Improved pectinase production in Penicillium griseoroseum recombinant strains

Abstract

Aims: To obtain recombinant strains of Penicillium griseoroseum that produce high levels of pectin lyase (PL) and polygalacturonase (PG) simultaneously. Methods and Results: A strain with high production of PL was transformed with the plasmid pAN52pgg2, containing the gene encoding PG of P. griseoroseum, under control of the gpd promoter gene from Aspergillus nidulans. Southern blot analysis demonstrated that all strain had at least one copy of pAN52pgg2 integrated into the genome. The recombinant strain P. griseoroseum T20 produced levels of PL and PG that were 266‐ and 27‐fold greater, respectively, than the wild‐type strain. Furthermore, the extracellular protein profile of recombinant T20 showed two protein bands of c. 36 and 38 kDa, associated with PL and PG, respectively. Conclusions: This recombinant strain T20 produces PL and PG using carbon sources of low costs, and an enzyme preparation that is free of cellulolytic and proteolytic activities. Significance and Impact of the Study: PL and PG play an important role in the degradation of pectin. Owing to their use in the juice and wines industries, there is a growing interest in the inexpensive production of these enzymes. This work describes an efficient system of protein expression and secretion using the fungus P. griseoroseum.

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

“…evaluation of plg1 and pgg2 gene expression in double-recombinant strain P. griseoroseum t20 the expression of the plg1 and pgg2 genes was analyzed to determine whether there were differences in expression due to the position in the genome or the number of copies. for this purpose, the double-recombinant strain t20 was selected because it has a single copy of the plg1 gene and two copies of the pgg2 gene, both of which are controlled by the gpd gene promoter from A. nidulans with different ectopic integration [31]. the strain P. griseoroseum PG63 was used as a control strain for the endogenous expression of the plg1 and pgg2 genes.…”

“…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].…”

“…Dna extraction and southern blot analysis for karyotype analysis using the telomeric regions of P. griseoroseum, total Dna was extracted following specht et al [29] with modifications [31]. the total Dna was cleaved with the restriction enzymes BamHI, EcorI, and SmaI, and the restriction fragments were separated by electrophoresis in a 0.8 % agarose gel.…”

“…the cDna synthesis was performed from 1.0 μg total rna using the Im Prom-II™ reverse transcription system (Promega) kit according to the manufacturer's instructions. to quantify plg1 and pgg2 gene expression, the cDna samples obtained from the PG63 and recombinant t20 strains of P. griseoroseum [31] were used. the γ-actin gene from P. griseoroseum was used as an endogenous control.…”

“…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].…”