LyGo: A platform for rapid screening of lytic polysaccharide monooxygenase production

Hernández-Rollán, Cristina; Falkenberg, Kristoffer Bach; Rennig, Maja; Bertelsen, Andreas Birk; Ipsen, Johan Ø.; Brander, Søren; Daley, Daniel O.; Johansen, Katja Salomon; Nørholm, Morten H. H.

doi:10.1101/2020.11.04.368555

Cited by 5 publications

(7 citation statements)

References 86 publications

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“…The LPMOs were heterologously expressed and purified as described previously (Simmons et al ., 2017; Tokin et al ., 2020). Tf LPMO10A (Kruer‐Zerhusen et al ., 2017) from Thermobifida fusca was expressed and purified as described elsewhere (Hernández‐Rollán et al ., 2020). Pa AA9E and Pa AA9H from Podospora anserina were cloned as described previously (Bennati‐Granier et al ., 2015) and were produced in a bioreactor and purified as described elsewhere (Filiatrault‐Chastel et al ., 2019).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of lytic polysaccharide monooxygenase by natural plant extracts

et al. 2021

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Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes of industrial and biological importance. In particular, LPMOs play important roles in fungal lifestyle. No inhibitors of LPMOs have yet been reported.In this study, a diverse library of 100 plant extracts was screened for LPMO activitymodulating effects. By employing protein crystallography and LC-MS, we successfully identified a natural LPMO inhibitor.Extract screening revealed a significant LPMO inhibition by methanolic extract of Cinnamomum cassia (cinnamon), which inhibited LsAA9A LPMO from Lentinus similis in a concentration-dependent manner. With a notable exception, other microbial LPMOs from families AA9 and AA10 were also inhibited by this cinnamon extract. The polyphenol cinnamtannin B1 was identified as the inhibitory component by crystallography. Cinnamtannin B1 was bound to the surface of LsAA9A at two distinct binding sites: one close to the active site and another at a pocket on the opposite side of the protein. Independent characterization of cinnamon extract by LC-MS and subsequent activity measurements confirmed that the compound inhibiting LsAA9A was cinnamtannin B1.The results of this study show that specific natural LPMO inhibitors of plant origin exist in nature, providing the opportunity for future exploitation of such compounds within various biotechnological contexts.

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Section: Methodsmentioning

confidence: 99%

Inhibition of lytic polysaccharide monooxygenase by natural plant extracts

et al. 2021

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“…Fragments constructed by PCR were made as described previously (22) and CRISPR-Cas9 vectors were assembled by USER fusion (45). The vectors for K. phaffii expression were constructed by LyGo cloning as described in Hernández-Rollán et al (36), and by USER fusion as described previously (46). Primers and synthetic gene fragments were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA), and are listed in Tables S6.…”

Section: Dna Fragment and Plasmid Constructions In A Nidulans And K P...mentioning

confidence: 99%

A membrane integral methyltransferase catalysing N-terminal histidine methylation of lytic polysaccharide monooxygenases

Batth

Simonsen

Hernández-Rollán

et al. 2022

Preprint

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Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in biotechnological purposes. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identified the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663. Targeted proteomics confirmed that NHMT was solely responsible for His1 methylation of LPMOs. NHMT is predicted to encode a unique seven-transmembrane segment anchoring a soluble methyltransferase domain. Co-localization studies showed endoplasmic reticulum residence of NHMT and co-expression in the industrial production yeast Komagataella phaffii with LPMOs resulted in His1 methylation of the LPMOs. This demonstrates the biotechnological potential of recombinant production of proteins and peptides harbouring this unique post-translational modification.

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“…Expression was induced at mid-late exponential phase with 1 mM IPTG and cells were harvested 20 h after induction. The periplasmic fraction was isolated as described previously [22], and filtered using a 0.22 μm filter (Frisenette, Knebel, Denmark). The periplasmic fraction was diluted in 20 mM Tris buffer at pH 8.3, and loaded onto a Q Sepharose column (GE Healthcare, Chicago, IL, USA).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…In total, 15 amino acid mutants were generated from PfCopC using uracil excision cloning (see Methods). Proteins were expressed heterologously in the Escherichia coli periplasm, followed by purification using conventional column chromatography [22]. The effects of the introduced mutants were investigated by measuring ascorbate turnover and copper-binding properties.…”

Section: Mutants Designed To Model His-braces Found In Naturementioning

confidence: 99%

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Copper binding and reactivity at the histidine brace motif: insights from mutational analysis of the Pseudomonas fluorescens copper chaperone CopC

et al. 2021

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The histidine brace (His-brace) is a copper-binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical and structural methods to characterize mutants of a Hisbrace-containing copper chaperone from Pseudomonas fluorescens (PfCopC). A total of 15 amino acid variants in primary and second-sphere residues were produced and characterized in terms of their copper binding and redox properties. PfCopC has a very high affinity for Cu(II) and also binds Cu(I). A high reorganization barrier likely prevents redox cycling and, thus, catalysis. In contrast, mutations in the conserved second-sphere Glu27 enable slow oxidation of ascorbate. The crystal structure of the variant E27A confirmed copper binding at the His-brace. Unexpectedly, Asp83 at the equatorial position was shown to be indispensable for Cu(II) binding in the His-brace of PfCopC. A PfCopC mutant that was designed to mimic the His-brace from lytic polysaccharide monooxygenase-like family X325 did not bind Cu(II), but was still able to bind Cu(I). These results highlight the importance of the proteinaceous environment around the copper His-brace for reactivity and, thus, the difference between enzyme and chaperone.

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LyGo: A platform for rapid screening of lytic polysaccharide monooxygenase production

Cited by 5 publications

References 86 publications

Inhibition of lytic polysaccharide monooxygenase by natural plant extracts

Inhibition of lytic polysaccharide monooxygenase by natural plant extracts

A membrane integral methyltransferase catalysing N-terminal histidine methylation of lytic polysaccharide monooxygenases

Copper binding and reactivity at the histidine brace motif: insights from mutational analysis of the Pseudomonas fluorescens copper chaperone CopC

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