Heinrich Volschenk

Heterologous expression of a Clostridium minicellulosome in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae was genetically modified to assemble a minicellulosome on its cell surface by heterologous expression of a chimeric scaffoldin protein from Clostridium cellulolyticum under the regulation of the phosphoglycerate kinase 1 (PGK1) promoter and terminator regulatory elements, together with the beta-xylanase 2 secretion signal of Trichoderma reesei and cell wall protein 2 (Cwp2) of S. cerevisiae. Fluorescent microscopy and Far Western blot analysis confirmed that the Scaf3p is targeted to the yeast cell surface and that the Clostridium thermocellum cohesin domain is functional in yeast. Similarly, functionality of the C. thermocellum dockerin domain in yeast is shown by binding to the Scaf3 protein in Far Western blot analysis. Phenotypic evidence for cohesin-dockerin interaction was also established with the detection of a twofold increase in tethered endoglucanase enzyme activity in S. cerevisiae cells expressing the Scaf3 protein compared with the parent strain. This study highlights the feasibility to future design of enhanced cellulolytic strains of S. cerevisiae through emulation of the cellulosome concept. Potentially, Scaf3p-armed yeast could also be developed into an alternative cell surface display strategy with various tailor-made applications.

Comparative secretome analysis of Trichoderma asperellum S4F8 and Trichoderma reesei Rut C30 during solid-state fermentation on sugarcane bagasse

BackgroundThe lignocellulosic enzymes of Trichoderma species have received particular attention with regard to biomass conversion to biofuels, but the production cost of these enzymes remains a significant hurdle for their commercial application. In this study, we quantitatively compared the lignocellulolytic enzyme profile of a newly isolated Trichoderma asperellum S4F8 strain with that of Trichoderma reesei Rut C30, cultured on sugarcane bagasse (SCB) using solid-state fermentation (SSF).ResultsComparison of the lignocellulolytic enzyme profiles of S4F8 and Rut C30 showed that S4F8 had significantly higher hemicellulase and β-glucosidase enzyme activities. Liquid chromatography tandem mass spectrometry analysis of the two fungal secretomes enabled the detection of 815 proteins in total, with 418 and 397 proteins being specific for S4F8 and Rut C30, respectively, and 174 proteins being common to both strains. In-depth analysis of the associated biological functions and the representation of glycoside hydrolase family members within the two secretomes indicated that the S4F8 secretome contained a higher diversity of main and side chain hemicellulases and β-glucosidases, and an increased abundance of some of these proteins compared with the Rut C30 secretome.ConclusionsIn SCB SSF, T. asperellum S4F8 produced a more complex lignocellulolytic cocktail, with enhanced hemicellulose and cellobiose hydrolysis potential, compared with T. reesei Rut C30. This bodes well for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail from T. asperellum for lignocellulosic feedstock hydrolysis.

Oxygen mass transfer for an immobilised biofilm of Phanerochaete chrysosporium in a membrane gradostat reactor

Abstract - A novel system, the membrane gradostat reactor (MGR), designed for the continuous production of secondary metabolites, has been shown to have higher production per reactor volume than batch culture systems. The MGR system mimics the natural environment in which wild occurring microorganism biofilms flourish. The biofilms are immobilised on the external surface of an ultrafiltration membrane where substrate distribution gradients are established across the biofilm. The hypothesis that, dissolved oxygen (DO) mass transfer parameters obtained in submerged pellets can be used to describe and model DO mass transfer parameters in the MGR, was refuted. Phanerochaete chrysosporium biofilms, immobilised on ultrafiltration capillary membranes in the MGR systems were used to quantify DO distribution using a Clark-type microsensor. The DO penetration depth decreased with increasing biofilm thickness, which resulted in the formation of anaerobic zones in the biofilms. Oxygen flux values of 0.27 to 0.7 g/(m

Sequence and structure‐based prediction of fructosyltransferase activity for functional subclassification of fungal GH32 enzymes

