RESUMEN
Many anaerobic microorganisms use the bifunctional aldehyde and alcohol dehydrogenase enzyme, AdhE, to produce ethanol. One such organism is Clostridium thermocellum, which is of interest for cellulosic biofuel production. In the course of engineering this organism for improved ethanol tolerance and production, we observed that AdhE was a frequent target of mutations. Here, we characterized those mutations to understand their effects on enzymatic activity, as well ethanol tolerance and product formation in the organism. We found that there is a strong correlation between NADH-linked alcohol dehydrogenase (ADH) activity and ethanol tolerance. Mutations that decrease NADH-linked ADH activity increase ethanol tolerance; correspondingly, mutations that increase NADH-linked ADH activity decrease ethanol tolerance. We also found that the magnitude of ADH activity did not play a significant role in determining ethanol titer. Increasing ADH activity had no effect on ethanol titer. Reducing ADH activity had indeterminate effects on ethanol titer, sometimes increasing and sometimes decreasing it. Finally, this study shows that the cofactor specificity of ADH activity was found to be the primary factor affecting ethanol yield. We expect that these results will inform efforts to use AdhE enzymes in metabolic engineering approaches.
RESUMEN
BACKGROUND: Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. RESULTS: In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. CONCLUSIONS: Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.
Asunto(s)
Anhidrasas Carbónicas , Biología Computacional , Escherichia coli , Solubilidad , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimología , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/genética , Biología Computacional/métodos , Filogenia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Dióxido de Carbono/metabolismoRESUMEN
Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis (Arabidopsis thaliana) xylan O-acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2-O-acetylation of xylan, followed by nonenzymatic acetyl migration to the O-3 position, resulting in products that are monoacetylated at both O-2 and O-3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the α/ß/α topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac-Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Polisacáridos/metabolismo , Acetilación , Acetiltransferasas/química , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arginina/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana , Modelos Moleculares , Mutación , Conformación Proteica , Xilanos/metabolismoRESUMEN
Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nkon ) and desorption (koff ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.
Asunto(s)
Celulosa , Protoplastos , Humanos , Protoplastos/metabolismo , Celulosa/metabolismo , Polisacáridos/metabolismo , Plantas/química , Metabolismo de los Hidratos de CarbonoRESUMEN
Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance.
Asunto(s)
Actinobacteria , Actinomycetales , Celulasa , Biomasa , Celulasa/química , Celulosa/química , HumanosRESUMEN
The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.
Asunto(s)
Modelos Moleculares , Conformación Proteica , Proteínas/química , Programas Informáticos , Secuencia de Aminoácidos , Biología Computacional , Microscopía por Crioelectrón , Cristalografía por Rayos X , Análisis de Secuencia de ProteínaRESUMEN
Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous ß-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. ß-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and ß-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular ß-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable ß-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan. IMPORTANCECaldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.
Asunto(s)
Actinobacteria/enzimología , Caldicellulosiruptor/metabolismo , Proteoma/metabolismo , Thermotoga maritima/enzimología , Xilanos/metabolismo , Xilosidasas/metabolismo , Actinobacteria/genética , Celobiosa/metabolismo , Escherichia coli/genética , Thermotoga maritima/genética , Xilosidasas/genéticaRESUMEN
Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases.IMPORTANCECaldicellulosiruptor has the ability to digest complex plant biomass without pretreatment and have been engineered to convert biomass, a sustainable, carbon neutral substrate, to fuels. Their strategy for deconstructing plant cell walls relies on an interesting class of cellulases consisting of multiple catalytic modules connected by linker regions and carbohydrate binding modules. The best studied of these enzymes, CelA, has a unique deconstruction mechanism. CelA is located in a cluster of genes that likely allows for optimal expression, secretion, and activity. One of the genes in this cluster is a putative isomerase that modifies the CelA protein. In higher eukaryotes, these isomerases are essential for the proper folding of glycoproteins in the endoplasmic reticulum, but little is known about the role of isomerization in cellulase activity. We show that the stability and activity of CelA is dependent on the activity of this isomerase.
