Genetic Modification Of Nemadectin Producing Strain And Optimization Of Fermentation Process

Nemadectin is a 16-member macrocyclic lactone antiparasitic antibiotic produced by S.cyaneogriseus.Moxidectin,obtained after the oximation of the 23-C site of nemadectin,can be widely used as a pesticide due to its broad-spectrum,highly efficient and safe anthelmintic activity.At present,because of its low fermentation yield and high production cost,moxidectin is mainly used as veterinary insecticide.With the further research and the improvement of nemadectin production,moxidectin is expected to be applied in a wide agricultural market as an agricultural antibiotic.Therefore,this paper aimed at exploring the improvement of nemadectin production of the wild-type strain S.cyaneogriseus MOX-101,which mainly included strain improvement and fermentation process optimization.Firstly,an efficient genetic operating system for MOX-101 was constructed after optimizing the operational conditions of E.coli-Streptomyces conjugation transfer.After optimization,the conjugation transfer efficiency of plasmids pSET 152 and pCL01 increased by 84 folds(7.2×10-6)and 55 folds(9.4×10-7)respectively,which can satisfy subsequent genetic modifications.Secondly,nemR was found in the nemadectin biosynthesis gene cluster by bioinformatics analysis.NemR,a LAL family regulator,is encoded by nemR and is involved in nemadectin biosynthesis in S.cyaneogriseus.In this paper,gene disruption and complementation experiments showed that nemR plays a positive role in the biosynthesis of nemadectin.The transcription levels of nemadectin biosynthetic genes in the nemR knockout strain were significantly decreased compared with those in the wild-type strain MOX-101.The overexpression of nemR under the control of both native and strong constitutive promoters increased the production of nemadectin by 56.5%and 73.5%,respectively,compared with that of MOX-101.Thirdly,in this paper,the method of biosynthetic gene cluster duplication was used to improve nemadectin production.We cloned the nemadectin biosynthetic gene cluster(nem)in vitro using the CRISPR-TAR technique.As the gene cluster is too large to be captured at a time,it was divided into two parts,namely,nem1(50 kb)and nem2(41.7 kb).The plasmids pCL-nem2 and pCL-nem2 containing these two DNA fragments were obtained,respectively.The gene clusters neml and nem2 were assembled by restriction enzyme digestion combined with the TAR method,thus generating the plasmid pCL-nem containing the gene cluster nem.The obtained plasmid was integrated into MOX-101 to obtain the engineered strain MX-n,which yieled 509 mg/L nemadectin,an increase of 108.6%.It indicated that overexpression of the nemadectin biosynthetic gene cluster could be used as an effective approach to improve nemadectin production.This is the first report to capture the complete nemadectin biosynthesis gene cluster in vitro and overexpressed in vivo for nemadectin producing strain.Fourthly,the mutant strain MX-13-46 was obtained by several rounds of natural isolation,ARTP mutagenesis and 60Co-y ray mutagenesis of the wild-type strain MOX101.Compared with that of MOX-101,the nemadectin yield of MX-13-46 was increased to 814 mg/L,a 169.5%increase.And then,MX-13-46 was modified through metabolic engineering,the results were as follows:(1)The strain MX-Knol was constructed by deleting the oligomycin biosynthetic pathway to remove the analogue LL-F28249ω,which showed the nemadectin yield was increased to 912 mg/L without LL-F28249ω production.(2)The strains MX-13-46 and MX-Knol were modified by knock-outing the gene nemD to generate MX-KD and double-knockouted strain MXKn-KD.The results showed that MX-KD no longer produced LL-F28249γ and LL-F28249λ,and nemadectin yield was increased to 913 mg/L.MXKn-KD no longer produced LLF28249ω,LL-F28249γ and LL-F28249λ,and nemadectin yield was increased to 878 mg/L,compared with that of MX-13-46.These results will simpify the downstream separation and purification process.(3)In order to enhance the supply of the precursors acyl-CoA,we attempted to overexpress acc or pcc genes involved in their biosynthesis pathways,respectively.The genes derived from S.coelicolor or S.cyaneogriseus were integrated into MX-13-46 separately to obtain the recombinant strains MX-sco-acc,MX-sco-pcc,MX-scy-acc,and MX-scy-pcc.Compared with that of MX-13-46,nemadectin yield of MX-sco-acc was improved to 883 mg/L,while the other three strains were no significant improvement.(4)More predicted regulatory genes 02400,MerR,TetR,MarR,nolRⅠ,and nolRⅡwere found by analysis of the genome of nemadectin producing strain using the antiSMASH.These regulatory genes including nemR were overexpressed in MX-1346,then generating the recombinant strains MX-nemR,MX-02400,MX-MerR,MXTetR,MX-MarR,MX-nolRⅠ,and MX-nolRⅡ.The results showed that nemadectin production of MX-nemR was increased by 24.5%(994 mg/L)compared with that of MX-13-46.However,nemadectin yield of MX-MerR decreased by 10.4%compared with that of MX-13-46,suggesting that MerR might be a negative regulatory gene.And overexpression of other regulatory genes in MX-13-46 had no significant effect on nemadectin production.(5)Enhancement of nemadectin biosynthesis by combinational genes modifications.In order to further improve nemadectin production,we overexpressed the gene nemR or the gene cluster nem in MX-13-46,MX-Knol,MX-KD,and MXKnKD respectively,to obtain eight engineering strains MX-nemR,MXKn-R,MXKD-R,MXKnD-R,MX-nem,MXKn-nem,MXKD-nem,and MXKnD-nem,and the results showed that nemadectin yields of these engineering strains were about 20%-45%higher than those of corresponding original strains.Among them,the yields of MXnem,MXKn-R,MXKD-R,and MXKnD-R were improved to 1177 mg/L,1108 mg/L,1088 mg/L,and 1069 mg/L.Finally,the fermentation medium and culture conditions were optimized for improvement of nemadectin production in shake flasker.Furthermore,the batch fermentation was carried out in 5 L fermentor,and the nemadectin production was up to 1779 mg/L by preliminary process optimzation.