No abstract

Epothilone F, 21-hydroxyl-epothilone B, is an intermediate in the synthesis of BMS-310705, an antitumor compound that has been evaluated in Phase I clinical trials. A bioconversion process utilizing the Gram-positive bacterium Amycolatopsis orientalis was used to prepare epothilone F from epothilone B. In order to improve the yield of epothilone F, a mutagenesis program was performed with the goal of engineering the epothilone-B hydroxylase (EBH) enzyme to improve the yield of epothilone F through oxidative biotransformation. The mutations in EBH increased the yield of epothilone F from 21% in the recombinant expression system to higher than 80% utilizing the best EBH mutants. The studies described here show how a homology model of EBH was used to obtain an understanding of the possible mechanism that led to improved yield of epothilone F in the mutated enzymes. A novel aspect of this study is that it provides some insight into how mutations distant from the binding site can affect enzyme activity.

Degenerate PCR primers were used to amplify cytochrome P450 gene fragments from the high-GC gram-negative bacteria Amycolatopsis orientalis, which catalyzes the hydroxylation of epothilone B to produce epothilone F. The amplified fragments were used as hybridization probes to identify and clone two intact cytochrome P450 genes. The expression of one of the cloned genes in a Streptomyces lividans transformant resulted in the biotransformation of epothilone B to epothilone F. The conversion of epothilone B to epothilone F by the S. lividans transformant was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy.

The microorganism, Amycolatopsis orientalis was incorrectly described as a high-GC Gram negative bacterium. This microorganism is a high-GC Gram positive bacterium. We regret this error.The online version of the original article can be found at http://dx

A recombinant fungal microorganism capable of producing deacetylcephalosporin C was constructed by transforming a cephalosporin C esterase gene from Rhodosporidium toruloides into Acremonium chrysogenum. The cephalosporin C esterase gene can be expressed from its endogenous R. toruloides promoter or from the Aspergillus nidulans trpC promoter under standard Acremonium chrysogenum fermentation conditions. The expression of an active cephalosporin C esterase enzyme in A. chrysogenum results in the conversion of cephalosporin C to deacetylcephalosporin C in vivo, a novel fermentation process for the production of deacetylcephalosporin C. The stability of deacetylcephalosporin C in the fermentation broth results in a 40% increase in the cephalosporin nucleus.