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Evidence for ligninolytic activity of the ascomycete fungus Podospora anserina
Erven, Gijs Van; Kleijn, Anne ; Patyshakuliyeva, Aleksandrina ; Falco, Marcos Di; Tsang, Adrian ; Vries, Ronald P. De; Berkel, Willem J.H. Van; Kabel, Mirjam A. - \ 2020
Wageningen University & Research
Biomass - Enzymes - Lignin - Laccase - py-GC-MS - NMR spectroscopy - Proteomics - Secretomics
Background The ascomycete fungus Podospora anserina has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether P. anserina might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome. Results Quantitative 13C lignin internal standard py-GC–MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted (58% w/w removal). Structural characterization of residual lignin by using py-GC–MS and HSQC NMR analyses demonstrated that Cα-oxidized substructures significantly increased through fungal action, while intact β-O-4′ aryl ether linkages, p-coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of P. anserina. This was particularly reflected in a strong reduction of cellulases and galactomannanases, while H2O2-producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis. Conclusions For the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus P. anserina and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologies by contributing to the quest for novel lignin-active enzymes and organisms.
Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence
Tong, Yapei ; Trajkovic, Milos ; Savino, Simone ; Berkel, Willem J.H. van; Fraaije, Marco W. - \ 2020
Journal of Biological Chemistry 295 (2020)47. - ISSN 0021-9258 - p. 16013 - 16022.
Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.
|Vanillyl alcohol oxidase-catalysed production of (R)-1-(4′-hydroxyphenyl) ethanol
Ewing, T.A. ; Berkel, W.J.H. van - \ 2020
In: Applied Biocatalysis / Whittall, John, Sutton, Peter W., Wiley - ISBN 9781119487012 - p. 312 - 319.
The introduction of a chiral centre into a non-chiral starting material is hard to achieve using traditional methods of chemical synthesis. Nevertheless, it is very important to obtain efficient methods by which to do so, as pure enantiomers of chiral compounds are of great importance to e.g. the pharmaceutical industry. One way in which chirality may be created is through the application of enzymes that are capable of enantioselectively functionalising a target molecule. For example, the flavin-dependent oxidoreductase vanillyl alcohol oxidase (VAO) can catalyse the enantioselective hydroxylation of 4-alkylphenols at the Cα position, leading to the formation of the (R)-enantiomer of the corresponding alcohol in high enantiomeric excess. To demonstrate that this enzyme can be used to produce chiral secondary alcohols on a synthetically relevant scale, we employed it in the enantioselective hydroxylation of the non-chiral aromatic compound 4-ethylphenol to yield (R)-1-(4′-hydroxyphenyl)ethanol. The protocol described here provides a detailed practical description of how this synthesis can be performed at a scale of 10 g of starting material.
Vanillyl alcohol oxidase
Ewing, Tom A. ; Gygli, Gudrun ; Fraaije, Marco W. ; Berkel, Willem J.H. van - \ 2020
In: Enzymes Academic Press (Enzymes )
Catalytic mechanism - Covalent flavinylation - Crystal structure - Enantioselectivity - Enzyme dynamics - Flavoprotein family - Lignin and phenols - Oxidase - Phylogenetics - Suicide inhibition
This review presents a historical outline of the research on vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum, one of the canonical members of the VAO/PCMH flavoprotein family. After describing its discovery and initial biochemical characterization, we discuss the physiological role, substrate scope, and catalytic mechanism of VAO, and review its three-dimensional structure and mechanism of covalent flavinylation. We also explain how protein engineering provided a deeper insight into the role of certain amino acid residues in determining the substrate specificity and enantioselectivity of the enzyme. Finally, we summarize recent computational studies about the migration of substrates and products through the enzyme's structure and the phylogenetic distribution of VAO and related enzymes.
Configuration of active site segments in lytic polysaccharide monooxygenases steers oxidative xyloglucan degradation
Sun, Peicheng ; Laurent, Christophe V.F.P. ; Scheiblbrandner, Stefan ; Frommhagen, Matthias ; Kouzounis, Dimitrios ; Sanders, Mark G. ; Berkel, Willem J.H. van; Ludwig, Roland ; Kabel, Mirjam A. - \ 2020
Biotechnology for Biofuels 13 (2020)1. - ISSN 1754-6834 - 19 p.
