Directed Evolution Of L-amino Acid Deaminase And Biotransformation Synthesis Of ?-ketoglutaric Acid

?-ketoglutaric acid??-KG?is a binary short-chain carboxylic acid,which is also a key node connecting carbon-nitrogen metabolism.It is an important precursor of many compounds in microorganisms and is widely used.Food,medicine,fine chemicals and other fields.At present,the production methods of?-KG mainly include chemical synthesis method,microbial fermentation method and enzyme catalytic conversion method.Among them,the chemical synthesis method uses a large number of harmful reagents,which seriously pollutes the environment and limits its application in food and other industries.The microbial fermentation method has many disadvantages such as excessive by-products and long fermentation cycle.The enzyme catalytic conversion method has the advantages of mild reaction conditions,high conversion rate,few reaction by-products,easy separation of products,and the like,and is suitable for the production of?-ketoglutaric acid.In this study,a recombinant strain of E.coli?DE3?derived from the L-amino acid deaminase gene?pm1?of Proteus mirabilis was used to catalyze the synthesis of?-KG,a substrate for sodium glutamate and was modified from molecular modification.The efficiency of L-amino acid deaminase synthesis of?-KG was improved in the optimization of expression vector,fermentation in shake flask and can and optimization of whole cell transformation conditions.The main contents of the paper are as follows:?1?Recombinant E.coli BL21?pET20b-pm1?heterologously expressing the L-amino acid deaminase gene pm1 derived from Proteus mirabilis was subjected to conditions for fermentation and whole cell transformation on a shake flask.The optimal fermentation conditions were as follows:seed culture time 10 h,induction temperature 20?,induction time12 h,IPTG concentration of inducer 0.06 mM,induction time was OD₆₀₀ value of 3.0;optimal whole cell transformation conditions were:transformation temperature was 30?,pH was 6.0,the cell concentration was 20 g·L^-1,and the final?-KG yield reached 61.80 g·L^-1,and the substrate molar conversion rate was 39.6%.?2?The L-amino acid deaminase crystal model from Proteus myxofaciens was obtained through the online website SWISS MODEL,and the homology with PM1 was 93.74%.After homology modeling,the molecular docking of the substrate was performed using the automatic docking software Auto Dock 4.0.By analyzing the structure of the model,the amino acids around the key sites were saturated and mutated.After multiple rounds of screening,the mutants with higher yields than the control were obtained,namely G206R,P272F,V276C,V283I,E340S,E340G,and the yields were increased by 12.5%,18.2%,14.8%,4.5%,27.6%and17.0%,respectively.Subsequently,multiple rounds of compound mutations were carried out,and the mutant G206R/P272F/V276C/V283I/E340S had the highest yield,reaching 100.96 g·L^-1,and the substrate molar conversion rate was 64.7%.?3?Verify optimal conversion conditions after molecular modification.The temperature,pH and cell mass during the whole cell transformation of the mutant G206R/P272F/V276C/V283I/E340S were optimized in shake flasks.The optimum temperature was determined to be 30?,the optimum pH was 6.0,and the optimum cell volume was 20 g·L^-?4?Optimization of expression vector copy number for recombinant colonic strains.Recombinant plasmids of different copy number expression vectors pRSFDuet-1,pETDuet-1,pCDFDuet-1,pACYCDuet-1 were constructed,and the amount of?-KG produced by fermentation and whole cell transformation was compared.The results showed that the recombinant strain containing pACYCDuet-1 expression vector had the highest yield,106.37g·L^-1,and the substrate molar conversion rate was 68.1%,and the concentration of bacteria at the end of the culture was 2.25 times that of the control,which was more conducive to the next step.Cell transformation.?5?Optimization of fermentation and whole cell transformation conditions and substrate addition methods on the 3 L fermentor.The optimum agitation speed during the fermentation was determined to be 400 rpm and the optimal aeration was 3.0 sL·min^-1.The optimal agitation speed during the whole cell transformation was 600 rpm and the optimal aeration was 3 sL·min^-1.The substrate was added in three portions with a total concentration of 150 g·L^-1,the highest yield was 93.46 g·L^-1,and the substrate molar conversion was 79.8%.