Development of a transformation system for Penicillium brevicompactum based on the Fusarium oxysporum nitrate reductase gene

Abstract

Penicillium brevicompactum is a filamentous fungus that presents a potential for industrial use due its efficient pectinase production. A heterologous transformation system was developed for P. brevicompactum based on the complementation of a nitrate reductase mutant. Nitrate reductase mutants were obtained by resistance to chlorate in a rate of 23.24% when compared to other mutations that lead to the chlorate resistance. One mutant named 4457-18X was chosen for the transformation experiments with the pNH24 vector containing de Fusarium oxysporum nitrate reductase gene. A frequency of approximately 3 transformants/µg DNA was obtained using the circular vector pNH24. This frequency was multiplied about 10 fold using the linearized vector with the Xba I restriction enzyme. Southern analysis of the transformants showed a tendency of the linearized vector to diminish the number of integrations compared to the use of the circular vector. The integration was random and stable in the analyzed transformants. The establishment of a transformation system for P. brevicompactum is fundamental for genetic manipulation of this microorganism.

Altered nitrogen metabolism in biocontrol strains of Penicillium rubens

“…This is the first time a mutation has been described in the NirA coding gene in P. rubens. Mutations in nirA have been isolated in P. chrysogenum (Birkett and Rowlands, 1981), and Penicillium brevicompactum (Varavallo et al, 2005), based on tolerance to chlorate. Also, nirA mutants which failed to grow in nitrate and nitrite were isolated from Penicillium griseoroseum and transformed with the nirA gene of (Pereira et al, 2004).…”

“…Impairment of CH8 to utilize nitrate or nitrite can be explained by the fact that crnA1 mutation must abolish the principal transport mechanism for these nitrogen sources. Mutations in crnA have been isolated in P. brevicompactum, but they were not analyzed (Varavallo et al, 2005). The crnA1 mutation causes the loss of the last two transmembrane domains of the 12 that are predicted in that transporter.…”

Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum

“…To improve the transformation efficiency in P. brevicompactum, we developed a reliable procedure based on the use of protoplasts. For this purpose, we tested two buffers: potassium phosphate [14] and 0.9 M sodium chloride [25] for CBS 257.29 and CBS 110068. Based on our results, we concluded that 0.9 M sodium chloride containing 40 mg/mL Vinotaste®Pro digestive enzyme was the only effective buffer for protoplasting of P. brevicompactum strains.…”

“…Because of the limited number of selection markers, few studies have demonstrated genetic engineering of P. brevicompactum. In one report, the nitrate reductase gene from Fusarium oxysporum was used as a selection maker in an appropriate nitrate reductase-deficient recipient [14]. Successful A. tumefaciens-mediated transformation of P. brevicompactum has been described, as well, in which hph was used as a selectable marker under the control of the gpdA promoter from Aspergillus nidulans [15].…”