Carbohydrate-active enzymes that modify the cell wall of Aspergillus niger:Biochemical properties and physiological functions during autolysis and differentiation

De schimmel Aspergillus niger wordt ingezet voor industriele productie van eiwitten en organische zuren. Schimmels zoals A. niger groeien door draad-achtige structuren te maken, welke worden omgeven door celwanden. Deze celwand wordt voortdurend veranderd doordat koolhydraat-actieve enzymen de koolhydraten in de celwand produceren, veranderen en afbreken. De focus van dit proefschrift ligt op de fysiologische rollen en biochemische kenmerken van koolhydraat-actieve enzymen die worden gemaakt gedurende koolstof-uithongering. In tijden van voedselschaarste kan de schimmel zijn eerder opgeslagen energie en koolstof – bijvoorbeeld in de celwand - hergebruiken om stress-resistente sporen te produceren. Er is in dit promotieonderzoek gekeken welke genen die coderen voor koolhydraat-actieve enzymen worden geactiveerd gedurende koolstof uithongering, en wat de invloed hierop is van twee belangrijke regulatoren. Twee van de geidentificeerde enzymen zijn vervolgens biochemisch gekarakteriseerd, chitinase CfcA en α-1,3-glucanase AgnB. Dit chitinase heeft eem rol in de celwand afbraak gedurende koolstofuithongering. Daarnaast is een set genen gevonden, coderen voor koolhydraat-actieve enzymen, die specifiek geactiveerd worden zodra de schimmel sporen maakt. Dit waren voornamelijk enzymen die voorspeld waren actief te zijn op de celwand koolhydraten chitine en β-1,3-glucan, onder andere de chitinases CfcI en CtcB. Biochemisch gekarakterisatie van chitinase CfcI liet zien dat het een voor schimmelchitinases nieuwe activiteit heeft; CfcI breekt korte chitine ketens af door er de suikermonomeer af te knippen. Een duidelijk begrip van de werking van deze enzymen zorgt voor een goede basis voor het verbeteren van enzymen en maakt potentieel het verbeteren van industriële groei van schimmels mogelijk

Transcriptional regulation and responses in filamentous fungi exposed to lignocellulose

Biofuels derived from lignocellulose are attractive alternative fuels but their production suffers from a costly and inefficient saccharification step that uses fungal enzymes. One route to improve this efficiency is to understand better the transcriptional regulation and responses of filamentous fungi to lignocellulose. Sensing and initial contact of the fungus with lignocellulose is an important aspect. Differences and similarities in the responses of fungi to different lignocellulosic substrates can partly be explained with existing understanding of several key regulators and their mode of action, as will be demonstrated for Trichoderma reesei, Neurospora crassa and Aspergillus spp. The regulation of genes encoding Carbohydrate Active enZymes (CAZymes) is influenced by the presence of carbohydrate monomers and short oligosaccharides, as well as the external stimuli of pH and light. We explore several important aspects of the response to lignocellulose that are not related to genes encoding CAZymes, namely the regulation of transporters, accessory proteins and stress responses. The regulation of gene expression is examined from the perspective of mixed cultures and models are presented for the nature of the transcriptional basis for any beneficial effects of such mixed cultures. Various applications in biofuel technology are based on manipulating transcriptional regulation and learning from fungal responses to lignocelluloses. Here we critically access the application of fungal transcriptional responses to industrial saccharification reactions. As part of this chapter, selected regulatory mechanisms are also explored in more detail

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose.

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production.ResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.ConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism

Enzymatic synthesis of N-acetyllactosamine from lactose enabled by recombinant β1,4-galactosyltransferases

Utilising a fast and sensitive screening method based on imidazolium-tagged probes, we report unprecedented reversible activity of bacterial β1,4-galactosyltransferases to catalyse the transgalactosylation from lactose to N-acetylglucosamine to form N-acetyllactosamine in the presence of UDP. The process is demonstrated by the preparative scale synthesis of pNP-β-LacNAc from lactose using β1,4-galactosyltransferase NmLgtB-B as the only biocatalyst

