Heterologous Production Of The Termite-killing Compound Cedarone In Escherichia Coli

There are more than 3000 species of termites in the world,and many countries suffer from different degrees of ant damage.Termite control methods include chemical control,physical control and biological control.At present,termites are mainly used chemical agents.Chemical agents have problems such as inefficiency and high toxic agent residues.With people's awareness of environmental protection and food safety enhancement,as well as the advancement and widespread application of biotechnology.There is an urgent need for efficient,low-toxicity,environmentally friendly,and economical termite control agents to improve current status of termite control.Biological control is the use of termites?predatory and parasitic?natural enemies or microorganisms?bacteria,fungi,viruses or nematodes,etc.?or microbial fermentation metabolites to control termites.Plant endophyte is a potential microbial resource,and its biological fermentation metabolites have antibacterial,anti-tumor,and anti-viral effects.The research group has screened endophytes that produce termiticide compounds from the natural termite-resistant trees Juniperus virginiana and Chamaecyparis lawsoniana..Purified from the strain's biological fermentation broth to five highly effective termite-repelling compounds,nootkatone,torreyol,?-terpineol,?-cadinene,and?-burt orange tomato alcohol.Due to the instability of plant endophyte and the low yield of metabolites.Therefore,the use of microorganisms to efficiently and stably produce compounds having termite-killing activity is an urgent problem to be solved.The Escherichia coli expression system is currently the most detailed and fastest-developing heterologous expression system.E.coli as a model strain has been widely used in genetic engineering and metabolic engineering.In this study,HDZK-BYSB7 isolated from Chamaecyparis lawsoniana were used as test strains.The endophyte can produce nootkatone,but the yield is low.In order to increase the yield of nootkatone,this experiment analyzed the biosynthetic pathway of nootkatone.The related enzymes in the biosynthetic pathway of nootkatone were synthesized,expressed and purified,and a heterologous expression vector for the enzyme related to nootkatone biosynthesis was constructed and heterologously expressed in E.coli.The result showed nootkatone was found by GC-MS.At the same time,the whole genome of strain HDZK-BYSB7 was sequenced in this experiment to find the homologous protein of YjiB in the HDZK-BYSB7 genome,construct a homologous protein expression vector,and express and purify homologous proteins.1?The results of intermediate Valencene feeding experiments showed that strain HDZK-BYSB7 contained genes that catalyzed the biosynthesis of Valencene into Nootkatone.2?Identification and synthesis of genes related to Nootkatone biosynthesis valS,yjiB,camA,camB.3?The expression vectors for valA,yjiB,camA,and camB were constructed,and the proteins YjiB,CamA,CamB,and ValS were successfully expressed and purified.4?The expression and purification conditions of protein ValA were determined for the first time,37?,220 r/min,cultured to an OD₆₀₀ of about 0.7,and induced by IPTG at a final concentration of 100?mol/L;cultured at 25?.and 200 r/min for 20h;4000r/min centrifugation.30min,ultrasonic disruption of bacteria?ultrasonication 2s,intermittent 6s?;25mmol/L imidazole eluting heteroprotein,100mmol/L imidazole eluting target protein.5?The constructed Duet series expression vectors can express proteins YjiB,CamA,CamB,ValS.6?The yield of the recombinant strain BL21/pCT28-camA-yjiB-camB-valS?DJW4?Valencene is the highest and there is production of Nootkatone,but the yield of Nootkatone is not high,indicating that the catalytic efficiency of YjiB is low.7?The whole genome sequence of strain HDZK-BYSB7 was obtained,and three cytochrome P450 enzymes were identified by protein function.These three cytochrome P450 enzymes are identical to the three homologous proteins obtained by homology comparison with yjiB and the whole genome.8?The expression and purification conditions of three homologous proteins were determined.The expression temperatures of H1,H2,and H3 were both 25?,and the expression time was 20h.H1 was elute the heteroprotein with 25 mmol/L imidazole and200 mmol/L imidazole elutes the target protein.H2 and H3 elute the heteroprotein with5mmol/L imidazole and 100mmol/L imidazole elutes the target protein.This test lays a new production method for the highly efficient,low-toxic,environmentally friendly termite control agent nootkatone?At the same time,it also provides new methods and ideas for biological control of termites,and has certain academic research value,commercial value and potential promotion and application prospects.