Degradation of Ester Linkages in Rice Straw Components By

Ohta Yukari (Orcid ID: 0000-0001-5645-2311) Degradation of ester linkages in rice straw components by Sphingobium species recovered from the sea bottom using a non- secretory tannase-family α/β hydrolase Yukari Ohta1, Madoka Katsumata1, Kanako Kurosawa2, Yoshihiro Takaki2, Hiroshi Nishimura3, Takashi Watanabe3, Ken-ichi Kasuya1,4 1 Gunma University Center for Food Science and Wellness, 4-2 Aramaki, Maebashi, Gunma 371-8510, Japan 2 Super-cutting-edge Grand and Advanced Research Program, JAMSTEC, 2-15, Natsushima, Yokosuka, Kanagawa 237-0061, Japan 3 Biomass Conversion, Research Institute for Sustainable Humanosphere, Kyoto University Gokasho, Uji, Kyoto 611-0011, Japan 4 Division of Molecular Science, Faculty of Science and Technology, Gunma University, 1-5-1 Tenjin, Kiryu, Gunma 376-8515, Japan Correspondence to: Yukari Ohta Gunma University Center for Food Science and Wellness, Gunma University, 4-2 Aramaki, Maebashi, Gunma 371-8510, Japan Telephone: +81-27-220-7641 E-mail: [email protected] Running title: Non-secretory α/β hydrolase for plant degradation This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.15551 This article is protected by copyright. All rights reserved. Originality-significance statement We show that an unrecognized non-secretory hydrolase of the Alphaproteobacteria strain functions as a ferulic acid esterase. Before our study, the gene for the enzyme was annotated as mono(2-hydroxyethyl) terephthalic acid hydrolase (MHETase). The MHETase is involved in the microbial biodegradation pathway of polyethylene terephthalate. This study demonstrated that the enzyme cleaved hydroxycinnamic acid esters, such as ferulic, p-coumaric esters but did not degrade mono(2-hydroxyethyl) terephthalate (MHET). Further, we showed that the enzyme was responsible for critical phenotypic features of the strain for degrading terrestrial plants in an aquatic environment. The findings are critically important to understand the global carbon cycle and assess the biodegradability of commodity plastics incidentally imported into aquatic environments. Also, the sunken wood used for screening of the biomass-degrading bacteria in this study was retrieved from the sea bottom in that region has been seriously influenced by the huge inputs of terrestrial organic materials from the Tohoku earthquake and the tsunami in 2011. This study uncovers the molecular bases of microbial actions on recalcitrant terrestrial plant components, increasing impact on global carbon cycle and climate change and environmental disturbances, such as heavy rains, typhoons, and discharge of anthropogenic harmful chemicals. Summary Microbial decomposition of allochthonous plant components imported into the aquatic environment is one of the vital steps of the carbon cycle on earth. To expand the knowledge of the biodegradation of complex plant materials in aquatic environments, we recovered a sunken wood from the bottom of Otsuchi Bay, situated in northeastern Japan in 2012. We isolated Sphingobium with high ferulic acid esterase activity. The strain, designated as OW59, grew on various aromatic compounds and sugars, occurring naturally in terrestrial plants. A genomic study of the strain suggested its role in degrading hemicelluloses. We identified a gene encoding a non-secretory tannase-family α/β hydrolase, which exhibited ferulic acid esterase activity. This enzyme shares the consensus catalytic triad (Ser-His-Asp) within the tannase family block X in the ESTHER database. The molecules, which had the same calculated elemental compositions, were produced consistently in both the enzymatic and microbial degradation of rice straw crude extracts. The non-secretory tannase-family α/β hydrolase activity may confer an important phenotypic feature on the strain to accelerate plant biomass degradation. Our study provides insights into the underlying biodegradation process of terrestrial plant polymers in aquatic environments. Introduction The components of terrestrial plants mainly comprise three polymers: cellulose, hemicellulose, and lignin. These components are associated with each other, have different degrees of modification, and function as physical and chemical barriers of degradation. Ferulic, p- coumaric, and p-hydroxybenzoic acids are the frequently detected hydroxycinnamic acids (HACs) in grass plants. The HACs incorporated into polysaccharides (Wong, 2006; Karlen et al. 2016; Iiyama et al., 1994) and lignin (Hatfield et al., 1999; Regner et al., 2018) provide plants with persistency (Hartley et al. 1972; Grabber et al., 2009; de Oliveira et al., 2015. Despite their recalcitrance, an array of microbial enzymes has evolved