Accessory Activities 3: Novel Flavoprotein Oxidoreductase from bacterial sources
SUPERVISOR: Clemens PETERBAUER
Lignocellulose is the main component of woody plants´ cell wall and has evolved to fulfil mechanical requirements as well as protect plants against herbivores and saprobionts (Janusz et al. 2017 and references therein). Studies on lignin degradation have focused on white-rot and brown-rot fungi so far (Bugg et al. 2011 and references therein). In recent years, a number of bacteria were observed to be able to degrade and metabolize lignin components and derivatives as well. It is not well understood if bacterial lignin depolymerization follows pathways comparable to those from white rot fungi. Genome sequencing projects have revealed the presence of genes hypothetically annotated as lignin modifying enzymes across a range of mostly α- and γ-Proteobacteria and Actinobacteria (Cragg et al. 2015; Janusz et al. 2017). Besides laccase genes, many genes annotated as peroxidases (putatively dye-decolorizing peroxidases, catalase-peroxidases or versatile peroxidases rather than lignin or manganese peroxidases) were identified in Actinobacteria genomes (Brown et al. 2012; Brown and Chang 2014).
This suggests that such bacteria should also contain auxiliary enzymes such as aryl alcohol oxidase, glucose dehydrogenase, pyranose oxidase or cellobiose dehydrogenase (Janusz et al., 2017). These enzymes are implicated to be involved in lignin degradation by providing hydrogen peroxide for activation of peroxidases and for reduction during redox cycling of quinones (Martinez et al. 2005; de Gonzalo et al. 2016). All belong to the GMC oxidoreductase family of flavoproteins. Pyranose oxidase catalyzes the oxidation of aldopyranoses to 2-ketoaldoses and hydrogen peroxide, but most pyranose oxidases also use benzoquinone as electron acceptor, often with a higher efficiency than oxygen (Leitner et al. 2001; Pisanelli et al. 2009; Salaheddin et al. 2010). Pyranose dehydrogenase oxidizes mono- and disaccharides and uses only (substituted) quinones or complexed metal ions as electron acceptors (Peterbauer and Volc 2010). Cellobiose dehydrogenases additionally contain a heme domain (Zamocky et al. 2014) and play a major role in oxidative cellulose degradation by transferring electrons to Lytic Polysaccharide Monoxygenases (LPMO; Kracher et al. 2016). Genome data reveal a number of genes putatively encoding such enzymes in bacteria, however, biochemical information is almost non-existent. There is a single report of a bacterial pyranose 2-oxidase, from Pseudarthrobacter siccitolerans. The recombinantly expressed enzyme is a 64-kDa monomer with an FAD cofactor that oxidizes D-glucose, oxygen and 1,4-benzoquinone act as an electron acceptor (Mendes et al. 2016). Another pyranose oxidase (from Kitasatospora aureofaciens) was recently expressed in E. coli and characterized (Herzog, Sützl and Peterbauer, manuscript in press, DOI: 10.1128/AEM.00390-19).
Aims and methods.
Many Actinomycetes contain genes putatively encoding peroxidases, laccases and a peroxide-providing system like pyranose oxidases (Brown and Chang 2014). We propose to select a limited number of Actinomycete model organisms with sequenced genomes containing genes encoding peroxidases, laccases and carbohydrate oxidoreductases and that are capable of degradation of lignin or lignin derivatives, namely from the genera Streptomyces, Amycolatopsis and Kitasatospora, and characterize their auxiliary enzyme system (Auxiliary Activities Family 3). A key point will be the subcellular location of the enzyme(s).
Our investigation will include interactions with other enzymes to be tested in vitro, namely the activation of peroxidases through hydrogen peroxide (or another mechanism). In case enzymes are encountered that fulfil the structural requirements for activation of LPMOs (like cellobiose dehydrogenases, which have not been shown to be present in bacteria), the interaction between these enzymes will be investigated as well.
