Sustainable biocatalytic synthesis of substituted muconic acids.

The development of sustainable routes to organic building blocks is a critical endeavor for reducing the environmental impact of chemical synthesis. Biocatalysts are poised to play an important role in sustainable synthesis, as they perform highly selective reactions under mild conditions. The application of enzymes to organic synthesis requires an approach which is operationally simple, inexpensive to prepare, and reasonably scalable. In this work, we demonstrated the utility of a Type I ring-cleaving dioxygenase CatA (P. putida KT2440) for preparative-scale synthesis of muconic acid derivatives. Muconic acids are important precursors in the synthesis of polymers and commodity chemicals. In this work, we optimized the performance of CatA under millimolar substrate concentrations and characterized the activity of the enzyme with an array of catechol substrates. Furthermore, we developed a scalable platform using cellular lysates to produce diverse muconic acids, generating up to a gram of the desired product. A simple trituration procedure was utilized for the purification of these muconic acids that obviated the need for chromatographic purification and reduced overall solvent waste.

Inactivation of Pseudomonas putida KT2440 pyruvate dehydrogenase relieves catabolite repression and improves the usefulness of this strain for degrading aromatic compounds.

Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss of PDH affects metabolism in Pseudomonas putida. PDH inactivation resulted in a strain unable to utilize compounds whose assimilation converges at pyruvate, including sugars and several amino acids, whereas compounds that generate acetyl-CoA supported growth. PDH inactivation also resulted in the loss of carbon catabolite repression (CCR), which inhibits the assimilation of non-preferred compounds in the presence of other preferred compounds. Pseudomonas putida can degrade many aromatic compounds, most of which produce acetyl-CoA, making it useful for biotransformation and bioremediation. However, the genes involved in these metabolic pathways are often inhibited by CCR when glucose or amino acids are also present. Our results demonstrate that the PDH-null strain can efficiently degrade aromatic compounds even in the presence of other preferred substrates, which the wild-type strain does inefficiently, or not at all. As the loss of PDH limits the assimilation of many sugars and amino acids and relieves the CCR, the PDH-null strain could be useful in biotransformation or bioremediation processes that require growth with mixtures of preferred substrates and aromatic compounds.

Bacterial surface display of PETase mutants and MHETase for an efficient dual-enzyme cascade catalysis.

Biological degradation of PET plastic holds great potential for plastic recycling. However, the high costs associated with preparing free enzymes for degrading PET make it unfeasible for industrial applications. Hence, we developed various cell catalysts by surface-displaying PETase mutants and MHETase using autotransporters in E. coli and P. putida. The efficiency of surface display was enhanced through modifying the host, co-expressing molecular chaperones, and evoluting the autotransporter. In strain EC9F, PET degradation rate was boosted to 3.85 mM/d, 51-fold and 23-fold increase compared to free enzyme and initial strain ED1, respectively. The reusability of cell catalyst EC9F was demonstrated with over 38 % and 30 % of its initial activity retained after 22 cycles of BHET degradation and 3 cycles of PET degradation. The highest reported PET degradation rate of 4.95 mM/d was achieved by the dual-enzyme cascade catalytic system EC9F+EM2+R, a mixture of cell catalyst EC9F and EM2 with surfactant rhamnolipid.

Biomolecular condensates of Chlorocatechol 1,2-Dioxygenase as prototypes of enzymatic microreactors for the degradation of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are molecules with two or more fused aromatic rings that occur naturally in the environment due to incomplete combustion of organic substances. However, the increased demand for fossil fuels in recent years has increased anthropogenic activity, contributing to the environmental concentration of PAHs. The enzyme chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is responsible for the breakdown of the aromatic ring of catechol, making it a potential player in bioremediation strategies. Pp 1,2-CCD can tolerate a broader range of substrates, including halogenated compounds, than other dioxygenases. Here, we report the construction of a chimera protein able to form biomolecular condensates with potential application in bioremediation. The chimera protein was built by conjugating Pp 1,2-CCD to low complex domains (LCDs) derived from the DEAD-box protein Dhh1. We showed that the chimera could undergo liquid-liquid phase separation (LLPS), forming a protein-rich liquid droplet under different conditions (variable protein and PEG8000 concentrations and pH values), in which the protein maintained its structure and main biophysical properties. The condensates were active against 4-chlorocatechol, showing that the chimera droplets preserved the enzymatic activity of the native protein. Therefore, it constitutes a prototype of a microreactor with potential use in bioremediation.

Synthesis of fluorinated amino acids by low-specificity, promiscuous aldolases coupled to in situ fluorodonor generation.

