Andrew R. Butler

Impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae.

BACKGROUND The polyketide lactone, tylactone, is produced in Streptomyces fradiae by the TylG complex of five multifunctional proteins. As with other type I polyketide synthases, the enzyme catalysing the final elongation step (TylGV) possesses an integral thioesterase domain that is believed to be responsible for chain termination and ring closure to form tylactone, which is then glycosylated to yield tylosin. In common with other macrolide producers, S. fradiae also possesses an additional thioesterase gene (orf5) located within the cluster of antibiotic biosynthetic genes. The function of the Orf5 protein is addressed here. RESULTS Disruption of orf5 reduced antibiotic accumulation in S. fradiae by at least 85%. Under such circumstances, the strain accumulated desmycosin (demycarosyl-tylosin) due to a downstream polar effect on the expression of orf6, which encodes a mycarose biosynthetic enzyme. High levels of desmycosin production were restored in the disrupted strain by complementation with intact orf5, or with the corresponding thioesterase gene, nbmB, from S. narbonensis, but not with DNA encoding the integral thioesterase domain of TylGV. CONCLUSIONS Polyketide metabolism in S. fradiae is strongly dependent on the thioesterase activity encoded by orf5 (tylO). It is proposed that the TylG complex might operate with a significant error frequency and be prone to blockage with aberrant polyketides. A putative editing activity associated with TylO might be essential to unblock the polyketide synthase complex and thereby promote antibiotic accumulation.

Multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae

BACKGROUND The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. RESULTS DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which (tylP) encodes a gamma-butyrolactone signal receptor for which tylQ is a probable target. Two other genes (tylS and tylT) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR, has been shown by mutational analysis to control various aspects of tylosin production. CONCLUSIONS The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.

The tylosin-biosynthetic genes of Streptomyces fradiae

The tylosin-biosynthetic (tyl) gene cluster occupies about 1% of the genome of Streptomyces fradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tylcluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of γ-butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.

Genetic engineering of aminodeoxyhexose biosynthesis in Streptomyces fradiae.

The antibacterial properties of macrolide antibiotics (such as erythromycin, tylosin, and narbomycin) depend ultimately on the glycosylation of otherwise inactive polyketide lactones. Among the sugars commonly found in such macrolides are various 6-deoxyhexoses including the 3-dimethylamino sugars mycaminose and desosamine (4-deoxymycaminose). Some macrolides (such as tylosin) possess multiple sugar moieties, whereas others (such as narbomycin) have only single sugar substituents. As patterns of glycosylation markedly influence a macrolides drug activity, there is considerable interest in the possibility of using combinatorial biosynthesis to generate new pairings of polyketide lactones with sugars, especially 6-deoxyhexoses. Here, we report a successful attempt to alter the aminodeoxyhexose-biosynthetic capacity of Streptomyces fradiae (a producer of tylosin) by importing genes from the narbomycin producer Streptomyces narbonensis. This engineered S. fradiae produced substantial amounts of two potentially useful macrolides that had not previously been obtained by fermentation.

Type II thioesterase from Streptomyces coelicolor A3(2)

Type I polyketide synthases (PKSs) are complexes of large, multimodular enzymes that catalyse biosynthesis of polyketide compounds via repetitive reaction sequences, during which each step is catalysed by a separate enzymic domain. Many type I PKSs, and also non-ribosomal peptide synthetase clusters, contain additional thioesterase genes located adjacent to PKS genes. These are discrete proteins called type II thioesterases (TE IIs) to distinguish them from chain-terminating thioesterase (TE I) domains that are usually fused to the terminal PKS module. A gene of a new TE II, scoT, associated with the cluster of putative type I PKS genes from Streptomyces coelicolor A3(2), was found. The deduced amino acid sequence of the gene product shows extensive similarity to other authentic thioesterase enzymes, including conservation of characteristic motifs and residues involved in catalysis. When expressed in the heterologous host Streptomyces fradiae, scoT successfully complemented the resident TE II gene (tylO), and, by restoring a significant level of macrolide production, proved to be catalytically equivalent to the TylO protein. S1 nuclease mapping of scoT revealed a single potential transcription start point with expression being switched on for a short period of time during a transition phase of growth.

Feedback control of polyketide metabolism during tylosin production

Tylosin is produced by Streptomyces fradiae via a combination of polyketide metabolism and synthesis of three deoxyhexose sugars, of which mycaminose is the first to be added to the polyketide aglycone, tylactone (protylonolide). Previously, disruption of the gene (tylMII) encoding attachment of mycaminose to the aglycone unexpectedly abolished accumulation of the latter, raising the possibility of a link between polyketide metabolism and deoxyhexose biosynthesis in S. fradiae. However, at that time, it was not possible to eliminate an alternative explanation, namely, that downstream effects on the expression of other genes, not involved in mycaminose metabolism, might have contributed to this phenomenon. Here, it is shown that disruption of any of the four genes (tylMI--III and tylB) specifically involved in mycaminose biosynthesis elicits a similar response, confirming that production of mycaminosyl-tylactone directly influences polyketide metabolism in S. fradiae. Under similar conditions, when mycaminose biosynthesis was specifically blocked by gene disruption, accumulation of tylactone could be restored by exogenous addition of glycosylated tylosin precursors. Moreover, certain other macrolides, not of the tylosin pathway, were also found to elicit qualitatively similar effects. Comparison of the structures of stimulatory macrolides will facilitate studies of the stimulatory mechanism.

Expression of tyIM genes during tylosin production: Phantom promoters and enigmatic translational coupling motifs

In the genome of Streptomyces fradiae, the three tyIM genes are codirectional with the upstream gene, tylGV. Although the introduction of transcriptional blocks into the tylM genes revealed that they are normally cotranscribed, expression of tylMI still persisted (albeit at a very low level) when either of the upstream genes, tylMII or tylMIII, was disrupted. Such expression apparently resulted from transcriptional initiation at spurious sites that probably contribute insignificantly, if at all, to promote activity in the wild type. Prior to the onset of tylosin production, tylMIII is transcribed independently of tylGV from an authentic promoter buried within tylGV. This latter observation is interesting given that the TGA stop codon of tylGV overlaps the GTG start codon of tylMIII. Evidently, terminally overlapping genes are not always translationally coupled. Journal of Industrial Microbiology & Biotechnology (2002) 28, 160–167 DOI: 10.1038/sj/jim/7000223