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In Vitro and in Vivo Exploration of the Cellobiose and Cellodextrin Phosphorylases Panel in Ruminiclostridium Cellulolyticum

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In Vitro and in Vivo Exploration of the Cellobiose and Cellodextrin Phosphorylases Panel in Ruminiclostridium Cellulolyticum In vitro and in vivo exploration of the cellobiose and cellodextrin phosphorylases panel in Ruminiclostridium cellulolyticum: implication for cellulose catabolism Nian Liu, Aurelie Fosses, Clara Kampik, Goetz Parsiegla, Yann Denis, Nicolas Vita, Henri-Pierre Fierobe, Stéphanie Perret To cite this version: Nian Liu, Aurelie Fosses, Clara Kampik, Goetz Parsiegla, Yann Denis, et al.. In vitro and in vivo exploration of the cellobiose and cellodextrin phosphorylases panel in Ruminiclostridium cel- lulolyticum: implication for cellulose catabolism. Biotechnology for Biofuels, BioMed Central, 2019, 12 (1), 10.1186/s13068-019-1549-x. hal-02312679 HAL Id: hal-02312679 https://hal-amu.archives-ouvertes.fr/hal-02312679 Submitted on 11 Oct 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Liu et al. Biotechnol Biofuels (2019) 12:208 https://doi.org/10.1186/s13068-019-1549-x Biotechnology for Biofuels RESEARCH Open Access In vitro and in vivo exploration of the cellobiose and cellodextrin phosphorylases panel in Ruminiclostridium cellulolyticum: implication for cellulose catabolism Nian Liu1, Aurélie Fosses1, Clara Kampik1, Goetz Parsiegla2, Yann Denis3, Nicolas Vita1, Henri‑Pierre Fierobe1 and Stéphanie Perret1* Abstract Background: In anaerobic cellulolytic micro‑organisms, cellulolysis results in the action of several cellulases gathered in extracellular multi‑enzyme complexes called cellulosomes. Their action releases cellobiose and longer cellodex‑ trins which are imported and further degraded in the cytosol to fuel the cells. In Ruminiclostridium cellulolyticum, an anaerobic and cellulolytic mesophilic bacteria, three cellodextrin phosphorylases named CdpA, CdpB, and CdpC, were identifed in addition to the cellobiose phosphorylase (CbpA) previously characterized. The present study aimed at characterizing them, exploring their implication during growth on cellulose to better understand the life‑style of cellulolytic bacteria on such substrate. Results: The three cellodextrin phosphorylases from R. cellulolyticum displayed marked diferent enzymatic char‑ acteristics. They are specifc for cellodextrins of diferent lengths and present diferent kcat values. CdpC is the most active enzyme before CdpA, and CdpB is weakly active. Modeling studies revealed that a mutation of a conserved histidine residue in the phosphate ion‑binding pocket in CdpB and CdpC might explain their activity‑level diferences. The genes encoding these enzymes are scattered over the chromosome of R. cellulolyticum and only the expression of the gene encoding the cellobiose phosphorylase and the gene cdpA is induced during cellulose growth. Char‑ acterization of four independent mutants constructed in R. cellulolyticum for each of the cellobiose and cellodextrin phosphorylases encoding genes indicated that only the cellobiose phosphorylase is essential for growth on cellulose. Conclusions: Unexpectedly, the cellobiose phosphorylase but not the cellodextrin phosphorylases is essential for the growth of the model bacterium on cellulose. This suggests that the bacterium adopts a “short” dextrin strategy to grow on cellulose, even though the use of long cellodextrins might be more energy‑saving. Our results suggest marked diferences in the cellulose catabolism developed among cellulolytic bacteria, which is a result that might impact the design of future engineered strains for biomass‑to‑biofuel conversion. Keywords: Cellobiose, Cellodextrins, Phosphorylase, Cellulolysis, Cellulose *Correspondence: [email protected] 1 Aix‑Marseille Univ, CNRS, LCB UMR 7283, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Biotechnol Biofuels (2019) 12:208 Page 2 of 14 Background located upstream of cuaABC-cbpA. In our previous Cellulose is the most abundant polysaccharide produced study, we constructed an R. cellulolyticum cuaD mutant on Earth and is constituted of linear chains of β-1,4- strain (MTLcuaD) in which a type II intron inactivates linked glucose units. It represents a large reservoir of