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Chun You - One of the best experts on this subject based on the ideXlab platform.
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Acceleration of Cellodextrin phosphorolysis for bioelectricity generation from cellulosic biomass by integrating a synthetic two-enzyme complex into an in vitro synthetic enzymatic biosystem
Biotechnology for biofuels, 2019Co-Authors: Dongdong Meng, Zhiguang Zhu, Juan Wang, Chun YouAbstract:Cellulosic biomass, the earth’s most abundant renewable resource, can be used as substrates for biomanufacturing biofuels or biochemicals via in vitro synthetic enzymatic biosystems in which the first step is the enzymatic phosphorolysis of Cellodextrin to glucose 1-phosphate (G1P) by Cellodextrin phosphorylase (CDP). However, almost all the CDPs prefer Cellodextrin synthesis to phosphorolysis, resulting in the low reaction rate of Cellodextrin phosphorolysis for biomanufacturing. To increase the reaction rate of Cellodextrin phosphorolysis, synthetic enzyme complexes containing CDP and phosphoglucomutase (PGM) were constructed to convert G1P to glucose 6-phosphate (G6P) rapidly, which is an important intermediate for biomanufacturing. Four self-assembled synthetic enzyme complexes were constructed with different spatial organizations based on the high-affinity and high-specific interaction between cohesins and dockerins from natural cellulosomes. Thus, the CDP–PGM enzyme complex with the highest enhancement of initial reaction rate was integrated into an in vitro synthetic enzymatic biosystem for generating bioelectricity from Cellodextrin. The in vitro biosystem containing the best CDP–PGM enzyme complex exhibited a much higher current density (3.35-fold) and power density (2.14-fold) than its counterpart biosystem containing free CDP and PGM mixture. Hereby, we first reported bioelectricity generation from cellulosic biomass via in vitro synthetic enzymatic biosystems. This work provided a strategy of how to link non-energetically favorable reaction (Cellodextrin phosphorolysis) and energetically favorable reaction (G1P to G6P) together to circumvent unfavorable reaction equilibrium and shed light on improving the reaction efficiency of in vitro synthetic enzymatic biosystems through the construction of synthetic enzyme complexes.
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Stoichiometric Conversion of Cellulosic Biomass by in Vitro Synthetic Enzymatic Biosystems for Biomanufacturing
ACS Catalysis, 2018Co-Authors: Dongdong Meng, Xinlei Wei, Yi-heng P. Job Zhang, Zhiguang Zhu, Chun YouAbstract:Cellulosic biomass is the earth’s most abundant renewable resource, which is considered to be a promising feedstock for manufacturing biofuels and biochemicals. In this study, stoichiometric enzymatic phosphorolysis of cellulosic biomass for manufacturing biochemicals or biofuels by in vitro synthetic enzymatic biosystems was designed. Three cascade phosphorolytic enzymes, Cellodextrin phosphorylase, cellobiose phosphorylase, and polyphosphate-dependent glucokinase, were used for the biotransformation of Cellodextrins to high-energy phosphorylated sugars (that is, glucose 1-phosphate and glucose 6-phosphate). A series of downstream exergonic reactions then converted these high-energy phosphorylated sugars into myo-inositol, resulting in a near-stoichiometric conversion of Cellodextrins with a high product yield of 98% (w/w). Moreover, this enzymatic biosystem can even work for the acid-treated biomass hydrolysate containing microorganism-toxic compounds. The construction of this in vitro synthetic enzymat...
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Stoichiometric Conversion of Cellulosic Biomass by in Vitro Synthetic Enzymatic Biosystems for Biomanufacturing
2018Co-Authors: Dongdong Meng, Xinlei Wei, Zhiguang Zhu, Yi-heng Job P. Zhang, Chun YouAbstract:Cellulosic biomass is the earth’s most abundant renewable resource, which is considered to be a promising feedstock for manufacturing biofuels and biochemicals. In this study, stoichiometric enzymatic phosphorolysis of cellulosic biomass for manufacturing biochemicals or biofuels by in vitro synthetic enzymatic biosystems was designed. Three cascade phosphorolytic enzymes, Cellodextrin phosphorylase, cellobiose phosphorylase, and polyphosphate-dependent glucokinase, were used for the biotransformation of Cellodextrins to high-energy phosphorylated sugars (that is, glucose 1-phosphate and glucose 6-phosphate). A series of downstream exergonic reactions then converted these high-energy phosphorylated sugars into myo-inositol, resulting in a near-stoichiometric conversion of Cellodextrins with a high product yield of 98% (w/w). Moreover, this enzymatic biosystem can even work for the acid-treated biomass hydrolysate containing microorganism-toxic compounds. The construction of this in vitro synthetic enzymatic biosystem provided an alternative method for the utilization of cellulosic biomass rather than cellulolytic enzyme hydrolysis to fermentative monomeric sugars followed by microorganism fermentation, showing potentials in the production of biocommodities such as hydrogen, rare sugars, and electricity from cellulosic biomass
Evelyne Forano - One of the best experts on this subject based on the ideXlab platform.
