Naoki Sunagawa

Structure of bacterial cellulose synthase subunit D octamer with four inner passageways

The cellulose synthesizing terminal complex consisting of subunits A, B, C, and D in Acetobacter xylinum spans the outer and inner cell membranes to synthesize and extrude glucan chains, which are assembled into subelementary fibrils and further into a ribbon. We determined the structures of subunit D (AxCeSD/AxBcsD) with both N- and C-terminal His6 tags, and in complex with cellopentaose. The structure of AxCeSD shows an exquisite cylinder shape (height: ∼65 Å, outer diameter: ∼90 Å, and inner diameter: ∼25 Å) with a right-hand twisted dimer interface on the cylinder wall, formed by octamer as a functional unit. All N termini of the octamer are positioned inside the AxCeSD cylinder and create four passageways. The location of cellopentaoses in the complex structure suggests that four glucan chains are extruded individually through their own passageway along the dimer interface in a twisted manner. The complex structure also shows that the N-terminal loop, especially residue Lys6, seems to be important for cellulose production, as confirmed by in vivo assay using mutant cells with axcesD gene disruption and N-terminus truncation. Taking all results together, a model of the bacterial terminal complex is discussed.

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum , tend to have few LPMOs and information on these enzymes is scarce. The genome of G. trabeum encodes four AA9 LPMOs, whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of Gt LPMO9A seem to be produced, a single domain variant, Gt LPMO9A-1, and a longer variant, Gt LPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct Gt LPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses, using HPAEC-PAD and MS, showed that Gt LPMO9A-2 is active on cellulose, carboxymethylcellulose and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, Gt LPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of Gt LPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, Nc LPMO9C from Neurospora crassa , revealed that Gt LPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurments also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of Nc LPMO9C. Experiments with co-polymeric substrates showed an inhibitory effect of hemicellulose-coating on cellulolytic LPMO activity and did not reveal additional activities of Gt LPMO9A-2. These results provide insight into the LPMO-potential of G. trabeum and provide a novel sensitive method, measurement of dynamic viscosity, for monitoring LPMO activity. Importance Currently, there are only a few methods available to analyze end-products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here we present an alternative and sensitive method based on measurement of dynamic viscosity, for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses such as xyloglucan. We have used both this novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown rot fungus. This enzyme, Gt LPMO9A-2, differs from previously characterized LPMOs, in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. Gt LPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibres together. The xyloglucan-degrading potential of Gt LPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown-rot, through degradation of the primary cell wall.ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.

Cellulose production by Enterobacter sp. CJF-002 and identification of genes for cellulose biosynthesis

Enterobacter sp. CJF-002, which had been isolated as a cellulose producer with saccharides as a carbon source, was shown to efficiently produce cellulose from beet molasses (B-Mol) and biodiesel fuel by-product (BDF-B), renewable non-edible and inexpensive biomasses. The cellulose production rates of Enterobacter sp. CJF-002 using B-Mol and BDF-B as carbon sources were faster than those of Acetobacter xylinum (A. xylinum) ATCC23769, a representative cellulose producing bacterium. To clarify the biosynthetic machinery of cellulose in the strain, genes responsible for cellulose biosynthesis were cloned. Six open reading frames (ORFs) were suggested to be clustered and their amino acid sequences had high similarities with those of BcsA, BcsB, BcsZ (endoglucanase), BcsC, YhjQ, and YhjK from Escherichia coli, respectively. Of these, the former four genes showed low similarities to corresponding orthologs in a cellulose biosynthetic gene cluster of A. xylinum. A bcsC-knockout mutant produced no cellulose, confirming that the gene is essential for cellulose production of Enterobacter sp. CJF-002. The predicted three-dimensional structure of BcsZEn from Enterobacter sp. CJF-002 had high similarity with that of CMCax (endoglucanase) from A. xylinum ATCC23769 in spite of the low similarity in their amino acid sequences. Taken together, A. xylinum and Enterobacter sp. CJF-002 might produce cellulose via a similar synthetic mechanism.

The plant cell-wall enzyme AtXTH3 catalyses covalent cross-linking between cellulose and cello-oligosaccharide

Cellulose is an economically important material, but routes of its industrial processing have not been fully explored. The plant cell wall – the major source of cellulose – harbours enzymes of the xyloglucan endotransglucosylase/hydrolase (XTH) family. This class of enzymes is unique in that it is capable of elongating polysaccharide chains without the requirement for activated nucleotide sugars (e.g., UDP-glucose) and in seamlessly splitting and reconnecting chains of xyloglucan, a naturally occurring soluble analogue of cellulose. Here, we show that a recombinant version of AtXTH3, a thus far uncharacterized member of the Arabidopsis XTH family, catalysed the transglycosylation between cellulose and cello-oligosaccharide, between cellulose and xyloglucan-oligosaccharide, and between xyloglucan and xyloglucan-oligosaccharide, with the highest reaction rate observed for the latter reaction. In addition, this enzyme formed cellulose-like insoluble material from a soluble cello-oligosaccharide in the absence of additional substrates. This newly found activity (designated “cellulose endotransglucosylase,” or CET) can potentially be involved in the formation of covalent linkages between cellulose microfibrils in the plant cell wall. It can also comprise a new route of industrial cellulose functionalization.

