Characterisation of a γ-butyrolactone receptor of Streptomyces tacrolimicus: effect on sporulation and tacrolimus biosynthesis

Abstract

Streptomyces tacrolimicus (ATCC 55098) was reported to produce the immunosuppressant tacrolimus. The wild-type strain sporulates sparsely and produces very low levels of this immunosuppressant. The lack of genetic knowledge of this strain has hampered strain improvement. In this work, we have cloned the gene encoding a γ-butyrolactone receptor protein (Gbr). The gbr gene is linked to two genes encoding two subunits of the dihydroxyacetone kinase, putatively involved in the biosynthesis of the dihydroxyacetone phosphate precursor of γ-butyrolactone but is not flanked by γ-butyrolactone synthetase genes. The Gbr protein was overexpressed in Escherichia coli and purified. Electrophoretic mobility shift assays showed that Gbr binds to a specific autoregulatory element sequence located 338 bp upstream of the gbr gene, indicating that its expression is self-regulated. The deletion mutant Δgbr showed a very early and intense sporulation in two different media. A phenotype similar to that of the wild-type strain was restored by complementation of the Δgbr mutant with a wild-type gbr allele. Duplication of the gbr gene resulted in a slower sporulation. The Δgbr mutant produced much lower amount (32%) of tacrolimus quantified by high performance liquid chromatography. This analysis, using an optimised system, allowed the resolution of tacrolimus from ascomycin and other contaminant metabolites. Our results indicate that the Gbr protein regulates negatively the sporulation and positively the production of tacrolimus.

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

“…These two gene clusters contain several additional CDSs, located in the “ all” group of genes involved in biosynthesis of allylmalonyl-CoA extender unit, when comparing them to the second group of gene clusters from Streptomyces tacrolimicus (formerly Streptomyces sp . ATCC 55098 [53,54]) and S. kanamyceticus KCTC 9225 [11,12]. Gene clusters of all published FK506-producing strains contain an fkbN regulatory gene homologue, but only the larger version of gene clusters from S. tsukubaensis NRRL 18488 and Streptomyces sp .…”

SbbR/SbbA, an Important ArpA/AfsA-Like System, Regulates Milbemycin Production in Streptomyces bingchenggensis

“…sbbR encodes a TetR (Tetracycline Repressor) family regulator that shows similarity to GBL receptors such as Gbr from Streptomyces tacrolimicus (53% identity) ( Salehi-Najafabadi et al, 2011 ), AvaR1 from Streptomyces avermitilis (31% identity) ( Kitani et al, 2011 ), and JadR3 from S. venezuelae (30% identity) ( Zou et al, 2014 ). sbbA , which is located upstream of the oppositely oriented sbbR gene, encodes a putative GBL biosynthesis protein that shows 32% identity with FarX from S. lavendulae ( Waki et al, 1997 ), 31% identity with SrrX from Streptomyces rochei ( Arakawa et al, 2007 ), 27% identity with AfsA from S. griseus ( Khokhlov et al, 1967 ), and 25% identity with JadW1 from S. venezuelae ( Zou et al, 2014 ).…”