The AbcCl1 transporter of Colletotrichum lindemuthianum acts as a virulence factor involved in fungal detoxification during common bean (Phaseolus vulgaris) infection.

Anthracnose, caused by Colletotrichum lindemuthianum, is a disease affecting the common bean plant, Phaseolus vulgaris. To establish infection, the phytopathogen must survive the toxic compounds (phytoanticipins and phytoalexins) that are produced by the plant as a defense mechanism. To study the detoxification and efflux mechanisms in C. lindemuthianum, the abcCl1 gene, which encodes an ABC transporter, was analyzed. The abcCl1 gene (4558 pb) was predicted to encode a 1450-amino acid protein. Structural analysis of 11 genome sequences from Colletotrichum spp. showed that the number of ABC transporters varied from 34 to 64. AbcCl1 was classified in the ABC-G family of transporters, and it appears to be orthologs to ABC1 from Magnaporthe grisea and FcABC1 from Fusarium culmorum, which are involved in pleiotropic drug resistance. A abcT3 (ΔabcCl1) strain showed reduction on aggressivity when inoculated on bean leaves that presented diminishing anthracnose symptoms, which suggests the important role of AbcCl1 as a virulence factor and in fungal resistance to host compounds. The expression of abcCl1 increased in response to different toxic compounds, such as eugenol, hygromycin, and pisatin phytoalexin. Together, these results suggest that AbcCl1 is involved in fungal resistance to the toxic compounds produced by plants or antagonistic microorganisms.

High-affinity transport, cyanide-resistant respiration, and ethanol production under aerobiosis underlying efficient high glycerol consumption by Wickerhamomyces anomalus.

Wickerhamomyces anomalus strain LBCM1105 was originally isolated from the wort of cachaça (the Brazilian fermented sugarcane juice-derived Brazilian spirit) and has been shown to grow exceptionally well at high amounts of glycerol. This paramount residue from the biodiesel industry is a promising cheap carbon source for yeast biotechnology. The assessment of the physiological traits underlying the W. anomalus glycerol consumption ability in opposition to Saccharomyces cerevisiae is presented. A new WaStl1 concentrative glycerol-H+ symporter with twice the affinity of S. cerevisiae was identified. As in this yeast, WaSTL1 is repressed by glucose and derepressed/induced by glycerol but much more highly expressed. Moreover, LBCM1105 aerobically growing on glycerol was found to produce ethanol, providing a redox escape to compensate the redox imbalance at the level of cyanide-resistant respiration (CRR) and glycerol 3P shuttle. This work is critical for understanding the utilization of glycerol by non-Saccharomyces yeasts being indispensable to consider their industrial application feeding on biodiesel residue.

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

The fungus Penicillium griseoroseum has the potential for application on an industrial scale as a host for the production of homologous and heterologous proteins, mainly because it does not produce some mycotoxins or secrete proteases under the growth conditions for pectinase production. However, for the fungus to be used effectively as an expression heterologous system, an understanding of the organization of its genome, as well as the mechanisms of gene expression and protein production, is required. In the present study, the size of the P. griseoroseum genome was estimated to be 29.8-31.5 Mb, distributed among four chromosomes. An analysis of plg1 and pgg2 pectinolytic genes expression and copy number in recombinant multi-copy strains of P. griseoroseum demonstrated that an increase in the number of gene copies could increase enzyme production, but the transcription could be affected by the gene integration position. Placing a copy of the plg1 gene under the control of the gpd promoter of Aspergillus nidulans yielded a 200-fold increase in transcription levels compared to the endogenous gene, and two copies of the pgg2 gene produced an 1100-fold increase compared with the endogenous gene. These results demonstrated that transcription, translation, and protein secretion in the fungus P. griseoroseum respond to an increased number of gene copies in the genome. The processing capacity and efficiency of protein secretion in P. griseoroseum are consistent with our premise that this fungus can be used for the industrial-scale production of several enzymes.

In vitro Scleroderma laeve and Eucalyptus grandis mycorrhization and analysis of atp6, 17S rDNA, and ras gene expression during ectomycorrhizal formation.