Sucrolytic enzymes catalyse sucrose hydrolysis or the synthesis of fructooligosaccharides (FOSs), a prebiotic in human and animal nutrition. FOS synthesis capacity differs between sucrolytic enzymes. Amino‐acid‐sequence‐based classification of FOS synthesizing enzymes would greatly facilitate the in silico identification of novel catalysts, as large amounts of sequence data lie untapped. The development of a bioinformatics tool to rapidly distinguish between high‐level FOSs synthesizing predominantly sucrose hydrolysing enzymes from fungal genomic data is presented. Sequence comparison of functionally characterized enzymes displaying low‐ and high‐level FOS synthesis revealed conserved motifs unique to each group. New light is shed on the sequence context of active site residues in three previously identified conserved motifs. We characterized two enzymes predicted to possess low‐ and high‐level FOS synthesis activities based on their conserved motif sequences. FOS data for the enzymes confirmed our successful prediction of their FOS synthesis capacity. Structural comparison of enzymes displaying low‐ and high‐level FOS synthesis identified steric hindrance between nystose and a long loop region present only in low‐level FOS synthesizers. This loop is proposed to limit the synthesis of FOS species with higher degrees of polymerization, a phenomenon observed among enzymes displaying low‐level FOS synthesis. Conserved sequence motifs surrounding catalytic residues and a distant structural determinant were identifiers of FOS synthesis capacity and allow for functional annotation of sucrolytic enzymes directly from amino acid sequence. The tool presented may also be useful to study the structure–function relationships of β‐fructofuranosidases by identifying mutations present in a group of closely related enzymes displaying similar function.

Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production

ABSTRACT The Aspergillus japonicus β-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the β-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semirational approach yielded a functionally enriched first-round library of 36 single-amino-acid-substitution variants with 58% retaining activity, and of these, 71% displayed improved activities compared to the parent. The substitutions yielding the five most improved variants subsequently were exhaustively combined and evaluated. A four-substitution combination variant was identified as the most improved and reduced the time to completion of an efficient industrial-like reaction by 22%. Characterization of the top five combination variants by isothermal denaturation assays indicated that these variants displayed improved thermostability, with the most thermostable variant displaying a 5.7°C increased melting temperature. The variants displayed uniquely altered, concentration-dependent substrate and product binding as determined by differential scanning fluorimetry. The altered catalytic activity was evidenced by increased specific activities of all five variants, with the most improved variant doubling that of the parent. Variant homology modeling and computational analyses were used to rationalize the effects of amino acid changes lacking direct interaction with substrates. Data indicated that targeting substitutions to loop regions resulted in improved enzyme thermostability, specific activity, and relief from product inhibition.

Overview of parameters influencing biomass and bioreactor performance used for extracellular ligninase production from Phanerochaete chrysosporium

The production of extracellular enzymes is gaining momentum as commercial interests seek alternative ways to improve the productivity in the biotechnology and pharmaceutical industries. Early research studies looked at improving batch bioreactor operational challenges; however, the use of continuous cultures was indicated to be favourable. This led to a new approach developed to produce extracellular enzymes continuously using fixed-film bioreactors from biofilms immobilised on polymeric and inorganic membranes. In this review, the performance of P. chrysosporium biomass, evaluated in terms of ligninase production using different bioreactor operation conditions, is highlighted. Furthermore, the limitations related to the implementation of optimised batch culture conditions to continuous fixed-film bioreactors are discussed. DO transportation, trace element toxicity and lipid peroxidation effects on P. chrysosporium biomass in fixed-film bioreactors operated for elongated periods, are also discussed.

