Multidomain chimeric enzymes as a promising alternative for biocatalysts improvement: a minireview.

Searching for new and better biocatalysts is an area of study in constant development. In nature, mechanisms generally occurring in evolution, such as genetic duplication, recombination, and natural selection processes, produce various enzymes with different architectures and properties. The recombination of genes that code proteins produces multidomain chimeric enzymes that contain two or more domains that sometimes enhance their catalytic properties. Protein engineering has mimicked this process to enhance catalytic activity and the global stability of enzymes, searching for new and better biocatalysts. Here, we present and discuss examples from both natural and synthetic multidomain chimeric enzymes and how additional domains heighten their stability and catalytic activity. Moreover, we also describe progress in developing new biocatalysts using synthetic fusion enzymes and revise some methodological strategies to improve their biological fitness.

Potencial de la levadura oleaginosa Clavispora lusitaniae Hi2 en la conversión de residuos agroindustriales a lípidos / Capacity of the oleaginous yeast Clavispora lusitaniae Hi2 to transform agroindustrial residues into lipids

AntecedentesLos lípidos obtenidos de microorganismos oleaginosos a partir de hidrolizados de residuos lignocelulósicos son una alternativa para la fabricación de biodiesel.ObjetivosAislar una levadura oleaginosa capaz de producir lípidos a partir de nejayote centrifugado (NC), hidrolizado de sólidos de nejayote (HSN) e hidrolizado de bagazo de caña de azúcar (HBC).MétodosPara identificar los aislamientos recuperados se secuenció el ADN ribosómico 26S. La capacidad metabólica se evaluó mediante tiras API20C AUX. La caracterización nutricional del NC, HSN y HBC se realizó cuantificando azúcares reductores, carbohidratos totales, almidón, proteína y nitrógeno total. La capacidad de producción de biomasa y lípidos de la cepa Clavispora lusitaniae Hi2 se evaluó mediante cinéticas de crecimiento en medios de cultivo formulados a partir de NC, HSN y HBC.ResultadosSe aislaron e identificaron seis cepas de levaduras oleaginosas, siendo C. lusitaniae Hi2 seleccionada para producir lípidos mediante el uso de nejayote. Dicha cepa puede utilizar glucosa, xilosa, arabinosa, galactosa y celobiosa como fuentes de carbono. Los cultivos de C. lusitaniae Hi2 en medio con NC y HSN (en relación 25:75) presentaron la mayor producción de biomasa, 5,6 ± 0,28 g/L; la mayor producción de lípidos, 0,99±0,09 g/L, se obtuvo con una relación 50:50 de estos residuos a las 20 h de incubación.ConclusionesLa utilización de NC, HSN y HBC para el crecimiento de C. lusitaniae Hi2 es una opción para el aprovechamiento de estos residuos y la generación de compuestos de interés biotecnológico. (AU)

BackgroundSingle-cell oils obtained from oleaginous microorganisms by using lignocellulosic waste hydrolysates are an alternative for producing biodiesel.AimsTo isolate a yeast strain able to produce lipids from centrifuged nejayote (CN), hydrolyzed nejayote solids (HNS) and hydrolyzed sugarcane bagasse (HSB).MethodsIn order to identify the yeasts recovered, 26S ribosomal DNA was sequenced. The metabolic profile was assessed by using API20C AUX strips. The nutritional characterization of CN, HNS and HSB was performed by quantifying reducing sugars, total carbohydrates, starch, protein and total nitrogen. The biomass and lipid production ability were evaluated by performing growth kinetics of Clavispora lusitaniae Hi2 in combined culture media.ResultsSix oleaginous yeast strains were isolated and identified, selecting C. lusitaniae Hi2 to study its lipids production by using nejayote. The C. lusitaniae Hi2 strain can use glucose, xylose, arabinose, galactose and cellobiose as carbon sources. Cultures of C. lusitaniae Hi2 presented the best biomass (5.6±0.28 g/L) and lipid production (0.99±0.09 g/L) at 20 h of incubation with the CN:HNS media in the 25:75 and 50:50 ratios, respectively.ConclusionsThe use of CN, HNS and HSB for the growth of C. lusitaniae Hi2 is an option to take advantage of these agro-industrial residues and generate compounds of biotechnological interest. (AU)

[Capacity of the oleaginous yeast Clavispora lusitaniae Hi2 to transform agroindustrial residues into lipids]. / Potencial de la levadura oleaginosa Clavispora lusitaniae Hi2 en la conversión de residuos agroindustriales a lípidos.

