Construction Of Short Branched-chain Fatty Acids Hyper-Producers And Fermentation Process Optimization In Bacillus Licheniformis

Short-chain fatty acids are an important multifunctional platform compound widely used in manufacturing,food,pharmaceutical and agricultural.The chemical synthesis of short branched-chain fatty acids is highly dependent on petrochemical products,and the reaction is severe,and the obtained toxic by-products are difficult to separate,which is easy to pollute the environment and does not conform to the green sustainable development concept.Compared with chemical synthesis,short branchedchain fatty acids producing by microbial fermentation have the advantages of renewable raw materials,mild reaction,environmental friendliness and easy product extraction.However,the production of SBCFAs by microbial fermentation is currently reported to be at a low level,which limits the industrial production and practical application of short branched-chain fatty acids.The research is divided into two parts.The first part of the research aims to achieve high-level production of short branched-chain fatty acids from waste raw bean dregs and crude glycerol by genetically modified Bacillus licheniformis.Firstly,the synthetic gene of short branched-chain fatty acids in Bacillus licheniformis was overexpressed by genetic means.The recombinant strain was obtained.Secondly,the fermentation process of the recombinant strain using crude glycerol and bean dregs as raw materials to produce short-chain branched fatty acids was optimized.Finally,the level analysis of 5 L fermenter was carried out under the conditions of the optimal process,and the high level production of short branched chain fatty acids was achieved.The second part of the research aims to enhance the ability of B.licheniformis to synthesize short branched-chain fatty acids from the "head" through metabolic engineering.Firstly,the yield of short branched-chain fatty acids was increased by enhancing the short branched-chain fatty acids biosynthesis pathway in Bacillus licheniformis.At the same time,the heterologous short branched-chain fatty acids synthesis pathway was introduced.Finally the short-chain fatty acid high-yield strain was obtained by combined metabolic engineering breeding to further increase the production of short branched-chain fatty acids.1?The main conclusions of the first part of the study are as follows:Firstly,four strong promoters(P43,PbacA,PsrfA,PalsSD)were screened to replace the native promoter of the bkd operon in Bacillus licheniformis DWc9n*,overexpressing the SBCFAs synthetic gene,and four promoter replacement strains were obtained,named DWc9n*-P43bkd,DWc9n*-PbacAbkd,DWc9n*-PsrfAbkd and DWc9n*-PalsSDbkd.Under the initial medium conditions,the recombinant strain DWc9n*-PbacAbkd got the highest concentration of SBCFAs using the crude glycerin and okara as the substrate,which was 4.68 g/L,increasing by 1.98 times compared with the wild-type strain(1.57 g/L).Secondly,the fermentation process of SBCFAs synthesized by Bacillus licheniformis DWc9n*-PbacAbkd was optimized through single factor experiment and orthogonal experiment.The optimal medium composition after optimization was:glycerol 60 g/L,bean dregs 80 g/L,CaCO3 6 g/L,ZnSO4 0.3 g/L and(NH4)2SO4 1 g/L.Under optimized conditions,the yield of SBCFAs was further increased to 7.85 g/L,which was 4.00 times higher than that of the wild type strain(1.57 g/L)under the initial medium conditions.Finally,in the 5 L fermentor,the optimized fermentation medium was used to investigate the dynamic changes of SBCFAs batch fermentation of DWc9n*PbacAbkd with pure glycerol and crude glycerol under the optimized medium.The concentration of SBCFAs synthesized by B.licheniformis DWc9n*-PbacAbkd reached 8.37 g/L,and the productivity was 0.20 g/L/h,which is the highest level of SBCFAs production based on low-value substrates fermentation.2?The main conclusions of the second part of the study are as follows:In order to enhance the ability of Bacillus licheniformis to synthesize short branched-chain fatty acids from the "head",the obtained recombinant strain DWc9n*PbacAbkd(DW2-SA1)was used as the research object,and the biosynthetic pathway of SBCFAs precursor branched ?-keto acid was firstly over-expressed via promoter replacement.The promoters of the ilvC-leu operon and the ilvD gene were replaced with the strong promoter PbacA,respectively,resulted in the separate promoter replacement strains DW2-SA2,DW2-SA3,and double promoter replacement strain DW2-SA4.It was showed that both single-promoter replacement strain and doublepromoter replacement strain could increase short branched-chain fatty acids production,and the yield of dual-promoter replacement strain DW2-SA4 was 4.41 g/L,increasing by 46.9%compared with the wild-type strain DW2-SA1(2.89 g/L).Secondly,in order to further increase the yield of short branched-chain fatty acids,the by-product acetoin synthesis pathway gene alsD was knocked out on the basis of strain DW2-SA4,obtained the strain DW2-SA5.The yield of strain DW2SA5 was 4.95 g/L,increasing by 12.2%compared to DW2-SA4.The increase was slightly,and the biomass of the cells was reduced after the deletion of the gene alsD.It is speculated that the biomass of the cells has a great influence on the fermentation of short branched-chain fatty acids by Bacillus licheniformis,which may be the reason for limiting the further increase of SBCFAs.In order to achieve heterologous synthesis of short branched-chain fatty acids in B.licheniformis,a ketoacid decarboxylase gene kivD derived from Lactococcus lactis was introduced to construct a heterologous SBCFAs synthetic strain,thus constructed the recombinant strain DWc9n*/pHY-P43kivD.The SBCFAs production of strain DWc9n*/pHY-P43kivD was 3.23 g/L,which achieved the heterologous synthesis of short branched-chain fatty acids.In addition,ketoacid decarboxylase expression strains DWc9n*/pHY-kivDS268T,DWc9n*/pHY-kivDV461I,DWc9n*/pHY-kivDF381W and DWc9n*/pHY-kivDM538W with different site mutations were constructed.The best ketoacid decarboxylase mutant strain was DWc9n*/pHY-kivDF381W,and the SBCFAs yield of it was 4.23 g/L,increasing by 30.9%compared with strain DWc9n*/pHYP43kivD,which further improved heterologous synthesis of short branched-chain fatty acids.Finally,the recombinant strain DW2-SA5/pHY-kivDF381W was constructed by combined metabolic engineering.The SBCFAs yield of strain DW2-SA5/pHYkivDF381W reached 7.85 g/L,which was 60.5%higher than that of DW2SA5/pHY300PLK.The combination of endogenous and exogenous synthetic pathways further increased production of short branched-chain fatty acids,suggesting the enhanced ability of B.licheniformis to synthesize short branched-chain fatty acids from the "head" by metabolic engineering.