Asunto(s)
Proteínas Bacterianas/genética , Caldicellulosiruptor/genética , Celulosa/metabolismo , Isomerasa de Peptidilprolil/genética , Proteínas Bacterianas/metabolismo , Caldicellulosiruptor/metabolismo , Eliminación de Gen , Glicosilación , Isomerasa de Peptidilprolil/metabolismo , Especificidad por SustratoRESUMEN
Modern biological tools generate a wealth of data on metabolite and protein concentrations that can be used to help inform new strain designs. However, learning from these data to predict how a cell will respond to genetic changes, a key need for engineering, remains challenging. A promising technique for leveraging omics measurements in metabolic modeling involves the construction of kinetic descriptions of the enzymatic reactions that occur within a cell. Parameterizing these models from biological data can be computationally difficult, since methods must also quantify the uncertainty in model parameters resulting from the observed data. While the field of Bayesian inference offers a wide range of methods for efficiently estimating distributions in parameter uncertainty, such techniques are poorly suited to traditional kinetic models due to their complex rate laws and resulting nonlinear dynamics. In this paper, we employ linear-logarithmic kinetics to simplify the calculation of steady-state flux distributions and enable efficient sampling and inference methods. We demonstrate that detailed information on the posterior distribution of parameters can be obtained efficiently at a variety of problem scales, including nearly genome-scale kinetic models trained on multiomics datasets. These results allow modern Bayesian machine learning tools to be leveraged in understanding biological data and in developing new, efficient strain designs.
Asunto(s)
Enzimas/metabolismo , Metabolismo/fisiología , Metabolómica/métodos , Algoritmos , Teorema de Bayes , Genómica/métodos , Cinética , Aprendizaje Automático , Ingeniería Metabólica/estadística & datos numéricos , Modelos BiológicosRESUMEN
The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.
Asunto(s)
Biología Computacional , Conformación Proteica , Proteínas/ultraestructura , Arabidopsis/química , Arabidopsis/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Proteínas/química , Proteínas/genéticaRESUMEN
Genomes of extremely thermophilic Caldicellulosiruptor species encode novel cellulose binding proteins, called tapirins, located proximate to the type IV pilus locus. The C-terminal domain of Caldicellulosiruptor kronotskyensis tapirin 0844 (Calkro_0844) is structurally unique and has a cellulose binding affinity akin to that seen with family 3 carbohydrate binding modules (CBM3s). Here, full-length and C-terminal versions of tapirins from Caldicellulosiruptor bescii (Athe_1870), Caldicellulosiruptor hydrothermalis (Calhy_0908), Caldicellulosiruptor kristjanssonii (Calkr_0826), and Caldicellulosiruptor naganoensis (NA10_0869) were produced recombinantly in Escherichia coli and compared to Calkro_0844. All five tapirins bound to microcrystalline cellulose, switchgrass, poplar, and filter paper but not to xylan. Densitometry analysis of bound protein fractions visualized by SDS-PAGE revealed that Calhy_0908 and Calkr_0826 (from weakly cellulolytic species) associated with the cellulose substrates to a greater extent than Athe_1870, Calkro_0844, and NA10_0869 (from strongly cellulolytic species). Perhaps this relates to their specific needs to capture glucans released from lignocellulose by cellulases produced in Caldicellulosiruptor communities. Calkro_0844 and NA10_0869 share a higher degree of amino acid sequence identity (>80% identity) with each other than either does with Athe_1870 (â¼50%). The levels of amino acid sequence identity of Calhy_0908 and Calkr_0826 to Calkro_0844 were only 16% and 36%, respectively, although the three-dimensional structures of their C-terminal binding regions were closely related. Unlike the parent strain, C. bescii mutants lacking the tapirin genes did not bind to cellulose following short-term incubation, suggesting a role in cell association with plant biomass. Given the scarcity of carbohydrates in neutral terrestrial hot springs, tapirins likely help scavenge carbohydrates from lignocellulose to support growth and survival of Caldicellulosiruptor species.IMPORTANCE The mechanisms by which microorganisms attach to and degrade lignocellulose are important to understand if effective approaches for conversion of plant biomass into fuels and chemicals are to be developed. Caldicellulosiruptor species grow on carbohydrates from lignocellulose at elevated temperatures and have biotechnological significance for that reason. Novel cellulose binding proteins, called tapirins, are involved in the way that Caldicellulosiruptor species interact with microcrystalline cellulose, and additional information about the diversity of these proteins across the genus, including binding affinity and three-dimensional structural comparisons, is provided here.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Celulosa/metabolismo , Firmicutes/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Celulosa/química , Firmicutes/química , Firmicutes/genética , Genoma Bacteriano , Manantiales de Aguas Termales/microbiología , Calor , Dominios ProteicosRESUMEN
The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of investigation for the consolidated processing of plant lignocellulosic biomass to biofuels and bioproducts. To investigate the synergy of enzymes involved and to further improve the ability of C. bescii to degrade cellulose, we introduced CAZymes that act synergistically with the C. bescii exoproteome in vivo and in vitro. We recently demonstrated that the Acidothermus cellulolyticus E1 endo-1,4-ß-D-glucanase (GH5) with a family 2 carbohydrate-binding module (CBM) increased the activity of C. bescii exoproteome on biomass, presumably acting in concert with CelA. The ß-glucanase, GuxA, from A. cellulolyticus is a multi-domain enzyme with strong processive exoglucanase activity, and the cellobiose phosphorylase from Thermotoga maritima catalyzes cellulose degradation acting synergistically with cellobiohydrolases and endoglucanases. We identified new chromosomal insertion sites to co-express these enzymes and the resulting strain showed a significant increase in the enzymatic activity of the exoproteome.
