C-type cytochrome-initiated reduction of bacterial lytic polysaccharide monooxygenases.

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanized new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyze a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria.

A Multiple-Technique Approach to Inferring Bio-electrochemical Reaction Parameters

Uncovering the secrets of the biological Faradaic reactions, essential to the understanding of complex metalloenzymes, requires an information recovery process that is robust, rapid and replicable. This paper is a description of the workflow we have developed over the course of inferring chemical reaction parameters for a simple protein system, a bacterial cytochrome domain from \textit{Cellvibrio japonicus}. This was a challenging task, as the signal-to-noise ratio in such protein-film voltammetry experiments is significantly lowered relative to the voltammetric data generated by simple chemicals. We have overcome these challenges by using a multiple-technique approach, which compensates for the difficulties inherent to analysis of the individual voltammetry experiments. We have shown that the parameters inferred are robust across multiple experiments performed for different preperations of the protein. This is an important proof-of-concept result for analysis of more complex metalloenzymes, which incorporate catalytic processes and multiple internal electron-transfer sites. <br>

A Multiple-Technique Approach to Inferring Bio-electrochemical Reaction Parameters

Uncovering the secrets of the biological Faradaic reactions, essential to the understanding of complex metalloenzymes, requires an information recovery process that is robust, rapid and replicable. This paper is a description of the workflow we have developed over the course of inferring chemical reaction parameters for a simple protein system, a bacterial cytochrome domain from \textit{Cellvibrio japonicus}. This was a challenging task, as the signal-to-noise ratio in such protein-film voltammetry experiments is significantly lowered relative to the voltammetric data generated by simple chemicals. We have overcome these challenges by using a multiple-technique approach, which compensates for the difficulties inherent to analysis of the individual voltammetry experiments. We have shown that the parameters inferred are robust across multiple experiments performed for different preperations of the protein. This is an important proof-of-concept result for analysis of more complex metalloenzymes, which incorporate catalytic processes and multiple internal electron-transfer sites. <br>

Characterization of a galactosyl-binding protein module from a Cellvibrio japonicus endo-xyloglucanase defines a new family of Carbohydrate Binding Modules

Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, e.g. by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is valuable source for CAZyme and CBM discovery and characterization, due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (“X181”), and a Glycoside Hydrolase Family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase [Attia et al. 2018. Biotechnol. Biofuels, 11: 45]. In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest product of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes.Importance This study reveals Carbohydrate Binding Module Family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the Carbohydrate-Active Enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.

Trehalose Degradation by Cellvibrio japonicus Exhibits No Functional Redundancy and Is Solely Dependent on the Tre37A Enzyme

ABSTRACT The α-diglucoside trehalose has historically been known as a component of the bacterial stress response, though it more recently has been studied for its relevance in human gut health and biotechnology development. The utilization of trehalose as a nutrient source by bacteria relies on carbohydrate-active enzymes, specifically those of the glycoside hydrolase family 37 (GH37), to degrade the disaccharide into substituent glucose moieties for entry into metabolism. Environmental bacteria using oligosaccharides for nutrients often possess multiple carbohydrate-active enzymes predicted to have the same biochemical activity and therefore are thought to be functionally redundant. In this study, we characterized trehalose degradation by the biotechnologically important saprophytic bacterium Cellvibrio japonicus. This bacterium possesses two predicted α-α-trehalase genes, tre37A and tre37B, and our investigation using mutational analysis found that only the former is essential for trehalose utilization by C. japonicus. Heterologous expression experiments found that only the expression of the C. japonicus tre37A gene in an Escherichia coli treA mutant strain allowed for full utilization of trehalose. Biochemical characterization of C. japonicus GH37 activity determined that the tre37A gene product is solely responsible for cleaving trehalose and is an acidic α-α-trehalase. Bioinformatic and mutational analyses indicate that Tre37A directly cleaves trehalose to glucose in the periplasm, as C. japonicus does not possess a phosphotransferase system. This study facilitates the development of a comprehensive metabolic model for α-linked disaccharides in C. japonicus and more broadly expands our understanding of the strategies that saprophytic bacteria employ to capture diverse carbohydrates from the environment. IMPORTANCE The metabolism of trehalose is becoming increasingly important due to the inclusion of this α-diglucoside in a number of foods and its prevalence in the environment. Bacteria able to utilize trehalose in the human gut possess a competitive advantage, as do saprophytic microbes in terrestrial environments. While the biochemical mechanism of trehalose degradation is well understood, what is less clear is how bacteria acquire this metabolite from the environment. The significance of this report is that by using the model saprophyte Cellvibrio japonicus, we were able to functionally characterize the two predicted trehalase enzymes that the bacterium possesses and determined that the two enzymes are not equivalent and are not functionally redundant. The results and approaches used to understand the complex physiology of α-diglucoside metabolism from this study can be applied broadly to other polysaccharide-degrading bacteria.

Complete Genome Sequences of Cellvibrio japonicus Strains with Improved Growth When Using α-Diglucosides

Cellvibrio japonicus is a saprophytic bacterium that has been studied for its substantial carbohydrate degradation capability. We announce the genome sequences of three strains with improved growth characteristics when utilizing α-diglucosides. These data provide additional insight into the metabolic flexibility of a biotechnologically relevant bacterium.

Enzyme-Catalyzed Production of Potato Galactan-Oligosaccharides and Its Optimization by Response Surface Methodology

This work shows an optimized enzymatic hydrolysis of high molecular weight potato galactan yielding pectic galactan-oligosaccharides (PGOs), where endo-β-1,4-galactanase (galactanase) from Cellvibrio japonicus and Clostridium thermocellum was used. For this, response surface methodology (RSM) by central composite design (CCD) was applied. The parameters varied were temperature (°C), pH, incubation time (min), and enzyme/substrate ratio (U/mg). The optimized conditions for the production of low degree of polymerization (DP) PGOs were obtained for each enzyme by spectrophotometric assay and confirmed by chromatography. The optimal conditions predicted for the use of C. japonicus galactanase to obtain PGOs of DP = 2 were T = 51.8 °C, pH 5, E/S = 0.508 U/mg, and t = 77.5 min. For DP = 3, they were T = 21 °C, pH 9, E/S = 0.484 U/mg, and t = 12.5 min; and for DP = 4, they were T = 21 °C, pH 5, E/S = 0.462 U/mg, and t = 12.5 min. The efficiency results were 51.3% for substrate hydrolysis. C. thermocellum galactanase had a lower yield (35.7%) and optimized conditions predicted for PGOs of DP = 2 were T = 60 °C, pH 5, E/S = 0.525 U/mg, and time = 148 min; DP = 3 were T = 59.7 °C, pH 5, E/S = 0.506 U/mg, and time = 12.5 min; and DP = 4, were T = 34.5 °C, pH 11, E/S = 0.525 U/mg, and time = 222.5 min. Fourier transformed infrared (FT-IR) and nuclear magnetic resonance (NMR) characterizations of PGOs are presented.