RESUMO
From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding 'higher' Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-microl environment can be.
Assuntos
Bactérias/metabolismo , Genoma Bacteriano/genética , Genômica , Intestinos/microbiologia , Isópteros/metabolismo , Isópteros/microbiologia , Madeira/metabolismo , Animais , Bactérias/enzimologia , Bactérias/genética , Bactérias/isolamento & purificação , Fontes de Energia Bioelétrica , Carbono/metabolismo , Domínio Catalítico , Celulose/metabolismo , Costa Rica , Genes Bacterianos/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hidrólise , Lignina/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Simbiose , Madeira/química , Xilanos/metabolismoRESUMO
Several emerging technologies are aiming to meet renewable fuel standards, mitigate greenhouse gas emissions, and provide viable alternatives to fossil fuels. Direct conversion of solar energy into fungible liquid fuel is a particularly attractive option, though conversion of that energy on an industrial scale depends on the efficiency of its capture and conversion. Large-scale programs have been undertaken in the recent past that used solar energy to grow innately oil-producing algae for biomass processing to biodiesel fuel. These efforts were ultimately deemed to be uneconomical because the costs of culturing, harvesting, and processing of algal biomass were not balanced by the process efficiencies for solar photon capture and conversion. This analysis addresses solar capture and conversion efficiencies and introduces a unique systems approach, enabled by advances in strain engineering, photobioreactor design, and a process that contradicts prejudicial opinions about the viability of industrial photosynthesis. We calculate efficiencies for this direct, continuous solar process based on common boundary conditions, empirical measurements and validated assumptions wherein genetically engineered cyanobacteria convert industrially sourced, high-concentration CO(2) into secreted, fungible hydrocarbon products in a continuous process. These innovations are projected to operate at areal productivities far exceeding those based on accumulation and refining of plant or algal biomass or on prior assumptions of photosynthetic productivity. This concept, currently enabled for production of ethanol and alkane diesel fuel molecules, and operating at pilot scale, establishes a new paradigm for high productivity manufacturing of nonfossil-derived fuels and chemicals.
Assuntos
Biocombustíveis , Clorófitas/metabolismo , Cianobactérias/metabolismo , Microbiologia Industrial/tendências , Fotossíntese , Biomassa , Clorófitas/crescimento & desenvolvimento , Técnicas de Cultura , Cianobactérias/crescimento & desenvolvimento , Eficiência , Mitocôndrias/metabolismo , Fótons , Luz SolarRESUMO
The use of enzymes in industrial catalysis continues to grow because of the considerable advantages of natural catalytic systems. The need for enantiomerically pure fine chemicals and the movement away from chemically burdened technologies will drive the acceptance of enzyme-assisted processes. New technologies for enzyme discovery and optimization have enabled the application of enzymes in harsh industrial conditions and in processes demanding stringent selectivity. These discovery and laboratory evolution methods entail genomic approaches that by their nature engender screening of extremely large numbers of gene types and variants. By extension, the fitness of an individual high-throughput screen requires an intelligent, process-targeted assay amenable to a chosen screening platform.
Assuntos
Biotecnologia/tendências , Enzimas/genética , Catálise , Estabilidade Enzimática , Enzimas/isolamento & purificação , Enzimas/metabolismo , Expressão Gênica , Biblioteca Gênica , Genômica , Mutagênese , Especificidade por SubstratoRESUMO
Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90 degrees C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61 degrees C to as high as 96 degrees C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild-type parent).
Assuntos
Evolução Molecular Direcionada/métodos , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Substituição de Aminoácidos , Endo-1,4-beta-Xilanases/química , Estabilidade Enzimática , Variação Genética/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Mapeamento de Peptídeos , Homologia de Sequência de Aminoácidos , Temperatura de TransiçãoAssuntos
Mutagênese/genética , Proteínas/genética , Sequência de Aminoácidos , Aminoácidos/genética , Códon/genética , Primers do DNA , Escherichia coli/genética , Técnicas Genéticas , Hidrolases/química , Hidrolases/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Mapeamento por Restrição/métodos , Rhodococcus/enzimologia , Rhodococcus/genéticaRESUMO
A unique multifunctional glycosyl hydrolase was discovered by screening an environmental DNA library prepared from a microbial consortium collected from cow rumen. The protein consists of two adjacent catalytic domains. Sequence analysis predicted that one domain conforms to glycosyl hydrolase family 5 and the other to family 26. The enzyme is active on several different beta-linked substrates and possesses mannanase, xylanase, and glucanase activities. Site-directed mutagenesis studies on the catalytic residues confirmed the presence of two functionally independent catalytic domains. Using site-specific mutations, it was shown that one catalytic site hydrolyzes beta-1,4-linked mannan substrates, while the second catalytic site hydrolyzes beta-1,4-linked xylan and beta-1,4-linked glucan substrates. Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE) also confirmed that the enzyme has discrete domains for binding and hydrolysis of glucan- and mannan-linked polysaccharides. Such multifunctional enzymes have many potential industrial applications in plant processing, including biomass saccharification, animal feed nutritional enhancement, textile, and pulp and paper processing.
