Directed Evolution Of Imine Reductase Red52 For Biosynthesis Of The Drug Candidate 6-IA-PTOX

Through the study of the tubulin binding site near the colchicine binding domain,it was found that the antitumor activity of the C-NH-linked podophyllotoxin derivative4β-NH(6-aminoindole)-4-deoxy-podophyllotoxin(6-IA-PTOX)was increased 1000 fold compared with the antitumor activity of the podophylloside and teniposide antitumor drugs that have been listed.From the previous micromolar level to the nanomolar level,6-IA-PTOX has great potential for drug candidate.So far,6-IA-PTOX can only be prepared by chemical synthesis,but this process involves the use of toxic reagents,such as boron trifluoride ether and triethylamine;meanwhile,the yield is less than 30%.Owing to the disadvantages of chemical synthesis,we tried to synthesize 6-IA-PTOX by enzymatic method.By analyzing the molecular structure of 6-IA-PTOX,we designed a two-step enzymatic synthesisroute including an alcohol dehydrogenase and animine reductase.The first step of this reaction has been established by our lab previously by oxidizing the commercial available podophyllotoxin to podophyllotoxone catalyzed by the alcohol dehydrogenase from sinopodophyllum hexandrum.The purpose of this dissertation is to use podophyllin and 6-aminoindole as substrates to explore suitable imine reductase elements catalyzing the reductive amination reaction for the synthesis of 6-IA-PTOX.By screening the existing 200 imine reductases in the laboratory,the imine reductases that can directly catalyze podophyllin and 6-aminoindole have not been screened.Based on the fact that imine reductase is more inclined to catalyze the reduction of imine intermediates,6-IA-PTOX imine intermediates were chemically synthesized and used as substrates to screen existing imine reductases.Finally,it was found that twenty kinds of imine reductases,such as Red52 and IR45,can catalyze the intermediate of 6-IA-PTOX to generate the final product 6-IA-PTOX.Directed evolution was carried out with the highest conversion rate of imine reductase Red52 as the parent to achieve efficient catalysis.A set of crystal data of the complex of imine reductase Red52 and cofactor NADPH was obtained by crystal culture and X-ray diffraction,and the crystal structure of the complex was solved by molecular replacement method.Through four rounds of semi-rational design guided by the crystal structure,the conversion rate of the Red52-M4 mutant to the 6-IA-PTOX intermediate was increased from the initial 3.0% to 88.3%.