Current Biotechnology - Volume 6, Issue 2, 2017

Background: Basidiomycete Trametes versicolor is one of the most studied white rot fungus due to its great genetic potential responsible for the expression of different and powerful capacities such as degrading lignocellulose in wood decay bioprocesses. T. versicolor whole genome is currently being sequenced due to the high interest in using its enzymatic complex systems as well as producing its polysaccharopeptides with important biological activities. Mehtod: T. versicolor fruiting bodies, their infusions, solid state fermentations and submerged cultures are increasingly used for multiple research studies and also on industrial biotechnological applications for obtaining commercial products. The oxireductive enzymatic activities found in T. versicolor were studied and the fungus was directly applied in Kraft pulp bleaching and for wood delignification. Furthermore it was applied with environmental objectives reducing pollutant content converting recalcitrant toxic substances. The biomass of this fungus was also used for biosorption in effluents cleaning processes. Extracellular enzymes produced by T. versicolor cultures have a wide range of applications and laccase was reported for wine and beer industries, pulp and paper and textile industries as well as for bioremediation. Result: The potential from T. versicolor polysaccharopeptides has been systematically investigated in human cancer, being the best commercially established mushroom-derived therapeutics. Production and isolation of polysaccharopeptide Krestin and another polysaccharopeptide, dark brown powders, with a light odor both soluble in hot water allowed their commercialization and their physiological activities are being researched and evaluated. Conclusion: Applied research based on this fungus is a challenge that can be very promissory in the future.

Background: The conversion of biomass to fuels and chemicals is an important technology to replace petroleum as a transportation fuel which will ease climate effects of burning fossil fuels. Recent advances in cellulosic ethanol production have enabled the establishment of commercial scale plants that produce ethanol for transportation fuel. Thermotolerant cellulase enzymatic mixtures from thermophilic fungi are an attractive alternative to currently available commercial cellulase cocktails. Methods: Thermoascus aurantiacus is a thermophilic ascomycete fungus within the order of Eurotiales that was first isolated by Miehe in 1907. Strains of T. aurantiacus have been isolated from a variety of terrestrial environments, which all have been shown to be homothallic and produce large amounts of ascopores with an optimal growth temperature at ~50130;°C. T. aurantiacus secretes high titers of cellulases (>1 g/L) when grown in the presence of plant biomass substrates and produces a remarkably simple cellulase mixture consisting of GH7 cellobiohydrolase, GH5 endoglucanase, AA9 lytic polysaccharide monooxygenase and GH3 beta-glucosidase. Results: In this mini-review, the biology and enzymology underlying cellulase production are described and an approach to developing T. aurantiacus strains for industrial cellulase production is outlined. Conclusion: The properties of T. aurantiacus and the thermotolerant cellulase mixture it produces may be the basis for new enzymatic cocktails to produce sugars from plant biomass that can be converted to biofuels.

Background: Oxalic acid is a common metabolite of wood-degrading basidiomycete fungi, and it has been proposed to possess several important roles in fungal physiology, including assistance in degradation of lignocellulose polymers. Because high concentrations of oxalic acid are toxic to fungi, they produce oxalate degrading enzymes, i.e. oxalate decarboxylases (ODC) and oxalate oxidases (OXO), to regulate the amount of oxalic acid in the vicinity of fungal hyphae. In addition, oxalate degrading enzymes are of interest for several biotechnological applications, such as in diagnostics to determine oxalic acid concentrations and in bioprocess industry to prevent the formation of oxalate deposits. Our previous studies have shown that the wood-rotting white rot fungus Dichomitus squalens actively secretes and degrades oxalic acid during growth both in liquid cultures and on natural spruce wood cultures. Methods: To study the biochemical properties of D. squalens ODCs, the five ODC encoding genes were expressed in Pichia pastoris under control of the methanol inducible alcohol oxidase I (AOXI) promoter. Results: The secretion of recombinant ODCs (rODCs) from P. pastoris cells was driven by both the Saccharomyces cerevisiae α-mating factor fused to mature ODCs and native leader peptides of ODC proteins, thus showing that the native signal sequences of D. squalens ODCs were functional. All the rODCs were produced as multimeric proteins, which had dissimilar biochemical characteristics. The rODC3 and rODC4 degraded oxalic acid at the widest pH and temperature range. Conclusion: All the rODCs were produced as active enzymes with rODC3 and rODC4 having the highest thermostability and showing activity over the broadest pH range, thus having potential as robust biocatalysts in various biotechnological processes.

