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Wu X.,Institute Biotecnologia INBIOTEC | Garcia-Estrada C.,Institute Biotecnologia INBIOTEC | Vaca I.,Institute Biotecnologia INBIOTEC | Vaca I.,University of Chile | And 2 more authors.
Biochimie | Year: 2012

The first step in the penicillin biosynthetic pathway is the non-ribosomal condensation of l-α-aminoadipic acid, l-cysteine and l-valine into the tripeptide δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV). This reaction is catalysed by the multienzyme ACV synthetase (ACVS), which is encoded in the filamentous fungus Penicillium chrysogenum by the pcbAB gene. This enzyme contains at least ten catalytic domains. The precise role of the C-terminal domain of this multidomain NRPS still remains obscure. The C-terminal region of ACVS bears the epimerase and the thioesterase domains and may be involved in the epimerization of LLL-ACV to LLD-ACV and in the hydrolysis of the thioester bond. In this work, the conserved motifs 3371EGHGRE 3376 (located in the putative epimerase domain) and 3629GWSFG 3633 (located in the thioesterase domain) were changed by site-directed-mutagenesis to LGFGLL and GWAFG, respectively. In addition, the whole thioesterase domain (230 amino acids) and the different parts of this domain were deleted. The activity of these mutant enzymes was assessed in vivo by two different procedures: i) through the quantification of bisACV produced by the fungus and ii) by quantifying the benzylpenicillin production using tailored strains of P. chrysogenum, which lack the pcbAB gene, as host strains. All indicated mutant enzymes showed lower or null activity than the control strain confirming that E3371, H3373, R3375 and E3376 belong to the epimerase active centre. Different fragments included in the C-terminal region of ACVS control thioester hydrolysis. Overexpression of the sequence encoding the ACVS integrated thioesterase domain as a separate (stand-alone) transcriptional unit complemented mutants lacking the integrated thioesterase domain, although with low ACV releasing activity, suggesting that the stand-alone thioesterease interacts with the other ACVS domains. © 2011 Elsevier Masson SAS. All rights reserved.

Vicente C.M.,University of Leon | Vicente C.M.,Institute Biotecnologia INBIOTEC | Santos-Aberturas J.,University of Leon | Santos-Aberturas J.,Institute Biotecnologia INBIOTEC | And 5 more authors.
Applied Microbiology and Biotechnology | Year: 2014

The DNA region encoding the filipin gene cluster in Streptomyces avermitilis (pte) contains a PAS-LuxR regulatory gene, pteF, orthologue to pimM, the final pathway-specific positive regulatory protein of pimaricin biosynthesis in Streptomyces natalensis. Gene replacement of the gene from S. avermitilis chromosome resulted in a severe loss of filipin production and delayed spore formation in comparison to that of the wild-type strain, suggesting that it acts as a positive regulator of filipin biosynthesis and that it may also have a role in sporulation. Complementation of the mutant with a single copy of the gene integrated into the chromosome restored wild-type phenotypes. Heterologous complementation with the regulatory counterpart from S. natalensis also restored parental phenotypes. Gene expression analyses in S. avermitilis wild-type and the mutant by reverse transcription-quantitative polymerase chain reaction of the filipin gene cluster suggested the targets for the regulatory protein. Transcription start points of all the genes of the cluster were studied by 5′-rapid amplification of complementary DNA ends. Transcription start point analysis of the pteF gene revealed that the annotated sequence in the databases is incorrect. Confirmation of target promoters was performed by in silico search of binding sites among identified promoters and the binding of the orthologous regulator for pimaricin biosynthesis PimM to gene promoters by electrophoretic mobility shift assays. Precise binding regions were investigated by DNAse I protection studies. Our results indicate that PteF activates the transcription from two promoters of polyketide synthase genes directly, and indirectly of other genes of the cluster. © 2014, Springer-Verlag Berlin Heidelberg.

Cepeda-Garcia C.,University of Leon | Cepeda-Garcia C.,Centro Andaluz Of Biologia Molecular Y Medicina Regenerativa Cabimer | Dominguez-Santos R.,University of Leon | Dominguez-Santos R.,Institute Biotecnologia INBIOTEC | And 7 more authors.
Applied Microbiology and Biotechnology | Year: 2014

The transcription factor CreA is the main regulator responsible for carbon repression in filamentous fungi. CreA is a wide domain regulator that binds to regulatory elements in the promoters of target genes to repress their transcription. Penicillin biosynthesis and the expression of penicillin biosynthetic genes are subject to carbon repression. However, evidence of the participation of CreA in this regulation is still lacking, and previous studies on the promoter of the pcbC gene of Aspergillus nidulans indicated the lack of involvement of CreA in its regulation. Here we present clear evidence of the participation of CreA in carbon repression of penicillin biosynthesis and expression of the pcbAB gene, encoding the first enzyme of the pathway, in Penicillium chrysogenum. Mutations in cis of some of the putative CreA binding sites present in the pcbAB gene promoter fused to a reporter gene caused an important increase in the measured enzyme activity in glucose-containing medium, whereas activity in the medium with lactose was not affected. An RNAi strategy was used to attenuate the expression of the creA gene. Transformants expressing a small interfering RNA for creA showed higher penicillin production, and this increase was more evident when glucose was used as carbon source. These results confirm that CreA plays an important role in the regulation of penicillin biosynthesis in P. chrysogenum and opens the possibility of its utilization to improve the industrial production of this antibiotic. © 2014 Springer-Verlag.

Feltrer R.,Institute Biotecnologia INBIOTEC | Alvarez-Rodriguez M.L.,University of Leon | Barreiro C.,Institute Biotecnologia INBIOTEC | Godio R.P.,Institute Biotecnologia INBIOTEC | Coque J.-J.R.,University of Leon
Fungal Genetics and Biology | Year: 2010

De novo sequencing of eight internal peptides of purified chlorophenol O-methyltransferase, or CMT1 (before named as CPOMT), from Trichoderma longibrachiatum was performed by MALDI-TOF/TOF and ESI-IT. A novel gene (cmt1) encoding CMT1 was cloned by using a PCR approach based on the amino acid sequence of two internal peptides. The gene (1637. bp) encoded a protein of 468 amino acids with a deduced molecular mass of 52.4. kDa, and a theoretical isoelectric point of 5.93. This gene contains four introns, whose location was confirmed by comparison of cDNA and chromosomal sequences. The expression of cmt1 gene was induced at transcriptional level by exposure of fungal mycelia to 2,4,6-trichlorophenol (2,4,6-TCP). Putative homologous genes were detected in many different fungal strains, including other Trichoderma species. Partial silencing of cmt1 gene resulted in a 48.9% (±5.2) decrease of CMT1 activity levels in a T. longibrachiatum At37 transformant strain by comparison with the wild type, whereas a decrease of up to 53.0% was observed in the levels of 2,4,6-TCA produced in liquid cultures. Efficient expression of cmt1 gene in Escherichia coli unequivocally confirmed that it encodes a CMT1 enzyme. © 2010 Elsevier Inc.

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