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NMR study of cellulose and wheat straw degradation by Ruminococcus albus 20.
The FEBS journal, 2008Co-Authors: M. Matulová, Anne-marie Delort, R. Nouaille, Peter Capek, Michel Péan, Evelyne ForanoAbstract:Cellulose and wheat straw degradation by Ruminococcus albus was monitored using NMR spectroscopy. In situ solid-state (13)C-cross-polarization magic angle spinning NMR was used to monitor the modification of the composition and structure of cellulose and (13)C-enriched wheat straw during the growth of the bacterium on these substrates. In cellulose, amorphous regions were not preferentially degraded relative to crystalline areas by R. albus. Cellulose and hemicelluloses were also degraded at the same rate in wheat straw. Liquid state two-dimensional NMR experiments were used to analyse in detail the sugars released in the culture medium, and the integration of NMR signals enabled their quantification at various times of culture. The results showed glucose and Cellodextrin accumulation in the medium of cellulose cultures; the Cellodextrins were mainly cellotriose and accumulated to up to 2 mm after 4 days. In the wheat straw cultures, xylose was the main soluble sugar detected (1.4 mm); arabinose and glucose were also found, together with some oligosaccharides liberated from hemicellulose hydrolysis, but to a much lesser extent. No Cellodextrins were detected. The results indicate that this strain of R. albus is unable to use glucose, xylose and arabinose for growth, but utilizes efficiently xylooligosaccharides. R. albus 20 appears to be less efficient than Fibrobacter succinogenes S85 for the degradation of wheat straw.
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Concurrent maltodextrin and Cellodextrin synthesis by Fibrobacter succinogenes S85 as identified by 2D NMR spectroscopy.
European journal of biochemistry, 2001Co-Authors: M. Matulová, Anne-marie Delort, R. Nouaille, Geneviève Gaudet, Evelyne ForanoAbstract:1D and 2D NMR experiments were used to analyse the synthesis of various metabolites by resting cells of Fibrobacter succinogenes S85 when incubated with [1-13C]glucose, in both extracellular and cellular media. Besides the expected glycogen, succinate, acetate, glucose-1-P and glucose-6-P, maltodextrins and Cellodextrins were detected. Maltodextrins were excreted into the external medium. They were found to have linear structures with a maximum degree of polymerization (DP) of about 6 or 7 units. Cellodextrins were located in the cells (cytoplasm and/or periplasm), and their DP was ≤ 4. Both labelled (1-13C and 6-13C) and unlabelled maltodextrins and Cellodextrins were detected, showing the contribution of carbohydrate cycling in F. succinogenes, including the reversal of glycolysis and the futile cycle of glycogen. The mechanisms of these oligosaccharide syntheses are discussed.
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Concurrent maltodextrin and Cellodextrin synthesis by fibrobacter succinogenes S85 as identified by 2D NMR spectroscopy
European Journal of Biochemistry, 2001Co-Authors: M. Matulová, Anne-marie Delort, R. Nouaille, Gérard Gaudet, Evelyne ForanoAbstract:1D and 2D NMR experiments were used to analyse the synthesis of various metabolites by resting cells of Fibrobacter succinogenes S85 when incubated with [1-C-13]glucose, in both extracellular and cellular media. Besides the expected glycogen, succinate, acetate, glucose-1-P and glucose-6-P, maltodextrins and Cellodextrins were detected. Maltodextrins were excreted into the external medium. They were found to have linear structures with a maximum degree of polymerization (DP) of about 6 or 7 units. Cellodextrins were located in the cells (cytoplasm and/or periplasm), and their DP was less than or equal to4. Both labelled (1-C-13 and 6-C-13) and unlabelled maltodextrins and Cellodextrins were detected, showing the contribution of carbohydrate cycling in F. succinogenes, including the reversal of glycolysis and the futile cycle of glycogen. The mechanisms of these oligosaccharide syntheses are discussed.