BACKGROUND: Single-cell oils obtained from oleaginous microorganisms by using lignocellulosic waste hydrolysates are an alternative for producing biodiesel. AIMS: To isolate a yeast strain able to produce lipids from centrifuged nejayote (CN), hydrolyzed nejayote solids (HNS) and hydrolyzed sugarcane bagasse (HSB). METHODS: In order to identify the yeasts recovered, 26S ribosomal DNA was sequenced. The metabolic profile was assessed by using API20C AUX strips. The nutritional characterization of CN, HNS and HSB was performed by quantifying reducing sugars, total carbohydrates, starch, protein and total nitrogen. The biomass and lipid production ability were evaluated by performing growth kinetics of Clavispora lusitaniae Hi2 in combined culture media. RESULTS: Six oleaginous yeast strains were isolated and identified, selecting C. lusitaniae Hi2 to study its lipids production by using nejayote. The C. lusitaniae Hi2 strain can use glucose, xylose, arabinose, galactose and cellobiose as carbon sources. Cultures of C. lusitaniae Hi2 presented the best biomass (5.6±0.28 g/L) and lipid production (0.99±0.09 g/L) at 20 h of incubation with the CN:HNS media in the 25:75 and 50:50 ratios, respectively. CONCLUSIONS: The use of CN, HNS and HSB for the growth of C. lusitaniae Hi2 is an option to take advantage of these agro-industrial residues and generate compounds of biotechnological interest.

Abasy Atlas: a comprehensive inventory of systems, global network properties and systems-level elements across bacteria.

The availability of databases electronically encoding curated regulatory networks and of high-throughput technologies and methods to discover regulatory interactions provides an invaluable source of data to understand the principles underpinning the organization and evolution of these networks responsible for cellular regulation. Nevertheless, data on these sources never goes beyond the regulon level despite the fact that regulatory networks are complex hierarchical-modular structures still challenging our understanding. This brings the necessity for an inventory of systems across a large range of organisms, a key step to rendering feasible comparative systems biology approaches. In this work, we take the first step towards a global understanding of the regulatory networks organization by making a cartography of the functional architectures of diverse bacteria. Abasy ( A: cross- BA: cteria SY: stems) Atlas provides a comprehensive inventory of annotated functional systems, global network properties and systems-level elements (global regulators, modular genes shaping functional systems, basal machinery genes and intermodular genes) predicted by the natural decomposition approach for reconstructed and meta-curated regulatory networks across a large range of bacteria, including pathogenically and biotechnologically relevant organisms. The meta-curation of regulatory datasets provides the most complete and reliable set of regulatory interactions currently available, which can even be projected into subsets by considering the force or weight of evidence supporting them or the systems that they belong to. Besides, Abasy Atlas provides data enabling large-scale comparative systems biology studies aimed at understanding the common principles and particular lifestyle adaptions of systems across bacteria. Abasy Atlas contains systems and system-level elements for 50 regulatory networks comprising 78 649 regulatory interactions covering 42 bacteria in nine taxa, containing 3708 regulons and 1776 systems. All this brings together a large corpus of data that will surely inspire studies to generate hypothesis regarding the principles governing the evolution and organization of systems and the functional architectures controlling them.Database URL: http://abasy.ccg.unam.mx.

Catechol biosynthesis from glucose in Escherichia coli anthranilate-overproducer strains by heterologous expression of anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1.

BACKGROUND: The aromatic compound catechol is used as a precursor of chemical products having multiple applications. This compound is currently manufactured by chemical synthesis from petroleum-derived raw materials. The capacity to produce catechol is naturally present in several microbial species. This knowledge has been applied to the generation of recombinant Escherichia coli strains that can produce catechol from simple carbon sources. RESULTS: Several strains derived from E. coli W3110 trpD9923, a mutant that overproduces anthranilate, were modified by transforming them with an expression plasmid carrying genes encoding anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. The additional expression of genes encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase and transketolase from E. coli, was also evaluated. Generated strains were characterized in complex or minimal medium in shake-flask and fed-batch bioreactor cultures and incubation temperatures ranging from 37 to 28°C. These experiments enabled the identification of culture conditions for the production of 4.47 g/L of catechol with strain W3110 trpD9923, expressing 1,2-dioxygenase, DAHP synthase and transketolase. When considering the amount of glucose consumed, a yield of 16% was calculated, corresponding to 42% of the theoretical maximum as determined by elementary node flux analysis. CONCLUSIONS: This work demonstrates the feasibility of applying metabolic engineering for generating E. coli strains for the production of catechol from glucose via anthranilate. These results are a starting point to further optimize environmentally-compatible production capacity for catechol and derived compounds.