to efficiently degrade plant biomasses (Guerriero et al., 2015). Decomposition of plant biomass is accelerated by treatment with HAC esterases, including ferulic acid esterases (Faes) (Wong, 2006, Faulds et al., 2010). Currently, dozens of Faes have been identified and characterized at the protein level. These enzymes release ferulic acid and p-coumaric acid from aquatic slurry or plant biomass extracts (Wong, 2006; Mathew and Abraham, 2004). The major part consists of enzymes from fungal strains belonging to the genera, such as Anaeromyces, Aspergillus, Neurospora, and Thermothelomyces and several bacterial strains of Cellulosilyticum, Cellvibrio, Prevotella, and Ruminiclostridium. In an earlier study (Crepin et al., 2004), Faes were classified into ABCD types based on substrate specificity. Hundreds of putative Fae sequences were deposited in databases, such as CAZy (Levasseur et al., 2013) and the ESTHER database (Lenfant et al., 2013) of the α/β hydrolase fold superfamily of proteins (Marchot and Chatonnet, 2012). In the ESTHER database, Faes are classified into three groups within block X, namely, A85-feruloyl- esterase, FaeC, and tannase families. A85-feruloyl-esterase is closely related to the CAZy CE family 1(Makela et al., 2018). The two families’ enzymes can hydrolyze the cross-links between plant polysaccharide chains releasing the diferulic acids (Dilokpimol et al., 2016). The FaeC group proteins often have non-catalytic domains, such as cellulose biding domains or Ricin B lectin motifs, and hydrolyze the feruloyl esters linked to the sugar moieties, which is usually arabinose in 'natural' substrates. The tannase-family enzymes show diverse substrate specificity for not only galloyl and feruloyl esters but also terephthalic acid monoester with ethylene glycol (mono(2-hydroxyethyl) terephthalic acid/MHTE) (Palm et al., 2019). Furthermore, more than 1000 of computationally predicted Faes are predicted from fungal genome sequences by database mining and divided into 13 subfamilies, including uncharacterized subfamilies (Banerjee et al., 2012; Underlin et al., 2020). These studies suggested the plausible potential of the discovery of new Faes for degrading complex natural substrates. Sunken wood is a large organic input from land and ubiquitously distributed in the ocean floor (Wolff, 1979) and is involved in microbial diversity and dispersal in the marine environment (Fagervold et al., 2012, Bienhold 2013). It develops high diversity in the microbial community, including primary degraders of wood components and successive generations of heterotrophic and chemosynthetic organisms. Earlier studies have suggested that dissolved organic carbon (DOC) in marine ecosystems is sourced from complex materials, including terrestrial organic matter from plants, which are discharged by rivers (McNichol and Aluwihare, 2007; Raymond and Bauer, 2001) and other sources, such as chemosynthesis, sedimentary methane, and anthropogenic activities. A significant part of the marine DOC is probably refractory carbon (Griffith et al., 2012). Their composition, transformation, and turnover remain poorly understood (Bianchi, 2012; Follett et al., 2014). The main processes for the decomposition of plants in shallow marine environments include photodegradation and biodegradation (Miller and Zepp, 1995; Benner and Opsahl, 2001; Ward et al., 2013; Fichot and Benner, 2014). Previous studies have shown that biodegradation dominates over photodegradation in river- influenced ocean margins (Ward et al., 2013; Fichot and Benner, 2014). Here, we detected dozens of bacteria capable of degrading feruloyl ester linkages from a sunken wood recovered from the sea bottom in the Tohoku district, northeastern Japan. To the best of our knowledge, only a few bacterial Faes have been identified in water-logged environments. We investigated the enzymatic and microbial properties of a selected isolate on synthetic esterase substrates and natural plant biomass components. This study will shed light on the microbial processes overlooked in terrestrial plant biomass degradation in aquatic environments. Results Screening of ethyl ferulate (EF)-degrading bacteria from sunken wood Bacteria living on the sunken wood were grown on an agar medium containing rice straw meal as the nutrient sources in artificial seawater. Sixty-six isolates were obtained by observing the differences in colony morphologies. Subsequently, these isolates were inoculated on a solid agar medium containing EF (Fig S1, compound 1). EF has low solubility in water; therefore, the medium becomes turbid. After a day of incubation, transparent regions were observed. About

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