Mining Of Bryophyte Sesquiterpene Synthases,Optimization Of Enzyme Activity And Biosynthesis Of Related Products

Bryophytes are rich in structurally diverse sesquiterpenes,most of which have antibacterial and insecticidal activities.Mylia taylorii and Jungermannia exsertifolia belong to the Jungermanniaceae family and their essential oils contain aromadendrenetype sesquiterpenes.In recent research,some bryophyte sesquiterpene synthases(STSs)were identified as microbial-type like sesquiterpene synthases(MTPSLs),which are a new clade of terpene synthases.So far,MTPSLs were only found in some nonseed land plants,such as liverworts,hornworts and lycophytes.In addition,studies of the catalytic mechanism and heterologous expression of MTPSLs are limited.Here,the transcriptome analyses of M.taylorii and J.exsertifolia were performed to identify M.taylorii sesquiterpene synthases(MtSTSs)and J.exsertifolia sesquiterpene synthases(JeSTSs).According to sequence alignment and homologous modeling,single-point and multi-point mutagenesis of STSs were conducted to enhance the yield of catalytic products.Based on metabolic engineering modification,the efficient biosynthesis of bryophyte sesquiterpenes were achieved in engineered Saccharomyces cerevisiae.Three MtSTSs and three JeSTSs were identified from transcriptome analyses.They were closely related to bryophyte and microbial STSs,and were thereby classified as MTPSLs.MtSTS1,MtSTS3,JeSTS1 and JeSTS2 catalyzed the conversion of FPP to nerolidol.MtSTS2 and JeSTS4 are promiscuous enzymes.MtSTS2 mainly producedβ-cadinene,cubenol and α-cadinol.JeSTS4 mainly produced bicyclogermacrene,γ-elemene and aromadendrene.Based on sequence alignment and homologous modeling of bicyclogermacrene synthase(LdTPS5)with known STS,site-directed mutagenesis was performed and the catalytic residues of LdTPS5 were identified.The mutation studies of MtSTSs and JeSTSs were carried out.The enzyme activities of mutants LdTPS5M33(I290V,I316 L and L454C)and JeSTS4M24(G91S)were improved.In addition,the yields of their mutant strains were increased by 52.7 and 39 times,respectively.These catalytic residues,which are called plasticity residues,are located far away from the active site pocket.We infer that these residues may remotely affect the properties of active sites to improve the release of pyrophosphate group.In addition,the deprotonation of the intermediate may also be enhanced.Three genes,ACS1,ERG20 and ADH1,were overexpressed in the engineered Saccharomyces cerevisiae.Combined with the modified LdTPS5M33 and JeSTS4M24,two engineered yeast strains Y-LdTPS5M33-1 and Y-JeSTS4M24-1 that could efficiently produce bicyclogermacrene were obtained and their fed-batch shake flask fermentation yields were 484.51 and 518.59 mg/L,respectively.Compared with the original strains Y-LdTPS5 and Y-JeSTS4,the main product yields were increased by 268 and 192 times,respectively.The high-efficiency biosynthesis of bicyclogermacrene was initially achieved through synthetic biology.