Heterologous Expression Of EpoK Gene And Analysis Of The Epoxidation Of4-monomethylated Epothilone D

Epothilones, the most potential anticancer agents after taxol, are a kind of polyketide produced by Sorangium cellulosm. The aim of the thesis is to establish a biological method to epoxidaze4-monomethylated epothilone D to4-monomethylated epothilone B.It had been discovered that the enzyme P450epoK in the mutated myxobcteria catalyzed4-monomethylated epthione D to4-monomethylated epothilone B in a very low efficiency and it could only recognize either epothilone D1(BG44A) or D2(BG44B). Thus there was little4-monomethylated epothilone B produced and the products were so complicated that the isolation of pure single component became rather difficult. It was hypothesized that P450epoK cannot recoganize BG44A and BG44B, so it was proposed to establish a platform firstly to heterologous express epoK and detect the function of P450epoK, then to modify the gene by site-directed mutagenesis, finally to screen the mutated enzymes by the platform to get the expected enzyme that can epoxidase4-monomethylated epothilone D efficiently.Firstly, epoK was transformed to E. coli and P450epoK was isolated. Enzyme catalysis was conducted in vitro by adding natural substrate epothilone D, spanich ferrodoxin, spanich ferrodoxin reductase, NADPH and NADPH regeneration system. It was rather difficult to get the activated enzyme due to the complexity of the catalyzation mechanism of P450. Then, epoK gene was transformed to S. lividans TK24, S. coelicolor M512and S. albus. Biotransformation was conducted by adding epothilone D to the culture media. The generation of products were detected by HPLC. Different resultes were observed and S. albus was considered the best host strain. P450epoK expressed in S. albus could epoxidaze epothilone D to epothilone B and no impurity produced.4-monomethylated epothilone D were biotransformed in S. albus and it was found that P450epoK could epoxidaze both BG44A and BG44B to BG44A-O and BG44B-O with a low ratio (about15%).Next, measures were taken from both internal and external aspects simultaneously. On the one hand, three mutants were constructed by mutating the amino acids Arg71, Lys92and Leu301to Ala. On the other hand, the conditions of biotransformation were optimized, including the choice of media for spore formation and biotransformation, the time of adding substrates and the time course of biotransformation. The results were rather intriguing that by optimizing the conditions of biotransformation, natural P450epoK enzyme could epoxidaze both natural substrate (epothilone D) and new substrates (BG44A and BG44B) effectively and efficiently (about97%). The structure of prodcts were identified by MS and HNMR. In the three mutants, the ratios of epothilone D retained97%, however, the ratios of BG44A and BG44B reduced:20%for BG44A and25%for BG44B in R71A,60%for BG44A and75%for BG44B in K92A,7%for BG44A and8%for BG44B in L301A.Though it was proved that our assumption was incorrect, we achieved our aim more directly. There were some advantages of biotransformation such as simple procedure, mild reaction conditions, high yields, little pollution and low price.