Effect Of Heterologous ABC Transporter Expression On The Synthesis Of Terpenoids In Saccharomyces Cerevisiae

Terpenoids are a group of high value compounds.However,the microbial synthesis levels of these compounds were restricted by their cytotoxicity.ATP-binding cassette transporter(ABC transporter)can transport the bound substrates across cell membrane by using the energy generated by ATP hydrolysis.Thus,this study intends to introduce heterologous ABC transporters into Saccharomyces cerevisiae hosts and investigate their effect on terpenoid synthesis.In this study,GcABCG1(from Grosmannia clavigera),YlABC2 and YlABC3(both from Yarrowia lipolytica)were introduced into a sabinene-producing S.cerevisiae strain(titer of 22.3 mg/L at shake-flask level).Compared with the initial strain,expressions of Gc ABCG2 and YlABC3 increased sabinene production by13.5% and 34.5%,respectively.However,single-phase cultivation and sabinene tolerance test have not found the sabinene tolerance of the strains was improved.In order to identify the target region which can be subsequently engineered,the amino acid sequences of the three transporters were split into three domains(A,B,C)by homologous sequence alignment.Replacing each domain in Gc ABCG2 and YlABC3 with the corresponding structural units of YlABC2 showed that the replacement of GcABCG1-B and YlABC3-A decreased the productivity most drastically,so these two domains are the most critical domains for their corresponding transporters to transport sabinene.Terpenoids have the same basic structural skeleton.To investigate the substrate selectivity of ABC transporter,we chose amorphadiene to represent sesquiterpene,geranylgeraniol(GGOH)to represent diterpene,squalene and ergosterol to reprent triterpene,and lycopene to represent tetraterpene.GcABCG1,YlABC2,and YlABC3 were introduced into the producing yeasts for the above compounds,respectively.For sesquiterpene,the expressions of all three transporters can increase the synthesis level of amorphadiene.Among them,the expression of YlABC2 achieved the highest amorphadiene production(increased by 24.6%),and its efflux rate was increased by 16.0%.For the synthesis and transport of diterpene,the expressions of GcABCG1 and YlABC2 both reduced the extracellular productions of GGOH.For linear triterpene and tetraterpene,although most of the products are accumulated in the cells,heterologous expression of GcABCG1 can effectively promote the synthesis of squalene and lycopene,which increased the production of them by 23.0% and 28.9%,respectively.The expressions of the three ABC transporters had no effect on the synthesis and transport of tetracyclic ergosterol.Above results shows that different ABC transporters have different substrate selectivity of terpenoids with different number of carbon atoms.This study systematically investigated the effect of heterologous expression of ABC transporter in S.cerevisiae on the synthesis of terpenoids,and provided a reference to synthesize other terpenoids in yeast.