Functions Of Statin Pump Protein In Heterologous Biosynthesis Of Lovastatin And Monacolin J In Yeast

Cardiovascular disease,as a type of disease with mortality exceeding cancer,has received much attention in recent years.Lovastatin is a polyketide isolated from Aspergillus terreus.It is widely used in lowering blood lipids that creating great medicinal and economic value.Compared with lovastatin,simvastatin has higher medicinal and economic value and less side effects.Monacolin J is an important intermediate in the natural biosynthesis pathway of lovastatin and a key intermediate in the semi-synthesis of simvastatin.With the development of synthetic biology,it has been reported that monacolin J and lovastatin were heterologously synthesized in yeast cells and converted to simvastatin in Escherichia coli.Heterologous expression of genes might impose overwhelming metabolic stress on the host cell and excessive accumulation of the bioactive compounds might cause toxicity to the cell.In our previous study,we have heterologously synthesized monacolin J and lovastatin in Pichia pastoris and found that heterologous synthesis of products had an adverse effect on the host.In this study we focused on the statin pump proteins from different species and analysis of the efflux function to lovastatin and monacolin J,which provides reference to the heterologous production of statin molecules.Firstly,a BLAST search with MIcE protein from Penicillium citrinum revealed that there is a hypothetical protein with 69%similarity to MlcE protein in the natural host of lovastatin,Aspergillus terreus wild-type strain NIH2624.Its function is similar to efflux pump,and we then named it as TapA.We also found that LovI protein and MokI protein are similar to MlcE protein.We compared the amino acid sequence of each protein and infered their conservative functional domain.Besides,we analysized the difference among four statin pump proteins in lovastatin resistance and monacolin J transport in the Pichia pastoris expression system.Secondly,the efflux function of statin pump protein TapA to lovastatin was analysized from different angles.Subcellular localization showed that TapA was localized in the cell membrane,which contributed to transport of compounds.The yeast wild-type GS115 strain was tolerant to lovastatin at a concentration of no more than 0.5 mmol/L,while the yeast strain overexpressing TapA can bear more than 25 mmol/L concentration of lovastatin.TapA was overexpressed in the yeast strains,which heterologously synthesized with lovastatin.Strains were cultured in a shake flask and a 5 L reactor.Overexpression of TapA can not only relieve the toxicity of lovastatin to cells,but also greatly improved the production of lovastatin.In addition,we confirmed that monacolin J has a poor transmembrane ability,and only about 2%of extracellular monacolin J can cross the cell membrane.The yeast strain,which heterologously synthesized with monacolin J,overexpressing TapA under the control of promoter PAOXl obviously increased the production of monacolin J in a shake flask,while the yield of monacolin J in the reactor decreased with methanol as sole carbon source.The ratio of intracellular monacolin J was about 49%in TapA overexpressing strain J#9-TapA but only 16%in the J#9.We speculated that there is a large difference between the intracellular and extracellular concentration of monacolin J because of the higher monacolin J titre in the reactor.This promotes the extracellular monacolin J transferring into the cell with the TapA protein to maintain a small difference in the concentration of monacolin J inside and outside the cell.It leads to an increase in the toxicity of intracellular monacolin J to cells and a decrease in the production of monacolin J.Finally,we tested the potential of statin pump protein to increase monacolin J production in an engineered yeast coculture system.Firstly,a high-level glucose repressed and low-level glucose inducible promoter PHXTl was used to control the expression of fatty acid synthase gene.Ethanol was used as a carbon source to efficiently enhance the synthesis ability of the product.The yield of the bioreactor fermentation of monacolin J was increased by 45%.Based on this,TapA was overexpressed in the downstream oxidative strain of the coculture system and the effect of the TapA protein expressed by different strength promoters showed different effects.Compared with the flora that did not overexpress the TapA protein,the yield of monacolin J of the flora that overexpressing the TapA protein fluctuated in the range of 9%to 140%in shake flask culture.The stronger the promoter expressing the TapA protein,the higher the monacolin J yield of the flora.The yield of monacolin J reached up to 1.4 g/L in bioreactor fermentation.The yield of monacolin J decreased after overexpression of TapA.We speculate that the difference between the intracellular and extracellular concentration of monacolin J causes the potential energy,which promotes TapA to function as a transporter.Moreover,we also explored the relationship between statin pump protein TapA and dihydromonacolin L,an upstream intermediate of monacolin J.TapA showed no pump function to dihydromonacolin L,which might ascribed to that the compounds own different structures.This study showed that the four statin pump proteins can work in the transport of monacolin J and lovastatin in the Pichia pastoris expression system.To some extent,they also alleviate the growth and metabolism pressure of heterologous expression strains and enhance the synthesis of statins.This study provided new ideas for the synthesis of other medicinally valuable compounds such as simvastatin.