B. Hofer, J. Schneider, P. Washausen; with M. Seeger (Uni Valparaiso) and K. Buchholz (TU-Braunschweig), Schering AG
Our expertise in biotransformation of small molecules originates in part from work of the previous GBF research group of Microbial Transformation (head Prof. Kieslich) which provided us a large collection of microbe strains and fermentation protocols in the form of a bio/data bank. The recognition of this expertise is documented by the fact that Schering AG has entered into a collaboration with us to derivatize some of their precious key scaffolds.
During our work in recent years on the genetic engineering of enzyme substrate specificities, we have obtained substantial expertise in biotransformations involving two different families of enzymes, aryl-hydroxylating dioxygenases and sucrose-utilizing glycosyltransferases. Both types of reactions are attractive with respect to the combinatorial synthesis of small molecules. Both enzyme families are able to hydroxylate or glycosylate, respectively, a quite diverse range of compounds (see Figure). Furthermore, hydroxylation by dioxygenases generates potential substrates for the glycosyltransferases.
Through enzyme engineering, applying a metagenomic approach as well as directed and random formats of mutagenesis, we were able to generate libraries of variants possessing diversified substrate ranges that expand our synthetic capabilities for combinatorial compound synthesis. In collaboration with the group of Prof. M. Seeger we have shown that our strains are able to hydroxylate various benzene derivatives, including compounds with unsaturated and cyclic substituents, e. g. biphenyls or diphenylmethanes, as well as compounds containing condensed rings and heteroatoms, like biphenylenes, benzofurans, indoles and bipyridyls, natural and synthetic isoflavones (57). Several of these compounds represent known „privileged“ substructures which are repeatedly found in natural products that modulate cellular functions.
For the research on glycosyltransferases, funded by DFG-SFB 578, recombinant high-level expression systems have been constructed and are used to investigate the acceptor substrate specificity of the glycosyltransferase R (GtfR). So far, it has been shown that not only various carbohydrates, but also alcohols and amino acids can be glycosylated. Furthermore, libraries of GtfR variants have been generated by random segmental mutagenesis and have been successfully screened for novel substrate specificities (56). Very recently, we were able to demonstrate for the first time for this family of enzymes the turnover of a sucrose analogue containing a modified glucose moiety. Future studies will include screenings of our collection of GtfR variants against our collection of secondary metabolites as potential acceptor substrates, with particular emphasis on those known to modulate cellular functions.