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In vivo 13C NMR study of glucose and cellobiose metabolism by four cellulolytic strains of the genus Fibrobacter
Biodegradation, 1998Co-Authors: Christelle Matheron, Anne-marie Delort, Geneviève Gaudet, Evelyne ForanoAbstract:The metabolism of glucose and cellobiose, products of cellulose hydrolysis, was investigated in four cellulolytic strains of the genus Fibrobacter: Fibrobacter succinogenes S85, 095, HM2 and Fibrobacter intestinalis NR9. In vivo 13C nuclear magnetic resonance was used to quantify the relative contribution of glucose and cellobiose to metabolite production, glycogen storage and Cellodextrins synthesis in these four strains. The same features were found in all four strains of the genus Fibrobacter metabolizing simultaneously glucose and cellobiose: i) differential metabolism of glucose and cellobiose; glucose seems preferentially used for glycogen storage and energy production, while part of cellobiose seems to be diverted from glycolysis, ii) synthesis of Cellodextrins, mainly from cellobiose not entering into glycolysis, iii) accumulation of glucose 6-phosphate, iv) simultaneous presence of cellobiose phosphorylase and cellobiase activities. Although genetically diverse, the Fibrobacter genus appears to possess a marked homogeneity in its carbon metabolism.
Dongdong Meng - One of the best experts on this subject based on the ideXlab platform.
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Acceleration of Cellodextrin phosphorolysis for bioelectricity generation from cellulosic biomass by integrating a synthetic two-enzyme complex into an in vitro synthetic enzymatic biosystem
Biotechnology for biofuels, 2019Co-Authors: Dongdong Meng, Zhiguang Zhu, Juan Wang, Chun YouAbstract:Cellulosic biomass, the earth’s most abundant renewable resource, can be used as substrates for biomanufacturing biofuels or biochemicals via in vitro synthetic enzymatic biosystems in which the first step is the enzymatic phosphorolysis of Cellodextrin to glucose 1-phosphate (G1P) by Cellodextrin phosphorylase (CDP). However, almost all the CDPs prefer Cellodextrin synthesis to phosphorolysis, resulting in the low reaction rate of Cellodextrin phosphorolysis for biomanufacturing. To increase the reaction rate of Cellodextrin phosphorolysis, synthetic enzyme complexes containing CDP and phosphoglucomutase (PGM) were constructed to convert G1P to glucose 6-phosphate (G6P) rapidly, which is an important intermediate for biomanufacturing. Four self-assembled synthetic enzyme complexes were constructed with different spatial organizations based on the high-affinity and high-specific interaction between cohesins and dockerins from natural cellulosomes. Thus, the CDP–PGM enzyme complex with the highest enhancement of initial reaction rate was integrated into an in vitro synthetic enzymatic biosystem for generating bioelectricity from Cellodextrin. The in vitro biosystem containing the best CDP–PGM enzyme complex exhibited a much higher current density (3.35-fold) and power density (2.14-fold) than its counterpart biosystem containing free CDP and PGM mixture. Hereby, we first reported bioelectricity generation from cellulosic biomass via in vitro synthetic enzymatic biosystems. This work provided a strategy of how to link non-energetically favorable reaction (Cellodextrin phosphorolysis) and energetically favorable reaction (G1P to G6P) together to circumvent unfavorable reaction equilibrium and shed light on improving the reaction efficiency of in vitro synthetic enzymatic biosystems through the construction of synthetic enzyme complexes.
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Stoichiometric Conversion of Cellulosic Biomass by in Vitro Synthetic Enzymatic Biosystems for Biomanufacturing
ACS Catalysis, 2018Co-Authors: Dongdong Meng, Xinlei Wei, Yi-heng P. Job Zhang, Zhiguang Zhu, Chun YouAbstract:Cellulosic biomass is the earth’s most abundant renewable resource, which is considered to be a promising feedstock for manufacturing biofuels and biochemicals. In this study, stoichiometric enzymatic phosphorolysis of cellulosic biomass for manufacturing biochemicals or biofuels by in vitro synthetic enzymatic biosystems was designed. Three cascade phosphorolytic enzymes, Cellodextrin phosphorylase, cellobiose phosphorylase, and polyphosphate-dependent glucokinase, were used for the biotransformation of Cellodextrins to high-energy phosphorylated sugars (that is, glucose 1-phosphate and glucose 6-phosphate). A series of downstream exergonic reactions then converted these high-energy phosphorylated sugars into myo-inositol, resulting in a near-stoichiometric conversion of Cellodextrins with a high product yield of 98% (w/w). Moreover, this enzymatic biosystem can even work for the acid-treated biomass hydrolysate containing microorganism-toxic compounds. The construction of this in vitro synthetic enzymat...