AA9 LPMO - Active site segments - Biomass - Biorefinery - Hemicellulose - Lignocellulose - Neurospora crassa - Phylogenetic tree - Plant cell wall - Xyloglucan
Background: Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave plant cell wall polysaccharides. LPMOs classified as fungal Auxiliary Activities family 9 (AA9) have been mainly studied for their activity towards cellulose; however, various members of this AA9 family have been also shown to oxidatively cleave hemicelluloses, in particularly xyloglucan (XG). So far, it has not been studied in detail how various AA9 LPMOs act in XG degradation, and in particular, how the mode-of-action relates to the structural configuration of these LPMOs. Results: Two Neurospora crassa (Nc) LPMOs were found to represent different mode-of-action towards XG. Interestingly, the configuration of active site segments of these LPMOs differed as well, with a shorter Segment 1 (−Seg1) and a longer Segment 2 (+Seg2) present in NcLPMO9C and the opposite for NcLPMO9M (+Seg1−Seg2). We confirmed that NcLPMO9C cleaved the non-reducing end of unbranched glucosyl residues within XG via the oxidation of the C4-carbon. In contrast, we found that the oxidative cleavage of the XG backbone by NcLPMO9M occurred next to both unbranched and substituted glucosyl residues. The latter are decorated with xylosyl, xylosyl-galactosyl and xylosyl-galactosyl-fucosyl units. The relationship between active site segments and the mode-of-action of these NcLPMOs was rationalized by a structure-based phylogenetic analysis of fungal AA9 LPMOs. LPMOs with a −Seg1+Seg2 configuration clustered together and appear to have a similar XG substitution-intolerant cleavage pattern. LPMOs with the +Seg1−Seg2 configuration also clustered together and are reported to display a XG substitution-tolerant cleavage pattern. A third cluster contained LPMOs with a −Seg1−Seg2 configuration and no oxidative XG activity. Conclusions: The detailed characterization of XG degradation products released by LPMOs reveal a correlation between the configuration of active site segments and mode-of-action of LPMOs. In particular, oxidative XG-active LPMOs, which are tolerant and intolerant to XG substitutions are structurally and phylogenetically distinguished from XG-inactive LPMOs. This study contributes to a better understanding of the structure-function relationship of AA9 LPMOs.
Flavin-dependent N-hydroxylating enzymes : distribution and application
Mügge, Carolin ; Heine, Thomas ; Baraibar, Alvaro Gomez ; Berkel, Willem J.H. van; Paul, Caroline E. ; Tischler, Dirk - \ 2020
Applied Microbiology and Biotechnology 104 (2020). - ISSN 0175-7598 - p. 6481 - 6499.
Bioactive compounds - Biocatalysis - Biotransformation - Flavoproteins - Monooxygenases - N-Hydroxylases - Phylogenetics - Siderophores
Amino groups derived from naturally abundant amino acids or (di)amines can be used as “shuttles” in nature for oxygen transfer to provide intermediates or products comprising N-O functional groups such as N-hydroxy, oxazine, isoxazolidine, nitro, nitrone, oxime, C-, S-, or N-nitroso, and azoxy units. To this end, molecular oxygen is activated by flavin, heme, or metal cofactor-containing enzymes and transferred to initially obtain N-hydroxy compounds, which can be further functionalized. In this review, we focus on flavin-dependent N-hydroxylating enzymes, which play a major role in the production of secondary metabolites, such as siderophores or antimicrobial agents. Flavoprotein monooxygenases of higher organisms (among others, in humans) can interact with nitrogen-bearing secondary metabolites or are relevant with respect to detoxification metabolism and are thus of importance to understand potential medical applications. Many enzymes that catalyze N-hydroxylation reactions have specific substrate scopes and others are rather relaxed. The subsequent conversion towards various N-O or N-N comprising molecules is also described. Overall, flavin-dependent N-hydroxylating enzymes can accept amines, diamines, amino acids, amino sugars, and amino aromatic compounds and thus provide access to versatile families of compounds containing the N-O motif. Natural roles as well as synthetic applications are highlighted.• N-O and N-N comprising natural and (semi)synthetic products are highlighted.• Flavin-based NMOs with respect to mechanism, structure, and phylogeny are reviewed.• Applications in natural product formation and synthetic approaches are provided. [Figure not available: see fulltext.].