cuaD. Te modifcation induced a polar efect on the glucose and an attractive renewable energy source. Nev- expression of the downstream genes cuaS (sensor) and ertheless, glucose molecules are scarcely available from cuaR (regulator), thus preventing the upregulation of cellulose because of the tight crystalline packing of the the expression of cuaABC-cbpA operon encoding the cellulosic chains which makes this material recalcitrant cellodextrins ABC transporter and the cellobiose phos- to enzymatic degradation. Its biological deconstruction phorylase A. In consequence, the MTLcuaD strain is, therefore, a limiting step in the carbon cycle on Earth was unable to grow on either cellobiose or cellulose. and also a bottleneck in the process of biofuel or bio- Te transformation of the strain with a vector contain- based chemicals production [1]. ing the ABC transporter genes but not the cellobiose Nonetheless, several anaerobic bacteria are able to phosphorylase-encoding gene cbpA restored growth on use this recalcitrant substrate as the sole carbon and cellulose but not on cellobiose. Tis observation sug- energy source [2]. Among them, Ruminiclostridium gests that cellodextrins of degree of polymerization cellulolyticum, a mesophilic, anaerobic model bacte- (DP) greater than 2 might be imported in the cytosol, rium raises special interest for years due to its ability thus ensuring growth on cellulose of this strain. Simi- to efciently degrade and use plant cell wall polysac- larly, another cellulolytic strain (Hungatei) Clostridium charides including cellulose and hemicellulose, and the thermocellum was reported to assimilate long cellodex- availability of genetic tools [3–8]. To achieve the enzy- trins of 5 and 6 glucose residues when grown on cel- matic degradation of plant cell wall polysaccharides, it lulose [11]. In general, the import of long cellodextrins produces multi-enzyme complexes called cellulosomes is believed to be more cost-efective compared to the by assembling on a scafolding protein diverse enzymes import of short ones, since for the same ATP transport belonging to families of glycoside hydrolase (GH), car- cost, long cellodextrins carry more glucose units and, bohydrate esterase (CE), or polysaccharide lyase (PL) therefore, generate more energy than short ones [11]. [6, 9]. Te released mono- and oligosaccharides are In anaerobic cellulolytic bacteria, the cytosolic degra- subsequently imported by the bacteria and catabolized. dation of cellodextrins is usually ensured by cellobiose/ For example, the uptake of xyloglucan and cellodextrins cellodextrin phosphorylases [12, 13]. Te cellodextrin was shown to be ensured by specifc ABC transporters, phosphorylases catalyze reversible phosphorolysis reac- the imported dextrins being further degraded into sim- tion in which a β-1,4-glycosidic bond of a cellodextrin ple monosaccharides by cytosolic GHs. Two distinct of n glucose units (called Gn with n ≥ 2) is cleaved in clusters of genes dedicated to either the catabolism the presence of inorganic phosphate, releasing one G-1P of xyloglucan or cellodextrins were shown to encode from the non-reducing end and one G n-1 molecule. Te ABC transporter components (including a solute- phosphorylated glucose can directly enter the glyco- binding protein collecting the solute to be imported lysis pathway after conversion into glucose 6-phosphate and two transmembrane domains forming a channel), (G-6P), without consumption of an ATP molecule for intracellular GH(s), and a signal transduction system its phosphorylation, in contrast to the unphosphoryl- [8, 10]. Te ABC transporter called CuaABC (for cel- ated glucoses generated by hydrolysis of cellodextrins. lulose utilization associated) has a solute-binding pro- Tis pathway, therefore, represents an energetically more tein which binds to cellodextrins with lengths ranging advantageous way of degrading oligosaccharides com- from cellobiose (G2) to cellopentaose (G5), suggest- pared to hydrolysis, which is especially benefcial for ing that at least these cellodextrins might be imported anaerobic organisms [12–14]. Te cellobiose phosphory- in the cytosol. CuaABC was shown to be essential for lase A from R. cellulolyticum belongs to the GH94 Fam- growth of the bacterium on both cellobiose and cel-
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