Evolutionary Engineering Of Bacillus For Acetoin Production

Acetoin is a platform chemical with a special creamy aroma,which is widely applised in the industries of food additives,pharmaceuticals and chemicals.Based on sustainable development,microbial fermentation of acetoin by has been paid more and more attention.Most bacillus are generally recognized as safe?GRAS?,which have natural metabolic pathways for the synthesis of acetoin,but the yield is limited by strain tolerance to acetoin,which can not satisfy the demand of industrial production.In this study,Bacillus amyloliquefaciens and Bacillus subtilis were modified by traditional compound mutagenesis and adaptive evolution which combined with evolutionary engineering strategies and metabolic engineering methods to enhance the tolerance and production performance of acetoin.The main contents are as follows:1.Traditional mutation and fermentation of bacillus for acetoin production.B.amyloliquefaciens FMME088 was treated by atmospheric pressure room temperature plasma?ARTP?and gamma rays.Two-round screening was adopted for obtaining positive mutants,and the best mutant B.amyloliquefaciens H-5 produced acetoin 68.2 g·L^-1 in shake flask.Then,culture conditions were optimized in 5-L fermentor to enhance acetoin production.Finally,85.2 g·L^-1 acetoin was produced by B.amyloliquefaciens H-5,which was increased by 26.8%compared with that of the original strain B.amyloliquefaciens FMME088.2.Designing and constructing of an autonomous evolution mutation system?AEMS?.First,to check whether the P_srfA promoter satisfied the self-induction property of quorum sensing?QS?,we monitored the EGFP expression kinetics with recombinant strain QS01-egfp,which varied depending on the cellvgrowth,and its expression level increasing substantively in the middle to late logarithmic phase?8-12 h?and the OD₆₀₀ was 1.0-2.2.Secondly,to improve P_srfA promoter strength,we replaced the-35 or-10 region of the P_srfA promoter with the conserved sequence TTGACA and TATAAT.The highest expression level of EGFP was obtained under promoter P_TH11,and its strength was increased by 40%.In addition,we truncated different lengths of its upstream sequence,the strength of P_JD15?-136⁺291?was markedly increased by 48%.Third,to further fine-tune promoter P_JD15 and alleviate its background leakiness,we designed a hybrid promoter P_srfAxylO^m that could be induced by cell density and xylose,which combined promoter P_JD15 with xylose.To expand its regulatory range,we established a hierarchical dynamic regulation system which combined promoter P_srfAxylO^m with IPTG-induced hybrid promoter P_graclacO to achieve self-monolayer regulation and switch between the two layers by means of an adaptor protein SspB and a modified ssrA tag which involved in the protease degradation process.Forth,we tested the effects of deletion of genes mutL,alkA,mutS,mutH and overexpression of genes recA,ssbA,bstG,polY1 on improving acetoin tolerance frequency.The results indicate that the double knockout strain HS114 and dual-gene overexpression strain HS119 increased the acetoin tolerance frequency by 14.6-fold and 26.5-fold.Therefore,mutL and alkA were selected as a high-fidelity module?HFM?,and ssbA and bstG were selected as a mutagenic module?MUM?.Finally,HFM and MUM were combined with the hierarchical dynamic regulation system to construct an autonomous evolutionary mutation system?AEMS?.3.Application of the autonomous evolution mutation system?AEMS?.First,we applied AEMS to adaptive evolution for screening the acetoin tolerance phenotype,with the positive mutation rate increased to 44.8%,and mutant HS013 was obtained with excellent tolerance.Second,AEMS was applied to adaptive evolution for screening acetoin high-production strains.We overexpressed three key enzymes PFKA,ALS and ALDC in HS013,and mutant HS016 was identified producing acetoin up to 64.5 g·L^-1 in shake flask.Next,we overexpressed the transcriptional regulators CcpA and AlsR that positively regulate acetoin synthesis in HS016,and mutant HS019 was identified producing acetoin reaching 76.3 g·L^-1.Finally,to obtain even higher production of acetoin,we tested the optimal mutant HS019 in5-L fed-batch cultivation for 52 h,the highest titer of acetoin was 69.2 g·L^-1,with yield and productivity of 0.39 g·g^-1 and 1.33 g·?L·h?^-1,respectively.To evaluate its fermentation performance of mutant HS019,a 30-L fermentor was run by fed-batch cultivation.The titer of acetoin reached a maximum of 82.5 g·L^-1,with yield and productivity reaching 0.46 g·g^-1 and1.59 g·?L·h?^-1;these were increased by 19.2%,17.5%,and 19.3%compared with those of 5-L fermentor.