Evidence for ligninolytic activity of the ascomycete fungus Podospora anserina
Erven, Gijs Van; Kleijn, Anne F. ; Patyshakuliyeva, Aleksandrina ; Falco, Marcos Di; Tsang, Adrian ; Vries, Ronald P. De; Berkel, Willem J.H. Van; Kabel, Mirjam A. - \ 2020
Biotechnology for Biofuels 13 (2020)1. - ISSN 1754-6834
Biomass - Enzymes - Laccase - Lignin - NMR spectroscopy - Proteomics - py-GC-MS - Secretomics
Background: The ascomycete fungus Podospora anserina has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether P. anserina might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome. Results: Quantitative 13C lignin internal standard py-GC-MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted (58% w/w removal). Structural characterization of residual lignin by using py-GC-MS and HSQC NMR analyses demonstrated that Cα-oxidized substructures significantly increased through fungal action, while intact β-O-4′ aryl ether linkages, p-coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of P. anserina. This was particularly reflected in a strong reduction of cellulases and galactomannanases, while H2O2-producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis. Conclusions: For the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus P. anserina and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologies by contributing to the quest for novel lignin-active enzymes and organisms.
Plant Aromatic Prenyltransferases : Tools for Microbial Cell Factories
Bruijn, Wouter J.C. de; Levisson, Mark ; Beekwilder, Jules ; Berkel, Willem J.H. van; Vincken, Jean Paul - \ 2020
Trends in Biotechnology 38 (2020)8. - ISSN 0167-7799 - p. 917 - 934.
bioactive compounds - isoprenoids - microbial cell factories - phytochemicals - prenylation - secondary metabolism
In plants, prenylation of aromatic compounds, such as (iso)flavonoids and stilbenoids, by membrane-bound prenyltransferases (PTs), is an essential step in the biosynthesis of many bioactive compounds. Prenylated aromatic compounds have various health-beneficial properties that are interesting for industrial applications, but their exploitation is limited due to their low abundance in nature. Harnessing plant aromatic PTs for prenylation in microbial cell factories may be a sustainable and economically viable alternative. Limitations in prenylated aromatic compound production have been identified, including availability of prenyl donor substrate. In this review, we summarize the current knowledge about plant aromatic PTs and discuss promising strategies towards the optimized production of prenylated aromatic compounds by microbial cell factories.
Tuning of pKa values activates substrates in flavin-dependent aromatic hydroxylases
Pitsawong, Warintra ; Chenprakhon, Pirom ; Dhammaraj, Taweesak ; Medhanavyn, Dheeradhach ; Sucharitakul, Jeerus ; Tongsook, Chanakan ; Berkel, Willem J.H. van; Chaiyen, Pimchai ; Miller, Anne Frances ; Banerjee, Ruma - \ 2020
Journal of Biological Chemistry 295 (2020)12. - ISSN 0021-9258 - p. 3965 - 3981.
Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19F NMR in conjunction with fluorinated substrate analogs to directly measure pKa values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hy-droxylase (3HB6H) depresses the pKa of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pKa of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C2 component of HPAH. 19F NMR also revealed a pKa of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions. Our implementation of 19F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.
Flavoenzyme-mediated regioselective aromatic hydroxylation with coenzyme biomimetics
Guarneri, Alice ; Westphal, Adrie ; Leertouwer, J. ; Lunsonga, J. ; Franssen, M.C.R. ; Opperman, D.J. ; Hollmann, F. ; Berkel, W.J.H. van; Paul, C.E. - \ 2020
ChemCatChem 12 (2020)5. - ISSN 1867-3880 - p. 1368 - 1375.
