumaric acid.(2)Enhancing(2S)-naringenin accumulation through optimizing(2S)-naringenin biosynthesis pathway genes expression level based on promoter adjustment.Pathway optimization plays an important role in the fine-tuning of metabolic pathways.Introducing heterologous pathways often leads to metabolic flux imbalance in host strains.To achieve the re-balance of metabolic flux in Saccharomyces cerevisiae,66 promoters with different strengths were selected primarily on the basis of RNA-Seq data base.Translation activities were verified on the basis of enhanced green fluorescent protein(EGFP)fluorescence strength through fluorescence activated cell sorting(FACS).On the basis of this promoter library,the(2S)-naringenin biosynthesis pathway,which consists of Pc4CL,PhCHS and MsCHI,was constructed and optimized.A total of 30 promoters were chosen to randomly assembly Pc4CL,PhCHS and MsCHI into vectors.With a high throughput (2S)-naringenin detection method,the titer of (2S)-naringenin reached 1.21 g·L^-1.(3)Efficient biosynthesis of (2S)-eriodictyol from (2S)-naringenin in Saccharomyces cerevisiae by combination of promoter adjustment and directed evolution.(2S)-Eriodictyol is derived from (2S)-naringenin through flavonoid 3?-hydroxylase(F3?H) catalyzation.F3?H is cytochrome P450 enzyme and requires a cytochrome P450 reductase(CPR) for its function.However,P450s have a limited application in chemical biosynthesis owing to its low activity and complex catalyzing mechanism.Here,an efficient SmF3?H and a matched SmCPR were identified from Silybum marianum.After a well optimized expression of SmF3?H/SmCPR through promoter adjustment,(2S)-eriodictyol production increased dramatically.The analyzation of promoter adjusted strains reveals that SmF3?H/SmCPR should under similar and strong expression level.Furthermore,directed evolution was applied to improve SmF3?H/SmCPR activity and a coupling variant SmF3?H^D284N/SmCPR^I453V with increased activity were achieved.A high-throughput screening method was established and applied in promoter adjustment and directed evolution assays.This results in a maximum (2S)-eriodictyol titer of 3.28 g·L^-1 from 5.00 g·L^-1 (2S)-naringenin.,the highest titer reported to date in any host.(4)Identification of flavonoids 3-hydroxylase from Silybum marianum and its application in enhanced production of (2S)-taxifolin.Flavone 3-hydroxylase (F3H)can catalyze the synthesis of (2S)-taxifolin and other 3-hydroxylated flavonoids from (2S)-eriodictyol.Due to the low catalytic efficiency of F3H,the titer of many 3-hydroxyflavones,such as taxifolin,synthesized by microbial method is relatively low.In this study,a SmF3H was identified from the transcriptome of Silybum marianum(L.)Gaertn.The results of fermentation showed that SmF3H can catalyze the flavone 3-hydroxylation reaction,and its catalytic efficiency was significantly higher than that of commonly used Sl F3H from Solanum lycopersicum.Six promoters with different transcription strength were selected to optimize the synthesis pathway from the flavonoid precursor(2S)-naringenin to(2S)-taxifolin.The results showed that the highest titer of(2S)-taxifolin(695.90 mg·L^-1 in shake flask)could be obtained when the P_GAL7 promoter was used to control the expression of SmF3H.The titer of(2S)-taxifolin was further improved to 3.54 g·L^-1 in a 5-L fermenter,which is the highest titer according to current available literatures.(5)Constructed a multiple integration toolbox for de novo(2S)-taxifolin biosynthesis in Saccharomyces cerevisiae.At least seven heterogenous genes and three shikimic pathway related genes are involved in(2S)-taxifolin Biosynthesis in Saccharomyces cerevisiae.Expressing these genes in plasmid was no longer suitable due to its unstable.To make sure these genes under efficient and stable expression,a vector based tool box for simultaneous and multiple genomic integrations in Saccharomyces cerevisiae was built.In this tool box,five Ty retrotransposons sites sequences and ten markers were chosen and combined,fifty plasmids were built and fit for each Ty site insertion under any markers.The integration and expression abilities of these plasmids were verified through report gene EGFP.Then,(2S)-taxifolin biosynthesis pathway related genes were integrated into different Ty sites,as well as enhanced malonyl-CoA pathway,resulted a de novo(2S)-taxifolin biosynthesis strain.After fermentation optimization in bioreactor,135.83 mg·L^-1(2S)-taxifolin was gained from glucose.