CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass

The CRISPR/Cas9 system has been successfully applied for gene editing in filamentous fungi. Previous studies reported that single stranded oligonucleotides can be used as repair templates to induce point mutations in some filamentous fungi belonging to genus Aspergillus. In Aspergillus niger, extensive research has been performed on regulation of plant biomass degradation, addressing transcription factors such as XlnR or GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. Single nucleotide mutations leading to constitutively active forms of XlnR and GaaR have been previously reported. However, the mutations were performed by the introduction of versions obtained through site-directed or UV-mutagenesis into the genome. Here we report a more time- and cost-efficient approach to obtaining constitutively active versions by application of the CRISPR/Cas9 system to generate the desired mutation on-site in the A. niger genome. This was also achieved using only 60-mer single stranded oligonucleotides, shorter than the previously reported 90-mer strands. In this study, we show that CRISPR/Cas9 can also be used to efficiently change functional properties of the proteins encoded by the target gene by on-site genomic mutations in A. niger. The obtained strains with constitutively active XlnR and GaaR versions resulted in increased production of plant biomass degrading enzymes and improved release of D-xylose and L-arabinose from wheat bran, and D-galacturonic acid from sugar beet pulp.Peer reviewe

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.Peer reviewe

Oxalate-Metabolising Genes of the White-Rot Fungus Dichomitus squalens Are Differentially Induced on Wood and at High Proton Concentration

Peer reviewe

Depolymerization of biorefinery lignin by improved laccases of the white-rot fungus Obba rivulosa

Fungal laccases are attracting enzymes for sustainable valorization of biorefinery lignins. To improve the lignin oxidation capacity of two previously characterized laccase isoenzymes from the white-rot fungus Obba rivulosa, we mutated their substrate-binding site at T1. As a result, the pH optimum of the recombinantly produced laccase variant rOrLcc2-D206N shifted by three units towards neutral pH. O. rivulosa laccase variants with redox mediators oxidized both the dimeric lignin model compound and biorefinery poplar lignin. Significant structural changes, such as selective benzylic alpha-oxidation, were detected by nuclear magnetic resonance analysis, although no polymerization of lignin was observed by gel permeation chromatography. This suggests that especially rOrLcc2-D206N is a promising candidate for lignin-related applications.Peer reviewe

Fungal Treatment Modifies Kraft Lignin for Lignin- and Cellulose-Based Carbon Fiber Precursors

The kraft lignin's low molecular weight and too high hydroxyl content hinder its application in bio-based carbon fibers. In this study, we were able to polymerize kraft lignin and reduce the amount of hydroxyl groups by incubating it with the white-rot fungus Obba rivulosa. Enzymatic radical oxidation reactions were hypothesized to induce condensation of lignin, which increased the amount of aromatic rings connected by carbon-carbon bonds. This modification is assumed to be beneficial when aiming for graphite materials such as carbon fibers. Furthermore, the ratio of remaining aliphatic hydroxyls to phenolic hydroxyls was increased, making the structure more favorable for carbon fiber production. When the modified lignin was mixed together with cellulose, the mixture could be spun into intact precursor fibers by using dry-jet wet spinning. The modified lignin leaked less to the spin bath compared with the unmodified lignin starting material, making the recycling of spin-bath solvents easier. The stronger incorporation of modified lignin in the precursor fibers was confirmed by composition analysis, thermogravimetry, and mechanical testing. This work shows how white-rot fungal treatment can be used to modify the structure of lignin to be more favorable for the production of bio-based fiber materials.Peer reviewe

The Synthetic Potential of Fungal Feruloyl Esterases : A Correlation with Current Classification Systems and Predicted Structural Properties

Twenty-eight fungal feruloyl esterases (FAEs) were evaluated for their synthetic abilities in a ternary system of n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0 forming detergentless microemulsions. Five main derivatives were synthesized, namely prenyl ferulate, prenyl caffeate, butyl ferulate, glyceryl ferulate, and l-arabinose ferulate, offering, in general, higher yields when more hydrophilic alcohol substitutions were used. Acetyl xylan esterase-related FAEs belonging to phylogenetic subfamilies (SF) 5 and 6 showed increased synthetic yields among tested enzymes. In particular, it was shown that FAEs belonging to SF6 generally transesterified aliphatic alcohols more efficiently while SF5 members preferred bulkier l-arabinose. Predicted surface properties and structural characteristics were correlated with the synthetic potential of selected tannase-related, acetyl-xylan-related, and lipase-related FAEs (SF1-2, -6, -7 members) based on homology modeling and small molecular docking simulations.Peer reviewe

The transcriptomic response of two basidiomycete fungi to plant biomass is modulated by temperature to a different extent

Many fungi show a strong preference for specific habitats and growth conditions. Investigating the molecular mechanisms of fungal adaptation to varying environmental conditions is of great interest to biodiversity research and is important for many industrial applications. In this study, we compared the transcriptome profiles of two previously genome-sequenced white-rot wood-decay fungi, Trametes pubescens and Phlebia centrifuga, during their growth on two common plant biomass substrates (wheat straw and spruce) at two temperatures (15 degrees C and 25 degrees C). The results showed that both fungi partially tailored their molecular responses to different types of carbon sources, differentially expressing genes encoding polysaccharide degrading enzymes, transporters, proteases and monooxygenases. Notably, more lignin modification related AA2 genes and cellulose degradation related AA9 genes were differentially expressed in the tested conditions of T. pubescens than P. centrifuga. In addition, we detected more remarkable transcriptome changes to different growth temperature in P. centrifuga than in T. pubescens, which reflected their different ability to adapt to the temperature fluctuations. In P. centrifuga, differentially expressed genes (DEGs) related to temperature response mainly encode protein kinases, trehalose metabolism, carbon metabolic enzymes and glycoside hydrolases, while the main temperature-related DEGs identified in T. pubescens are only the carbon metabolic enzymes and glycoside hydrolases. Our study revealed both conserved and species-specific transcriptome changes during fungal adaptation to a changing environment, improving our understanding of the molecular mechanisms underlying fungal plant biomass conversion at varying temperatures.Peer reviewe

Comparative analysis of basidiomycete transcriptomes reveals a core set of expressed genes encoding plant biomass degrading enzymes

Basidiomycete fungi can degrade a wide range of plant biomass, including living and dead trees, forest litter, crops, and plant matter in soils. Understanding the process of plant biomass decay by basidiomycetes could facilitate their application in various industrial sectors such as food & feed, detergents and biofuels, and also provide new insights into their essential biological role in the global carbon cycle. The fast expansion of basidiomycete genomic and functional genomics data (e.g. transcriptomics, proteomics) has facilitated exploration of key genes and regulatory mechanisms of plant biomass degradation. In this study, we comparatively analyzed 22 transcriptome datasets from basidiomycetes related to plant biomass degradation, and identified 328 commonly induced genes and 318 repressed genes, and defined a core set of carbohydrate active enzymes (CAZymes), which was shared by most of the basidiomycete species. High conservation of these CAZymes in genomes and similar regulation pattern in transcriptomics data from lignocellulosic substrates indicate their key role in plant biomass degradation and need for their further biochemical investigation