In recent years, many secondary metabolite gene clusters, including polyketide biosynthetic genes, have been uncovered through fungal genome sequencing. Although approximately 33 polyketide biosynthetic genes can be identified in the genome of a fungus Chaetomium globosum, only 11 fungal polyketide scaffolds1 can be isolated from cultures grown under typical growth conditions.2 Transcription factors (TFs) located within a gene cluster often determine whether its respective secondary metabolites can be biosynthesized, and it is this class of regulators that is responsible for the low expression level of biosynthetic genes and less-than-desirable yield of the corresponding natural products.3, 4 Here, we explored the feasibility of activating a silent gene cluster through overexpression of a TF encoded in a putative polyketide biosynthetic gene cluster in C. globosum for which a stably propagating expression vector is unavailable.

Next, we examined whether transcription of cgsA encoding for a PKS can be activated when the transcriptional regulator CgsG is overexpressed. Total RNA (1 μg) isolated from CGKW1 was subjected to reverse transcription-PCR for cgsA cDNA synthesis with a random hexamer primer (Applied Biosystems, Life Technologies). The cDNA formation was detected by agarose gel electrophoresis (Figure 1b, cgsA) following PCR amplification of cDNA using the following primer set: CHGG8793coreFw, 5′-TCCATGGATCCCCAGCAGAGG-3′ and CHGG8793coreRv, 5′-ACCCGTCCCATGAGCCTCCAC-3′. As a negative control, the absence of transcription of cgsA was confirmed in C. globosum transformed with pKW12203 lacking the cgsG insertion (Figure 1c, WT). Transcription of the β-tubulin gene was used as a positive control to confirm that general gene transcription remained similar in both the wild-type and the CGKW1 strain (Figure 1c, β-tubulin). Similar investigations revealed successful activation of cgsB and cgsF genes upon overexpression of an extraneous copy of cgsG (Figure 1c, cgsB and cgsF).

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