Cristina Hernández‐Rollán

Technical University of Denmark

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Morten H. H. Nørholm

Technical University of Denmark

Andreas Birk Bertelsen

Technical University of Denmark

Maja Rennig

Novo Nordisk Foundation

Kristoffer Bach Falkenberg

Technical University of Denmark

Katja S. Johansen

University of Copenhagen

Søren Brander

University of Copenhagen

JØ

Johan Ø. Ipsen

University of Copenhagen

Leila Lo Leggio

University of Copenhagen

Tanveer S. Batth

University of Copenhagen

Jesper V. Olsen

University of Copenhagen

Cooperative Institutions

University of Copenhagen

Technical University of Denmark

Novo Nordisk Foundation

Foundation Center

Aarhus University

Chalmers University of Technology

University of Cambridge

IT University of Copenhagen

Danish National Research Foundation

Capital Region of Denmark

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LyGo cloning v1

Kristoffer Bach Falkenberg Cristina Hernández‐Rollán Maja Rennig Andreas Birk Bertelsen Morten H. H. Nørholm

Lytic polysaccharide monooxygenses (LPMOs) are enzymes that play a critical role in breaking the chemical bonds of the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. LyGo cloning (Lytic Polysaccharide Monooxygenase Golden Gate cloning) is a versatile heterologous expression platform for LPMOs, which is compatible with cloning both PCR products and synthetic gene fragments with a simple 15-minute assembly step. The method allows for parallel construction of multiple expression vectors, enabling exploration of several expression strategies. The open-source LyGo collection consists of vectors for some of the most relevant model organisms used for protein production in both academic and industrial settings. This protocol describes how to clone LyGo fragments into LyGo vectors.

Cloning (programming)

Lytic cycle

Heterologous

Heterologous expression

clone (Java method)

Cloning vector

10.17504/protocols.io.bdmqi45w

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Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding

IUCrJ (2022)

Tobias Tandrup Sebastian J. Muderspach Sanchari Banerjee Gianluca Santoni Johan Ø. Ipsen

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes capable of degrading polysaccharide substrates oxidatively. The generally accepted first step in the LPMO reaction is the reduction of the active-site metal ion from Cu 2+ to Cu + . Here we have used a systematic diffraction data collection method to monitor structural changes in two AA9 LPMOs, one from Lentinus similis ( Ls AA9_A) and one from Thermoascus aurantiacus ( Ta AA9_A), as the active-site Cu is photoreduced in the X-ray beam. For Ls AA9_A, the protein produced in two different recombinant systems was crystallized to probe the effect of post-translational modifications and different crystallization conditions on the active site and metal photoreduction. We can recommend that crystallographic studies of AA9 LPMOs wishing to address the Cu 2+ form use a total X-ray dose below 3 × 10 4 Gy, while the Cu + form can be attained using 1 × 10 6 Gy. In all cases, we observe the transition from a hexacoordinated Cu site with two solvent-facing ligands to a T-shaped geometry with no exogenous ligands, and a clear increase of the θ 2 parameter and a decrease of the θ 3 parameter by averages of 9.2° and 8.4°, respectively, but also a slight increase in θ T . Thus, the θ 2 and θ 3 parameters are helpful diagnostics for the oxidation state of the metal in a His-brace protein. On binding of cello-oligosaccharides to Ls AA9_A, regardless of the production source, the θ T parameter increases, making the Cu site less planar, while the active-site Tyr—Cu distance decreases reproducibly for the Cu 2+ form. Thus, the θ T increase found on copper reduction may bring Ls AA9_A closer to an oligosaccharide-bound state and contribute to the observed higher affinity of reduced Ls AA9_A for cellulosic substrates.

Lytic cycle

10.1107/s2052252522007175

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A seven-transmembrane methyltransferase catalysing N-terminal histidine methylation of lytic polysaccharide monooxygenases

Research Square (Research Square) (2022)

Tanveer S. Batth Jonas Simonsen Cristina Hernández‐Rollán Søren Brander Jens Preben Morth

Abstract 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.

