Construction Of Genetically Engineered Bacteria With Polyisoprene-like Products

The polyisoprene-like product is a polymerization product formed by the polymerization of isoprene pyrophosphate,and the polyisoprene-like product includes an oligomerization product and a high polymerization product,which have wide application value.Oligomeric products such as squalene are widely used as moisturizers and emollients in cosmetics,and have a variety of health promoting functions,including inhibition of tumors and enhancement of immunity.Highly polymerized products such as polyisoprene are mainly used as natural rubber in the manufacture of tires,latex,rubber shoes,tapes,and the like.However,both of them are currently limited in mass production,so the biosynthesis method for developing polyisoprene-like products has important application value.This project builds metabolic engineering on the basis of synthetic biology,mainly using Escherichia coli as the host strain to construct a complete pathway of the mevalonate pathway(MVA pathway).The target products of squalene and polyisoprene are produced by microbial cells.The main work of this topic:(1)Optimization of the mevalonate strain.Based on the construction of the upstream metabolic pathway of mevalonate,pET28a-ES,it was digested and screened to optimize the host strain.Firstly,the E.coli DH5a and E.coli BL21 host strains were optimized for the E.coli host bacteria,and the E.coli BL21 was better adapted to the mevalonate metabolic pathway.Then the fermentation medium was used.Three mediums of LB medium,M9 medium and X medium were selected for comparison and optimization,and the fermentation strains were best grown in the X medium.Among them,the experimental strain BLES had the highest MVA yield in X medium,reaching 1.25 g/L.Finally,BL21 was selected as a host strain and fermentation was carried out in X medium.(2)Construction of metabolic pathways of squalene.According to the optimized upstream pathway of mevalonate,a downstream pathway was constructed to improve the metabolic pathway,and the target product squalene was subjected to fermentation detection products.Mainly based on ERG12,ERG8,ERG19,IDI1,fds,the splicing of the same tail enzyme was ligated to the vector plasmid of pET22 to form a basic downstream pathway,namely the recombinant plasmid pET22b-X.ERG9 derived from Yarrowia lipolytic yeast was further added for the synthesis of squalene,the recombinant plasmid pET22b-XQ.The pET28a-ES and pET22b-XQ were co-transformed into the E.coli BL21 competent state to obtain the squalene engineered strain BLESXQ.Fermentation culture,the product is tested.The product squalene was measured,indicating that the constructed squalene metabolic pathway was feasible,and the yield of strain BLESXQ in X medium reached a maximum of 16.12 mg/L,which further proved that the constructed mevalonate was substantially downstream.The pathway is feasible in E.coli.(3)Construction of a polyisoprene metabolic pathway.Based on the above constructed recombinant plasmid of pET22b-X,Arabidopsis thaliana-derived CPT and Brazilian rubber tree SRPP were added to form a target metabolic pathway.Two sets of polyisoprene metabolic pathway pET22b-XC recombinant plasmid and pET22b-XS recombinant plasmid were obtained.The two recombinant plasmids were co-transformed with pET28a-ES into E.coli BL21 competent state to obtain polyisoprene engineering strains BLESXC and BLESXS.The product was verified by fermentation,and then the enzyme activity of CPT enzyme was measured.Enzyme activity and polyisoprene.It is indicated that the enzyme expressed by the CPT and SRPP genes does not play a separate role in the production of polyisoprene.It is speculated that the enzymes from the plant may not be compatible with the host and may lack some cofactor.