White-rot fungus Phlebia floridensis ITM 12: Laccase production, oxidoreductase profile, and hydrogen-peroxide independent activity.

Phlebia genus is a relevant group of fungi with a crucial role in numerous ecosystems. In tropical and subtropical areas this genus allows the efficient degradation of lignin and carbon recovery; however, the majority of these fungal species remain undiscovered. The main purpose of this work was to determine the enzymatic activity of extracellular proteins of a novel Phlebia floridensis strain isolated in Yucatan Peninsula, Mexico. The results that are reported here demonstrate that the soluble protein extract of P. floridensis can degrade a broad spectrum of recalcitrant compounds. This induced protein extract is able to modify not only phenolic and nonphenolic compounds, but also anthroquinone dyes, even without the addition of exogenous hydrogen peroxide. Using liquid chromatography-mass spectrometry (LC-MS), we were able to identify a novel chloroperoxidase in enzymatic extract. As far as we know, this is the first report about the presence of this type of enzyme in the Phlebia genus.

Protein extract of Bromelia karatas L. rich in cysteine proteases (ananain- and bromelain-like) has antibacterial activity against foodborne pathogens Listeria monocytogenes and Salmonella Typhimurium.

Bromelia karatas L. is a plant species from the Americas. The presence of proteases in fruits of B. karatas has been reported but scarcely studied in detail. Proteolytic enzymes from Ananas comosus have displayed antifungal and antibacterial activity. Thus, novel proteases present in B. karatas may be useful as a source of compounds against microorganisms in medicine and food production. In this work, the protein extract from the fruits of B. karatas was characterized and its antibacterial activity against Salmonella Typhimurium and Listeria monocytogenes was determined for the first time. Proteins highly similar to ananain and the fruit bromelain from A. comosus were identified as the main proteases in B. karatas fruits using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The soluble protein extract (SPE) at a concentration of 2.0 mg/mL displayed up to 80% of antibacterial activity against S. Typhimurium. Complete inhibition of L. monocytogenes was reached with up to 1.65 mg/mL of SPE. Plant protease extract containing ananain-like enzyme inhibited up to 90% against S. Typhimurium and up to 85% against L. monocytogenes using only 10 µg/mL of the partial-purified enzyme.

Effects of phenolic and protein extracts from Melipona beecheii honey on pathogenic strains of Escherichia coli and Staphylococcus aureus.

Antimicrobial effects of Melipona beecheii honey have been attributed to diverse factors, in this sense, certain components such as proteins and phenolics could explain relevant aspects of its antimicrobial activity. The aim of this study was to evaluate the antibacterial activity of phenolic and protein extracts from M. beecheii honey against two bacterial pathogens: Staphylococcus aureus and Escherichia coli. With respect to phenolic content, HPLC analysis allowed the identification of phenolic acids like chlorogenic acid, caffeic acid, and flavonoids like catechin, myricetin, quercetin and apigenin. On the other hand, seven bands with molecular weight from 7.6 to 95 kDa were detected in protein extract by SDS-PAGE system. It was determined the antibacterial activity of both extracts, with MICs lower than 145 µg/mL and 60 µg/mL for the phenolic and protein extracts respectively. These results indicate that phenolic and protein components of M. beecheii honey contribute significantly to the antibacterial activity.

Extracellular Expression in Aspergillus niger of an Antibody Fused to Leishmania sp. Antigens.

Nucleoside hydrolase and sterol 24-c-methyltransferase, two antigenic proteins of Leishmania sp., were expressed in Aspergillus niger. Genetic transformation of conidia was achieved using underwater shock waves. scFv antibody addressed to DEC205, a receptor of dendritic cells, was fused to two proteins of Leishmania sp. Receptor 205 has a relevant role in the immune system in mammals; it can modulate T cell response to different antigens. Extracellular expression strategy of recombinant antibody was achieved using a fragment of native glucoamylase A (514 aa) as a carrier. Fermentations in shake flasks showed that the recombinant protein (104 kDa) was expressed and secreted only when maltose was used as carbon source; on the contrary, the expression was highly repressed in presence of xylose. Noteworthy, recombinant protein was secreted without glucoamylase-carrier and accumulation at intracellular level was not observed. The results presented here demonstrate the high value of Aspergillus niger as biotechnological platform for recombinant antibodies against Leishmania sp. at low cost. To the best of our knowledge, this is the first report about the recombinant expression of antigenic proteins of Leishmania sp. in filamentous fungi. The protein obtained can be used to explore novel strategies to induce immunity against Leishmania sp. or it can be employed in diagnostic kits to detect this neglected disease.

