| Pyruvic acid is an important organic acid and a key intermediate in the process of cell metabolism.It has been widely used in chemical,pharmaceutical and food fields.The current production methods of pyruvate mainly include chemical synthesis,microbial fermentation,enzyme conversion and whole-cell biocatalysis.Among them,the chemical synthesis method has serious environmental pollution,the product components in the microbial fermentation are complex and difficult to separate,the enzyme conversion method has high cost and is not easy to produce on a large scale,while the whole-cell biocatalysis has the advantages of low environmental pollution,high substrate conversion rate and simple product separation,etc.Therefore,the synthesis of pyruvate by whole-cell biocatalysis has broad prospects.In this study,E.coli recombinant expressing L-amino acid deaminase（Pm1）derived from Proteus mirabilis were used to catalyze the synthesis of pyruvate and to obtain D-alanine by chiral resolution of D,L-alanine;Then,N-terminal optimization of Pm1 was performed to enhance its protein expression;A high-throughput screening method for pyruvate was established,and the enzyme activity of Pm1 was improved by semi-rational design;Gene editing was used to reduce the uptake and utilization of pyruvate by cells,and the whole-cell catalytic conditions was optimized.The main results are as follows:（1）Recombinant E.coli BL21（DE3）（p ET20b-pm1）expressing PM1 was constructed to establish a whole-cell biocatalytic method for pyruvate production.The N-terminal coding sequence of PM1 was further optimized.When the pH of the reaction system was 7.0,the concentration of substrate D,L-alanine was 80 g·L-1,and the cell concentration was 1.5 g·L-1,the pyruvate yield of the BLKP00-N6 optimized by the N-terminal sequence reached 15.13g·L-1,and the catalytic activity was 0.36 g·h-1·g DCW-1.Semi-rational design strategy was carried out to engineer Pm1.Pm1 was homologously modeled by SWISS MODEL,and molecular docking was performed by Discovery Studio.Gln99,Val437,Tyr97,Leu278,Val411 and Trp438 were selected for site-specific saturation mutation.A high-throughput screening method for pyruvate based on 2,4-dinitrophenylhydrazine detection was established.Using this method,the mutant strain V437I with improved catalytic ability was screened out,and its pyruvate yield was 18.64 g·L-1,which was 26.0%higher than the control strain.The catalytic activity of V437I reached 0.44 g·h-1·g DCW-1.（2）Using CRISPR/Cas9 gene editing technology to knock out the genes cst A and bts T encoding the pyruvate transporter in E.coli BL21（DE3）,reducing the uptake of pyruvate by the whole-cell catalyst itself,and the yield of pyruvate reached 23.46 g·L-1.The key genes pps,pox B,pfl B,ldh A and ace EF that use pyruvate in the host bacteria were further knocked out,and the BLKP07 strain was obtained.Then,the plasmid p ET20b-N6-pm1-V437I was transferred into BLK07 to obtain BLKP07-N6-V437I.The strain’s consumption of pyruvate during the catalysis process was greatly reduced,and the yield of pyruvate reached 32.58 g·L-1,which was 38.9%higher than the control strain.（3）Optimized pH,dissolved oxygen,cell concentration and substrate concentration during whole-cell biocatalysis.When pH=7.0,rotation speed of Erlenmeyer flasks was 220r·min-1,cell concentration was 4.0 g·L-1,substrate concentration was 110 g·L-1,pyruvate yield reached 43.50 g·L-1,the catalytic activity reached 1.36 g·h-1·g DCW-1,the conversion rate of L-alanine reached 80.0%,and the resolution rate of D-alanine was 84.0%.The N6-pm1-V437I gene expression cassette was integrated into the genome of BLK07,and the plasmid-free E.coli recombinant BLK07P1-N6-V437I was constructed.The pyruvate yield of the strain was 42.20 g·L-1. |
PDF Full Text Request