Construction Of A Cell Factory For Efficient Production Of D-pantothenic Acid Based On ATP Regeneration

D-pantothenic acid（vitamin B5）is a water-soluble vitamin widely used in fields such as food,feed,medicine,and chemical engineering.By coupling L-aspartate-α-decarboxylase（Pan D）,L-aspartate enzyme（Asp A）and pantothenic acid synthase（Pan C）,this study developed a process in Escherichia coli BL21 for catalyzing the conversion of fumaric acid and D-pantoic acid to D-pantothenic acid.The production of D-pantothenic acid was fine-tuned by optimizing the expression levels of the three enzymes through dual plasmid expression,p ET expression plasmid improvement,and gene duplication strategy.The major results are listed as follow:（1）Design,construct,and validate a three enzyme cascade pathway for D-pantothenic acid production.First,L-aspartase（Cg Asp A）from Corynebacterium glutamicum and pantothenate synthase（Bb Pan C）from Bavibacterium brevis,and L-aspartate-α-decarboxylase(Bs Pan D^E56S)from Bacillus subtilis were used as pathway enzymes for the cascade reaction;Then,the feasibility of the Cg Asp A,Bs Pan D^E56S and Bb Pan C enzyme cascade reaction pathway was validated in vitro,and the production of the product D-pantothenic acid was detected;Finally,by adjusting the enzyme activity ratio of the three enzymes,the optimal in vitro enzyme activity ratio of the three enzymes was determined to be 1.56:1:1.47.（2）In vivo reconstruction and optimization of the D-pantothenic acid biosynthesis pathway.First,Cg Asp A,Bs Pan D^E56S and Bb Pan C were coexpressed in plasmid p ETDuet-1R to construct strain PL-01.The in vivo feasibility of the cascade reaction was validated using whole cell transformation.However,the difference in enzyme activity between Bs Pan D^E56S（rate limiting enzyme）,Cg Asp A and Bb Pan C（35.00:1:33.54）caused an imbalance in the reaction,resulting in a accumulation of intermediate product L-aspartic acid;Then,Bs Pan D^E56S was expressed separately on p ET-28a（+）,Cg Asp A and Bb Pan C were coexpressed on plasmid p ETDuet-1R to construct PL-02 strain.The enzyme activity ratio of the three enzymes was 5.60:1:5.61;Finally,further optimize the Asp A/Pan D/Pan C ratio to effectively produce D-pantothenic acid.The T7promoter and translation initiation region（TIR）of the p ET-28a（+）-Bs Pan D^E56S plasmid were modified to obtain PL-04 strain with an enzyme activity ratio of 4.52:1:4.39.In addition,the expression level of Bs Pan D^E56S was improved through gene duplication strategy.When the gene duplication number was increased to 3,the enzyme activity ratio of the three enzymes was1.67:1:1.56,which was similar to the optimal enzyme activity ratio in vitro.（3）Enhancing intracellular ATP supply by introducing an ATP regeneration system based on polyphosphate kinase（PPK）.Introducing PPK from Pseudomonas aeruginosa,PL-11 strain was obtained.Without the addition of ATP,70 g·L^-1 fumaric acid and 89 g·L^-1 D-pantoic acid was transformed into 74.5 g·L^-1D-pantothenic acid with a productivity of 84.9%.（4）Optimization and scaling up of the production system for D-pantothenic acid.Optimize the conversion conditions of PL-11 strain and determine the optimal conversion conditions as follows:catalyst concentration of 30 g·L^-1,conversion p H 7.0,and conversion temperature of35℃;Finally,in 1 L transformation system,a batch feeding culture strategy was adopted to achieve the transformation of 70 g·L^-1 fumaric acid and 89 g·L^-1 D-pantoic acid,resulting in128.6 g·L^-1 D-pantothenic acid with a productivity of 97.3%.