Genome Engineering For High Yield Shenqinmycin Cell Factory

Shenqinmycin,a secondary metabolite produced by Pseudomonas,consists largely of phenazine-1-carboxylic acid(PCA).1% Shenqinmycin suspending agent prevents a series of crucial crop diseases,such as rice sheath blight,cucumber seedling damping-off and pepper blight.Shenqinmycin is characterized by safe,high efficiency and good environmental compatibility.Currently,microbial fermentation is the best approach to produce Shenqinmycin.Since the low industrial fermentation titer of Shenqinmycin,the production costs of this biopesticide are higher than conventional chemical pesticides.Therefore,carrying out basic and applied research is necessary to improve biosynthesis efficiency and boost application of Shenqinmycin in practical production.Pseudomonas aeruginosa PA1201 has two highly homologous PCA biosynthesis gene clusters,which carry the phzC gene.However,the biological function of the phzC gene is not clear.In this study,we found that PhzC belongs to the type II 3-deoxy-d-arabinoheptulosonate 7-phosphate(DAHP)synthase family.The production of Shenqinmycin was significantly raised in phzC overexpression mutant,whereas it was remarkably reduced in phzC deletion mutant.PhzC contains two DAHP synthase domains.Overexpression of Cterminal domain of PhzC resulted in increasing Shenqinmycin production by 38%,while overexpression of N-terminal domain had no influence on PCA biosynthesis.Besides,7 putative active sites and 1 conservative amino acid residue were essential to the enzyme activity of PhzC via site-directed mutational analysis.Moreover,the DAHP synthase deficient mutant could be complemented by pBBR-phzC.The resulting strain resumed growth and also grew well in minimal medium within different concentration of Shenqinmycin,suggesting that PhzC was a DAHP synthase and the enzyme activity of PhzC was free from feedback inhibition.After modifying the PCA biosynthesis and secondary metabolite pathways of P.aeruginosa PA1201,the Shenqinmycin production of engineering strain PA-IV was increased 56-fold in this study.The used strategies are as follows:(1)blocking Shenqinmycin conversion pathways through deletion of phenazinemodifying genes(phzM,phzS and phzH);(2)augmenting carbon metabolic flux to shikimate pathway through alternating the original promoter of aroG and single-copy insertion of the phzC gene;(3)maximizing chroismate flux to PCA synthesis pathway through decreasing the metabolic flux to other chorismateutilizing pathways;(4)enhancing the expression of two PCA biosynthesis gene clusters(phz1 and phz2)through deletion of the 5'-UTR region and promoter replacement;(5)accelerating Shenqinmycin efflux to extra-cellular through engineering the promoter of MexGHI-OpmD efflux pump;(6)reducing the redundant metabolism through blocking the synthesis of 21 secondary metabolites.Using fed-batch fermentation,high yield strain PA-IV produced 9882 mg/L Shenqinmycin.P.aeruginosa M18 is used for industrial production of Shenqinmycin.The complete genome of M18 is composed of a single chromosome of 6327754-bp.Using rational design,the genome of M18 was reduced to optimize genetic manipulation and improve PCA production.First,restriction modification system and clustered regularly interspaced short palindromic repeats(CRISPR)loci were deleted to improve the transformation efficiency,laying the foundation for subsequently genetic modification and genome reduction.Second,five strain-specific genomic islands,two prophages and 20 secondary metabolite biosynthesis gene clusters were deleted to decrease redundant metabolism and regulatory network.Third,the gene expression profiles of M18 were analyzed by strand-specific transcriptome resequencing.Combined with essential gene data,22 candidate large DNA fragments were selected for deletion.The reads per kb per million reads(RPKMs)of these regions were lower than the average transcription level.After sequential deletion of 13 large DNA fragments,the genome size of M18 reduced 740306-bp(11.7% of the total 6.3-Mb genome size).The resulting strain MDS44 had a little better growth performance and high transformation efficiency.The Shenqinmycin production of MDS44 increased by 445%,compared with that of wild type strain.Besides,the evaluation of other properties of MDS44 is in progress.In conclusion,the high yield Shenqinmycin-producing strain PA-IV will notably reduce production costs and promote the feasibility of Shenqinmycin application.With a good property,MDS44 is a good starting material for constructing a better dominant minimum genome factory.