Transcriptomic responses of mixed cultures of ascomycete fungi to lignocellulose using dual RNA-seq reveal inter-species antagonism and limited beneficial effects on CAZyme expression

Gaining new knowledge through fungal monoculture responses to lignocellulose is a widely used approach that can lead to better cocktails for lignocellulose saccharification (the enzymatic release of sugars which are subsequently used to make biofuels). However, responses in lignocellulose mixed cultures are rarely studied in the same detail even though in nature fungi often degrade lignocellulose as mixed communities. Using a dual RNA-seq approach, we describe the first study of the transcriptional responses of wild-type strains of Aspergillus niger, Trichoderma reesei and Penicillium chrysogenum in two and three mixed species shake-flask cultures with wheat straw. Based on quantification of species-specific rRNA, a set of conditions was identified where mixed cultures could be sampled so as to obtain sufficient RNA-seq reads for analysis from each species. The number of differentially-expressed genes varied from a couple of thousand to fewer than one hundred. The proportion of carbohydrate active enzyme (CAZy) encoding transcripts was lower in the majority of the mixed cultures compared to the respective straw monocultures. A small subset of P. chrysogenum CAZy genes showed five to ten-fold significantly increased transcript abundance in a two-species mixed culture with T. reesei. However, a substantial number of T. reesei CAZy transcripts showed reduced abundance in mixed cultures. The highly induced genes in mixed cultures indicated that fungal antagonism was a major part of the mixed cultures. In line with this, secondary metabolite producing gene clusters showed increased transcript abundance in mixed cultures and also mixed cultures with T. reesei led to a decrease in the mycelial biomass of A. niger. Significantly higher monomeric sugar release from straw was only measured using a minority of the mixed culture filtrates and there was no overall improvement. This study demonstrates fungal interaction with changes in transcripts, enzyme activities and biomass in the mixed cultures and whilst there were minor beneficial effects for CAZy transcripts and activities, the competitive interaction between T. reesei and the other fungi was the most prominent feature of this study

Construction of arsB and tetH Mutants of the Sulfur-Oxidizing Bacterium Acidithiobacillus caldus by Marker Exchange▿

Acidithiobacillus caldus is a moderately thermophilic, acidophilic bacterium that has been reported to be the dominant sulfur oxidizer in stirred-tank processes used to treat gold-bearing arsenopyrite ores. It is also widely distributed in heap reactors used for the extraction of metals from ores. Not only are these bacteria commercially important, they have an interesting physiology, the study of which has been restricted by the nonavailability of defined mutants. A recently reported conjugation system based on the broad-host-range IncW plasmids pSa and R388 was used to transfer mobilizable narrow-host-range suicide plasmid vectors containing inactivated and partially deleted chromosomal genes from Escherichia coli to A. caldus. Through the dual use of a selectable kanamycin resistance gene and a hybridization probe made from a deleted portion of the target chromosomal gene, single- and double-recombinant mutants of A. caldus were isolated. The functionality of the gene inactivation system was shown by the construction of A. caldus arsB and tetH mutants, and the effects of these mutations on cell growth in the presence of arsenic and by means of tetrathionate oxidation were demonstrated

Recent advances in enzymatic synthesis of β-glucan and cellulose

Bottom-up synthesis of β-glucans such as callose, fungal β-(1,3)(1,6)-glucan and cellulose, can create the defined compounds that are needed to perform fundamental studies on glucan properties and develop applications. With the importance of β-glucans and cellulose in high-profile fields such as nutrition, renewables-based biotechnology and materials science, the enzymatic synthesis of such relevant carbohydrates and their derivatives has attracted much attention. Here we review recent developments in enzymatic synthesis of β-glucans and cellulose, with a focus on progress made over the last five years. We cover the different types of biocatalysts employed, their incorporation in cascades, the exploitation of enzyme promiscuity and their engineering, and reaction conditions affecting the production as well as in situ self-assembly of (non)functionalised glucans. The recent achievements in the application of glycosyl transferases and β-1,4- and β-1,3-glucan phosphorylases demonstrate the high potential and versatility of these biocatalysts in glucan synthesis in both industrial and academic contexts