Background: Wood rotting white-rot and litter-decomposing basidiomycetes form a huge reservoir of oxidative enzymes, needed for applications in the pulp and paper and textile industries and for bioremediation. Objective: The aim was (i) to achieve higher throughput in enzyme screening through miniaturization and automatization of the activity assays, and (ii) to discover fungi which produce efficient oxidoreductases for industrial purposes. Methods: Miniaturized activity assays mostly using dyes as substrate were carried out for lignin peroxidase, versatile peroxidase, manganese peroxidase and laccase. Methods were validated and 53 species of basidiomycetes were screened for lignin modifying enzymes when cultivated in liquid mineral, soy, peptone and solid state oat husk medium. Results: Manganese peroxidases were the most common enzymes produced by 96% of the species. They typically had acidic pH optima, although Hyphodontia sp., Pleurotus pulmonarius and Trametes ochracea produced enzymes highly active at pH 7. Versatile peroxidase was produced by 66% of the fungi with efficient production from Phlebia radiata, P. pulmonarius and Galerina marginata. Novel lignin peroxidase producing fungi Cylindrobasidium evolvens and Daedaleopsis septentrionalis were found among the 26% of the species showing here lignin peroxidase production. Laccase was shown in 92% of the species. Several fungi produced laccase active at pH 7, which is noteworthy because usually laccases of white-rot fungi are efficient and relevant for many industrial applications. Conclusion: Automated screening allowed us to monitor many specific enzyme activities and extend the range of assay conditions from relatively small fungal cultivation sample volumes.

Background: Manganese peroxidases (MnP) and lignin peroxidases (LiP) are haem-including fungal secreted class-II peroxidases, which are interesting oxidoreductases in protein engineering aimed at designing of biocatalysts for lignin and lignocellulose conversion, dye compound degradation, activation of aromatic compounds, and biofuel production. Objective. Recombinant short-type MnP (Pr-MnP3) of the white rot fungus Phlebia radiata, and its manganese- binding site (E40, E44, D186) directed variants were produced and characterized. To allow catalytic applications, enzymatic bleaching of Reactive Blue 5 and conversion of lignin-like compounds by engineered class- II peroxidases were explored. Method: Pr-MnP3 and its variants were expressed in Escherichia coli. The resultant body proteins were lysed, purified and refolded into haem-including enzymes in 6-7% protein recovery, and examined spectroscopically and kinetically. Results: Successful production of active enzymes was attained, with spectral characteristics of high-spin class-II peroxidases. Recombinant Pr-MnP3 demonstrated high affinity to Mn2+, which was noticeably affected by single (D186H/N) and double (E40H+E44H) mutations. Without addition of Mn2+, Pr- MnP3 was able to oxidize ABTS and decolorize Reactive Blue 5. Pc-LiPH8, its Trp-radical site variants, and engineered CiP-LiP demonstrated conversion of veratryl alcohol and dimeric non-phenolic lignin-model compounds (arylglycerol-β-aryl ethers) with production of veratraldehyde, which is evidence for cation radical formation with subsequent Cα-Cβ cleavage. Pc-LiPH8 and CiP variants were able to effectively oxidize and convert the phenolic dimer (guaiacylglycerol-β-guaiacyl ether). Conclusion: Our results demonstrate suitability of engineered MnP and LiP peroxidases for dyedecolorizing, and efficiency of LiP and its variants for activation and degradation of phenolic and nonphenolic lignin-like aryl ether-linked compounds.

