Molecular characterization and expression profile of pectin-lyase-encoding genes fromPenicillium griseoroseum

Abstract

Penicillium griseoroseum has been studied by our group because of its good pectinase production. Attempts have been done to clone pectinolytic genes, aiming to obtain pectinase-overproducing strains for industrial purposes. Here, two genes coding for pectin lyase were isolated from the P. griseoroseum genome. The plg1 gene has an open reading frame of 1341 bp coding for a putative protein of 374 amino acids with a calculated molecular mass of 40.1 kDa. The plg2 gene is characterized by an open reading frame of 1400 nucleotides and codes for a polypeptide of 383 amino acids. The plg1 gene 5'-flanking region contains putative binding sites for the transcription factors involved in regulation by ambient pH and catabolite repression. The primary structure of Plg1 and Plg2 proteins showed a relatively high homology (varying between 32.4% and 74.8%) to fungal pectin lyases characterized to date. Southern blotting analysis revealed that both genes are present as single copies in the fungus genome. Expression studies revealed a differing pattern of gene expression of plg1 and plg2 when mycelium was cultivated on medium containing different pectic components. Citric pectin followed by apple pectin were the carbon sources that best induced plg1 expression, and transcripts were detected from 24 to 76 h. The expression of the plg2 gene was monitored by reverse transcriptase - polymerase chain reaction, since Northern analysis failed to detect hybridization signals. The differential expression of these genes may provide means for the fungus to adapt to various growth conditions.

Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum

“…Furthermore, it oVers the advantage of producing great amounts of PL using sugar cane juice as the sole carbon source, which would lower production costs. Moreover, the PL enzyme has an estimated pI (9.46) higher than those reported for the Aspergillus PLs (varying from 3.65 to 5.9) [6,23], which, in association with its working pH, renders great industrial value to this enzyme. Further experiments will be carried out in order to optimize the growth conditions of this strain for scaling up PL production in bench bioreactors.…”

“…4). Total extracellular proteins [6]. On the other hand, the commercial pectinase preparation showed several bands of moderate intensity, none of them presenting the same size of the putative pectin lyase produced by strain 105.…”

“…P. griseoroseum niaD mutant (PG63) and plasmid pNPG1, containing the nitrate reductase (niaD) gene of P. griseoroseum as selective marker were developed by Pereira et al [31]. Plasmid pPlg1, containing the complete sequence of plg1 gene from P. griseoroseum was constructed by Bazzolli et al [6]. Plasmid pAN52-1-GFP carrying the SGFP-TYG version of the green Xuorescent protein (GFP) under control of the A. nidulans gpdA promoter was kindly provided by Dr. Corinne Clavé, of the Institut de Biochimie et de Genetique Cellulaires, Bordeaux, France.…”

“…Penicillium griseoroseum CCT6421 has proven to be a good pectinase producer [4]. Multiple forms of pectin lyases are produced by Wlamentous fungi and several pectin lyase genes have been isolated and characterized, which constitute gene families in many fungal species, such as, Aspergillus niger [14,15,23,24], Aspergillus oryzae [20,21], and P. griseoroseum [6]. Two pectin lyase-encoding genes (plg1 and plg2) from P. griseoroseum CCT6421 were isolated, characterized, and their expression regulation evaluated by northern hybridization and RT-PCR analyzes.…”

“…Two pectin lyase-encoding genes (plg1 and plg2) from P. griseoroseum CCT6421 were isolated, characterized, and their expression regulation evaluated by northern hybridization and RT-PCR analyzes. Both genes are regulated at transcriptional level, being induced by pectin and substantially repressed by glucose [6]. The requisite of induction by pectin for the expression of PL genes constitutes a major limitation to the enzymatic production process at industrial level due to the high cost of this substrate.…”