Heterologous Distribution And Optimization Of Lovastatin Biosynthetic Pathway In Pichia Pastoris

With the development of synthetic biology,heterologous synthesis of bioactive rare natural products has drawn wide attention,which can solve problems of traditional microbial fermentation like long culture phase,complex process,numerous byproducts and many other issues.However,the traditional heterologous synthesis is usually carried out in a single strain,facing the problems of heavy metabolism burden,single cell environment,poor compatibility between synthesis pathway and metabolically engineered cells,etc..Using synthetic pathway modular and multiple-cell heterologous assembly to synthesize natural products may solve these problems.Lovastatin is a kind of statin drugs that is widely used in lowering blood lipids and treating cardiovascular disease.It is also a polyketide with a complex biosynthetic pathway produced by Aspergillus terreus.In this study,lovastatin and its key intermediate monacolin J were used as the target product and Pichia pastoris was used as chasiss cells for heterologous construction of synthesis pathway.Then multiplecell heterologous assembly was involved to ease the heavy metabolic burden,balance the pathway,and then improve the production of target products through the optimization of multiple-cell co-culture system.It was reported that the heterologous biosynthetic pathway of lovastatin involved 8 genes of lovB,lovC,lovG,npgA,lovA,cpr,lovF and lovD.In the preliminary study in our group,the genes lovA and lovD were optimized to fit with P.pastoris.The optimized genes of slovA,slovD and other six genes were constructed into three expression vectors,respectively,and then the pathway of lovastatin was reconstructed successfully through the combinatorial expression.Based on this,in this work the three plasmids were transformed into P.pastoris based on the steps of the product synthesis pathway,and single copy strains were screened.Finally,a single copy of the synthesis pathway was constructed in P.pastoris and monaclin J and lovastatin was synthesized successfully.However,results showed that single cell system existed issues of heavy cell pressure,accumulation of intermediate product,imbalance of synthesis pathway.To alleviate these problems,this study further explored the multiple-cell heterologous assembly of the synthetic pathway and improved the synthesis of monaklin J and lovastatin through optimizations of multiple-cell co-culture system.Firstly,the synthesis pathway of Monacolin J was divided into two parts at the pathway node of dehydromonacolin L,and then the divided pathways were constructed in two strains of GS-BCGN-G and GS-AC-Y,respectively.The two strains were marked by green and yellow fluorescent protein in order to facilitate the flow cytometry analysis of the cell-to-cell ratio in co-culture system.Two strategies of dual-cell coculture were designed to synthesize monacolin J,and the yield of monacolin J was improved by 37%when the ratio of GS-BCGN-G and GS-AC-Y was 1:2.The results showed that the metabolic stress could be effectively alleviated by the dual-cell co-culture.Based on this,the multicellular synthesis of lovastatin was further explored.The synthesis pathway was divided into three parts at the pathway nodes of dihydromonacolin L and monacolin J,and constructed in three strains of GS-BCGN-G,GS-AC-Y and GS-FND-R marked by green,yellow and red fluorescent proteins,respectively.The results indicated that the difficulty of monacolin J crossing cell membrane limited the synthesis of lovastatin in triple-cell co-culture.The synthesis of lovastatin was then promoted when the synthesis pathway of lovastatin was distributed into two strains of GS-BCGN-G and GS-ACFND-R at the pathway node of dehydromonacolin L,respectively.It is consistent with the results of the synthesis of monacolin J by co-culture.Three different lovastatin dual-cell systems were further designed for the syhthesis of lovastatin.It was proved that the pressure of cells could be relieved by dual-cell co-culture,and the production of lovastatin was increased by 71%after optimization of the inoculation ratio.