Fluorine (F) is an important element in the synthesis of molecules broadly used in medicine, agriculture, and materials. F addition to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to produce fluorometabolites (such as fluorinated amino acids, key building blocks for synthesis) are relatively scarce. This chapter discusses the use of L-threonine aldolase enzymes (LTAs), a class of enzymes that catalyze reversible aldol addition to the α-carbon of glycine. The C-C bond formation ability of LTAs, together with their known substrate promiscuity, make them ideal for in vitro F biocatalysis. Here, we describe protocols to harness the activity of the low-specificity LTAs isolated from Escherichia coli and Pseudomonas putida on 2-fluoroacetaldehyde to efficiently synthesize 4-fluoro-L-threonine in vitro. This chapter also provides a comprehensive account of experimental protocols to implement these activities in vivo. These methods are illustrative and can be adapted to produce other fluorometabolites of interest.

Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6

Background: Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. Results: In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2+ substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. Conclusion: The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments.

GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium.

"El hecho de que sean más mujeres, no garantiza nada": feminización y experiencias de las mujeres en la ginecobstetricia en México / "The fact that there are more women doesn't guarantee anything": The feminization of obstetrics and gynecology and the experiences of female medical professionals in Mexico

En el marco de la creciente feminización de la profesión médica en México, el artículo indaga sobre las características de este proceso para el caso de la ginecobstetricia. Considerando la feminización como un proceso de cambio, que se analiza cuantitativa y cualitativamente, el artículo se detiene en especial en las experiencias de las mujeres ginecobstetras, experiencias que se dan en el seno de una especialidad que, desde sus orígenes, funcionó como un dispositivo de control del cuerpo de las mujeres. Basado en una investigación etnográfica, el artículo combina fuentes estadísticas, de archivo y de observación de campo. El material que surge de las entrevistas muestra las experiencias y tensiones que viven las ginecobstetras en este contexto.

In the framework of an increasing feminization of the medical profession in Mexico, this article explores the characteristics of this process in the obstetrics and gynecology specialty. Understanding feminization as a process of change to be analyzed both quantitatively and qualitatively, the article focuses special attention on the experiences of female obstetrician-gynecologists within a medical specialty that has since its origins functioned as a mechanism of control over women's bodies. Based on ethnographic research, the article combines statistical and archival sources and field observation. The interviews reveal the experiences and tensions women obstetrician-gynecologists encounter in this context.

Health impact assessment of decreases in PM10 and ozone concentrations in the Mexico City Metropolitan Area: A basis for a new air quality management program / Evaluación de impacto en salud ante reducciones de PM10 y ozono en la Zona Metropolitana del Valle de México: Base para un nuevo programa de calidad del aire

Objective. To conduct a health impact assessment (HIA) to quantify health benefits for several PM and O3 air pollution reduction scenarios in the Mexico City Metropolitan Area (MCMA). Results from this HIA will contribute to the scientific support of the MCMA air quality management plan (PROAIRE) for the period 2011-2020. Materials and methods. The HIA methodology consisted of four steps: 1) selection of the air pollution reduction scenarios, 2) identification of the at-risk population and health outcomes for the 2005 baseline scenario, 3) selection of concentration-response functions and 4) estimation of health impacts. Results. Reductions of PM10 levels to 20 μg/m³ and O3 levels to 0.050ppm (98 µg/m³) would prevent 2300 and 400 annual deaths respectively. The greatest health impact was seen in the over-65 age group and in mortality due to cardiopulmonary and cardiovascular disease. Conclusion. Improved air quality in the MCMA could provide significant health benefits through focusing interventions by exposure zones.

Objetivo. Realizar una evaluación de impacto en salud (EIS) que documente los beneficios en salud ante diversos escenarios de reducción de PM10 y O3 en el aire de la Zona Metropolitana del Valle de México (ZMVM). Los resultados contribuyen al sustento científico del plan de gestión de calidad del aire (PROAIRE 2011-2020). Material y métodos. La metodología de EIS comprende cuatro pasos: 1) selección de los escenarios de reducción, 2) identificación de la población en riesgo y de los eventos en salud para el año basal 2005, 3) selección de las funciones de concentración-respuesta y 4) estimación del impacto en la salud. Resultados. Reducciones de PM10 a 20μg/m³ y de O3 a 0.050ppm (98 µg/m³) evitarían, respectivamente, cerca de 2 300 y 400 muertes por año. El mayor impacto se observa en el grupo de más de 65 años y en la mortalidad por causas cardiopulmonares y cardiovasculares. Conclusiones. Mejorar la calidad del aire en la ZMVM podría reflejar importantes beneficios para la salud focalizados por zonas o áreas de exposición.