Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum

Abstract

The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml(-1) respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.

Biosynthesis of Industrially Relevant Extracellular Pectinase from Aspergillus terreus NCFT 4269.10 Using Orange Peels as Substrate

“…Remarkably, they have the potentiality for best suited in different fermentation processes, biosynthesis of bulk amount of commercially significant enzymes, cultivation possibilities using cheapest substrates and low-cost-high valued biosynthesis in large bioreactors (De Vries and Visser, 2001;Aro et al, 2005). Among many filamentous fungi, most exploited fungal genera for the biosynthesis of polymer-degrading enzymes are Trichoderma and a variety of strains of Aspergillus and Penicillium (Aro et al, 2005;Cardoso et al, 2008). Furthermore, the biosynthesis of microbial pectinase by fungi is solely dependent on the type of strain and substrate implemented for fermentation, growth conditions like pH, temperature, incubation period, methods of cultivation regimen, cell growth pattern, requirement of nutrients and minimization of cross-contamination during the entire fermentation.…”

Genome organization and assessment of high copy number and increased expression of pectinolytic genes from Penicillium griseoroseum: a potential heterologous system for protein production

“…since both genes are under control of the same regulatory region and responds to the same transcriptional factors. It has been reported that the variation in gene expression level for multi-copy strains may be due to the effect of genome position and not to the control of the regulatory promoter region [9,11]. this finding may explain the reduction in Pl and PG activities that was primarily observed for strains t09, t3.4, and t143 ( fig.…”

“…the recombinant strains (t) that overproduce Pl, PG, or Pl and PG were obtained by araújo et al [3], cardoso et al [9] and teixeira et al [31]. these recombinant strains were previously characterized and showed ectopic integration of the expression vectors in the genome [9,31,32]. the mutant strain P. griseoroseum PG63 niaD − has a 122-bp deletion in the niaD gene, which encodes nitrate reductase [23].…”

“…recombinant strains of P. griseoroseum with increased Pl and PG production have been obtained using integrative expression vectors containing the target gene under the control of a strong constitutive promoter. these recombinant strains have additional copies of the plg1 and pgg2 genes that are under the control of a strong constitutive promoter for the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) and the termination region of the gene encoding tryptophan synthase (trpc) from Aspergillus nidulans [3,4,9,31,32]. the recombinant strains of P. griseoroseum exhibited a 15-fold increase in the enzymatic activity of PG and a 132-fold increase in Pl activity compared with the wild-type strain when cultured in sucrose or sugar cane juice [3,9,31,32].…”

Biotechnological potential of pectinolytic complexes of fungi

“…several grams per liter in strains of Aspergillus), the feasibility of cultivation, and the low-cost of production in large bioreactors (de Vries and Visser 2001;Aro et al 2005). The filamentous fungi most frequently used for the production of polymer-degrading enzymes are Trichoderma reesei and a number of strains of Aspergillus and Penicillium (Aro et al 2005;Cardoso et al 2007). Here we emphasize the biotechnological importance of pectinolytic enzymes produced by filamentous fungi and the strategies for their application and improvement of efficiency in the food industry.…”