Background: Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. Objective: To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity Methods: We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Results: Colorimetric assays for general glycosyl hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate β-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and β - glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of β -glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of β-glucosidase activity. Conclusions: While β-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific glycosyl hydrolase activities. It remains unclear whether loss of endocellulase activity to lignin binding is problematic for biomass conversion.

Background: A major bottleneck in DNA-mediated transformation of fungi is the limited number of available selective marker genes, especially when sequential transformations of the same strain are necessary. In the model basidiomycete Coprinopsis cinerea, the trp1+ gene is a commonly used dominant selection marker for transformation to complement trp1 auxotrophies. The trp1+ encoded tryptophan synthase is a bifunctional enzyme that catalyzes the final two reactions in the biosynthetic pathway of tryptophan. The Nterminal A-domain is responsible for the conversion of indole-3-glycerol-phosphate into indole, while the Cterminal B-domain catalyzes the subsequent production of tryptophan from serine and indole. The trp1-1,1-6 mutant allele used in C. cinerea hosts in transformation carries a mutation in each gene half, both of which prevent the strain from completing tryptophan biosynthesis. Methods: Taking advantage of this situation, we developed a new set of vectors containing either just the A- or the B-domain encoding trp1+ sequences (referred to as trpA+ and trpB+). The constructs were used in single-vector transformations and in co-transformations with other vectors containing genes which are not directly selectable (C. cinerea lcc1+ for laccase, egfp for green fluorescent protein). The trpB+-construct pYBdom and the trpA+-construct pYAdom can be applied in series in subsequent rounds of transformations. This allows successive introduction of different genes into the same genetic background in succeeding rounds of co-transformations. Results: For the first time in fungi, the functionality of independent tryptophan synthase domains TrpA and TrpB were documented. The new marker set enables the independent complementation of the distinct trp1 mutations in separate transformations. First, trpB+ needs to be transformed and indole must be added to the regeneration medium for production of tryptophan. The subsequent transformation with trpA+ results in the production of indole which is then used by TrpB in tryptophan synthesis. Double transformants are prototrophic but differ in growth rates as compared to transformants with the complete trp1+ gene. The two new dominant selection markers have successfully been used in sequential cotransformations with C. cinerea lcc1+ and egfp as additional genes. Plate assays helped in the detection of co-transformants expressing these additional genes. Co-transformation frequencies of 21-23% were obtained. Conclusions: Application of vectors pYBdom and pYAdom opens novel possibilities to add different genes in series into a same genetic background to manipulate metabolic or developmental processes in the fungus.

Background: Methanogens utilize low carbon molecules in anaerobic environments and produce CH4 that serves as a key component in the global carbon cycle in the atmosphere. Genome-scale metabolic modeling is a proficient computational tool for integrative analysis of their cellular and metabolic processes. Thus, genome-scale models of methanogens have gained great importance in environmental and biotechnological applications. Methods: The study explored literature databases in-depth for peer-reviewed research papers related to the methanogens and their ecological importance, particularly enteric methane emission. Metabolic information for these organisms was collected from MetaCyc database. Genome-scale metabolic modeling information was collected from Systems Biology Research Group and qualitative content was deduced from corresponding literature. This paper used standard tools to assess the validity of retrieved papers for highlighting the overall hypothesis. The conceptual framework was designed by using a deductive qualitative content analysis of screened papers. Results: In this review, 100 peer reviewed research papers were included out of which 60 papers were related to the methanogens. The present review describes the significance of genomic and metabolic features of genome-scale metabolic models and some potential rumen methanogens to reveal their environmental contributions in assorted ecosystem. Six genome-scale metabolic models (iMM518, iMG746, iMB745, iVS941, iAF692, iMAC868) have been constructed with detailed biochemical information for the methanogens. These models were further refined by comparing predicted growth yield with experimental growth data to validate the model’s consistency. Metabolic flux balance analysis was used to assess their biological impact on the carbon balance of methanogenic communities. The effect of the present ecosystem on global CH4 cycling, particularly reverse methanogenesis has been studied with genome- scale models of methanogens. Conclusion: Genome-scale models related to the experimental growth data would improve the prediction accuracy of metabolic flux coefficients and specific growth rate of diverse methanogens under the nutrient-deprivation conditions in anaerobic environment. Using genome-scale models, chemogenomic and vaccine targets for CH4 mitigation have also been identified and evaluated.