Anti-Sigma Factors in E. coli: Common Regulatory Mechanisms Controlling Sigma Factors Availability.

In bacteria, transcriptional regulation is a key step in cellular gene expression. All bacteria contain a core RNA polymerase that is catalytically competent but requires an additional σ factor for specific promoter recognition and correct transcriptional initiation. The RNAP core is not able to selectively bind to a given σ factor. In contrast, different σ factors have different affinities for the RNAP core. As a consequence, the concentration of alternate σ factors requires strict regulation in order to properly control the delicate interplay among them, which favors the competence for the RNAP core. This control is archived by different σ/anti-σ controlling mechanisms that shape complex regulatory networks and cascades, and enable the response to sudden environmental cues, whose global understanding is a current challenge for systems biology. Although there have been a number of excellent studies on each of these σ/anti-σ post-transcriptional regulatory systems, no comprehensive comparison of these mechanisms in a single model organism has been conducted. Here, we survey all these systems in E. coli dissecting and analyzing their inner workings and highlightin their differences. Then, following an integral approach, we identify their commonalities and outline some of the principles exploited by the cell to effectively and globally reprogram the transcriptional machinery. These principles provide guidelines for developing biological synthetic circuits enabling an efficient and robust response to sudden stimuli.

Prokaryotic regulatory systems biology: Common principles governing the functional architectures of Bacillus subtilis and Escherichia coli unveiled by the natural decomposition approach.

Escherichia coli and Bacillus subtilis are two of the best-studied prokaryotic model organisms. Previous analyses of their transcriptional regulatory networks have shown that they exhibit high plasticity during evolution and suggested that both converge to scale-free-like structures. Nevertheless, beyond this suggestion, no analyses have been carried out to identify the common systems-level components and principles governing these organisms. Here we show that these two phylogenetically distant organisms follow a set of common novel biologically consistent systems principles revealed by the mathematically and biologically founded natural decomposition approach. The discovered common functional architecture is a diamond-shaped, matryoshka-like, three-layer (coordination, processing, and integration) hierarchy exhibiting feedback, which is shaped by four systems-level components: global transcription factors (global TFs), locally autonomous modules, basal machinery and intermodular genes. The first mathematical criterion to identify global TFs, the κ-value, was reassessed on B. subtilis and confirmed its high predictive power by identifying all the previously reported, plus three potential, master regulators and eight sigma factors. The functionally conserved cores of modules, basal cell machinery, and a set of non-orthologous common physiological global responses were identified via both orthologous genes and non-orthologous conserved functions. This study reveals novel common systems principles maintained between two phylogenetically distant organisms and provides a comparison of their lifestyle adaptations. Our results shed new light on the systems-level principles and the fundamental functions required by bacteria to sustain life.

Molecular characterization of chloranilic acid degradation in Pseudomonas putida TQ07.

Pentachlorophenol is the most toxic and recalcitrant chlorophenol because both aspects are directly proportional to the halogenation degree. Biological and abiotic pentachlorophenol degradation generates p-chloranil, which in neutral to lightly alkaline environmental conditions is hydrolyzed to chloranilic acid that present a violet-reddish coloration in aqueous solution. Several genes of the degradation pathway, cadR-cadCDX, as well as other uncharacterized genes (ORF5 and 6), were isolated from a chloranilic acid degrading bacterium, Pseudomonas putida strain TQ07. The disruption by random mutagenesis of the cadR and cadC genes in TQ07 resulted in a growth deficiency in the presence of chloranilic acid, indicating that these genes are essential for TQ07 growth with chloranilic acid as the sole carbon source. Complementation assays demonstrated that a transposon insertion in mutant CAD82 (cadC) had a polar effect on other genes contained in cosmid pLG3562. These results suggest that at least one of these genes, cadD and cadX, also takes part in chloranilic acid degradation. Based on molecular modeling and function prediction, we strongly suggest that CadC is a pyrone dicarboxylic acid hydrolase and CadD is an aldolase enzyme like dihydrodipicolinate synthase. The results of this study allowed us to propose a novel pathway that offers hypotheses on chloranilic acid degradation (an abiotic by-product of pentachlorophenol) by means of a very clear phenotype that is narrowly related to the capability of Pseudomonas putida strain TQ07 to degrade this benzoquinone.

