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

  • Applied Genetics and Molecular Biotechnology
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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.

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Acknowledgments

This work was supported by a grant from the CICYT Ministry of Innovation and Science (Madrid, Spain) [BIO2006-14853-C02-01 (CONSOLIDER)]. C. Barreiro was supported by the Program ‘Personal Técnico de Apoyo’ of the Ministry of Science and Innovation of Spain and the European Social Fund (ESF) (PTA-2003-01-00689). M. Martínez-Castro received a PFU fellowship from the Ministry of Education and Science and E. Solera received a PFI fellowship from the Ministry of Education and Science. We acknowledge the kind technical assistance of A. Rodríguez-García and S. Albillos and the excellent technical support of B. Martín, J. Merino, A. Casenave and A. Mulero (INBIOTEC).

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Authors and Affiliations

  1. Área de Microbiología, Departamento de Biología Molecular, Facultad de CC. Biológicas y Ambientales, Universidad de León, Campus de Vegazana s/n, 24071, León, Spain

    Zahra Salehi-Najafabadi, Miriam Martínez-Castro & Juan F. Martín

  2. Instituto de Biotecnología de León (INBIOTEC), Parque Científico de León, Avda. Real, no. 1, 24006, León, Spain

    Carlos Barreiro, Elena Solera & Juan F. Martín

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  1. Zahra Salehi-Najafabadi
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  2. Carlos Barreiro
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  3. Miriam Martínez-Castro
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  4. Elena Solera
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  5. Juan F. Martín
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Correspondence to Juan F. Martín.

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Supplementary Figure S1

Degenerated primers used for amplification of the γ-butyrolactone receptor gene of S. tacrolimicus. a Alignment of nucleotide sequences of γ-butyrolactone receptors from different strains. Star symbols indicate identical nucleotides. Conserved regions are indicated by black boxes. The nucleotide regions which are identity-conserved (70–80%) are indicated by grey boxes. Arrows represent the regions used to design primers. Γ-Butyrolactone receptors which were used for alignment are farA of S. lavendulae, sngR of S. natalensis, scbR of S. coelicolor A3(2), barA of S. virginiae, tarA of S. tendae, srrA of S. rochei, brp of S. clavuligerus, avaR of S. avermitilis, spbR of S. pristinaespiralis and tylP of S. fradiae. b PCR products obtained using degenerated primers GbrE-1 and GbrE-3. The arrowhead shows the 150-bp band corresponding to the gbr gene (PDF 95 kb)

Supplementary Figure S2

Secondary structure of transcriptional terminators present in the 8,722-bp region surrounding the gbr gene based on the ΔG value (Unniraman et al. 2002). DHK2 is an abbreviation of ‘dihydroxyacetone kinase subunit 2’ (PDF 37 kb)

Supplementary Figure S3

Expression of the GST–Gbr fusion protein at different concentrations of LB. a Decrease of LB concentration until 4 g l−1 results in a larger amount of soluble GST–Gbr fusion protein. M Precision Plus Protein standard (Bio-Rad), S soluble phase, P pellet phase. b SDS-PAGE analysis of different fractions of purified GST–Gbr fusion protein after affinity chromatography in comparison with crude extract of the cells without IPTG (negative control) and crude extract of the cells containing IPTG (positive control). M Precision Plus Protein standard, A expressed protein in the absence of IPTG, B expressed proteins before elution using FPLC. The arrowhead indicates the band of the protein (PDF 366 kb)

Supplementary Figure S4

Lack of binding of purified Gbr protein to the GbrE-2 region. Electrophoretic mobility shift assays was done using the promoter region named GbrE-2, which includes the A–T-rich region located 70 bp upstream of the translation start codon of gbr (see Fig. 2) (PDF 53 kb)

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Salehi-Najafabadi, Z., Barreiro, C., Martínez-Castro, M. et al. Characterisation of a γ-butyrolactone receptor of Streptomyces tacrolimicus: effect on sporulation and tacrolimus biosynthesis. Appl Microbiol Biotechnol 92, 971–984 (2011). https://doi.org/10.1007/s00253-011-3466-1

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