Cloning And Analysis Of DhaT Gene Encoded For 1,3-propanediol Oxidoreductase And Preparatory Research Of Its Directed Evolution

1,3-Propanediol is a compound of great potential application in many synthesis reactions. Now interest has been focused on the production of 1,3-PD by microbial fermentation. The yield of 1,3-propanediol is affected by the ability of 1,3-propanediol oxidoreductase encoded by dhaT in the substrate with high concentration of 1,3-propanediol.We cloned dhdT gene from K. pneumoniae successfully. Using Primer to design two oligonucleotides P1:5'- CGAAGCTTCCGCATTATAACCTGAAGCG-3' and P2: 5'-CGGAATTCATGAGTGAGGGCTATCTCGC-3', and using single-factor and Uniform Design to optimize PCR. The PCR mixture consisted of 50 ng dha T gene template, 2 mmol.L-1 MgCl2, 160umol.L-1 each dNTP, 1.1 nmol.L-1 P1 and P2, 2 uL Taq DNA buffer and 1 U Taq DNA polymerase in 20 u L, and carried out at 95℃ for 5 min, followed by 30 cycles at 95℃ for 60 s, 64℃ for 70 s, 72℃ for 120 s, and then 72℃ for 5 min. Then used corresponding bioinformatics software to analyze the cloned DNA sequence and its amino acid sequence deduced from which. Its homology is high at the nucleic acid, as well as at the amino acid level. Its pI value was 5.94, and its molecular weight was 41.5 kD. In addition, there obviously were six continuous Beta-Strand, which appears in many members of glycerol dehydrogenase family. The analytical results of the dhdl gene fragment and the amino acid sequence showed that PDOR belongs to NAD(P)-dependent glycerol dehydrogenase group III iron-activated dehydrogenase family.The thesis investigated the methods for constructing mutant library, error-prone PCR and DNA shuffling, by using uniform design. It's fit for error-prone PCR that mixture consisted of 7 mmol.L-1 MgCl2, 0.5 mmol.L-1 MnCl2, dATP: dGTP: dTTP: dCTP=0.2 mmol.L-1:0.2 mmol.L-1:1mmol.L-1:1 mmol.L-1, 37.5 pmol.L-1 primer, 2 uLTaq DNA buffer and 1 U Tag DNA polymerase in 20 u L, carried out at 94℃ 30 s, 55℃ 30 s, 72℃ 20 min, and then 72℃ 10 min. At the same time, we found that digesting about 200 ng DNA by DNase I needs 0.25 mmol.L-1MgCl2, 50 mmol.L-1 Tris-HCl (pH7.4), 0.75 mmol.L-1 MnCl2,0.0007 U DNase I in 20 uL, carried at 15℃ for 14min, then 80℃ for 10min. Based on the character of dehydrogenase, we constructed the screening system, which combined NAD+/NADH+H+ coenzyme cycled and selectivities color reaction, and selected 20 g.L-1 1,3-propanediol as the original point of screening pressure.