Construction Of Pulcherrimin Hyper-producers And Fermentation Process Optimization In Bacillus Licheniformis

Pulcherrimin is a cyclodipeptide produced by microorganisms.Due to immobilizing the iron,pulcherrimin has strong antagonistic activities on other microorganisms.As a potential biocontrol agent,it has promising applications in agriculture and medical areas.However,the production of pulcherrimin on a commercial scale has not been realized because of its low-level yield,and the aim of this work is to achieve the high-level production of pulcherrimin by Bacillus licheniformis DW2 through optimization of culture conditions and metabolic engineering.The main results are as follows:1.Identifition of the pulcherrimin produced by B.licheniformis DW2In this study,the red pigment produced by B.licheniformis DW2 was extracted,purified and identified.The purified pigment exhibited the maxima absorption at 243 nm,282 nm and410 nm in the UV and visible ranges,which was identical with those of pulcherriminin from Bacillus species and yeast.Furthermore,the yvmC and cypX deletion strains and complementation strains were constructed respectively.Based on our results,the deficience of either yvmC or cypX made strains deficient in pigment synthesis and the complementation strains could resume the synthesis capability pigmentation.The above results implied that this red pigment was pulcherriminin.2.Optimization of the fermentation process for enhanced pulcherrimin productionThe fermentation process were optimized based on the single factor experiment.Among different carbon sources,glucose resulted in the highest pulcherrimin yield,and（NH₄）₂SO₄was the best nitrogen sourcen for cell growth and pulcherrimin synthesis.It was also found that additional yeast extract and Tween-80 could stimulate the production of pulcherrimin.The optimal medium compositions were obtained by the orthogonal experimental method（g/L）:glucose 40,（NH₄）₂SO₄ 6.2,yeast extract 1.0,Tween-80 1.0,K₂HPO₄·3H₂O 0.6,MgSO₄·7H₂O 0.5,CaCl₂·2H₂O 0.2,FeCl₃·6H₂O 0.04,MnSO₄·H₂O 0.02.And the optimal culture conditions were 37°C,inoculum age 10 h,inoculum amount 2%（v/v）and broth volume 50 m L/250 m L.Under the optimal fermentation conditions,DW2 could produce the highest pulcherrimin concentration of 313.17 mg/L,a 5.95-fold increase compared to the initial yield.3.Establishment of the pulcherrimin-producing mutant strainPulcherrimin is synthesized from L-leucine via a series of enzymatic and non-enzymatic reactions.Firstly,Leucine-tRNA synthetase catalyses L-leucine to leucyl tRNAs.Then,the cyclodipeptide synthase YvmC catalyzes two molecules of leucyl tRNAs to formation of cyclo-L-leucyl-L-leucyl（cLL）.Further,CypX,encodes a cytochrome P450,oxidizes cLL into pulcherriminic acid.Finally,pulcherriminic acid is secreted and chelated ferric ions by a non-enzymatic reaction to form pulcherrimin.If improving leucine biosynthesis maybe beneficial to the pulcherrimin production,and enhancing the expression of Leu-tRNA synthetase coding gene leuS and yvmC-cypX might improve the accumulation of pulcherrimin.In the present study,the wild type B.licheniformis DW2 was used as the objective strain,and the encoding genes of acetolactate synthase（alss and ilvB）,Leu-tRNA synthetase（leuS）and cyclodipeptide synthetase（yvmC）were over-expressed to improve the pulcherrimin systhesis,respectively.Based on our results,amplification of alss,ilvB,leuS and yvmC led to the enhanced production of pulcherrimin by 0.91%,7.80%,19.40%and 12.90%,respectively.BkdAB is a key enzyme for the synthesis of branched-chain fatty acids,and to weaken the synthesis of branched-chain fatty acids and improve leucine accumulation.The enzymes encoding genes,bkdAB,was deleted in DW2.These results showed that the deficience of bkdAB could improve the pulcherrimin production,and the pulcherrimin yield of DW2ΔbkdAB was 10.00%higher than that of DW2,and DW2ΔbkdAB mutant strain had a decreased growth rate.Further improvement was achieved by overexpression alss,leuS and yvmC genes in DW2ΔbkdAB through manipulating promoters.Substituting the native ilvB promoter,leuS promoter and yvmC promoter promoter with the bacABC operon promoter（PbacA）,respectively.Meanwhile,the yields of 349.73 mg/L and 339.79 mg/L were produced in DW2ΔbkdAB-PbacA（yvmC）and DW2ΔbkdAB-PbacA（leuS）,increased by 14.5 mg/L and4.52 mg/L compared with DW2ΔbkdAB（335.26 mg/L）strain,respectively.The pulcherrimin yield produced by DW2ΔbkdAB-PbacA（yvmC）-PbacA（ilvB）/pHY-leuS was 379.93 mg/L,a7.22-fold higher than the initial yield.