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Stoichiometric Conversion of Cellulosic Biomass by in Vitro Synthetic Enzymatic Biosystems for Biomanufacturing
2018Co-Authors: Dongdong Meng, Xinlei Wei, Zhiguang Zhu, Yi-heng Job P. Zhang, Chun YouAbstract:Cellulosic biomass is the earth’s most abundant renewable resource, which is considered to be a promising feedstock for manufacturing biofuels and biochemicals. In this study, stoichiometric enzymatic phosphorolysis of cellulosic biomass for manufacturing biochemicals or biofuels by in vitro synthetic enzymatic biosystems was designed. Three cascade phosphorolytic enzymes, Cellodextrin phosphorylase, cellobiose phosphorylase, and polyphosphate-dependent glucokinase, were used for the biotransformation of Cellodextrins to high-energy phosphorylated sugars (that is, glucose 1-phosphate and glucose 6-phosphate). A series of downstream exergonic reactions then converted these high-energy phosphorylated sugars into myo-inositol, resulting in a near-stoichiometric conversion of Cellodextrins with a high product yield of 98% (w/w). Moreover, this enzymatic biosystem can even work for the acid-treated biomass hydrolysate containing microorganism-toxic compounds. The construction of this in vitro synthetic enzymatic biosystem provided an alternative method for the utilization of cellulosic biomass rather than cellulolytic enzyme hydrolysis to fermentative monomeric sugars followed by microorganism fermentation, showing potentials in the production of biocommodities such as hydrogen, rare sugars, and electricity from cellulosic biomass
Jamie H D Cate - One of the best experts on this subject based on the ideXlab platform.
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Enhanced cellobiose fermentation by engineered Saccharomyces cerevisiae expressing a mutant Cellodextrin facilitator and cellobiose phosphorylase
Journal of biotechnology, 2018Co-Authors: Heejin Kim, Jamie H D Cate, Won-heong Lee, Stephan Lane, Yong-su JinAbstract:Abstract To efficiently ferment intermediate Cellodextrins released during cellulose hydrolysis, Saccharomyces cerevisiae has been engineered by introduction of a heterologous Cellodextrin utilizing pathway consisting of a Cellodextrin transporter and either an intracellular β-glucosidase or a cellobiose phosphorylase. Among two types of Cellodextrin transporters, the passive facilitator CDT-2 has not enabled better cellobiose fermentation than the active transporter CDT-1, which suggests that the CDT-2 might be engineered to provide energetic benefits over the active transporter in cellobiose fermentation. We attempted to improve cellobiose transporting activity of CDT-2 through laboratory evolution. Nine rounds of a serial subculture of S. cerevisiae expressing CDT-2 and cellobiose phosphorylase on cellobiose led to the isolation of an evolved strain capable of fermenting cellobiose to ethanol 10-fold faster than the original strain. After sequence analysis of the isolated CDT-2, a single point mutation on CDT-2 (N306I) was revealed to be responsible for enhanced cellobiose fermentation. Also, the engineered strain expressing the mutant CDT-2 with cellobiose phosphorylase showed a higher ethanol yield than the engineered strain expressing CDT-1 and intracellular β-glucosidase under anaerobic conditions, suggesting that CDT-2 coupled with cellobiose phosphorylase may be better choices for efficient production of cellulosic ethanol with the engineered yeast.
