Biosynthesis Of Ansacarbamitocins In Amycolatopsis Alba DSM44262

Maytansinoids belong to a group of 19-member macrolactam ansamycin antibiotics with potent antibacterial and antitumor activities,which are synthesized by type I polyketide synthases(PKS)using 3-amino-5-hydroxybenzoic acid(AHBA)as starter unit.They are widely used in the field of antibody drug conjugates(ADC),and ado trastuzumab entansine(commodity name Kadcyla)has been approved for the treatment of HER2 positive metastatic breast cancer.In addition,there are more than 10 kinds of coupling the toxin of ADC in drug clinical research.The core precursor of ADC,maytansinol,is currently obtained by semi-synthetic preparation of ansamitocins,which are made of microbial.But this method is high cost,low yield,and difficulty to separate and remove byproducts.Therefore,looking for new methods to produce maytansinol is of great significance.Previously,five ansacarbamitocins(1-5)were isolated from Amycolatopsis.alba DSM44262 by our laboratory.Through the analysis of its genome DNA,we located the synthetic gene cluster abm of ansacarbamitocins.Through further analysis,it was found that abm cluster contains two carbamyl acyltransferase homologous genes ahm21a and abm21b,a N-glycosyltransferase homologous gene abm25,speculating that they might be respectively responsible for ammonia formylation of C3-OH,C7-OH,and N-glycosylation.So it is expected to construct bacteria which producing maytansinol analogue by knocking out the gene for C3-OH ammonia formylation and abm25.So,we firstly established A.alba DSM44262 mycelium electricity genetic operation system;and then investigated the knockout of abm21a,abm2lb and abm25 in vivo.Through separation and identification of accumulation intermediates of abm21a and abm21b knockout mutants,it was found that accumulation intermediates of two knockout mutants are the same,a lot of 19-chloroproansamitocin and a small amount of 14-OH-19-chloroproansamitocin.Thus we speculate that Abm21a and Abm21b may together take part in ammonia formylation of C3 and C7 hydroxyl.By separation and identification of accumulation intermediate of abm25 knockout mutant,the N-deglucosyl-ansacarbamitocin A was obtained,showing that abm25 participates N-glycosylation of ansacarbamitocins.In addition,antibiotic N-glycosyltransferases slowly progress due to achieving the soluble and active protein very difficult,therefore Abm25 was researched in vitro.We obtained soluble and enzymatic active Abm25 by heterologous expression,which is the first time to get soluble and enzymatic active N-glycosyltransferase protein.By researching the influence on Abm25 enzyme activity of pH and temperature:the optimum pH and temperature are 8.0 and 30?,respectively.Then kinetic parameters of Abm25 was measured,under the concentration of UDP-Glc 500 ?M,Km is 2.44±0.26 ?M and kcat is 0.24±0.01 min-1 for 19-chloroproansamitocin;under the concentration of 19-chloroproansamitocin 400 ?M,Km,is 10.22±1.36 ?M and kcat is 0.15±0.01 min-1 UDP-Glc.At the same time,we also studied substrate broad spectrum of Abm25,finding that Abm25 has strict specificity for sugar receptors,but can use UDP-glucuronic acid as sugar donor,enriching the antibiotic N-glycosyltransferase tools library.This study identified ansacarbamitocins synthetic gene cluster abm from Amycolatopsis alba DSM44262,mainly researching three post-modification genes(abm21a,abm21b and abm25).Further work can be done by recovering asm21 gene of asm cluster upon abm21a or abm21b knockout mutant,in order to construct the strain which producing maytansinol analogues.These researches will lay foundation for antibody coupling drug research.