Regioselective aromatic hydroxylation is desirable for the production of valuable compounds. External flavin‐containing monooxygenases activate and selectively incorporate an oxygen atom in phenolic compounds through flavin reduction by the nicotinamide adenine dinucleotide coenzyme and subsequent reaction with molecular oxygen. This study provides the proof of principle of flavoenzyme‐catalyzed selective aromatic hydroxylation with coenzyme biomimetics. The carbamoylmethyl‐substituted biomimetic in particular affords full conversion in less than two hours for the selective hydroxylation of 5 mM 3‐ and 4‐hydroxybenzoates, displaying similar rates as with NADH, achieving a 10 mM/h enzymatic conversion of the medicinal product gentisate. This biomimetic appears to generate less uncoupling of hydroxylation that typically leads to undesired hydrogen peroxide. Therefore, we show these flavoenzymes have the potential to be applied in combination with biomimetics.
Mass spectrometric fragmentation patterns discriminate C1- and C4-oxidised cello-oligosaccharides from their non-oxidised and reduced forms
Sun, Peicheng ; Frommhagen, Matthias ; Kleine Haar, Maloe ; Erven, Gijs van; Bakx, Edwin J. ; Berkel, Willem J.H. van; Kabel, Mirjam A. - \ 2020
Carbohydrate Polymers 234 (2020). - ISSN 0144-8617
Biomass conversion - Cello-oligosaccharides - HILIC-ESI-CID-MS/MS - Lignocellulose - LPMOs - Mass spectrometric fragmentation - Oxidation - Reduction
Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that degrade recalcitrant polysaccharides, such as cellulose. However, the identification of LPMO-generated C1- and/or C4-oxidised oligosaccharides is far from straightforward. In particular, their fragmentation patterns have not been well established when using mass spectrometry. Hence, we studied the fragmentation behaviours of non-, C1- and C4-oxidised cello-oligosaccharides, including their sodium borodeuteride-reduced forms, by using hydrophilic interaction chromatography and negative ion mode collision induced dissociation - mass spectrometry. Non-oxidised cello-oligosaccharides showed predominantly C- and A-type cleavages. In comparison, C4-oxidised ones underwent B-/Y- and X-cleavage close to the oxidised non-reducing end, while closer to the reducing end C-/Z- and A-fragmentation predominated. C1-oxidised cello-oligosaccharides showed extensively A-cleavage. Reduced oligosaccharides showed predominant glycosidic bond cleavage, both B-/Y- and C-/Z-, close to the non-reducing end. Our findings provide signature mass spectrometric fragmentation patterns to unambiguously elucidate the catalytic behaviour and classification of LPMOs.
Uniformly 13C Labeled Lignin Internal Standards for Quantitative Pyrolysis-GC-MS Analysis of Grass and Wood
Erven, Gijs Van; Visser, Ries De; Waard, Pieter De; Berkel, Willem J.H. Van; Kabel, Mirjam A. - \ 2019
ACS sustainable chemistry & engineering 7 (2019)24. - ISSN 2168-0485 - p. 20070 - 20076.
biomass - isotope labeling - lignin content - lignin quantification - NMR spectroscopy - py-GC-MS
With the ever-advancing lignocellulose valorization strategies, lignin analyses need to advance as well. However, lignin quantification still heavily relies on unspecific, time- and sample-consuming gravimetric, and spectrophotometric analyses. Here, we demonstrate that lignin isolates from uniformly 13C-labeled wheat straw, willow, and douglas fir serve as "ideal" internal standards for pyrolysis gas chromatography high-resolution mass spectrometry (py-GC-HR-MS) analyses of plant biomass, allowing the accurate and precise quantification and structural characterization of lignin in grasses, hardwoods, and softwoods. The 13C lignin internal standards were comprehensively structurally characterized by HSQC NMR and py-GC-HR-MS analyses, and their application for lignin quantification was validated in biomass model systems and in actual plant biomass. For all botanical origins and species, the lignin content was determined within 5% relative deviation of the Klason benchmark. These results establish the capability of the developed analytical platform to selectively quantify and structurally characterize lignin simultaneously and demonstrate a valuable addition to the lignin analysis toolbox.