A Novel Pyrroloquinoline Quinone-Dependent 2-Keto-d-Glucose Dehydrogenase from Pseudomonas aureofaciens

A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-d-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl2 were added. (1)H nuclear magnetic resonance ((1)H-NMR) analysis of reaction products revealed 2-keto-d-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.

Characterization of an LPMO from the brown-rot fungus Gloeophyllum trabeum with broad xyloglucan specificity, and its action on cellulose-xyloglucan complexes

Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum , tend to have few LPMOs and information on these enzymes is scarce. The genome of G. trabeum encodes four AA9 LPMOs, whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of Gt LPMO9A seem to be produced, a single domain variant, Gt LPMO9A-1, and a longer variant, Gt LPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct Gt LPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses, using HPAEC-PAD and MS, showed that Gt LPMO9A-2 is active on cellulose, carboxymethylcellulose and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, Gt LPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of Gt LPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, Nc LPMO9C from Neurospora crassa , revealed that Gt LPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurments also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of Nc LPMO9C. Experiments with co-polymeric substrates showed an inhibitory effect of hemicellulose-coating on cellulolytic LPMO activity and did not reveal additional activities of Gt LPMO9A-2. These results provide insight into the LPMO-potential of G. trabeum and provide a novel sensitive method, measurement of dynamic viscosity, for monitoring LPMO activity. Importance Currently, there are only a few methods available to analyze end-products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here we present an alternative and sensitive method based on measurement of dynamic viscosity, for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses such as xyloglucan. We have used both this novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown rot fungus. This enzyme, Gt LPMO9A-2, differs from previously characterized LPMOs, in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. Gt LPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibres together. The xyloglucan-degrading potential of Gt LPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown-rot, through degradation of the primary cell wall.ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.

Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris.

BackgroundRandom mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule. Error-prone PCR is often employed for random mutagenesis in bacterial protein expression systems, but has rarely been used in the methylotrophic yeast Pichia pastoris system, despite its significant advantages, mainly because large (μg-level) amounts of plasmids are required for transformation.ResultsWe developed a quick and easy technique for random mutagenesis in P. pastoris by sequential Phi29 DNA polymerase-based amplification methods, error-prone rolling circle amplification (RCA) and multiple displacement amplification (MDA). The methodology was validated by applying it for random mutation of the gene encoding cellulase from the basidiomycete Phanerochaete chrysosporium (PcCel6A), a key enzyme in degradation of cellulosic biomass. In the error-prone RCA step, the concentrations of manganese ion (Mn2+) and cellulase gene-containing plasmid were varied, and the products obtained under each condition were subjected to the second MDA step in the absence of Mn2+. The maximum error rate was 2.6 mutations/kb, as evaluated from the results of large-scale sequencing. Several μg of MDA products was transformed by electroporation into Pichia cells, and the activities of extracellularly expressed PcCel6A mutants towards crystalline and amorphous celluloses were compared with those of wild-type enzyme to identify key amino acid residues affecting degradation of crystalline cellulose.ConclusionsWe present a rapid and convenient random mutagenesis method that does not require laborious steps such as ligation, cloning, and synthesis of specific primers. This method was successfully applied to the protein expression system in P. pastoris.

Kojima, Y., Várnai, A., Ishida, T., Sunagawa, N., Petrovic, D. M., Igarashi, K., ... & Eijsink, V. G. (2016). A lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus Gloeophyllum trabeum and its action on cellulose-xyloglucan complexes. Applied and environmental microbiology, 82(22), 6557-6572.

Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum , tend to have few LPMOs and information on these enzymes is scarce. The genome of G. trabeum encodes four AA9 LPMOs, whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of Gt LPMO9A seem to be produced, a single domain variant, Gt LPMO9A-1, and a longer variant, Gt LPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct Gt LPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses, using HPAEC-PAD and MS, showed that Gt LPMO9A-2 is active on cellulose, carboxymethylcellulose and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, Gt LPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of Gt LPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, Nc LPMO9C from Neurospora crassa , revealed that Gt LPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurments also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of Nc LPMO9C. Experiments with co-polymeric substrates showed an inhibitory effect of hemicellulose-coating on cellulolytic LPMO activity and did not reveal additional activities of Gt LPMO9A-2. These results provide insight into the LPMO-potential of G. trabeum and provide a novel sensitive method, measurement of dynamic viscosity, for monitoring LPMO activity. Importance Currently, there are only a few methods available to analyze end-products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here we present an alternative and sensitive method based on measurement of dynamic viscosity, for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses such as xyloglucan. We have used both this novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown rot fungus. This enzyme, Gt LPMO9A-2, differs from previously characterized LPMOs, in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. Gt LPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibres together. The xyloglucan-degrading potential of Gt LPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown-rot, through degradation of the primary cell wall.ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.