Assuntos
Glicosídeo Hidrolases , Complexos Multienzimáticos , Rúmen/microbiologia , Animais , Sequência de Bases , Bovinos , Biblioteca Gênica , Glucanos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Microbiologia Industrial , Mananas/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutagênese Sítio-Dirigida , Análise de Sequência de DNA , Xilanos/metabolismoRESUMO
There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.
Assuntos
Fibra de Algodão , Polissacarídeo-Liases/metabolismo , Bactérias/classificação , Bactérias/enzimologia , Evolução Molecular Direcionada , Biblioteca Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Polissacarídeo-Liases/química , Polissacarídeo-Liases/genética , Conformação Proteica , Proteínas Recombinantes/metabolismoRESUMO
High throughput screening of microbial DNA libraries was used to identify alpha-amylases with phenotypic characteristics compatible with large scale corn wet milling process conditions. Single and multiorganism DNA libraries originating from various environments were targeted for activity and sequence-based screening approaches. After initial screening, 15 clones were designated as primary hits based upon activity at pH 4.5 or 95 degrees C without addition of endogenous Ca(2+). After further characterization, three enzyme candidates were chosen each with an exceptional expression of one or more aspects of the necessary phenotype: temperature stability, pH optimum, lowered reliance on Ca(2+) and/or enzyme rate. To combine the best aspects of the three phenotypes to optimize process compatibility, the natural gene homologues were used as a parental sequence set for gene reassembly. Approximately 21,000 chimeric daughter sequences were generated and subsets screened using a process-specific, high throughput activity assay. Gene reassembly resulted in numerous improved mutants with combined optimal phenotypes of expression, temperature stability, and pH optimum. After biochemical and process-specific characterization of these gene products, one alpha-amylase with exceptional process compatibility and economics was identified. This paper describes the synergistic approach of combining environmental discovery and laboratory evolution for identification and optimization of industrially important biocatalysts.
Assuntos
Tecnologia de Alimentos/métodos , Amido/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismo , Sequência de Aminoácidos , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Zea mays , alfa-Amilases/isolamento & purificaçãoRESUMO
The discovery, from nature, of a diverse set of microbial epoxide hydrolases is reported. The utility of a library of epoxide hydrolases in the synthesis of chiral 1,2-diols via desymmetrization of a wide range of meso-epoxides, including cyclic as well as acyclic alkyl- and aryl-substituted substrates, is demonstrated. The chiral (R,R)-diols were furnished with high ee's and yields. The discovery of the first microbial epoxide hydrolases providing access to complementary (S,S)-diols is also described.
Assuntos
Álcoois/química , Epóxido Hidrolases/química , Compostos de Epóxi/química , Catálise , Epóxido Hidrolases/metabolismo , EstereoisomerismoRESUMO
The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62 degrees C for 1 h and 27% of its initial activity after 10 min at 85 degrees C, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.
Assuntos
6-Fitase/genética , 6-Fitase/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Suco Gástrico/enzimologia , Temperatura Alta , 6-Fitase/química , Fosfatase Ácida/química , Ração Animal , Animais , Suplementos Nutricionais , Estabilidade Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosfatos/metabolismo , Mutação PuntualRESUMO
The discovery, from Nature, of a large and diverse set of nitrilases is reported. The utility of this nitrilase library for identifying enzymes that catalyze efficient production of valuable hydroxy carboxylic acid derivatives is demonstrated. Unprecedented enantioselectivity and substrate scope are highlighted for three newly discovered and distinct nitrilases. For example, a wide array of (R)-mandelic acid derivatives and analogues were produced with high rates, yields, and enantiomeric excesses (95-99% ee). We also have found nitrilases that provide direct access to (S)-phenyllactic acid and other aryllactic acid derivatives, again with high yields and enantioselectivities. Finally, different nitrilases have been discovered that catalyze enantiotopic hydrolysis of 3-hydroxyglutaronitrile to afford either enantiomer of 4-cyano-3-hydroxybutyric acid with high enantiomeric excesses (>95% ee). The first enzymes are reported that effect this transformation to furnish the (R)-4-cyano-3-hydroxybutyric acid which is a precursor to the blockbuster drug Lipitor.
Assuntos
Aminoidrolases/química , Aminoidrolases/genética , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/química , Catálise , Biblioteca Gênica , Hidrólise , Hidroxiácidos/síntese química , Lactatos/síntese química , Ácidos Mandélicos/síntese química , EstereoisomerismoRESUMO
Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10(6) to 10(10) members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.
Assuntos
Aminoidrolases/genética , Aminoidrolases/metabolismo , Catálise , Microbiologia Ambiental , Biblioteca Gênica , Dados de Sequência Molecular , Nitrilas/química , Nitrilas/metabolismo , Filogenia , Estereoisomerismo , Especificidade por SubstratoRESUMO
Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted beta-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.