Background: Many investigations have attributed bio-functional properties to a range of conjugated fatty acids (CFAs) of which the conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) isomers have displayed potent health beneficial effects in a large number of in vitro and in vivo studies. CLNA isomers are naturally present in high concentrations in a variety of seed oils but relatively in small amount in meat and dairy products. However, some reports have shown the microbial production of such enzymes by lactic acid bacteria that convert linolenic acid (LNA) into CLNA. Present study aims to isolate CLNA producing lactobacilli of different origins and their potential to produce different CLNA isomers in MRS media. Methods: LNA containing MRS media was used for the isolation of lactobacilli and genotypic screening was done for the linoleate isomerase gene (LAI). Spectrophotometer was used to know the initial total fatty acid production and finally CLNA isomers were confirmed by the gas chromatography and their amounts were calculated. For this study, a total of 274 samples were collected from human milk, feces and dairy based fermented and non-fermented products, lab isolates and 178 lactobacilli strains were taken for CLNA screening. For conjugated isomers production by isolates, MRS broth with LNA at concentration of 0.5 mg/ml was used and incubated at 37°C for 48h. Results: Out of 178; only 40 isolates were able to survive at the tested LNA concentration. Finally, these 40 isolates were screened for conjugated double bond using spectrophotometer at 234nm. The highest conjugated fat producing isolates were further analyzed for c9, t11, c15 and t9, t11, c15 isomers using gas chromatography. Nine isolates in presence of LNA confirmed the production of CLNA isomers, C18:3 (c9, t11, c15 and t9, t11, c15). Only three isolates produced CLNA from LNA in MRS in a range of 52 to 80μg/ml and the rest 29 to 45μg/ml. Conclusion: Most likely, this is the first report describing CLNA production from lactobacilli isolated from human feces and fermented dairy products. Bioconversion of LNA into CLNA isomers opens newer options for the treatment of diseases. Therefore, it would be beneficial to get promising CLNA producing lactobacilli for improved absorbability and cure gastrointestinal disorders.

Background: Celite- immobilized lipase was used to catalyze the esterification reaction between different alcohol of carbon chain in increasing order viz. methanol, ethanol, propanol and butanol to form cinnamate esters in DMSO (solvent) at different temperatures for different time periods in a chemical reactor. Methods: The molar ratio of 1:1 (cinnamic acid: alcohol) was found to be optimum for the syntheses of all cinnamate esters when incubated for 10-12 h at 55ºC with 1-1.5% biocatalyst load in the presence of 20 and 30 mg/ml of molecular sieves for methyl, ethyl cinnamate and propyl and butyl cinnamate, respectively. Results: All the respective esters that were synthesized were characterized by FTIR spectroscopy followed by NMR spectroscopy. Conclusion: The positive inference in favour of esters formation comes from the presence of peaks of the functional groups (C=O stretching in ester) and (-COC- stretching) and absence of peak due to –OH stretching in the FTIR spectra of esters. 1H NMR spectra were also recorded on (INOVA 400 MHz spectrophotometer) in deuterated chloroform (CDCl3) solution with internal standard TMS (0 ppm), and chemical shifts in parts per million (δ/ppm) confirmed the ester syntheses.