Heterosoluble Expression Of Key Genes For Aklavinone Biosynthesis

Aklavinone is an economically important intermediate for biosynthesis of anthracyclines.Due to its low level in the fermentation broth of Streptomyces galilaeus and Streptomyces peucetius,it is highly desirable to select a suitable host for hetero-biosynthesis of aklavinone.Of reported host strains,Escherichia coli is advantageous due to rapid growth,easy product purification,and available molecular biology tools.However,aklavinone is a catalytic product of type II polyketide synthase(PKS),and the expressed PKS in E.coli in most cases presents in the form of inclusion body.Therefore,it is challenging to reconstruct PKS pathway in E.coli.Elucidating the heterosoluble expression of key enzyme genes in aklavinone biosynthesis pathway could provide insight for rewiring aklavinone pathway and even the pathways of DXR and ACM-A in E.coli.Below are details of the heterosoluble expression of dpsA,dpsB,dpsC,dpsD,and dpsG,the key genes for aklavinone biosynthesis pathway.1.Attempts were made to increase the solubility of proteins DpsA,DpsB,DpsC,and DpsD by lowering expression temperature,decreasing inducer concentration,replacing expression hosts,introducing chaperone proteins,and replacing promoter.Results showed that temperature,inducer concentration,and expression strain are not the main causes of protein inclusion body formation.2.To uncover the modification protein that enables enzymatic function of DpsG,dpsG was co-expressed with holo-ACP synthetase encoding gene acp S in E.coli.The results of protein purification and SDS-PAGE analysis showed that DpsG was post-translationally modified by Acp S.3.To clarify the effects of coupled transcription,protein fusion,and coexpression on protein solubility,three methods were adopted.First,the vectors were redesigned so that dpsB and dpsC were overlapped with four-nucleotide;Second,the dpsB and dpsC genes were linked with a flexible Linker;Third,the genes dpsB,dpsC,dpsG and acp S were arranged in an expression vector to ensure the integrity of the ACP-KS-CLF complex.Results indicated that coupled transcription and infusion protein are not suitable for improving solubility of DpsB and DpsC.Meanwhile,there was no mutual promotion folding effect between min PKS.4.Point mutations of DpsA,DpsB,DpsC,and DpsD were designed based on Solubis and AGGRESCAN3D(A3D).Meanwhile,multiple point mutations of DpsC were performed by PROSS to improve protein solubility by remodeling aggregation-prone regions.The mutation sites selected by Solubis and A3 D did not significantly improve protein solubility.The PROSSdependent re-design of DpsC led to greatly increased solubility.In this work,we investigated the effects of protein expression reduction,co-expression of chaperone proteins,as well as gene expression redesign on protein expression.On the basis of previous experiment,protein point mutation designed by Solubis and A3 D was implemented to improve protein solubility.Fortunately,a DpsC mutant,whose solubility were greatly enhanced in E.coli,was screened out by PROSS.This work provides basis for reconstruction of aklavinone biosynthesis pathway,and also suggests a method for heterosoluble expression of other type II PKS in E.coli.