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

Abstract

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.

Figure 1

(A) Structures of FK506 and FK520. (B) Schematic representation of the FK506 biosynthetic cluster. The genes located on the left and right side from the FK506 core PKS region are presented in more detail. Putative regulatory gene homologues allN, fkbN and fkbR are represented by white arrows. Promoters used in the rppA reporter studies, deleted regions and RT-PCR amplified regions are marked.

Figure 2

Schematic representation of disruption plasmids and inactivated fkbN (A) and fkbR (B) genes after secondary recombination.

Figure 3

Yield of FK506 by different strains of S. tsukubaensis. Bars encompass 95% of the sample population. Horizontal line representing the median values, and perpendicular lines indicating extreme values (min, max). Asterisks where representing statistically significant differences between different samples compared to control wild type samples (WT). The data were analyzed using SAS/STAT program as described in Methods. Introduction of additional “in trans” copies of target putative regulatory genes using phiC31-based integrative vector [WT-wild type, WT:R-fkbR over-expressed, WT:N-1 (shorter version of fkbN over-expressed), WT:N-fkbN over-expressed, inactivation of target putative regulatory S. tsukubaensis genes (ΔR-fkbR inactivated, ΔN-fkbN inactivated) and complementation experiments (ΔR:R-fkbR mutant complemented with fkbR, ΔRN:N-fkbR, fkbN double mutant complemented only with fkbN, ΔN:N-fkbN mutant complemented with fkbN)].

Figure 4

Promoter activity represented as expression of the reporter gene rppA in S. tsukubaensis wild type and mutant strains (light gray – WT, dark gray – ΔfkbR, white – ΔfkbN). The ΔA values represent the difference in absorbance at 270 nm, between the sample with an active promoter and the sample derived from the same mutant strain which was transformed by a promoterless plasmid (blank).

Figure 5

(A) Time course for FK506 production in the SPM2 medium. (B) Gene expression analysis by RT-PCR. Results of transcript analysis from three strains are presented WT-wild type, ΔR-fkbR inactivated, ΔN-fkbN inactivated. Total mycelial RNA was extracted after 36, 72 and 103 hours of fermentation.