We further propose to study the inactivation of encoding genes and a phenotypical assessment for degradation of lignin in at least one model strain, to establish whether carbohydrate-oxidizing flavoproteins and similar auxiliary activities are involved in lignin modification and degradation. Unlike fungi, bacteria are less likely to contain genes or gene families encoding enzymes with identical, complementary or overlapping activities. This will make investigations into the biological role of individual enzymes more straight-forward, as a phenotypic effect can be expected upon inactivation of a single gene.
3.1. Expression and characterization of bacterial pyranose oxidoreductases
DNA sequences encoding putative pyranose oxidases and related enzymes from Actinomycetes will be synthesized by commercial service providers. We will use established platforms like pET-21 for E. coli, as has been published for fungal enzymes (Pisanelli et al. 2009). We will, however, consider using gram-positive secretory expression systems based on Bacillus spp. or Streptomyces spp. (Anne et al. 2014), if sequence analysis suggests that the target genes encode secretory proteins, as this will more closely resemble the natural situation as well as facilitate downstream processing. Recombinant proteins will be expressed containing a His6 tag for easy purification based on metal affinity chromatography (IMAC). The enzymes will be biochemically characterized with respect to electron donor (carbohydrate substrate), electron acceptor and basic kinetic characteristics (e.g., Pisanelli et al. 2009; Salaheddin et al. 2010; Brugger et al. 2014).
3.2. Transcription and location of GMC oxidoreductases
Pyranose oxidases often do not contain recognizable signal peptides and their subcellular localization is unclear, even though activation of extracellular peroxidases through hydrogen peroxide suggests an extracellular location. We will cultivate selected species on glucose, cellulose (derivatives), lignocellulose and lignin model substrates and determine the expression profiles of peroxidase and oxidoreductase genes, including LPMO genes, in order to assess the biological role of individual enzymes. Previously obtained biochemical data will allow us to establish the localization of the enzymes in the original organisms, as enzymatic assays can be performed on the supernatant, the cell pellet fraction and cell free extracts after homogenization.
Streptomycetes are amenable to genetic manipulation (Anne et al. 2014). We will use established protocols to express the respective genes in the original organism as a cell fusion with a fluorescent marker, e.g., the Green Fluorescent Protein, and detect the localization during growth on lignin-containing substrates in vivo.
3.3. Interaction with other enzymes
Peroxidase activation, which happens indirectly through hydrogen peroxide, will be studied in vitro using purified enzymes with appropriate controls (addition of catalase) as was shown for lignin and manganese peroxidases. LPMOs were shown to be activated through interdomain electron transfer from cellobiose dehydrogenases (Courtade et al. 2016, Kracher and Scheiblbrandner et al. 2017). Available information to date does not suggest that such enzymes are present in bacteria. However, activation of LPMO has been shown through unspecific reduction by plant phenolic compounds and lignin fractions, as well as by plant-derived diphenols or quinones acting as redox mediators (Kracher and Scheiblbrandner et al. 2017). This could be a link between lignin and cellulose degradation, and we will investigate bacterial oxidoreductases if they can fulfil that role. Lastly there is the hypothesis that LPMO activity depends on hydrogen peroxide as a required co-substrate (Bissaro et al. 2017), which could point to yet another function for carbohydrate active oxidoreductases in the context of biomacromolecule degradation.
3.4. Functional genomic approach
In order to complement the biochemical data generated in the first work packages we will utilize the bacterial propensity for small and parsimonious genomes and their – relative – lack of multigenicity. The highly versatile CRISPR/Cas9 system for genome editing has been established in Streptomyces spp. (Cobb et al. 2015). This will allow us to selectively inactivate/knock out genes encoding GMC-oxidoreductases singly and in combination in much shorter time and with less effort than previously using integrative knock-out-cassettes, and study the phenotypic consequences particularly with respect to growth on lignocellulosic substrates, production of characteristic lignin degradation products and depolymerization of crystalline cellulose: depending on the hypothesis, knock-out of a (all) hydrogen peroxide-producing oxidoreductases should result in a lack of peroxidase activation and lignin degradation, perhaps also in a lowered LPMO reduction for cellulose attack and reduced capacity to degrade crystalline cellulose fractions. This approach will shed light on the particularly enigmatic role of GMC-oxidoreductases during lignocellulose depolymerization.
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