Structural Motifs of Wheat Straw Lignin Differ in Susceptibility to Degradation by the White-Rot Fungus Ceriporiopsis subvermispora
Erven, Gijs Van; Wang, Jianli ; Sun, Peicheng ; Waard, Pieter De; Putten, Jacinta Van Der; Frissen, Guus E. ; Gosselink, Richard J.A. ; Zinovyev, Grigory ; Potthast, Antje ; Berkel, Willem J.H. Van; Kabel, Mirjam A. - \ 2019
ACS sustainable chemistry & engineering 7 (2019)24. - ISSN 2168-0485 - p. 20032 - 20042.
biological pretreatment - lignin quantification - ligninolysis - NMR spectroscopy - oxidation - selective delignification - single-electron transfer - stereoselectivity
The white-rot fungus Ceriporiopsis subvermispora delignifies plant biomass extensively and selectively and, therefore, has great biotechnological potential. We previously demonstrated that after 7 weeks of fungal growth on wheat straw 70% w/w of lignin was removed and established the underlying degradation mechanisms via selectively extracted diagnostic substructures. In this work, we fractionated the residual (more intact) lignin and comprehensively characterized the obtained isolates to determine the susceptibility of wheat straw lignin's structural motifs to fungal degradation. Using 13C IS pyrolysis gas chromatography-mass spectrometry (py-GC-MS), heteronuclear single quantum coherence (HSQC) and 31P NMR spectroscopy, and size-exclusion chromatography (SEC) analyses, it was shown that β-O-4′ ethers and the more condensed phenylcoumarans and resinols were equally susceptible to fungal breakdown. Interestingly, for β-O-4′ ether substructures, marked cleavage preferences could be observed: β-O-4′-syringyl substructures were degraded more frequently than their β-O-4′-guaiacyl and β-O-4′-tricin analogues. Furthermore, diastereochemistry (threo > erythro) and γ-acylation (γ-OH > γ-acyl) influenced cleavage susceptibility. These results indicate that electron density of the 4′-O-coupled ring and local steric hindrance are important determinants of oxidative β-O-4′ ether degradation. Our findings provide novel insight into the delignification mechanisms of C. subvermispora and contribute to improving the valorization of lignocellulosic biomass.
Antitumor astins originate from the endophyte Cyanodermella asteris living within the medicinal plant Aster tataricus
Schafhauser, T. ; Jahn, L. ; Kirchner, Norbert ; Kulik, Andreas ; Flor, L. ; Lang, A. ; Caradec, T. ; Fewer, D.P. ; Sivonen, K. ; Berkel, W.J.H. van; Jacques, P. ; Weber, T. ; Gross, H. ; Pée, K.H. van; Wohlleben, W. ; Ludwig-Müller, L. - \ 2019
Proceedings of the National Academy of Sciences of the United States of America 116 (2019)52. - ISSN 0027-8424 - p. 26909 - 26917.
Medicinal plants are a prolific source of natural products with remarkable chemical and biological properties, many of which have considerable remedial benefits. Numerous medicinal plants are suffering from wildcrafting, and thus biotechnological production processes of their natural products are urgently needed. The plant Aster tataricus is widely used in traditional Chinese medicine and contains unique active ingredients named astins. These are macrocyclic peptides showing promising antitumor activities and usually containing the highly unusual moiety 3,4-dichloroproline. The biosynthetic origins of astins are unknown despite being studied for decades. Here we show that astins are produced by the recently discovered fungal endophyte Cyanodermella asteris. We were able to produce astins in reasonable and reproducible amounts using axenic cultures of the endophyte. We identified the biosynthetic gene cluster responsible for astin biosynthesis in the genome of C. asteris and propose a production pathway that is based on a nonribosomal peptide synthetase. Striking differences in the production profiles of endophyte and host plant imply a symbiotic cross-species biosynthesis pathway for astin C derivatives, in which plant enzymes or plant signals are required to trigger the synthesis of plant-exclusive variants such as astin A. Our findings lay the foundation for the sustainable biotechnological production of astins independent from aster plants.