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Analysis of Cellodextrin transporters from Neurospora crassa in Saccharomyces cerevisiae for cellobiose fermentation
Applied Microbiology and Biotechnology, 2014Co-Authors: Heejin Kim, Jamie H D Cate, Jonathan M. Galazka, Won-heong Lee, Yong-su JinAbstract:Saccharomyces cerevisiae can be engineered to ferment Cellodextrins produced by cellulases as a product of cellulose hydrolysis. Direct fermentation of Cellodextrins instead of glucose is advantageous because glucose inhibits cellulase activity and represses the fermentation of non-glucose sugars present in cellulosic hydrolyzates. To facilitate Cellodextrin utilization by S . cerevisiae , a fungal Cellodextrin-utilizing pathway from Neurospora crassa consisting of a Cellodextrin transporter and a Cellodextrin hydrolase has been introduced into S . cerevisiae . Two Cellodextrin transporters (CDT-1 and CDT-2) were previously identified in N . crassa , but their kinetic properties and efficiency for cellobiose fermentation have not been studied in detail. In this study, CDT-1 and CDT-2, which are hypothesized to transport Cellodextrin with distinct mechanisms, were introduced into S . cerevisiae along with an intracellular β-glucosidase (GH1-1). Cellobiose transport assays with the resulting strains indicated that CDT-1 is a proton symporter while CDT-2 is a simple facilitator. A strain expressing CDT-1 and GH1-1 (DCDT-1G) showed faster cellobiose fermentation than the strain expressing CDT-2 and GH1-1 (DCDT-2G) under various culture conditions with different medium compositions and aeration levels. While CDT-2 is expected to have energetic benefits, the expression levels and kinetic properties of CDT-1 in S . cerevisiae appears to be optimum for cellobiose fermentation. These results suggest CDT-1 is a more effective cellobiose transporter than CDT-2 for engineering S . cerevisiae to ferment cellobiose.
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evidence for transceptor function of Cellodextrin transporters in neurospora crassa
Journal of Biological Chemistry, 2014Co-Authors: Elizabeth A Znameroski, Jonathan M. Galazka, Xin Li, Jordan C Tsai, N L Glass, Jamie H D CateAbstract:Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two Cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and Cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa Cellodextrin transporters act as “transceptors” with dual functions - Cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.
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Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant Cellodextrin transporters
Metabolic engineering, 2012Co-Authors: Jonathan M. Galazka, Heejin Kim, Yong-su Jin, Vesna Kordić, Jamie H D CateAbstract:Anaerobic bacteria assimilate Cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a Cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular β-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using Cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (ΔG°=+3.6kJmol(-1)). A thermodynamic "push" from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae.
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cofermentation of cellobiose and galactose by an engineered saccharomyces cerevisiae strain
Applied and Environmental Microbiology, 2011Co-Authors: Sukjin Ha, Jamie H D Cate, Jonathan M. GalazkaAbstract:We demonstrate improved ethanol yield and productivity through cofermentation of cellobiose and galactose by an engineered Saccharomyces cerevisiae strain expressing genes coding for Cellodextrin transporter (cdt-1) and intracellular -glucosidase (gh1-1) from Neurospora crassa. Simultaneous fermentation of cellobiose and galactose can be applied to producing biofuels from hydrolysates of marine plant biomass.
Stéphanie Perret - One of the best experts on this subject based on the ideXlab platform.
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In vitro and in vivo exploration of the cellobiose and Cellodextrin phosphorylases panel in Ruminiclostridium cellulolyticum: implication for cellulose catabolism
Biotechnology for Biofuels, 2019Co-Authors: Nian Liu, Aurélie Fosses, Yann Denis, Henri-pierre Fierobe, Clara Kampik, Goetz Parsiegla, Nicolas Vita, Stéphanie PerretAbstract:Background: In anaerobic cellulolytic microorganisms , 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 identified 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 lifestyle of cellulolytic bacteria on such substrate. Results: The three Cellodextrin phosphorylases from R. cellulolyticum displayed marked different enzymatic characteristics. They are specific for Cellodextrins of different lengths and present different k cat 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 differences. 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. Characterization 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 differences 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.
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In vitro and in vivo exploration of the cellobiose and Cellodextrin phosphorylases panel in Ruminiclostridium cellulolyticum: implication for cellulose catabolism
Biotechnology for biofuels, 2019Co-Authors: Nian Liu, Aurélie Fosses, Yann Denis, Henri-pierre Fierobe, Clara Kampik, Goetz Parsiegla, Nicolas Vita, Stéphanie PerretAbstract: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 Cellodextrins 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 identified 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. The three Cellodextrin phosphorylases from R. cellulolyticum displayed marked different enzymatic characteristics. They are specific for Cellodextrins of different lengths and present different 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 differences. 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. Characterization 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. 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 differences 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.
