Control of phenotypic diversification based on serial cultivations on different carbon sources leads to improved bacterial xylanase production

peer reviewedThermobacillus xylanilyticus is a thermophilic and hemicellulolytic bacterium of interest for the production of thermostable hemicellulases. Enzymes’ production by this bacterium is challenging, because the proliferation of a cheating subpopulation of cells during exponential growth impairs the production of xylanase after serial cultivations. Accordingly, a strategy of successive cultivations with cells transfers in stationary phase and the use of wheat bran and wheat straw as carbon sources were tested. The ratio between subpopulations and their corresponding metabolic activities were studied by flow cytometry and the resulting hemicellulases production (xylanase, acetyl esterase and β-xylosidase) followed. During serial cultivations, the results pointed out an increase of the enzymatic activities. On xylan, compared to the first cultivation, the xylanase activity increases by 7.15-fold after only four cultivations. On the other hand, the debranching activities were increased by 5.88-fold and 57.2-fold on wheat straw and by 2.77-fold and 3.34-fold on wheat bran for β-xylosidase and acetyl esterase, respectively. The different enzymatic activities then stabilized, reached a plateau and further decreased. Study of the stability and reversibility of the enzyme production revealed cell-to-cell heterogeneities in metabolic activities which could be linked to the reversibility of enzymatic activity changes. Thus, the strategy of successive transfers during the stationary phase of growth, combined with the use of complex lignocellulosic substrates as carbon sources, is an efficient strategy to optimize the hemicellulases production by T. xylanilyticus, by preventing the selection of cheaters. © 2022, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature

Wheat Bran Pretreatment by Room Temperature Ionic Liquid-Water Mixture: Optimization of Process Conditions by PLS-Surface Response Design

International audienceRoom Temperature Ionic Liquids (RTILs) pretreatment are well-recognized to improve the enzymatic production of platform molecules such as sugar monomers from lignocellulosic biomass (LCB). The conditions for implementing this key step requires henceforth optimization to reach a satisfactory compromise between energy saving, required RTIL amount and hydrolysis yields. Wheat bran (WB) and destarched wheat bran (DWB), which constitute relevant sugar-rich feedstocks were selected for this present study. Pretreatments of these two distinct biomasses with various 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc])-water mixtures prior to hydrolysis catalyzed by hemicellulolytic cocktail (Cellic CTec2) were finely investigated. The main operating conditions such as pretreatment temperature (25–150°C), time (40–180 min), WB and DWB loading (2–5% w/v) and concentration of [C2mim][OAc] in water [10–100% (v/v)] were screened through glucose and xylose yields and then optimized through a Partial Least Square (PLS)—Second Order Design. In an innovative way, the PLS results showed that the four factors and their interactions could be well-fitted by a second-order model (p < 0.05). The quadratic PLS models were used to predict optimal pretreatment conditions. Thus, maximum glucose (83%) and xylose (95%) yields were obtained from enzymatic hydrolysis of WB pretreated at 150°C for 40 min with 10% of [C2mim][OAc] in water and 5% of WB loading. For DWB, maximum glucose (100%) and xylose (57%) yields were achieved for pretreatment temperatures of 150°C and 25°C, respectively. The required duration was still 40 min, with 20% of [C2mim][OAc] in water and a 5% DWB loading. Then, Multiple Response Optimization (MRO) performed by Nelder-Mead Simplex Method displayed sugar yields similar to those obtained by individual PLS optimization. This complete statistical study confirmed that the established models were appropriate to predict the sugar yields achieved after different pretreatment conditions from WB and DWB biomasses. Finally, Scanning Electron microscopy (SEM) studies allowed us to establish clearer link between structural changes induced by pretreatment and the best enzymatic performances obtained

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane

Identification of Surprisingly Diverse Type IV Pili, across a Broad Range of Gram-Positive Bacteria