Influence of lytic polysaccharide monooxygenase active site segments on activity and affinity
Laurent, Christophe V.F.P. ; Sun, Peicheng ; Scheiblbrandner, Stefan ; Csarman, Florian ; Cannazza, Pietro ; Frommhagen, Matthias ; Berkel, Willem J.H. van; Oostenbrink, Chris ; Kabel, Mirjam A. ; Ludwig, Roland - \ 2019
International Journal of Molecular Sciences 20 (2019)24. - ISSN 1422-0067
Enzyme engineering - Lytic polysaccharide monooxygenase - Phylogenetic analysis - Regioselectivity - Substrate binding - Substrate specificity
In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO. This study investigates the changes in activity induced by shortening the second active site segment (Seg2) or removing the CBM1 from Neurospora crassa LPMO9C. NcLPMO9C and generated variants have been tested on regenerated amorphous cellulose (RAC), carboxymethyl cellulose (CMC) and xyloglucan (XG) using activity assays, conversion experiments and surface plasmon resonance spectroscopy. The absence of CBM1 reduced the binding affinity and activity of NcLPMO9C, but did not affect its regioselectivity. The linker was found important for the thermal stability of NcLPMO9C and the CBM1 is necessary for efficient binding to RAC. Wild-type NcLPMO9C exhibited the highest activity and strongest substrate binding. Shortening of Seg2 greatly reduced the activity on RAC and CMC and completely abolished the activity on XG. This demonstrates that Seg2 is indispensable for substrate recognition and the formation of productive enzyme-substrate complexes.
Elucidation of in Situ Ligninolysis Mechanisms of the Selective White-Rot Fungus Ceriporiopsis subvermispora
Erven, Gijs van; Hilgers, Roelant ; Waard, Pieter de; Gladbeek, Erik Jan ; Berkel, Willem J.H. van; Kabel, Mirjam A. - \ 2019
ACS sustainable chemistry & engineering 7 (2019)19. - ISSN 2168-0485 - p. 16757 - 16764.
Lignin - NMR spectroscopy - Oxidation - py-GC-MS - Selective delignification
Lignin degradation by white-rot fungi is an essential step in terrestrial carbon cycling and has great potential for biotechnological applications. Selective white-rot fungi have been recognized for their ability to effectively delignify lignocellulose, but the underlying mechanisms, particularly in situ, have largely remained elusive to date. In this work, we elucidate specific degradation routes of β-O-4 aryl ethers in actual lignocellulosic biomass for the industrially relevant selective white-rot fungus Ceriporiopsis subvermispora. Multidimensional NMR and py-GC-MS analyses together with enzymatically synthesized model compounds enabled, for the first time, the identification of various diagnostic lignin cleavage products in residual wheat straw. Our results support that in situ ligninolysis by C. subvermispora is initiated by single-electron transfer, which then cascades into the cleavage of Cα-Cβ, Cβ-O, and O-4-aryl bonds of β-O-4 aryl ethers. The high abundance of 1-(benzyl)-2,3-dihydroxypropan-1-ones indicated that β-O-|4 cleavage is a more important pathway than previously considered. Our approach highlights key diagnostic substructures for providing mechanistic insight into fungal ligninolysis.
