van Munster, J. M. | Search bioRxiv

bioRxiv2020

In vitro biosynthesis of poly-β-1,4-glucan derivatives using a promiscuous glycosyltransferase

The {beta}-1,4-glucose linkage of cellulose is the most abundant polymeric linkage on earth and as such is of considerable interest in biology and biotechnology. It remains challenging to synthesize this linkage in vitro due to a lack of suitable biocatalysts; the natural cellulose biosynthetic machinery is a membrane-associated complex with processive activity that cannot be easily manipulated to synthesize tailor-made oligosaccharides and their derivatives. Here we identify a promiscuous activity of a soluble recombinant biocatalyst, Neisseria meningitidis glycosyltransferase LgtB, suitable for the polymerization of glucose from UDP-glucose via the generation of {beta}-1,4-glycosidic linkages. We employed LgtB to synthesize natural and derivatized cello-oligosaccharides and we demonstrate how LgtB can be incorporated in biocatalytic cascades and chemo-enzymatic strategies to synthesize cello-oligosaccharides with tailored functionalities. We also show how the resulting glycan structures can be applied as chemical probes to report on activity and selectivity of plant cell wall degrading enzymes, including lytic polysaccharide monooxygenases. We anticipate that this biocatalytic approach to derivatized cello-oligosaccharides via glucose polymerization will open up new applications in biology and nanobiotechnology.

biochemistry↗

bioRxiv2019

Succession of physiological stages hallmarks the transcriptomic response of 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 Aspergilllus niger over its life time to six substrates important for biofuel production.\n\nResultsWe 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.\n\nConclusionIn 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.

microbiology↗