Efficient transformation of Mycosphaerella fijiensis by underwater shock waves.

Black leaf streak disease, also known as black Sigatoka, causes dramatic losses in production of banana and plantains fruits. The disease is caused by the pathogenic fungus Mycosphaerella fijiensis (anamorph Pseudocercospora fijiensis; Mycosphaerellaceae). Genetic transformation of M. fijiensis would allow a better understanding of molecular basis of pathogenicity and design novel approaches to control the infection caused by this pathogen. However, transformation of this fungus has not been easy. We report here a protocol for genetic transformation of M. fijiensis employing underwater shock waves and intact conidia. The recombinant strains recovered showed genetic stability over >10 generations. The frequency of transformation obtained was between 75 and 150 times higher than the efficiency reported in the only article published on transformation of M. fijiensis using spheroplasts. This improvement allowed the use of a thousand times less cells than the amount employed before, avoiding the need for cumbersome successive batch cultures. Our protocol is simple, highly efficient, fast and reproducible and together with the available genomes of M. fijiensis and Musa acuminata, it offers new possibilities to study the diverse mechanisms of pathogenesis of the fungus.

High-yield production of manganese peroxidase, lignin peroxidase, and versatile peroxidase in Phanerochaete chrysosporium.

The white-rot fungus Phanerochaete chrysosporium secretes extracellular oxidative enzymes during secondary metabolism, but lacks versatile peroxidase, an enzyme important in ligninolysis and diverse biotechnology processes. In this study, we report the genetic modification of a P. chrysosporium strain capable of co-expressing two endogenous genes constitutively, manganese peroxidase (mnp1) and lignin peroxidase (lipH8), and the codon-optimized vpl2 gene from Pleurotus eryngii. For this purpose, we employed a highly efficient transformation method based on the use of shock waves developed by our group. The expression of recombinant genes was verified by PCR, Southern blot, quantitative real-time PCR (qRT-PCR), and assays of enzymatic activity. The production yield of ligninolytic enzymes was up to four times higher in comparison to previously published reports. These results may represent significant progress toward the stable production of ligninolytic enzymes and the development of an effective fungal strain with promising biotechnological applications.

Tandem shock waves to enhance genetic transformation of Aspergillus niger.

Filamentous fungi are used in several industries and in academia to produce antibiotics, metabolites, proteins and pharmaceutical compounds. The development of valuable strains usually requires the insertion of recombinant deoxyribonucleic acid; however, the protocols to transfer DNA to fungal cells are highly inefficient. Recently, underwater shock waves were successfully used to genetically transform filamentous fungi. The purpose of this research was to demonstrate that the efficiency of transformation can be improved significantly by enhancing acoustic cavitation using tandem (dual-pulse) shock waves. Results revealed that tandem pressure pulses, generated at a delay of 300 µs, increased the transformation efficiency of Aspergillus niger up to 84% in comparison with conventional (single-pulse) shock waves. This methodology may also be useful to obtain new strains required in basic research and biotechnology.

Physical methods for genetic transformation of fungi and yeast.

The production of transgenic fungi is a routine process. Currently, it is possible to insert genes from other fungi, viruses, bacteria and even animals, albeit with low efficiency, into the genomes of a number of fungal species. Genetic transformation requires the penetration of the transgene through the fungal cell wall, a process that can be facilitated by biological or physical methods. Novel methodologies for the efficient introduction of specific genes and stronger promoters are needed to increase production levels. A possible solution to this problem is the recently discovered shock-wave-mediated transformation. The objective of this article is to review the state of the art of the physical methods used for genetic fungi transformation and to describe some of the basic physics and molecular biology behind them.

A novel and highly efficient method for genetic transformation of fungi employing shock waves.

Genetic transformation of filamentous fungi is an essential tool in many areas such as biotechnology, medicine, phytopathology and genetics. However, available protocols to transform fungi are inefficient, laborious and have low reproducibility. We report the use of underwater shock waves as a novel method to transform filamentous fungi. An experimental piezoelectric shock wave generator was designed to expose fungal conidia to heterologous DNA. The device was successfully tested in Aspergillus niger, Fusarium oxysporum, Trichoderma reesei and Phanerochaete chrysosporium. The transformation frequency per number of conidia was between two and four orders of magnitude higher in comparison to previously published methods. For example, the frequency of transformation in A. niger was improved up to 5400-fold as compared with Agrobacterium protocols. Transformation was verified by expression of the green fluorescent protein, PCR and Southern blot. Our method offers new possibilities for fast, easy and efficient genetic manipulation of diverse fungal species.