Gene Mining, Characterization And Fermentation Optimization Of A Recombinant Diketone Reductase

Chiral diol is a kind of valuable building block for the synthesis of fine chemicals, pharmaceuticals and chiral compounds because of the existence of two chiral centers. Particularly,(2S,3S)-2,3-butanediol plays an important role for the asymmetric synthesis of chiral compounds which contain two vicinal chiral centers. The reduction of diketones to chiral diols using carbonyl reductase is one of the important methods for the synthesis of chiral diols.Six reported carbonyl reductases which exhibit excellent stereoselectivity in the reduction of prochiral ketones were chosen as probes. After BLAST in Uni Prot protein database using the protein sequences, we chose nineteen proteins which share 40%–90% identity with its respective probe. Then the candidate genes were heterogeneously expressed in Escherichia coli BL21(DE3). After SDS-PAGE analysis and enzymatic activity analysis, we chose the enzyme which displayed the highest activity in the reduction of diacetyl and acetoin to be further studied. This reductase had 260 amino acids in length, and was originated from Bacillus licheniformis under the Uniprot accession number of R9TV22. The phylogenetic tree and the amino acid alignment showed that it was a meso-2,3-butanediol dehydrogenase(meso-2,3-BDH) and belonged to short-chain dehydrogenase family. We named this enzyme as Bl BDH.Then we purified and characterized Bl BDH. The molecular weight of Bl BDH was calculated to be about 125 k Da, suggesting it was a homotetramer. Then the substrate specificity showed that it could not only catalyze the reduction of diketones containing two vicinal carbonyl groups and acetoin, but also oxidization of 2,3-butanediol. We studied the optimal p H and temperature of Bl BDH. The optimal p H was 5.0 for diacetyl reduction while it was 10.0 for 2,3-butanediol oxidation. Uniquely, it had a wide range of p H optimum(5.0–8.0) for acetoin reduction. So it functioned mainly as a redutase in acid buffer. The optimal temperature was about 37°C for both reduction and oxidation reactions. The kinetic parameters showed that towards its substrate acetoin, Bl BDH had the smallest Km value of 0.47 mmol·L-1 and largest kcat/Km value of 432.41 L·mmol-1·s-1, implying that acetoin was the best substrate for Bl BDH. It decreased to half of the initial activity in 40 min under 50°C showing no extraordinary stability under 50°C. Moreover, no metal ion was proven to activate the enzyme. In the end, the stereoselectivity showed that it could reduce diacetyl to(S)-acetoin with an ee value of 97%, then further to(2S,3S)-2,3-butanediol with an optical purity of about 95%.To reduce the fermentation cost, we optimized the medium components and induction conditions of a Bl BDH-producing recombinant E. coli strain in flasks. The optimal medium composition for the production of Bl BDH was listed as follows: glycerol 5 g·L-1, yeast extract 10 g·L-1, peptone 5 g·L-1,(NH4)2SO4 1.5 g·L-1, sodium citrate 5 g·L-1, KH2PO4 1 g·L-1, Mg SO4·7H2O 0.5 g·L-1 and Na Cl 2 g·L-1. The optimal induction conditions were listed as follows: 37°C and 0.1 mmol·L-1 IPTG. Under these optimized conditions, the enzyme activities reached 13.84 k U·g-1 dry cell weight and 33.65 k U·L-1, which were 2.04 and 4.23 folds of those in basic medium, 1.16 and 1.71 folds of those in LB medium. In a 3-L fermenter, under the optimized conditions, the maximum enzyme activities achieved at 14.09 k U·g-1 and 64.62 k U·L-1 after cultivation for 8 h.