Astin C Production by the Endophytic Fungus Cyanodermella asteris in Planktonic and Immobilized Culture Conditions
Vassaux, Antoine ; Tarayre, Cédric ; Arguëlles-Arias, Anthony ; Compère, Philippe ; Delvigne, Frank ; Fickers, Patrick ; Jahn, Linda ; Lang, Alexander ; Leclère, Valérie ; Ludwig-Müller, Jutta ; Ongena, Marc ; Schafhauser, Thomas ; Telek, Samuel ; Théatre, Ariane ; Berkel, Willem J.H. van; Vandenbol, Micheline ; Pée, Karl Heinz van; Willems, Luc ; Wohlleben, Wolfgang ; Jacques, Philippe - \ 2019
Biotechnology Journal 14 (2019)8. - ISSN 1860-6768
astin C - biofilms - Cyanodermella asteris - immobilized-cell cultures - secondary metabolites
The fungal endophyte Cyanodermella asteris (C. asteris) has been recently isolated from the medicinal plant Aster tataricus (A. tataricus). This fungus produces astin C, a cyclic pentapeptide with anticancer and anti-inflammatory properties. The production of this secondary metabolite is compared in immobilized and planktonic conditions. For immobilized cultures, a stainless steel packing immersed in the culture broth is used as a support. In these conditions, the fungus exclusively grows on the packing, which provides a considerable advantage for astin C recovery and purification. C. asteris metabolism is different according to the culture conditions in terms of substrate consumption rate, cell growth, and astin C production. Immobilized-cell cultures yield a 30% increase of astin C production, associated with a 39% increase in biomass. The inoculum type as spores rather than hyphae, and a pre-inoculation washing procedure with sodium hydroxide, turns out to be beneficial both for astin C production and fungus development onto the support. Finally, the influence of culture parameters such as pH and medium composition on astin C production is evaluated. With optimized culture conditions, astin C yield is further improved reaching a five times higher final specific yield compared to the value reported with astin C extraction from A. tataricus (0.89 mg g−1 and 0.16 mg g−1 respectively).
Editorial: Actinobacteria, a source of biocatalytic tools
Tischler, Dirk ; Berkel, Willem J.H. Van; Fraaije, Marco W. - \ 2019
Frontiers in Microbiology 10 (2019). - ISSN 1664-302X
Actinomycetes - Biocatalysis - Biotechnology - Extremophile actinobacteria - Germination - High GC genetics - Novel biocatalysts - Secondary metabolites
Dimerization of Proline Dehydrogenase from Thermus thermophilus Is Crucial for Its Thermostability
Huijbers, Mieke M.E. ; Wu, Jenny W. ; Westphal, Adrie H. ; Berkel, Willem J.H. van - \ 2019
Biotechnology Journal 14 (2019)5. - ISSN 1860-6768
flavoprotein - protein oligomerization - thermostability - Thermus thermophilus - triosephosphate isomerase barrel
Thermus thermophilus proline dehydrogenase (TtProDH) catalyzes the first step in proline catabolism. The thermostable flavoenzyme consists of a distorted triosephosphate isomerase (TIM) barrel and three N-terminal helices: αA, αB, and αC. Using maltose-binding protein (MBP) fused constructs, it has been recently demonstrated that helix αC is crucial for TtProDH catalysis and for tetramerization through positioning of helix α8. Here, the structural features that determine the thermostability of TtProDH are reported. Selective disruption of two ion pairs in the dimerization interface of several MBP-TtProDH variants result in the formation of monomers. The newly created monomers have improved catalytic properties but their melting temperatures are decreased by more than 20 °C. Sequence comparison suggests that one of the ion-pairs involved in dimerization is unique for ProDHs from Thermus species. In summary, intermolecular ion-pairs improve the thermostability of TtProDH and a trade-off is made between thermostability and catalytic activity.
Hydrocarbon Synthesis via Photoenzymatic Decarboxylation of Carboxylic Acids
Zhang, Wuyuan ; Ma, Ming ; Huijbers, Mieke M.E. ; Filonenko, Georgy A. ; Pidko, Evgeny A. ; Schie, Morten van; Boer, Sabrina de; Burek, Bastien O. ; Bloh, Jonathan Z. ; Berkel, Willem J.H. van; Smith, Wilson A. ; Hollmann, Frank - \ 2019
Journal of the American Chemical Society 141 (2019)7. - ISSN 0002-7863 - p. 3116 - 3120.
A recently discovered photodecarboxylase from Chlorella variabilis NC64A ( CvFAP) bears the promise for the efficient and selective synthesis of hydrocarbons from carboxylic acids. CvFAP, however, exhibits a clear preference for long-chain fatty acids thereby limiting its broad applicability. In this contribution, we demonstrate that the decoy molecule approach enables conversion of a broad range of carboxylic acids by filling up the vacant substrate access channel of the photodecarboxylase. These results not only demonstrate a practical application of a unique, photoactivated enzyme but also pave the way to selective production of short-chain alkanes from waste carboxylic acids under mild reaction conditions.