Protein methylation

Lytic cycle

Aspergillus nidulans

10.21203/rs.3.rs-2289157/v1

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Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli v1

Cristina Hernández‐Rollán Kristoffer Bach Falkenberg Maja Rennig Andreas Birk Bertelsen Morten H. H. Nørholm

E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available. This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs. The protocol is adapted from Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. .

Lytic cycle

10.17504/protocols.io.bdr2i58e

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Protein expression on the surface of Escherichia coli v1

Maja Rennig Kristoffer Bach Falkenberg Cristina Hernández‐Rollán Andreas Birk Bertelsen Morten H. H. Nørholm

Display of proteins on the bacterial cell surface has always been an attractive technique for the production of functional cell-anchored proteins, thereby reducing the cost, time and effort related to enzyme purification. Thus, surface display can enable fast and easy screening of protein libraries, e.g. activity variants, or screening of different enzyme substrates, while maintaining a connection between the phenotype and the genotype. Additionally, cells displaying functional enzymes can potentially be used as whole-cell catalysts. This protocol describes how to express proteins on the surface of E. coli BL21 (DE3) using a L-rhamnose inducible expression system.

Surface protein

10.17504/protocols.io.bfh4jj8w

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Protein expressionof hard-to-produce proteins in the cytoplasm of Escherichia coli v1

Cristina Hernández‐Rollán Kristoffer Bach Falkenberg Maja Rennig Andreas Birk Bertelsen Morten H. H. Nørholm

This protocol describes the expression and extraction of heterologously cytoplasmically expressed proteins in the bacterium E.coli, for T7 based expression systems. Specifically, it describes how to produce "hard-to-produce", as for example, LPMOs. It is based on a modified version of: "IPTG Induction and Extraction of Proteins from Bacteria" by Swathi Arur and Sudhir Nayak, Schedl Lab. Washington University Genetics, St. Louis. Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. The extraction of cytoplasmatic proteins is followed by a chemical lysis protocol, alternatively, sonication can also be employed.

Lytic cycle

Protein purification

10.17504/protocols.io.bfbwjipe

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Transformation of heterolous DNA in Bacillus subtilis v1

Kristoffer Bach Falkenberg Cristina Hernández‐Rollán Maja Rennig Andreas Birk Bertelsen Morten H. H. Nørholm

B. subtilis is a gram-positive bacteria used by both academia and industry as a protein production workhorse. This is due to its' excellent fermentation properties, high production titers, and capacity to secrete proteins into the extracellular medium. This protocol describes transformation of B. subtilis by natural competence. The method utilizes the natural stress-induced competence of B. subtilis to take up heterologous DNA. The protocol requires the cells to be grown for a specific amount of time in starvation media (SM). The protocol is adapted from Vojcic, L., Despotovic, D., Martinez, R., Maurer, K., & Schwaneberg, U. (2012). An efficient transformation method for Bacillus subtilis DB104. Applied Microbiology and Biotechnology, 94(2), 487–493. https://doi.org/10.1007/s00253-012-3987-2.

Heterologous

10.17504/protocols.io.bdmti46n

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Epigenetics: An overview

Cristina Hernández‐Rollán

Source

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LyGo protocol collection v1

Kristoffer Bach Falkenberg Maja Rennig Cristina Hernández‐Rollán Andreas Birk Bertelsen Morten H. H. Nørholm

10.17504/protocols.io.9nvh5e6

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Protonation State of an Important Histidine from High Resolution Structures of Lytic Polysaccharide Monooxygenases

Biomolecules (2022)

Sanchari Banerjee Sebastian J. Muderspach Tobias Tandrup Kristian E. H. Frandsen Raushan Kumar Singh

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the Nε2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

Glycosidic bond

10.3390/biom12020194

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Citations (25)