Roles of the crotonyl-CoA carboxylase/reductase homologues in acetate assimilation and biosynthesis of immunosuppressant FK506 in Streptomyces tsukubaensis.

BACKGROUND: In microorganisms lacking a functional glyoxylate cycle, acetate can be assimilated by alternative pathways of carbon metabolism such as the ethylmalonyl-CoA (EMC) pathway. Among the enzymes converting CoA-esters of the EMC pathway, there is a unique carboxylase that reductively carboxylates crotonyl-CoA, crotonyl-CoA carboxylase/reductase (Ccr). In addition to the EMC pathway, gene homologues of ccr can be found in secondary metabolite gene clusters that are involved in the provision of structurally diverse extender units used in the biosynthesis of polyketide natural products. The roles of multiple ccr homologues in the same genome and their potential interactions in primary and secondary metabolic pathways are poorly understood. RESULTS: In the genome of S. tsukubaensis we have identified two ccr homologues; ccr1 is located in the putative ethylmalonyl-CoA (emc) operon and allR is located on the left fringe of the FK506 cluster. AllR provides an unusual extender unit allylmalonyl-CoA (ALL) for the biosynthesis of FK506 and potentially also ethylmalonyl-CoA for the related compound FK520. We have demonstrated that in S. tsukubaensis the ccr1 gene does not have a significant role in the biosynthesis of FK506 or FK520 when cultivated on carbohydrate-based media. However, when overexpressed under the control of a strong constitutive promoter, ccr1 can take part in the biosynthesis of ethylmalonyl-CoA and thereby FK520, but not FK506. In contrast, if ccr1 is inactivated, allR is not able to sustain a functional ethylmalonyl-CoA pathway (EMC) and cannot support growth on acetate as the sole carbon source, even when constitutively expressed in the chimeric emc operon. This is somewhat surprising considering that the same chimeric emc operon results in production of FK506 as well as FK520, consistent with the previously proposed relaxed specificity of AllR for C4 and C5 substrates. CONCLUSIONS: Different regulation of the expression of both ccr genes, ccr1 and allR, and their corresponding pathways EMC and ALL, respectively, in combination with the different enzymatic properties of the Ccr1 and AllR enzymes, determine an almost exclusive role of ccr1 in the EMC pathway in S. tsukubaensis, and an exclusive role of allR in the biosynthesis of FK506/FK520, thus separating the functional roles of these two genes between the primary and secondary metabolic pathways.

Draft Genome Sequence of Streptomyces rapamycinicus Strain NRRL 5491, the Producer of the Immunosuppressant Rapamycin.

Streptomyces rapamycinicus strain NRRL 5491 produces the important drug rapamycin. It has a large genome of 12.7 Mb, of which over 3 Mb consists of 48 secondary metabolite biosynthesis clusters.

FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis.

BACKGROUND: FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses. RESULTS: Three putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively. CONCLUSIONS: Our results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of S. tsukubaensis for improving the yield of fermentative processes for production of FK506.

Annotation of the modular polyketide synthase and nonribosomal peptide synthetase gene clusters in the genome of Streptomyces tsukubaensis NRRL18488.

The high G+C content and large genome size make the sequencing and assembly of Streptomyces genomes more difficult than for other bacteria. Many pharmaceutically important natural products are synthesized by modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). The analysis of such gene clusters is difficult if the genome sequence is not of the highest quality, because clusters can be distributed over several contigs, and sequencing errors can introduce apparent frameshifts into the large PKS and NRPS proteins. An additional problem is that the modular nature of the clusters results in the presence of imperfect repeats, which may cause assembly errors. The genome sequence of Streptomyces tsukubaensis NRRL18488 was scanned for potential PKS and NRPS modular clusters. A phylogenetic approach was used to identify multiple contigs belonging to the same cluster. Four PKS clusters and six NRPS clusters were identified. Contigs containing cluster sequences were analyzed in detail by using the ClustScan program, which suggested the order and orientation of the contigs. The sequencing of the appropriate PCR products confirmed the ordering and allowed the correction of apparent frameshifts resulting from sequencing errors. The product chemistry of such correctly assembled clusters could also be predicted. The analysis of one PKS cluster showed that it should produce a bafilomycin-like compound, and reverse transcription (RT)-PCR was used to show that the cluster was transcribed.