Study On Pyrrolidone Production From L-glutamate Via A Tri-enzymatic Cascade Pathway

Pyrrolidone is an important medical intermediate,widely used in chemistry,medicine and other fields.Methods for the synthesis of pyrrolidone mainly include chemical,biological-chemical and biological methods,among which chemical and biological-chemical methods require harsh chemical conditions and use corrosive chemicals and catalysts,while microbial fermentation is limited by long fermentation time and by-product accumulation.Therefore,the enzyme conversion method with mild reaction conditions,high conversion rate and low environmental pollution is a potential production method of pyrrolidone.In this study,a three-enzyme cascade pathway with L-glutamate as substrate was developed to produce pyrrolidone.The catalytic efficiency of carnitine Co A ligase（EcCaiC）was improved by protein engineering,and the yield of pyrrolidone was further improved by optimizing the ribosome-binding site（RBS）sequence regulation,induction conditions and transformation conditions.The main research results are as follows:（1）Design,construction and validation of pyrrolidone synthesis paths.First,the glutamate decarboxylase（Bm GAD）from Bacillus gigantus,carnitine Co A ligase（EcCaiC）from Escherichia coli and poly phosphokinase 2（Dego PPK2）from Deinococcus geothermalis were screened to catalyzed the reaction as path enzymes.Then,the production of the product was monitored by in vitro catalytic reaction,and the feasibility of the pathway was verified by cationic mass spectrometry.Finally,the rate-limiting enzyme was determined to be EcCaiC by further in vitro conversion experiment and determination of the best specific enzyme activity of the three enzymes.（2）Protein engineering to improve the catalytic efficiency of EcCaiC:Based on the analysis of substrate binding and reaction mechanism of EcCaiC,we proposed two strategies to improve the catalytic activity of EcCaiC:1)improve the pre-catalytic conformation of the intermediate 1（IM1）by removing the steric hindrance of the loop ring and 2)stabilize the closed conformation of the protein by fixing the hinge region.The optimal doublet mutant EcCaiC^F380M/N430D was obtained by high-throughput screening.The enzyme properties of EcCaiC^F380M/N430D were determined to be 1.2 times higher than that of EcCaiC,and k_cat/K_m value was 1.5 times higher than that of EcCaiC.We performed MD simulations on the EcCaiC^F380M/N430D-IM1 complex.Compared with EcCaiC-IM1,the active conformation for pyrrolidone formation is increased in the EcCaiC^F380M/N430D-IM1 complex,which may because F380M expands the binding cavity in the loop region,the amino group of IM1 is easier to attack the scissile phospholipid bond.Furthermore,the aspartic acid of N430D in the hinge region forms an electrostatic interaction with K425,as well as the introduction of the salt bridge reduces the flexibility of the hinge region and increases the conformation of enzyme in closed state,thus increasing the enzyme activity.（3）One-pot production of pyrrolidone by assembling the three enzymes.Four engineering strains were successfully constructed by assembling three pathway enzymes using plasmids with different copy numbers,and the best co-expression strain E.coli 03 was obtained by whole cell transformation experiment.The best mutant EcCaiC^F380M/N430D was replaced by EcCaiC to construct the co-expressing strain E.coli 05.The expression levels of the pathway enzymes were optimized by RBS strategy to construct the co-expressing strain E.coli 08,and the enzymic and reaction conditions of transformation of L-glutamic acid to produce pyrrolidone were optimized at the shaking flask level.Under the optimum induction:the initial induction OD₆₀₀ was 0.8,and 0.4 m M IPTG for enzyme induction for 16 h at 25°C and transformation condition:10 m M AMP,140 m M Mg²⁺,80 m M Poly P,100 m M PBS buffer（p H 6.5）and 40℃),the initial concentration of L-glutamate was 15 g·L^-1,5 g·L^-1 was added every 1h and three batches were fed in total.Finally,pyrrolidone was produced at the shaker level with a yield of16.6 g·L^-1 and a molar conversion of 95.2%.