Characterization of a novel 2,4,6-trichlorophenol-inducible gene encoding chlorophenol O-methyltransferase from Trichoderma longibrachiatum responsible for the formation of chloroanisoles and detoxification of chlorophenols.

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.

The global regulator LaeA controls penicillin biosynthesis, pigmentation and sporulation, but not roquefortine C synthesis in Penicillium chrysogenum.

The biosynthesis of the beta-lactam antibiotic penicillin is an excellent model for the study of secondary metabolites produced by filamentous fungi due to the good background knowledge on the biochemistry and molecular genetics of the beta-lactam producing microorganisms. The three genes (pcbAB, pcbC, penDE) encoding enzymes of the penicillin pathway in Penicillium chrysogenum are clustered, but no penicillin pathway-specific regulators have been found in the genome region that contains the penicillin gene cluster. The biosynthesis of this beta-lactam is controlled by global regulators of secondary metabolism rather than by a pathway-specific regulator. In this work we have identified the gene encoding the secondary metabolism global regulator LaeA in P. chrysogenum (PcLaeA), a nuclear protein with a methyltransferase domain. The PclaeA gene is present as a single copy in the genome of low and high-penicillin producing strains and is not located in the 56.8-kb amplified region occurring in high-penicillin producing strains. Overexpression of the PclaeA gene gave rise to a 25% increase in penicillin production. PclaeA knock-down mutants exhibited drastically reduced levels of penicillin gene expression and antibiotic production and showed pigmentation and sporulation defects, but the levels of roquefortine C produced and the expression of the dmaW involved in roquefortine biosynthesis remained similar to those observed in the wild-type parental strain. The lack of effect on the synthesis of roquefortine is probably related to the chromatin arrangement in the low expression roquefortine promoters as compared to the bidirectional pbcAB-pcbC promoter region involved in penicillin biosynthesis. These results evidence that PcLaeA not only controls some secondary metabolism gene clusters, but also asexual differentiation in P. chrysogenum.

RNA-silencing in Penicillium chrysogenum and Acremonium chrysogenum: validation studies using beta-lactam genes expression.

In this work we report the development and validation of a new RNA interference vector (pJL43-RNAi) containing a double-stranded RNA expression cassette for gene silencing in the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum. Classical targeted gene disruption in these fungi is very laborious and inefficient due to the low frequency of homologous recombination. The RNAi vector has been validated by testing the attenuation of two different genes of the beta-lactam pathway; pcbC in P. chrysogenum and cefEF in A. chrysogenum. Quantification of mRNA transcript levels and antibiotic production showed knockdown of pcbC and cefEF genes in randomly isolated transformants of P. chrysogenum and A. chrysogenum, respectively. The process is efficient; 15 to 20% of the selected transformants were found to be knockdown mutants showing reduced penicillin or cephalosporin production. This new RNAi vector opens the way for exploring gene function in the genomes of P. chrysogenum and A. chrysogenum.

Post-translational enzyme modification by the phosphopantetheinyl transferase is required for lysine and penicillin biosynthesis but not for roquefortine or fatty acid formation in Penicillium chrysogenum.

NRPSs (non-ribosomal peptide synthetases) and PKSs (polyketide synthases) require post-translational phosphopantetheinylation to become active. This reaction is catalysed by a PPTase (4'-phosphopantetheinyl transferase). The ppt gene of Penicillium chrysogenum, encoding a protein that shares 50% similarity with the stand-alone large PPTases, has been cloned. This gene is present as a single copy in the genome of the wild-type and high-penicillin-producing strains (containing multiple copies of the penicillin gene cluster). Amplification of the ppt gene produced increases in isopenicillin N and benzylpenicillin biosynthesis. A PPTase-defective mutant (Wis54-PPT(-)) was obtained. It required lysine and lacked pigment and penicillin production, but it still synthesized normal levels of roquefortine. The biosynthesis of roquefortine does not appear to involve PPTase-mediated modification of the synthesizing enzymes. The PPT(-) mutant did not require fatty acids, which indicates that activation of the fatty acid synthase is performed by a different PPTase. Complementation of Wis54-PPT(-) with the ppt gene restored lysine biosynthesis, pigmentation and penicillin production, which demonstrates the wide range of processes controlled by this gene.