The interaction between fungi and plants that form ectomycorrhizae (ECM) promotes alterations in the gene expression profiles of both organisms. Fungal genes expression related to metabolism were evaluated at the pre-symbiotic stage and during the ECM development between Scleroderma laeve and Eucalyptus grandis. Partial sequences of ATP synthase (atp6), translation elongation factor (ef1α), the RAS protein (ras), and the 17S rDNA genes were isolated. The expression of the atp6 and 17S rDNA genes during the pre-symbiotic stage showed an approximately threefold increase compared to the control. During ECM development, the expression of the 17S rDNA gene showed a 4.4-fold increase after 3 days of contact, while the expression of the atp6 gene increased 7.23-fold by the 15th day, suggesting that protein synthesis and respiratory chain activities are increased during the formation of the mantle and the Hartig net. The ras gene transcripts were only detected by RT-PCR 30 days after fungus-plant contact, suggesting that RAS-mediated signal transduction pathways are functional during the establishment of symbiosis. The present study demonstrates that alterations in gene expression occur in response to stimuli released by the plant during ECM association and increases the understanding of the association between S. laeve and E. grandis.

pH-dependent effect of pectinase secretion in Penicillium griseoroseum recombinant strains.

A number of parameters, including culture medium pH, affect growth and enzyme production by microorganisms. In the present study, the production and secretion of pectin lyase (PL) and polygalacturonase (PG) by recombinant strains of Penicillium griseoroseum cultured in mineral-buffered media (MBM; initial pH 6.8) and mineral-unbuffered medium (MUM; initial pH 6.3) were evaluated. Under these culture conditions, no change in the transcriptional levels of plg1 and pgg2 was observed. However, the levels of secreted total protein ranged from 7.80 ± 1.1 to 3.25 ± 1.50 µg ml(-1) in MBM and MUM, respectively, and were evaluated by SDS-PAGE. PL and PG enzymatic activities decreased 6.4 and 3.6 times, respectively, when P. griseoroseum was cultivated under acidic pH conditions (MUM). Furthermore, differences were observed in the hypha and mycelium morphology. These findings suggest that acidic growing conditions affect PL and PG secretion, even though the transcription and translation processes are successful. The data obtained in this study will help to establish optimal culture conditions that increase production and secretion of recombinant proteins by filamentous fungi.

Overproduction of polygalacturonase by Penicillium griseoroseum recombinant strains and functional analysis by targeted disruption of the pgg2 gene.

Inactivation of the pgg2 gene, a polygalacturonase-encoding gene from Penicillium griseoroseum, reduced the total activity of polygalacturonase (PG) by 90 % in wild-type P. griseoroseum, which indicates that the pgg2 gene is the major gene responsible for PG production in this species. To increase PG production, the coding region of the pgg2 gene was cloned under the control of the glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter and the terminator region of the tryptophan synthase (trpC) gene from Aspergillus nidulans (pAN52pgg2 vector). This vector was then used to transform P. griseoroseum. The transformed strains were characterized according to PG production using glucose, sucrose, or sugar cane juice as the carbon sources. The recombinant P. griseoroseum T146 strain contained an additional copy of the pgg2 gene, which resulted in a 12-fold increase in PG activity when compared with that detected in the supernatant of the control PG63 strain. The proteins secreted by the recombinant strain T146 showed a strong band at 38 kDa, which corresponds to the molecular weight of PG of the P. griseoroseum. The results demonstrate the significant biotechnological potential of recombinant P. griseoroseum T146 for use in PG production.

Pectin lyase production by recombinant Penicillium griseoroseum strain 105.

Recombinant Penicillium griseoroseum strain 105 overproduces an extracellular pectin lyase (PL) under the transcriptional control of the strong gpdA promoter of Aspergillus nidulans. Our aim was to evaluate PL production by recombinant P. griseoroseum strain 105 in submerged fermentation system bioreactors BioFloIII and BioFloIV using 2 or 10 L working volumes under different growth conditions and to analyze the production of cellulase, polygalacturonase, pectin methylesterase, and protease. PL overproduction by recombinant P. griseoroseum strain 105 was 112 times higher than that of P. griseoroseum PG63 grown in sugarcane juice. Cellulases and proteases were not detected in the culture filtrate, and evaluation for extracellular proteins in the culture medium by SDS-PAGE showed the presence of a 36 kDa predominant band, similar to the molecular mass estimated from the nucleotide sequence of plg1 gene for PL of P. griseoroseum strain 105. This recombinant strain provides the advantage of PL production, which predominates over other extracellular proteins usually present in most commercial pectinase preparations, using sugarcane juice as a substrate of low cost.

Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum.

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.