The Study About Using Transporter To Improve Yeast's Tolerability To Terpenes

Terpenes,which are usually extracted from plants,are a kind of abundant natural secondary metabolites and have diverse biological activities in anti-inflammatory,antimalarial and anti-tumor,et al.In recent years,it is considered to be the most potential method to produce terpenes in recombinant microbes with the synthetic biology tools,such as artemisinic acid,farnesene and other substances fermentation production.However,a considerable portion of terpenes are synthesized to defend exogenous microbe invation in plants and these kinds of terpenes are toxic to microbe hosts,thus their accumulation will definitely affect synthesis efficiency.On the other hand,as a result of lipid-soluble characteristics and the lack of corresponding transporters to pump them out,these terpenes are usually considered to be localized in capacity limited membranes,which will definitely limt production and affect products'purification.Therefore,it is the key step to mine and applicate functional proteins that could enhance the corresponding terpene tolerance of the microbial host.In this study,we firstly evaluated different terpenes' inhibition effects towards Saccharomyces cerevisiae CEN.PK2-1D.A plenty of terpenes,such as monoterpenes,sesquiterpenes,diterpenes and terpene oils,could inhibit CEN.PK2-1D'S growth and among the tested terpenes,sesquiterpenes was the most significant(IC50 varied from 5.0 to 80.0 mg/L and Gernacrene A's IC50 was 5.0 mg/L).Secondly,a transporter mutant library was constructed to screen for a functional protein which could enhance tolerance of terpenenes.When using Germacrene A as inhibitor,ScABC1,the transporter related to Germacrene A gene was screened out:when 5.0 mg/L Germacrene A was added,the ScABC1 deficient strain was significantly inhibited(cell growth decreased 82%compared to the control strain)and the ScABC1 overexpressing strain was restored at the cell growth level(decreased 2.8%compared to the control strain).Further experiments proved that ScABC1 could also work against other sesquiterpenes,diterpenes and terpene oils.To test whether overexpressing ScABC1 could increase terpene cell factories'production,we constructed cell factories for production of Geraniol ester,Nerolidol,Sclareol in Saccharomyces cerevisiae.Next,ScABC1 was overexpressed in the terpene producing strains.Results showed that overexpression ScABC1 could result in 44.4%,7.9%,24.6%increase of revelent terpene's production,independently.ScABC1,a transporter related mainly to transport sesquiterpenes,was obtained on the basis of the experiments of terpenes inhibition to yeast.Overexpression of the gene could enhance the host' tolerance to terpenes and increase the yield of engineering strain.This study lays the foundation of constructing efficient cell factories for producingnatural products.