Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium

Russo, David A.; Zedler, Julie A Z; Wittmann, Daniel; Möllers, Benedikt Kb; Singh, Raushan Kumar; Batth, Tanveer S.; Oort, Bart van; Olsen, Jesper V.; Bjerrum, Morten J.; Jensen, Poul Erik

doi:10.1186/s13068-019-1416-9

Cited by 26 publications

(31 citation statements)

References 64 publications

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“…cDNA encoding the AA10 modules, including the native SEC secretion signal peptide, of Cfi LPMO10, Cfla LPMO10A, Cfla LPMO10B, and Cfla LPMO10C, was cloned from genomic DNA with the addition of a C-terminal His 6 purification tag and transformed into E. coli for expression. Secretion of LPMO proteins through the SEC pathway in E. coli has previously resulted in accumulation of recombinant protein in the periplasm [ 91 , 92 ] and the culture medium [ 93 , 94 ], with the former necessitating periplasmic fractionation. We observed that the majority of the recombinant Cellulomonas LPMOs were found in the media, with negligible amounts in the periplasm.…”

Section: Resultsmentioning

confidence: 99%

Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

Solhi

Goddard‐Borger

et al. 2021

Biotechnol Biofuels

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Background The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Results Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Conclusions Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.

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Section: Resultsmentioning

confidence: 99%

Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

Solhi

Goddard‐Borger

et al. 2021

Biotechnol Biofuels

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“…Bacterial LPMOs are usually overexpressed in the periplasm of Escherichia coli [33,45,49]. However, other strategies for AA10 LPMO production in the cytoplasm of E. coli with a cleavable N-terminal tag [17,19,20] or as secreted protein using Gram-positive bacteria (Brevibacillus choshinensis SP3 or Bacillus subtilis) [18,50], cyanobacteria (Synechococcus elongatus UTEX 2973) [51], or yeast (Pichia pastoris) [52] as expression host have also been successfully developed. Using E. coli as recombinant expression system, we chose the N-terminal pelB signal peptide to direct PlAA10 production to the periplasm.…”

Section: Plaa10 Binds Copper Similarly To Other Chitin-active Lpmosmentioning

confidence: 99%

Characterization of a bacterial copper‐dependent lytic polysaccharide monooxygenase with an unusual second coordination sphere

et al. 2020

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Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes involved in the degradation of recalcitrant polysaccharides such as cellulose or chitin. LPMOs act in synergy with glycoside hydrolases such as cellulases and chitinases by oxidatively cleaving a number of glycosidic bonds at the surface of their crystalline substrate(s). Besides their role in biomass degradation, some bacterial LPMOs have been found to be virulence factors in some human and insect pathogens. Photorhabdus luminescens is a nematode symbiont bacterium that is pathogenic to a wide range of insects. A single gene encoding a LPMO is found in its genome.In this work, we report the characterization of this LPMO, referred to as PlAA10. Surprisingly, PlAA10 lacks the conserved alanine residue (substituted by an isoleucine) found in the second coordination sphere of the copper active site in bacterial LPMOs. PlAA10 was found to be catalytically active on both α-and β-chitin, and exhibits a C1-oxidation regiospecificity, similarly to other chitin-active LPMOs. The 1.6 Å X-ray crystal structure confirmed that PlAA10 adopts the canonical immunoglobulin-like fold typical for LPMOs. The geometry of the copper active site is not affected by the nearby isoleucine, as also supported by electron paramagnetic resonance. Nevertheless, the bulkier side chain of isoleucine protrudes from the substrate-binding surface. A bioinformatic study on putative bacterial LPMOs unveiled that they exhibit some variability at the conserved active site alanine position with a substitution in about 15% of all sequences analyzed. Author contributionCD designed the research and coordinated the project. Molecular biology (cloning) was done by AM and CD. AM and BEK expressed and purified the protein. BEK performed fluorescence experiments. AM and AJS recorded EPR spectra. AJS simulated the EPR spectra. AM and BEK ran activity assays on chitin. MF performed mass spectrometry experiments. MF, HR, and DR analyzed mass spectrometry data. CD and BEK crystallized the protein. AR collected diffraction data at the synchrotron. CD and AR solved the crystal structure. Bioinformatic analysis was carried out by AM and CD. All the data were compiled and analyzed by CD, JS, and MR. All authors contributed to the writing and approved the final version of the manuscript.Acknowledgements CNRS and Aix Marseille Université are acknowledged for their financial support. AM is a recipient of a French Ministère de l'Enseignement Supérieur et de la Recherche PhD fellowship. The ESRF is acknowledged for access to beamline ID29 via its in-house research program.

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“…In our previous study, we demonstrated the secretion of the LPMO TfAA10A from T. fusca in the cyanobacterium S. elongatus UTEX 2973 (24). TfAA10A is a copper-containing enzyme with a complex catalytic cycle not suitable for high-throughput analysis.…”

Section: Nluc Is Efficiently Secreted In Synechocystis Sp Pcc 6803mentioning

confidence: 99%

“…Previously we have shown that the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 can secrete the heterologously expressed enzyme TfAA10A, a lytic polysaccharide monooxygenase (LPMO) from the gram-positive bacterium Thermobifida fusca. TfAA10A was secreted via a two-step process with the cleavage of its N-terminal Sec signal peptide occurring when crossing the cytoplasmic membrane (24). To address whether the T4aPS can secrete non-pilin proteins, we developed a NanoLuc luciferase (NLuc)-based secretion reporter harboring the TfAA10A signal peptide and genetically transferred the reporter into a collection of cyanobacterial T4aPS mutants.…”

Section: Introductionmentioning

confidence: 99%

Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria

et al. 2022

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Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multi-protein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however little is known regarding their function, regulation and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of non-pilin proteins, we developed a NanoLuc-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. PCC 6803. The NLuc reporter presented a wide dynamic range with at least one order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to explore protein secretion in cyanobacteria further. Importance Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here we introduce a NanoLuc (NLuc)-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge on cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

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Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium

Cited by 26 publications

References 64 publications

Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

Characterization of a bacterial copper‐dependent lytic polysaccharide monooxygenase with an unusual second coordination sphere

Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria

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