Metabolic Engineering Of Bacillus Amyloliquefaciens HZ-12 For High-level Production Of Iturin A

Iturin A is a kind of lipopeptide biosurfactant mainly produced by Bacillus sp.,and it has good antimicrobial activity,low toxicity,good biodegradability and environmental compatibility.Therefore,iturin A has wide application prospects in agricultural production,drug development and environmental management.However,the low yield of iturin A limits its industrial production and practical application.In this study,Bacillus amyloliquefaciens HZ-12Pbac A（named as Pbac A）was used as the original strain,and engineered strain with high-level of iturin A was constructed via enhancing the utilization of substrate starch,strengthening the synthesis pathway of lipopeptide precursors（proline and fatty acids）,interrupting the synthesis pathway of metabolic byproduct glycan,regulating the synthesis of lipopeptides,and enhancing the expression of lipopeptide transporters,the following conclusions were obtained.1.In this study,the engineering strain Pbac AΔgab T was constructed via knocking out gab T,a key gene for synthesis of 1-deoxynojirimycin.Engineered strains Pbac A/p HY-mal R and Pbac A-Pbac A_{（mal R）}were constructed via overexpression of transcription factor Mal R relating maltose.Utilizing maize starch as the substrate,the iturin A production was determined after 72 h fermentation.The results showed that the iturin A production of strains Pbac AΔgab T,Pbac A/p HY-mal R and Pbac A-Pbac A_{（mal R）}were 2.58 g/L,2.45 g/L and 2.27 g/L,respectively.Compared with the control strain Pbac A（1.98 g/L）,strains Pbac AΔgab T and Pbac A-Pbac A_{（mal R）}increased by 30.30%and 14.65%,respectively.Strains Pbac A/p HY-mal R increased by 21.89%compared with the control strain Pbac A/p HY-300（2.01 g/L）.2.Through the precursor amino acids addition experiment,this study confirmed that the exogenous addition of proline can improve the yield of iturin A.Subsequently,the overexpression strains of pro A（5-semialdehyde dehydrogenase）,pro B（glutamate 5-kinase）,pro C（pyrroline-5-carboxylate reductase）,and oc D（ornithine cyclic deaminase）were constructed.After 72 h of fermentation,iturin A production was measured.The results showed that:compared with Pbac A/p HY-300,the production of iturin A was increased after overexpression of oc D,but there was no significant change after overexpression of pro A,pro B and pro C.Then,the oc D integrated expression strain,Pbac AΔgab T::oc D was constructed based on the strain Pbac AΔgab T,and the production of iturin A was increased to 2.98 g/L,which was 50.51%higher than that of original strain Pbac A.In this study,malonyl carboxylase-enhancing strains Pbac AΔgab T/p HY-yng H,Pbac AΔgab T/p HY-acc A and Pbac AΔgab T/p HY-acc C were also constructed.After 72 h of fermentation,iturin A production was determined.The results showed that after overexpression of malonyl carboxylase,iturin A production was 2.87g/L,2.93 g/L and 2.83 g/L,respectively,which were 12.55%,14.90%and 10.98%higher than that of the control strain Pbac AΔgab T/p HY-300（2.55 g/L）.3.In this study,the engineered strains Pbac AΔsip W,Pbac AΔsac B and Pbac AΔeps AB with gene deletion related to glycan synthesis were constructed.After 72 h of fermentation,iturin A production was determined.The results showed that the iturin A production of strains Pbac AΔsip W,Pbac AΔsac B and Pbac AΔeps AB were 2.45 g/L,2.54 g/L and 2.38 g/L,respectively,which were increased by 21.89%,26.37%and18.41%compared with the control strain Pbac A.At the same time,engineered strains with gene deletion related to lipopeptide synthesis,Pbac AΔfen C,Pbac AΔlic A and Pbac AΔbac A were constructed.The yield of iturin A after fermentation was only 0.52g/L,2.07 g/L and 2.03 g/L,respectively.The iturin A production of Pbac AΔfen C was significantly lower than that of control strain,while the production of engineered strains Pbac AΔlic A and Pbac AΔbac A showed no significant change compared with that of control strain.4.In this study,the transcription factor Sig A overexpression strain Pbac A/p HY-sig A and carbon metabolic transcription factor Ccp C deletion strain Pbac AΔccp C were constructed.After 72 h of fermentation,iturin A production was determined.The results showed that the iturin A production of strains Pbac A/p HY-sig A and Pbac AΔccp C were2.48 g/L and 2.37 g/L,which were 23.38%and 19.69%higher than those of control strains Pbac A/p HY-300（2.01 g/L）and Pbac A（1.98 g/L）,respectively.5..In this study,the overexpression strains of lipopeptide transporter Swr C,Ycx A,Pbac A/p HY-swr C and Phbac A/p HY-ycx A were constructed.The production of iturin A was determined after 72 h fermentation.The results showed that iturin A production of strains Pbac A/p HY-swr C and Pbac A/p HY-ycx A were 2.51 g/L and 2.27 g/L,which were 26.13%and 14.07%higher than that of control strain Pbac A/p HY-300,respectively.6.In this study,through the combined metabolic engineering breeding strategy,we constructed the engineered strains Pbac AΔgab T::oc DΔsip WΔsac BΔccp C-Pbac A_{-mal R},iturin A production increased to 3.87 g/L,which was 95.45%higher than the starting strain Pbac A.Combining the above results,the following conclusions are drawn:1.Enhancing starch utilization of substrate maize can improve iturin A production via knocking out the gene gab T and enhancing the expression of transcription factor Mal R.2.Increasing the supply of precursors is conducive to improving the level of iturin A synthesis via enhancing the synthesis pathway of lipopeptide precursors（proline and fatty acids）.3.Interrupting the glycan synthesis pathway can improve iturin A production via knocking out the glycan synthesis gene of by-product.4.Regulating the synthesis of lipopeptides via enhancing the expression of transcription factor Sig A and knocking out transcription factor Ccp C is conducive to iturin A synthesis.5.Increasing the level of lipopeptide transport via enhancing the expression of lipopeptide transporters Swr C and Ycx A is conducive to iturin A synthesis.6.In this study,an engineered strain of B.amyloliquefaciens producing iturin A was constructed by metabolic engineering to provide technical support for the scale production and application of iturin A.