Forward genetics by genome sequencing uncovers the central role of the aspergillus niger goxB locus in hydrogen peroxide induced glucose oxidase expression

Aspergillus niger is an industrially important source for gluconic acid and glucose oxidase (GOx), a secreted commercially important flavoprotein which catalyses the oxidation of b-D-glucose by molecular oxygen to D-glucolactone and hydrogen peroxide. Expression of goxC, the GOx encoding gene and the concomitant two step conversion of glucose to gluconic acid requires oxygen and the presence of significant amounts of glucose in the medium and is optimally induced at pH 5.5. The molecular mechanisms underlying regulation of goxC expression are, however, still enigmatic. Genetic studies aimed at understanding GOx induction have indicated the involvement of at least seven complementation groups, for none of which the molecular basis has been resolved. In this study, a mapping-by-sequencing forward genetics approach was used to uncover the molecular role of the goxB locus in goxC expression. Using the Illumina and PacBio sequencing platforms a hybrid high quality draft genome assembly of laboratory strain N402 was obtained and used as a reference for mapping of genomic reads obtained from the derivative NW103:goxB mutant strain. The goxB locus encodes a thioredoxin reductase. A deletion of the encoding gene in the N402 parent strain led to a high constitutive expression level of the GOx and the lactonase encoding genes required for the two-step conversion of glucose in gluconic acid and of the catR gene encoding catalase R. This high constitutive level of expression was observed to be irrespective of the carbon source and oxidative stress applied. A model clarifying the role of GoxB in the regulation of the expression of goxC involving hydrogen peroxide as second messenger is presented

Bioethanol Production from Glycerol by Mixed Culture System

AbstractGlycerol, a by-product from biodiesel industry, is a promising feedstock for subsequent bioconversion to higher-value products. Potential application of a mixed microbial consortium on the fermentative conversion of glycerol to ethanol was demonstrated in this study. Maximum ethanol concentration of 11.1 g l-1 was produced after 72 h fermentation from the initial pure glycerol concentration of 45 g l-1, at 30 ̊C, and pH 7 under anaerobic conditions, corresponding to the ethanol production rate and yield of 0.34 g l-1 h-1, and 0.81 mol ethanolmol-1 glycerol, respectively. The microbial consortium yielded lower ethanol concentration (6.5 g l-1) but similar ethanol yield (0.85 mol ethanol mol-1 glycerol) when crude glycerol of 45 g l-1 was fermented at the same condition. At the optimum fermentative condition of the pure glycerol, phylogenetic analysis of microbial consortium based on 16S rRNA gene sequences indicated that Gammaproteobacteria represented 95% of the microbial diversity in the consortium while the rest belonged to Betaproteobacteria. The consortium was dominated by bacteria closely related to genera Enterobacter and Klebsiella, which could play the role on conversion of glycerol to ethanol in this system

Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei

Background: Global climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from Aspergillus Niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes. Results: A hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. Niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. Niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. Niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization. Conclusions: This study revealed the existence of a variety of xylose transporters in the cell factories A. Niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. Niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. Niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.</p

Insight into the molecular mechanisms of organic acid pathways in Aspergillus niger

The filamentous fungus Aspergillus niger is well-known as an industrial producer of a number of organic acids. However, despite decades of research, there are still bottlenecks and, with respect to the molecular mechanism underlying A. niger organic acids production there are still a number of unclarities. This thesis aims to gain a better understanding of the molecular mechanisms underlying organic acid production in A. niger and is mostly focused on active transport of carbon sources, organic acids and reactive oxygen species via transmembrane transport proteins. Additionally, the regulation of glucose oxidase expression system in gluconate pathways as well as response of A. niger to environmental factors during organic acid fermentation were also studied.</p

Identification of Aspergillus niger Aquaporins Involved in Hydrogen Peroxide Signaling

Aspergillus niger is a robust microbial cell factory for organic acid production. However, the regulation of many industrially important pathways is still poorly understood. The regulation of the glucose oxidase (Gox) expression system, involved in the biosynthesis of gluconic acid, has recently been uncovered. The results of that study show hydrogen peroxide, a by-product of the extracellular conversion of glucose to gluconate, has a pivotal role as a signaling molecule in the induction of this system. In this study, the facilitated diffusion of hydrogen peroxide via aquaporin water channels (AQPs) was studied. AQPs are transmembrane proteins of the major intrinsic proteins (MIPs) superfamily. In addition to water and glycerol, they may also transport small solutes such as hydrogen peroxide. The genome sequence of A. niger N402 was screened for putative AQPs. Seven AQPs were found and could be classified into three main groups. One protein (AQPA) belonged to orthodox AQP, three (AQPB, AQPD, and AQPE) were grouped in aquaglyceroporins (AQGP), two (AQPC and AQPF) were in X-intrinsic proteins (XIPs), and the other (AQPG) could not be classified. Their ability to facilitate diffusion of hydrogen peroxide was identified using yeast phenotypic growth assays and by studying AQP gene knock-outs in A. niger. The X-intrinsic protein AQPF appears to play roles in facilitating hydrogen peroxide transport across the cellular membrane in both Saccharomyces cerevisiae and A. niger experiments

Survey of Microbial Diversity in Flood Areas during Thailand 2011 Flood Crisis Using High-Throughput Tagged Amplicon Pyrosequencing.

The Thailand flood crisis in 2011 was one of the largest recorded floods in modern history, causing enormous damage to the economy and ecological habitats of the country. In this study, bacterial and fungal diversity in sediments and waters collected from ten flood areas in Bangkok and its suburbs, covering residential and agricultural areas, were analyzed using high-throughput 454 pyrosequencing of 16S rRNA gene and internal transcribed spacer sequences. Analysis of microbial community showed differences in taxa distribution in water and sediment with variations in the diversity of saprophytic microbes and sulfate/nitrate reducers among sampling locations, suggesting differences in microbial activity in the habitats. Overall, Proteobacteria represented a major bacterial group in waters, while this group co-existed with Firmicutes, Bacteroidetes, and Actinobacteria in sediments. Anaeromyxobacter, Steroidobacter, and Geobacter were the dominant bacterial genera in sediments, while Sulfuricurvum, Thiovirga, and Hydrogenophaga predominated in waters. For fungi in sediments, Ascomycota, Glomeromycota, and Basidiomycota, particularly in genera Philipsia, Rozella, and Acaulospora, were most frequently detected. Chytridiomycota and Ascomycota were the major fungal phyla, and Rhizophlyctis and Mortierella were the most frequently detected fungal genera in water. Diversity of sulfate-reducing bacteria, related to odor problems, was further investigated using analysis of the dsrB gene which indicated the presence of sulfate-reducing bacteria of families Desulfobacteraceae, Desulfobulbaceae, Syntrobacteraceae, and Desulfoarculaceae in the flood sediments. The work provides an insight into the diversity and function of microbes related to biological processes in flood areas

Map of sample collection sites.

<p>The sampling areas are in Bangkok, Pathumthani, Nonthaburi, and Nakornpathom provinces.</p

Comparative distribution of fungal diversity in sediment and water.

<p>(A) Principal component analysis (PCA) based on percent relative abundance of fungal taxa at genus level in sediment (orange) and water (blue). (B) Bar chart with extended error bar for pair-wise comparisons of fungal genera between sediment (orange) and water (blue) samples.</p

Comparison of fungal diversity in sediment and water.

<p>Percent relative abundance of fungal phyla based on ITS sequence abundance are shown. Left and right bars represent relative abundance collected from sediment and water, respectively.</p