Molecular Cloning And Differential Expression Of The Global Regulator Pci-laeA In Penicillium Citrinum

Mevastatin (compactin or ML-236B), a potent and competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, is a polyketide compound produced by Penicillium citrinum and has been used as a substrate for microbial conversion to pravastatin. Pravastatin has been widely used as a pharmaceutical product for the therapy of hypercholesterolemia. biosynthesis in P. citrinum has been identified. Among them, mlcR is a pathway-specific zinc cluster transcriptional activator of the mevastatin biosynthetic genes. However, the global regulators of mevastatin biosynthesis in P. citrinum have not been identified.In filamentous fungi, LaeA is a major global regulator for secondary etmabolism. It was a first discovered in Aspergillus nidulans. LaeA regulated the expressions of multiple secondary metabolism gene clusters and production of secondary metabolites, such as sterigmatocysin,aflatoxin,lovastatin,penicillin and gliotoxin. Because it was widely existed in filamentous fungi and its conservative function, laeA would to be a target for strain improvement and the first antimicrobial target specific to pathogenic filamentous fungi.Therefore, it is important to determine the role of LaeA in the secondary metabolism of mevastatin producer P. citrinum. In this work, we identified and characterized a P. citrinum laeA (Pci-laeA) gene encoding a protein very similar to that of Aspergillus nidulans, Aspergillus fumigatus and Penicillium chrysogenum, Based on the sequences of several known LaeA proteins deposited in the NCBI database, two degenerate primers D1 and D2 were synthesized. Using degenerate primers D1 and D2, a 782-bp cDNA fragment was obtained from P. citrinum by RT-PCR (Fig.1). The nucleotide sequence of the internal fragment of laeA cDNA showed high similarity to that of A. nidulans, A. fumigatus and P. chrysogenum. By 3' RACE technique, we obtained a 3'end fragment of P. citrinum laeA. The fragment was 1136bp in length (Fig. 2). Moreover, we also obtained a 5'-flanking fragment of 981bp (Fig.3) by TAIL-PCR using a primer set (the degenerate arbitrary primer AD1 and nested gene-specific primers R1-3). The nucleotide sequence of the 5'-flanking fragment contained the expected overlapping sequence with the middle fragment.Assembly of the internal and flanking fragments was carried out to form the full-length sequence of laeA gene by the SeqMan program of DNAstar software. Based on the full-length sequence of laeA, we designed a pair of primers:LAF1 and LAR1 for cloning of the coding region of laeA DNA and cDNA. We obtained a genomic DNA fragment encoding a 1340-bp ORF (Fig.4), which was identified to be the P. citrinum laeA(Pci-laeA) gene.. We obtained the Pci-laeA cDNA of 1284bp (Fig.4). An examination of P. citrinum laeA genomic and cDNA sequences revealed that P. citrinum laeA gene had a 56-bp intron spanning nucleotides 390-445 of the Pci-laeA gDNA.Pci-laeA cDNA encoded a protein of 427 amino acids. Alignment of the amino acid sequences of Pci-laeA revealed a conserved S-adenosylmethionine (SAM) binding site demonstrating that LaeA was a putative methyltransferase. The results of sequences homology analysis indicated that Pci-LaeA showed 95%,61%and 60% identity with LaeA protein of P. chrysogenum, A. fumigatus and A. nidulans.The expression difference of Pci-laeA gene in different growth stages of P. citrinum was evaluated by semi-quantitative RT-PCR using pgk as inner control. The optimized PCR cycle numbers of pgk and Pci-laeA were 28,30, respectively. The semi-quantitative RT-PCR analysis was performed with samples taken after 24h,96h, 120, and 168h from cultures of P. citrinum grown in mevastatin production medium. The result indicated that the expression levels of Pci-laeA gene were almost constitutively throughout different fermentation stages, with an increase in the later stages. The change trend of Pci-laeA gene transcription is consistent with that of mevastatin production in P. citrinum.The discovery of Pci-laeA will provide clues for understanding the regulatory mechanism in mevastatin biosynthesis and offer a potential target for strain improvement.