In Gram-negative bacteria, type IV pili (TFP) have long been known to play important roles in such diverse biological phenomena as surface adhesion, motility, and DNA transfer, with significant consequences for pathogenicity. More recently it became apparent that Gram-positive bacteria also express type IV pili; however, little is known about the diversity and abundance of these structures in Gram-positives. Computational tools for automated identification of type IV pilins are not currently available.To assess TFP diversity in Gram-positive bacteria and facilitate pilin identification, we compiled a comprehensive list of putative Gram-positive pilins encoded by operons containing highly conserved pilus biosynthetic genes (pilB, pilC). A surprisingly large number of species were found to contain multiple TFP operons (pil, com and/or tad). The N-terminal sequences of predicted pilins were exploited to develop PilFind, a rule-based algorithm for genome-wide identification of otherwise poorly conserved type IV pilins in any species, regardless of their association with TFP biosynthetic operons (http://signalfind.org). Using PilFind to scan 53 Gram-positive genomes (encoding >187,000 proteins), we identified 286 candidate pilins, including 214 in operons containing TFP biosynthetic genes (TBG+ operons). Although trained on Gram-positive pilins, PilFind identified 55 of 58 manually curated Gram-negative pilins in TBG+ operons, as well as 53 additional pilin candidates in operons lacking biosynthetic genes in ten species (>38,000 proteins), including 27 of 29 experimentally verified pilins. False positive rates appear to be low, as PilFind predicted only four pilin candidates in eleven bacterial species (>13,000 proteins) lacking TFP biosynthetic genes.We have shown that Gram-positive bacteria contain a highly diverse set of type IV pili. PilFind can be an invaluable tool to study bacterial cellular processes known to involve type IV pilus-like structures. Its use in combination with other currently available computational tools should improve the accuracy of predicting the subcellular localization of bacterial proteins

Le système d'adhésion de ruminococcus albus (implication de pili de type IV et de deux glycosl-hydrolases)

Ruminococcus albus est une des bactéries cellulolytiques majeures du rumen. L'adhésion de cette bactérie à la cellulose est une première étape indispensable dans le processus de dégradation des fibres végétales. L'objectif de ce travail est d'élucider les mécanismes impliqués dans l'adhésion de R. albus à la cellulose. Ce travail a permis de caractériser une glycoprotéine de 25 kDa (GP25) sous-produite par un mutant non adhérent de la souche 20 de R. albus. GP25 est la sous-unité majeure des pili de type IV observés à la surface de R. albus 20. Ces pili sont nécessaires à l'adhésion de la bactérie à la cellulose. En aval du gène gp25, deux gènes pilA2 et secD-F codant une deuxième protéine homologue aux pilines de type IV et des protéines impliquées dans la sécrétion, sont identifiées. Deux protéines affines pour la cellulose (CBP1 et CBP2) sous-produites par le mutant ont également été caractérisées par électrophorèse bidimensionnelle et spectrométrie de masse (MALDI-TOF). Ces protéines homologues à deux endoglucanases (Cel9E et Cel48A) de R. albus 8 seraient impliquées dans l'adhésion. Ainsi, le système d'adhésion de R. albus est multi-factoriel et ferait intervenir des pili de type IV et des glycosyl-hydrolasesLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Aga1, the first alpha-galactosidase from the human bacteria Ruminococcus gnavus E1, efficiently transcribed in gut conditions

Differential gene expression analysis was performed in monoxenic mice colonized with Ruminococcus gnavus strain El, a major endogenous member of the gut microbiota. RNA arbitrarily primed-PCR fingerprinting assays allowed to specifically detect the in vivo expression of the agal gene, which was further confirmed by RT-PCR. The agal gene encoded a protein of 744 residues with calculated molecular mass of 85,207 Da. Agal exhibited significant similarity with previously characterized alpha-Galactosidases of the GH 36 family. Purified recombinant protein demonstrated high catalytic activity (104 +/- 7 U mg(-1)) and efficient p-nitrophenyl-alpha-D-galactopyranoside hydrolysis [k(cat)/K-m = 35.115 +/- 8.82 s(-1) mM(-1) at 55 degrees C and k(cat)/K-m = 17.48 +/- 4.25 s(-1) mM(-1) at 37 degrees C]

Enzymatic deconstruction of wheat bran xylans by a thermophylic bacterium, Thermobacillus xylanilyticus, to produce valuable molecules

Enzymatic deconstruction of wheat bran xylans by a thermophylic bacterium, [i]Thermobacillus xylanilyticus[/i], to produce valuable molecules. 3. International Symposium on Green Chemistr

D-Xylose and L-arabinose laurate esters: Enzymatic synthesis, characterization and physico-chemical properties

Efficient enzymatic synthesis of D-xylose and L-arabinose lauryl mono- and diesters has been achieved by transesterification reactions catalysed by immobilized Candida antarctica lipase B as biocatalyst, in organic medium in the presence of D-xylose or L-arabinose and vinyllaurate at 50°C. In case of L-arabinose, one monoester and one diester were obtained in a 57% overall yield. A more complex mixture was produced for Dxylose as two monoesters and two diesters were synthesized in a 74.9% global yield. The structures of all these pentose laurate esters was solved. Results demonstrated that the esterification first occurred regioselectively onto the primary hydroxyl groups. Pentose laurate esters exhibited interesting features such as low critical aggregation concentrations values all inferior to 25μM. Our study demonstrates that the enzymatic production of L-arabinose and D-xylose-based esters represents an interesting approach for the production of green surfactants from lignocellulosic biomass-derived pentoses