Direct, simultaneous quantification of fructooligosaccharides by FT-MIR ATR spectroscopy and chemometrics for rapid identification of superior, engineered β-fructofuranosidases

AbstractFructooligosaccharides (FOS) are popular components of functional foods produced by the enzymatic transfer of fructose units to sucrose. Improving β-fructofuranosidase traits by protein engineering is restricted by the absence of a rapid, direct screening method for the fructooligosaccharide products produced by enzyme variants. The use of standard high-performance liquid chromatography (HPLC) methods involves time-consuming sample preparation and chromatographic and data analysis steps. To overcome these limitations, this work presents a rapid method for screening β-fructofuranosidase variant libraries using Fourier transform mid-infrared attenuated total reflectance (FT-MIR ATR) spectroscopy and calibration using partial least squares (PLS) regression. The method offers notable improvements in terms of sample analysis times and cost, with the added benefit of the absence of toxic eluents. Wavenumber interval selection methods were tested to develop optimised PLS regression models that were successfully applied to quantify of glucose, fructose, sucrose, 1-kestose and nystose, the substrates and products of β-fructofuranosidase activity. To the best of our knowledge, this is the first report on the use of infrared spectroscopy and PLS calibration for the quantification of 1-kestose and nystose. Independent test set-validated results indicated that optimal wavenumber selection by interval PLS (iPLS) served to provide the best models for all sugars, bar glucose. Application of this screening method will facilitate the engineering of β-fructofuranosidases and other glycosyltransferase enzymes by random mutagenesis strategies, as it provides, for the first time, a rapid, direct assay for transferase products that may be adapted to a high-throughput set-up. Graphical Abstractᅟ

Limitations of a membrane gradostat bioreactor designed for enzyme production from biofilms of Phanerochaete chrysosporium

Growing interest has been shown in the continuous production of high-value products such as extracellular secondary metabolites used in the biotechnology, bioremediation and pharmaceutical industries. These high-value extracellular secondary metabolites are mostly produced in submerged fermentations. However, the use of continuous membrane bioreactors was determined to be highly productive. A novel membrane bioreactor, classified as a membrane gradostat reactor (MGR) was developed to immobilize biofilms to produce extracellular secondary metabolites continuously using an externally unskinned and internally skinned membrane. Anaerobic zones were identified in the MGR system when air was used for aeration. To improve the MGR system, limitations related to the performance of the bioreactor were determined using P. chrysosporium. A DO penetration depth of +/-450 microm was identified after 264 h, with the anaerobic zone thickness reaching approximately 1,943 microm in the immobilised biofilms. The penetration ratio, decreased from 0.42 after 72 h to 0.14 after 264 h. This led to the production of ethanol in the range of 10 to 56 mg/L in the MCMGR and 7 to 54 mg/L in SCMGR systems. This was attributed to an increase in beta-glucan within immobilised biofilms when an oxygen enriched aeration source was used. Increasing lipid peroxidation and trace element accumulation was observed with the use of an oxygen enriched aeration source.

Proteome response of two natural strains of Saccharomyces cerevisiae with divergent lignocellulosic inhibitor stress tolerance

Strains of Saccharomyces cerevisiae with improved tolerance to plant hydrolysates are of utmost importance for the cost-competitive production of value-added chemicals and fuels. However, engineering strategies are constrained by a lack of understanding of the yeast response to complex inhibitor mixtures. Natural S. cerevisiae isolates display niche-specific phenotypic and metabolic diversity, encoded in their DNA, which has evolved to overcome external stresses, utilise available resources and ultimately thrive in their challenging environments. Industrial and laboratory strains, however, lack these adaptations due to domestication. Natural strains can serve as a valuable resource to mitigate engineering constraints by studying the molecular mechanisms involved in phenotypic variance and instruct future industrial strain improvement to lignocellulosic hydrolysates. We, therefore, investigated the proteomic changes between two natural S. cerevisiae isolates when exposed to a lignocellulosic inhibitor mixture. Comparative shotgun proteomics revealed that isolates respond by regulating a similar core set of proteins in response to inhibitor stress. Furthermore, superior tolerance was linked to NAD(P)/H and energy homeostasis, concurrent with inhibitor and reactive oxygen species detoxification processes. We present several candidate proteins within the redox homeostasis and energy management cellular processes as possible targets for future modification and study. Data are available via ProteomeXchange with identifier PXD010868.