Enantioselective Reduction of -Keto Acids with Engineered Streptomyces coelicolor

摘要

The soil bacterium Streptomyces coelicolor has long been studied owing to its production of antibiotics, the most significant of which is the dimeric benzoisochromanequinone actinorhodin (ACT, 1). S. coelicolor is generally regarded as the best genetically characterized of all the streptomycete bacteria. Recently its complete genomic sequence was published.[1] Over the years a clear picture has emerged for the biosynthesis of the skeleton of 1.[2] It is generally accepted that 2 (or the phenol tautomer of 2) is assembled from eight malonate units by a type II polyketide synthase (PKS). Whether 2 is released at this stage from the PKS as the free -keto acid (R=OH) or is subject to further biotransformations as the enzyme-bound thiolate (R=SEnz) is a matter for speculation (see below). Reduction of 2 is thought to lead to the hydroxy acid 3, which then undergoes cyclization followed by dehydration to give the yellow pigment (S)-DNPA (4, 4,10-dihydro-9-hydroxy-1-methyl-10-oxo-3H-naphtho[2,3c]pyran-3-(S)-acetic acid). Significantly, 4 has been isolated from both mutant[3] and recombinant[4] strains of S. coelicolor, thus strongly implicating its role as a true intermediate in ACT biosynthesis. It is thought that 4 then undergoes a series of enzyme-controlled reduction, oxidation, hydroxylation, and dimerization steps, which result in the formation of 1 (Scheme 1). A number of genes thought to encode the various enzymes have been identified (act tailoring genes), but at this stage the likely intermediates in the biosynthesis of 4 have yet to be confirmed. The reduction of 2 is believed to occur by the action of a reductase (RED1) encoded by the actVI-ORF-1 gene. Good evidence for this hypothesis was first obtained by Floss and co-workers,[3] who found that the B1 mutant strain of S. coelicolor accumulated significant quantities of 4. More recently we showed[4] that a recombinant strain (CH999/pIJ5660) of S. coelicolor containing actPKS and actVI-ORF-1 genes resulted in the production of 4 on fermentation in a liquid medium. A further strain was engineered from the plasmid pIJ5660, from which the ketosynthase (KS) gene actI-ORF-1 had been deleted. This new strain (CH999/pIJ5675) did not have the ability to synthesize the polyketide skeleton from which 2 is derived, but could still express RED1 and thus serve as a whole-cell reduction medium. A number of N-acetylcysteamine -ketothioester (SNAC) substrates were fed to this strain. They underwent enantioselective reduction to the corresponding -hydroxy acids with moderate to high ee values (analyzed as the methyl esters 6, R=Me). The fact that a range of nonnatural SNAC derivatives proved to be substrates for RED1, but that other -ketoesters were not, provided convincing evidence at the time for the likely existence of 2 as the thiolate (R=SEnz). These results led us to conclude that NAC thioesters are required for recognition by the actVIORF-1 reductase and/or they are required for successful transport across the cell membrane.[5] Herein we present results from further study that demonstrate that this statement is likely to be incorrect and that CH999/pIJ5675 is a much more general biotransformation system than previously thought.