Asunto(s)
Celulosa/química , Glucosiltransferasas/biosíntesis , Glicósido Hidrolasas/biosíntesis , Thermoanaerobacterium/enzimología , beta-Glucanos/química , Actinomycetales/metabolismo , Biomasa , Celobiosa , Celulasa/metabolismo , Clostridiales/metabolismo , Ingeniería Genética , Técnicas Genéticas , Hidrólisis , Microbiología Industrial , Plantas/microbiología , Proteoma , Proteómica , Azúcares/químicaRESUMEN
BACKGROUND: In recent years, various substrates have been tested to increase the sustainable production of biomethane. The effect of these substrates on methanogenesis has been investigated mainly in small volume fermenters and were, for the most part, focused on studying the diversity of mesophilic microorganisms. However, studies of thermophilic communities in large scale operating mesophilic biogas plants do not yet exist. METHODS: Microbiological, biochemical, biophysical methods, and statistical analysis were used to track thermophilic communities in mesophilic anaerobic digesters. RESULTS: The diversity of the main thermophile genera in eight biogas plants located in the Czech Republic using different input substrates was investigated. In total, 19 thermophilic genera were detected after 16S rRNA gene sequencing. The highest percentage (40.8%) of thermophiles was found in the Modrice biogas plant where the input substrate was primary sludge and biological sludge (50/50, w/w %). The smallest percentage (1.87%) of thermophiles was found in the Cejc biogas plant with the input substrate being maize silage and liquid pig manure (80/20, w/w %). CONCLUSIONS: The composition of the anaerobic consortia in anaerobic digesters is an important factor for the biogas plant operator. The present study can help characterizing the impact of input feeds on the composition of microbial communities in these plants.
Asunto(s)
Biocombustibles , Consorcios Microbianos/fisiología , Aguas del Alcantarillado/microbiología , Anaerobiosis , ARN Bacteriano/genética , ARN Ribosómico 16S/genéticaRESUMEN
The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely because of the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase 1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2-fucosyl residues to xyloglucan side chains - a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X-ray crystallography, mutagenesis experiments and molecular simulations. Mammalian cell culture expressions enable the sufficient production of the enzyme for X-ray crystallography, which reveals the structural architecture of AtFUT1 in complex with bound donor and acceptor substrate analogs. The lack of an appropriately positioned active site residue as a catalytic base leads us to propose an atypical water-mediated fucosylation mechanism facilitated by an H-bonded network, which is corroborated by mutagenesis experiments as well as detailed atomistic simulations.
Asunto(s)
Arabidopsis/enzimología , Fucosiltransferasas/química , Glucanos/química , Modelos Estructurales , Xilanos/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cristalografía por Rayos X , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Glicosilación , Simulación de Dinámica Molecular , Mutagénesis , Agua/metabolismo , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
Members of the genus Caldicellulosiruptor have the ability to deconstruct and grow on lignocellulosic biomass without conventional pretreatment. A genetically tractable species, Caldicellulosiruptor bescii, was recently engineered to produce ethanol directly from switchgrass. C. bescii contains more than 50 glycosyl hydrolases and a suite of extracellular enzymes for biomass deconstruction, most prominently CelA, a multidomain cellulase that uses a novel mechanism to deconstruct plant biomass. Accumulation of cellobiose, a product of CelA during growth on biomass, inhibits cellulase activity. Here, we show that heterologous expression of a cellobiose phosphorylase from Thermotoga maritima improves the phosphorolytic pathway in C. bescii and results in synergistic activity with endogenous enzymes, including CelA, to increase cellulolytic activity and growth on crystalline cellulose.IMPORTANCE CelA is the only known cellulase to function well on highly crystalline cellulose and it uses a mechanism distinct from those of other cellulases, including fungal cellulases. Also unlike fungal cellulases, it functions at high temperature and, in fact, outperforms commercial cellulase cocktails. Factors that inhibit CelA during biomass deconstruction are significantly different than those that impact the performance of fungal cellulases and commercial mixtures. This work contributes to understanding of cellulase inhibition and enzyme function and will suggest a rational approach to engineering optimal activity.