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A seven-gene cluster in Ruminiclostridium cellulolyticum is essential for signalization, uptake and catabolism of the degradation products of cellulose hydrolysis
Biotechnology for Biofuels, 2017Co-Authors: Aurélie Fosses, Maria Maté, Nathalie Franche, Yann Denis, Romain Borne, Pascale Philip, Henri-pierre Fierobe, Stéphanie PerretAbstract:Background Like a number of anaerobic and cellulolytic Gram-positive bacteria, the model microorganism Ruminiclostridium cellulolyticum produces extracellular multi-enzymatic complexes called cellulosomes, which efficiently degrade the crystalline cellulose. Action of the complexes on cellulose releases cellobiose and longer Cellodextrins but to date, little is known about the transport and utilization of the produced Cellodextrins in the bacterium. A better understanding of the uptake systems and fermentation of sugars derived from cellulose could have a major impact in the field of biofuels production. Results We characterized a putative ABC transporter devoted to Cellodextrins uptake, and a cellobiose phosphorylase (CbpA) in R. cellulolyticum . The genes encoding the components of the ABC transporter (a binding protein CuaA and two integral membrane proteins) and CbpA are expressed as a polycistronic transcriptional unit induced in the presence of cellobiose. Upstream, another polycistronic transcriptional unit encodes a two-component system (sensor and regulator), and a second binding protein CuaD, and is constitutively expressed. The products might form a three-component system inducing the expression of cuaABC and cbpA since we showed that CuaR is able to recognize the region upstream of cuaA . Biochemical analysis showed that CbpA is a strict cellobiose phosphorylase inactive on longer Cellodextrins; CuaA binds to all Cellodextrins (G2–G5) tested, whereas CuaD is specific to cellobiose and presents a higher affinity to this sugar. This results are in agreement with their function in transport and signalization, respectively. Characterization of a cuaD mutant, and its derivatives, indicated that the ABC transporter and CbpA are essential for growth on cellobiose and cellulose. Conclusions For the first time in a Gram-positive strain, we identified a three-component system and a conjugated ABC transporter/cellobiose phosphorylase system which was shown to be essential for the growth of the model cellulolytic bacterium R. cellulolyticum on cellobiose and cellulose. This efficient and energy-saving system of transport and phosphorolysis appears to be the major cellobiose utilization pathway in R. cellulolyticum , and seems well adapted to cellulolytic life-style strain. It represents a new way to enable engineered strains to utilize Cellodextrins for the production of biofuels or chemicals of interest from cellulose.
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A seven-gene cluster in Ruminiclostridium cellulolyticum is essential for signalization, uptake and catabolism of the degradation products of cellulose hydrolysis.
Biotechnology for biofuels, 2017Co-Authors: Aurélie Fosses, Nathalie Franche, Yann Denis, Romain Borne, Pascale Philip, Henri-pierre Fierobe, Nian Liu, María J. Maté, Stéphanie PerretAbstract:Like a number of anaerobic and cellulolytic Gram-positive bacteria, the model microorganism Ruminiclostridium cellulolyticum produces extracellular multi-enzymatic complexes called cellulosomes, which efficiently degrade the crystalline cellulose. Action of the complexes on cellulose releases cellobiose and longer Cellodextrins but to date, little is known about the transport and utilization of the produced Cellodextrins in the bacterium. A better understanding of the uptake systems and fermentation of sugars derived from cellulose could have a major impact in the field of biofuels production. We characterized a putative ABC transporter devoted to Cellodextrins uptake, and a cellobiose phosphorylase (CbpA) in R. cellulolyticum. The genes encoding the components of the ABC transporter (a binding protein CuaA and two integral membrane proteins) and CbpA are expressed as a polycistronic transcriptional unit induced in the presence of cellobiose. Upstream, another polycistronic transcriptional unit encodes a two-component system (sensor and regulator), and a second binding protein CuaD, and is constitutively expressed. The products might form a three-component system inducing the expression of cuaABC and cbpA since we showed that CuaR is able to recognize the region upstream of cuaA. Biochemical analysis showed that CbpA is a strict cellobiose phosphorylase inactive on longer Cellodextrins; CuaA binds to all Cellodextrins (G2–G5) tested, whereas CuaD is specific to cellobiose and presents a higher affinity to this sugar. This results are in agreement with their function in transport and signalization, respectively. Characterization of a cuaD mutant, and its derivatives, indicated that the ABC transporter and CbpA are essential for growth on cellobiose and cellulose. For the first time in a Gram-positive strain, we identified a three-component system and a conjugated ABC transporter/cellobiose phosphorylase system which was shown to be essential for the growth of the model cellulolytic bacterium R. cellulolyticum on cellobiose and cellulose. This efficient and energy-saving system of transport and phosphorolysis appears to be the major cellobiose utilization pathway in R. cellulolyticum, and seems well adapted to cellulolytic life-style strain. It represents a new way to enable engineered strains to utilize Cellodextrins for the production of biofuels or chemicals of interest from cellulose.