| Isobutanol,an excellent alternative biofuel and a platform compound has been widely used in the fields of food,industrial,medicine,etc.An increased attention have been made to enhance the production of isobutanol in microbial.The biosynthesis of isobutanol was achieved by combining the branched-chain amino acids synthetic pathway of host strain and exogenous Ehrlich pathway.The capability of Bacillus licheniformis to tolerate isobutanol toxicity was evaluated.The isobutanol biosynthesis pathway in B.licheniformis was constructed by metabolic engineering.Moreover,the yield of isobutanol was improved by metabolic pathway transformation.In this study,we compared the capability of B.licheniformis,Bacillus subtilis and Escherichia coli to tolerate isobutanol,The relative growth and viable rates of the strains were determined by exposing these organism to isobutanol at different concentrations(0%,0.4%,0.8%,1.2%,1.6% and 2.0%),The results showed that B.licheniformis displayed higher viability in comparison with B.subtilis and E.coli,and was suitable for the host strain of isobutanol biosynthesis.Due to without isobutanol synthesis pathway,an efficient heterologous Ehrlich pathway need to be introduced into B.licheniformis for the isobutanol biosynthesis.This study the effects of overexpression of three alcohol dehydrogenase from different strains on catalyzing the isobutyraldehyde to isobutanol in B.licheniformis were compared.The results showed that the yqhD gene from E.coli was the most efficient.In order to improve its stability,the gene was integrated into the phage protein gene xkdI site of B.licheniformis chromosomes,and the recombinant strain DW2-01 was obtained.The kivD gene encoding the ketoacid decarboxylase derived from L.lactis CICC 6246 was expressed on DW2-01,and the recombinant strain DW2-02 was obtained.The results showed that isobutanol was detected and the yield of isobutanol of DW2-02 strain was 550 mg/L.The biosynthesis of isobutanol in B.licheniformis was first reported.BkdB can convert aldehydes into acyl-CoA,it can reduce the accumulation of isobutyraldehyde in the Ehrlich pathway,and further affects the synthesis of isobutanol.In this study,the bkdB mutant strain DW2-02ΔbkdB was obtained and subjected to shake flask fermentation.The results showed that the yield of isobutanol in DW2-02ΔbkdB strain was 964 mg/L,which was 75.3% higher than that of DW2-02.The bkdB mutant improved the production of isobutanol.Blocking the bypass pathway could re-direct carbon flow into the isobutanol synthesis.We used DW2-02ΔbkdB as the objective strain and gene knockout technology to delete the encoding gene of lactate dehydrogenase(ldh),acetolactate decarboxylase(alsD)and α-isopropylmalate synthase(leuA),respectively and a series of recombinant strains were obtained.The results of isobutanol fermentation showed that the production of acetoin and 2,3-butanediol in DW2-02ΔbkdBΔalsD strain was significantly decreased,the cell biomass and glucose consumption rate were lower than those of DW2-02ΔbkdB,the DW2-02ΔbkdBΔals D produced 523 mg/L isobutanol,decreasing by 45.7% compared with DW2-02ΔbkdB.The isobutanol yield of DW2-02ΔbkdBΔleuA strain was 843 mg/L,which was lower than that of DW2-02ΔbkdB strain.The isobutanol accumulation rate of DW2-02ΔbkdBΔldh is higher than DW2-02ΔbkdB and the yield of isobutanol was 1014 mg/L,which was 5.2% higher than that of DW2-02ΔbkdB. |
