Biocatalytic Cascade Reaction For Preparation Of Pyruvate And L-tyrosine Derivatives

Pyruvate,as an important raw material and intermediate,is widely used in chemistry,medicine,agriculture and other fields.The traditional pyruvate production process of dehydration and decarboxylation of tartaric acid is easy to operate,but there are still many problems,such as serious pollution,low yield and large consumption of raw materials,which lead to high prices of pyruvate and its derivatives.In recent years,the production of pyruvate by biological methods,including fermentation of sugar raw materials and enzymatic oxidation of lactate,have attracted wide attention due to its low cost of raw materials,mild reaction conditions and safety and reliability of products.Due to the active metabolic pathway related to pyruvate in cells,which limits the improvement of pyruvate fermentation titer,and the high solubility of pyruvate in aqueous solution,the cost of separating pyruvate from complex fermentation broth is high,thus the production of pyruvate by fermentation could not achieve large-scale industrialization.The biocatalytic method based on the short chain L-2-hydroxy acid oxidase has a good industrial application prospect because it does not need expensive coenzyme,the catalytic system is simple and the product separation and purification is convenient.However,hydrogen peroxide,a by-product of the catalytic process,can easily generate acetate and carbon dioxide from pyruvate,resulting in the decrease of yield.Optically pure L-tyrosine and its derivatives are often used to synthesize some important drugs,such as Parkinson’s drug L-DOPA and anti-tumor drug(-)-saframycin A.Chemical de novo synthesis of L-tyrosine derivatives requires many steps,and the final product is racemate,which needs to be resolved later.Tyrosine phenol-lyase can directly catalyze pyruvate,phenol derivatives and ammonia to form L-tyrosine derivatives,which is a very promising green synthetic way of L-tyrosine derivatives.However,due to the high cost and instability of pyruvate,the method is still lack of competition.Biocatalytic cascade has been widely used in the synthesis of high value-added chemicals because it can overcome the toxicity and instability of intermediate products,reduce the cost of substrate and improve the efficiency of catalysis.To solve the above problems,two kinds of biocatalytic cascade reaction systems were developed in this paper:1)By coupling the oxidative dehydrogenation of lactate catalyzed by short chain L-2-hydroxy acid oxidase with the decomposition of hydrogen peroxide catalyzed by catalase,a whole cell catalyst for the oxidation of L-lactate,which is cheap and biobased material,to pyruvate was constructed.2)Combining the above system with tyrosine phenol-lyase,another cascade reaction system including L-2-hydroxy acid oxidase,catalase and tyrosine phenol-lyase was constructed.L-tyrosine derivatives were prepared by one-pot using L-lactate and phenol derivatives as starting substrates.In present thesis,a systematic research was done to develop these biocatalytic cascade process,the main contents are as follows:First,six kinds of short chain L-2-hydroxy acid oxidase and 10 kinds of monofunctional catalase were cloned by means of literature research and gene mining,and lactate oxidase from Aerococcus viridans(AvLOX)and catalase from Ureibacillus thermosaphaericus(UtCAT)with high catalytic activity and good stability were obtained.In order to improve AvLOX activity,the strategies of reducing the expression temperature and concentration of IPTG,RBS sequence directed evolution and co-expression with molecular chaperone were used to enhance its soluble expression.By reducing the expression temperature and the concentration of IPTG,the solubility of AvLOX was increased by 3.4 times,and the enzyme activity was increased by 48.9%.Through directed evolution of RBS sequence,the RBS mutant GGGGGC which was beneficial to AvLOX soluble expression was successfully obtained,and AvLOX in recombinant E.coli-pET28a-T7rbsAvLOX increased by 1.7 times in solubility and 62.7%in activity.On this basis,further co-expression with molecular chaperone GroES-GroEL increased by 1.8 times in solubility and 43.1%in enzyme activity.Finally,AvLOX solubility increased from 8.5%to 89.2%and enzyme activity increased from 4.5 U/ml to 15.6 U/ml.The in vitro enzyme cascade reaction system was constructed by UtCAT and AvLOX crude extracts.The optimal activity ratio of UtCAT and AvLOX was determined to be 300:1.The yield of 0.5 M L-lactate was 92.3%,which was 12.6 times of that without catalase(7.3%).In order to simplify the process,improve the coupling efficiency and reduce the cost,AvLOX and UtCAT were co-expressed in E.coli BL21(DE3),and pET28a-T7rbsAvLOX was used as the plasmid skeleton to construct an in vivo cascade whole cell catalyst.The co-expression of two enzymes was successfully realized by three different co-expression strategies,and they showed different enzyme activity and protein expression.The co-expression strain E.coli-pET28a-T7rbsAvLOX-rbsUtCAT showed the best expression level.In order to further improve catalase activity,RBS sequence in front of UtCAT gene was designed by RBS Calculator,and expression was enhanced by increasing translation initiation rate.The results showed that the RBS3 sequence was the best,and the co-expression strains E.coli-pET28a-7T7rbsAvLOX-rbs3UtCAT was obtained,which UtCAT and AvLOX activity reached 4127.3 and 12.6 U/mL,respectively(activity ratio 328:1).Then,the whole cell catalytic reaction of co-expression strain was optimized.The optimum conditions were:reaction temperature 37℃,pH 7.0,cell dosage 20 g/L and O2 pressure 0.7 MPa,in which oxygen concentration was the limiting factor of L-lactate oxidation.Under this condition,0.5 ml lactic acid can be transformed in 1.5 h,and the yield is 92.2%.At the same time,considering that maintaining oxygen pressure has certain requirements on the equipment,the substrate fed batch catalytic process was established under atmospheric pressure.The pyruvate concentration reached 544.8 mM with yield of 90.8%in the reaction with the total concentration of L-lactate of 600 mM.Then,a thermostable tyrosine phenol-lyase TTPL from Symbiobacterium toebii was expressed in E coli and its substrate spectrum was analyzed.It was found that the wild-type TTPL could only catalyze phenol,catechol and 2-fluorophenol.However,when the ortho substituents were replaced with Cl,Br,CH3 and OCH3,TTPL lost its activity.To expand the substrate spectrum of TTPL,rational design was carried out.Based on homology modeling and molecular docking simulation analysis,the pocket which plays a decisive role in substrate specificity in TTPL was expanded by site directed mutagenesis,so that it can accommodate the larger substituent group in the ortho position of phenol.Three mutants F37V,T126S and M380V with high catalytic activity for 2-methylphenol were obtained.The substrate specificity studies showed that the mutant F37V and M380V showed wider substrate spectrum and good catalytic activity.Finally,a three-enzyme cascade reaction system based on AvLOX,UtCAT and TTPL M3 80V was constructed with 2-fluorophenol as the model substrate.The optimal reaction conditions were:reaction temperature 40℃,pH 8.0,the ratio of E.coli-pET28a-T7rbsAvLOX-rbs3UtCAT to E.coli-pET28a-TTPLM380V was 1:2,the total amount of wet cells was 25 g/L,and the substrate concentration was 46 mM.Under this condition,the yield of 3-fluoro-L-tyrosine was increased from 83.1%to 97.1%,with ee value>99%.On this basis,five other L-tyrosine derivatives can be synthesized by changing the types of thermostable tyrosine phenol lyase mutants.The yield is 81.1-96.3%,and the ee value>99%.In the 0.5 L scale reaction,the conversion of phenols and the concentration of product were 77.4-94.3%and 48.0-60.4g/l,respectively.The problems of substrate inhibition,catalyst instability and low yield were successfully solved,which showed a good prospect of industrial application.