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

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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- ily. It is specifc for cellobiose as well as other cellobiose lulose growth substrate [10]. We have formerly shown phosphorylases described so far, with the exception of that imported cellobiose is subsequently converted into the cellobiose phosphorylase from Termosipho afri- glucose and α-glucose 1-phosphate (G-1P) by the cello- canus which is active on both cellobiose and long cello- biose phosphorylase A (CbpA) [10]. Te gene encoding dextrins [10, 15–19]. If R. cellulolyticum is able to import this enzyme forms with the genes cuaABC an operon long cellodextrins, enzyme(s) other than the cellobiose named (cuaABC-cbpA). Tis operon is regulated by a phosphorylase A might be implicated in their degrada- predicted three-component system CuaDSR encom- tion. We analyzed the genome of R. cellulolyticum and passing a binding protein CuaD, a sensor (CuaS), and identifed three genes encoding putative cytosolic cel- a regulator (CuaR) encoded by the operon cuaDSR lodextrin phosphorylases belonging to the GH94 family. Liu et al. Biotechnol Biofuels (2019) 12:208 Page 3 of 14 We characterized the three cellodextrin phosphorylases tree was generated based on the amino acid sequence and addressed the question of their role in the cellulose from characterized bacterial GH94 phosphorylases. It catabolism achieved by R. cellulolyticum. encompasses phosphorylases
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    Europäisches Patentamt (19) European Patent Office & Office européen des brevets (11) EP 1 117 822 B1 (12) EUROPÄISCHE PATENTSCHRIFT (45) Veröffentlichungstag und Bekanntmachung des (51) Int Cl.: Hinweises auf die Patenterteilung: C12P 19/18 (2006.01) C12N 9/10 (2006.01) 03.05.2006 Patentblatt 2006/18 C12N 15/54 (2006.01) C08B 30/00 (2006.01) A61K 47/36 (2006.01) (21) Anmeldenummer: 99950660.3 (86) Internationale Anmeldenummer: (22) Anmeldetag: 07.10.1999 PCT/EP1999/007518 (87) Internationale Veröffentlichungsnummer: WO 2000/022155 (20.04.2000 Gazette 2000/16) (54) HERSTELLUNG VON POLYGLUCANEN DURCH AMYLOSUCRASE IN GEGENWART EINER TRANSFERASE PREPARATION OF POLYGLUCANS BY AMYLOSUCRASE IN THE PRESENCE OF A TRANSFERASE PREPARATION DES POLYGLUCANES PAR AMYLOSUCRASE EN PRESENCE D’UNE TRANSFERASE (84) Benannte Vertragsstaaten: (56) Entgegenhaltungen: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU WO-A-00/14249 WO-A-00/22140 MC NL PT SE WO-A-95/31553 (30) Priorität: 09.10.1998 DE 19846492 • OKADA, GENTARO ET AL: "New studies on amylosucrase, a bacterial.alpha.-D-glucosylase (43) Veröffentlichungstag der Anmeldung: that directly converts sucrose to a glycogen- 25.07.2001 Patentblatt 2001/30 like.alpha.-glucan" J. BIOL. CHEM. (1974), 249(1), 126-35, XP000867741 (73) Patentinhaber: Südzucker AG Mannheim/ • BUTTCHER, VOLKER ET AL: "Cloning and Ochsenfurt characterization of the gene for amylosucrase 68165 Mannheim (DE) from Neisseria polysaccharea: production of a linear.alpha.-1,4-glucan" J. BACTERIOL. (1997), (72) Erfinder: 179(10), 3324-3330, XP002129879 • GALLERT, Karl-Christian • DE MONTALK, G. POTOCKI ET AL: "Sequence D-61184 Karben (DE) analysis of the gene encoding amylosucrase • BENGS, Holger from Neisseria polysaccharea and D-60598 Frankfurt am Main (DE) characterization of the recombinant enzyme" J.
  • Downloaded from the Ribosomal Database Project Website [22], and Aligned Using MUSCLE [23]

    Downloaded from the Ribosomal Database Project Website [22], and Aligned Using MUSCLE [23]

    Diversity 2013, 5, 627-640; doi:10.3390/d5030627 OPEN ACCESS diversity ISSN 1424-2818 www.mdpi.com/journal/diversity Article Untangling the Genetic Basis of Fibrolytic Specialization by Lachnospiraceae and Ruminococcaceae in Diverse Gut Communities Amy Biddle 1, Lucy Stewart 1, Jeffrey Blanchard 2 and Susan Leschine 3,* 1 Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA; E-Mails: [email protected] (A.B.); [email protected] (L.S.) 2 Department of Biology, University of Massachusetts, Amherst, MA 01003, USA; E-Mail: [email protected] 3 Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-413-545-0673; Fax: +1-413-545-6326. Received: 17 May 2013; in revised form: 21 June 2013 / Accepted: 26 June 2013 / Published: 9 August 2013 Abstract: The Lachnospiraceae and Ruminococcaceae are two of the most abundant families from the order Clostridiales found in the mammalian gut environment, and have been associated with the maintenance of gut health. While they are both diverse groups, they share a common role as active plant degraders. By comparing the genomes of the Lachnospiraceae and Ruminococcaceae with the Clostridiaceae, a more commonly free-living group, we identify key carbohydrate-active enzymes, sugar transport mechanisms, and metabolic pathways that distinguish these two commensal groups as specialists for the degradation of complex plant material. Keywords: Clostridiales; Ruminococcaceae; Lachnospiraceae; carbohydrate-active enzymes; comparative genomics; plant degradation Diversity 2013, 5 628 1. Introduction 1.1. Taxonomic Revision of the Clostridiales Is a Work in Progress Classically, the genus Clostridium was described as comprising spore-forming, non-sulfate reducing obligate anaerobic bacteria with a gram-positive cell wall.
  • 55856192.Pdf

    55856192.Pdf

    Members of the jury: Prof. dr. ir. Frank Devlieghere (chairman) Prof. dr. ir. Wim Soetaert (promotor) Prof. dr. ir. Erick Vandamme (promotor) Prof. dr. Els Vandamme Prof. dr. Savvas Savvides Dr. Tom Desmet Dr. Henk-Jan Joosten Promotors: Prof. dr. ir. Wim SOETAERT (promotor) Prof. dr. ir. Erick VANDAMME (promotor) Centre of expertise – Industrial Biotechnology and Biocatalysis Department of Biochemical and Microbial Technology Ghent University, Belgium Dean: Prof. dr. ir. Guido Van Huylenbroeck Rector: Prof. dr. Paul Van Cauwenberge The research was conducted at the Centre of expertise - Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University (Ghent, Belgium) ir. An CERDOBBEL ENGINEERING THE THERMOSTABILITY OF SUCROSE PHOSPHORYLASE FOR INDUSTRIAL APPLICATIONS Thesis submitted in fulfilment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Dutch translation of the title: Engineering van de thermostabiliteit van sucrose phosphorylase voor industriële toepassingen Cover illustration: “Three-dimensional structure of sucrose phosphorylase colored by B-factor.” Printed by University Press, Zelzate To refer to this thesis: Cerdobbel, A. (2011). Engineering the thermostability of sucrose phosphorylase for industrial applications. PhD thesis, Faculty of Bioscience Engineering, Ghent University, Ghent, 200 p. ISBN-number: 978-90-5989-414-3 The author and the promoter give the authorization to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. WOORD VOORAF Er wordt wel eens gezegd dat het woord vooraf het meest gelezen stukje is van een proefschrift.