Functional architecture of Escherichia coli: new insights provided by a natural decomposition approach.

BACKGROUND: Previous studies have used different methods in an effort to extract the modular organization of transcriptional regulatory networks. However, these approaches are not natural, as they try to cluster strongly connected genes into a module or locate known pleiotropic transcription factors in lower hierarchical layers. Here, we unravel the transcriptional regulatory network of Escherichia coli by separating it into its key elements, thus revealing its natural organization. We also present a mathematical criterion, based on the topological features of the transcriptional regulatory network, to classify the network elements into one of two possible classes: hierarchical or modular genes. RESULTS: We found that modular genes are clustered into physiologically correlated groups validated by a statistical analysis of the enrichment of the functional classes. Hierarchical genes encode transcription factors responsible for coordinating module responses based on general interest signals. Hierarchical elements correlate highly with the previously studied global regulators, suggesting that this could be the first mathematical method to identify global regulators. We identified a new element in transcriptional regulatory networks never described before: intermodular genes. These are structural genes that integrate, at the promoter level, signals coming from different modules, and therefore from different physiological responses. Using the concept of pleiotropy, we have reconstructed the hierarchy of the network and discuss the role of feedforward motifs in shaping the hierarchical backbone of the transcriptional regulatory network. CONCLUSIONS: This study sheds new light on the design principles underpinning the organization of transcriptional regulatory networks, showing a novel nonpyramidal architecture composed of independent modules globally governed by hierarchical transcription factors, whose responses are integrated by intermodular genes.

RegulonDB (version 6.0): gene regulation model of Escherichia coli K-12 beyond transcription, active (experimental) annotated promoters and Textpresso navigation.

RegulonDB (http://regulondb.ccg.unam.mx/) is the primary reference database offering curated knowledge of the transcriptional regulatory network of Escherichia coli K12, currently the best-known electronically encoded database of the genetic regulatory network of any free-living organism. This paper summarizes the improvements, new biology and new features available in version 6.0. Curation of original literature is, from now on, up to date for every new release. All the objects are supported by their corresponding evidences, now classified as strong or weak. Transcription factors are classified by origin of their effectors and by gene ontology class. We have now computational predictions for sigma(54) and five different promoter types of the sigma(70) family, as well as their corresponding -10 and -35 boxes. In addition to those curated from the literature, we added about 300 experimentally mapped promoters coming from our own high-throughput mapping efforts. RegulonDB v.6.0 now expands beyond transcription initiation, including RNA regulatory elements, specifically riboswitches, attenuators and small RNAs, with their known associated targets. The data can be accessed through overviews of correlations about gene regulation. RegulonDB associated original literature, together with more than 4000 curation notes, can now be searched with the Textpresso text mining engine.

The phosphotransferase system-dependent sucrose utilization regulon in enteropathogenic Escherichia coli strains is located in a variable chromosomal region containing iap sequences.

The capacity to utilize sucrose as a carbon and energy source (Scr(+) phenotype) is a highly variable trait among Escherichia coli strains. In this study, seven enteropathogenic E. coli (EPEC) strains from different sources were studied for their capacity to grow using sucrose. Liquid media cultures showed that all analyzed strains have the Scr(+) phenotype and two distinct groups were defined: one of five and another of two strains displaying doubling times of 67 and 125 min, respectively. The genes conferring the Scr(+) phenotype in one of the fast-growing strains (T19) were cloned and sequenced. Comparative sequence analysis revealed that this strain possesses the scr regulon genes scrKYABR, encoding phosphoenolpyruvate:phosphotransferase system-dependent sucrose transport and utilization activities. Transcript level quantification revealed sucrose-dependent induction of scrK and scrR genes in fast-growing strains, whereas no transcripts were detected in slow-growing strains. Sequence comparison analysis revealed that the scr genes in strain T19 are almost identical to those present in the scr regulon of prototype EPEC E2348/69 and in both strains, the scr genes are inserted in the chromosomal intergenic region of hypothetical genes ygcE and ygcF. Comparison of the ygcE-ygcF intergenic region sequence of strains MG1655, enterohemorrhagic EDL933, uropathogenic ECFT073 and EPEC T19-E2348/69 revealed that the number of extragenic highly repeated iap sequences corresponded to nine, four, two and none, respectively. These results show that the iap sequence-containing chromosomal ygcE-ygcF intergenic region is highly variable in E. coli.