Asunto(s)
Celulasa/metabolismo , Celulosa/metabolismo , Glucosiltransferasas/genética , Redes y Vías Metabólicas/genética , Thermotoga maritima/genética , Proteínas Bacterianas/metabolismo , Biomasa , Celobiosa/metabolismo , Celulasas/metabolismo , Glucosiltransferasas/metabolismo , Hidrólisis , Plantas/metabolismo , Thermotoga maritima/enzimologíaRESUMEN
Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways-namely, acetate and formate production-and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe.IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic acid production improvements and identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in A. succinogenes metabolism.
Asunto(s)
Actinobacillus/genética , Actinobacillus/metabolismo , Ácido Succínico/metabolismo , Reactores Biológicos/microbiología , Fermentación , Formiatos/metabolismo , Glucosa/metabolismo , Ingeniería MetabólicaRESUMEN
The use of microbial cells to convert plant biomass directly to fuels and chemicals is referred to as consolidated bioprocessing (CBP). Members of the bacterial genus, Caldicellulosiruptor (Gram-positive, anaerobic hyperthermophiles) are capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This is accomplished by the production and secretion of free, multi-domain enzymes that outperform commercial enzyme cocktails on some substrates. Here, we show that the exoproteome of Caldicellulosiruptor bescii may be enhanced by the heterologous expression of enzymes from Acidothermus cellulolyticus that act synergistically to improve sugar release from complex substrates; as well as improve cell growth. In this work, co-expression of the A. cellulolyticus Acel_0615 ß-glucanase (GH6 and GH12) and E1 endoglucanase (GH5) enzymes resulted in an increase in the activity of the exoproteome on Avicel; as well as an increase in growth of C. bescii on Avicel compared to the parental strain or the strain expressing the ß-glucanase alone. Our ability to engineer the composition and effectiveness of the exoproteome of these bacteria provides insight into the natural mechanism of plant cell wall deconstruction, as well as future directions for improving CBP. Biotechnol. Bioeng. 2017;114: 2474-2480. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Actinobacteria/genética , Celulosa/metabolismo , Mejoramiento Genético/métodos , Glicósido Hidrolasas/genética , Proteoma/metabolismo , Thermoanaerobacter/enzimología , Actinobacteria/enzimología , Activación Enzimática/genética , Hidrólisis , Thermoanaerobacter/genéticaRESUMEN
CelA is the most abundant enzyme secreted by Caldicellulosiruptor bescii and has been shown to outperform mixtures of commercially available exo- and endoglucanases in vitro. CelA contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. Here, repeated aspartate residues were introduced into the N-terminal ends of CelA GH9 and GH48 domains to improve secretion efficiency and/or catalytic efficiency of CelA. Among several constructs, the highest activity on carboxymethylcellulose (CMC), 0.81 ± 0.03 mg/mL was observed for the C. bescii strain containing CelA with 5-aspartate tag at the N-terminal end of GH9 domain-an 82% increase over wild type CelA. In addition, expression of CelA with N-terminal repeated aspartate residues in C. bescii results in a dramatic increase in its ability to grow on Avicel. Biotechnol. Bioeng. 2017;114: 945-950. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Proteínas Bacterianas/metabolismo , Celulasa/metabolismo , Celulosa/metabolismo , Firmicutes/metabolismo , Ingeniería Metabólica/métodos , Proteínas Recombinantes de Fusión/metabolismo , Ácido Aspártico/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biomasa , Celulasa/química , Celulasa/genética , Escherichia coli/genética , Firmicutes/genética , Dominios Proteicos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, ß-D-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. In spite of its ability to grow efficiently on crystalline cellulose, no extracellular ß-D-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted ß-D-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable ß-D-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that ß-D-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.
Asunto(s)
Celulosa/metabolismo , Clostridiales/genética , Clostridiales/metabolismo , Glucosidasas/genética , Glucosidasas/metabolismo , Proteoma/metabolismo , Actinomycetales/enzimología , Actinomycetales/genética , Celobiosa/metabolismo , Celulosa/química , Clostridiales/crecimiento & desarrollo , Estabilidad de Enzimas , FermentaciónRESUMEN
Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the product inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. The theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.