La microbiota ruminal como fuente de enzimas industriales

Resumen el español e inglésTítulo en inglés: Ruminal microbiota as a source of industrial enzymesEl rumen constituye una gran cámara de fermentación colonizada por una compleja comunidad microbiana que está compuesta mayoritariamente por especies que no pueden cultivarse. Por ello, la microbiota ruminal representa una fuente inexplorada de enzimas para la industria biotecnológica. La diversidad funcional de estos microorganismos tiene un alto potencial para el desarrollo de aplicaciones industriales más sostenibles y eficientes (p. ej., para la producción de biodiesel, la degradación de sustancias plásticas o la fabricación de ingredientes de origen biológico). Nuestros equipos de investigación están trabajando con contenido digestivo del rumen de ovejas. Mediante un cribado de alta capacidad, buscamos nuevas enzimas relacionadas con el metabolismo lipídico (hidratasas y deshidrogenasas). Entre sus posibles aplicaciones estaría la de contribuir a la biotransformación; por ejemplo, convirtiendo los ácidos grasos de diversos residuos, como los aceites usados de fritura, en productos con mayor valor añadido (hidroxi- y cetoácidos)The rumen is a large fermentation chamber colonised by a complex mi crobial community that is mostly composed of as-yet uncultured species. Therefore, rumen microbiota represents an unexplored source of enzymes for the biotechnology industry. The functional diversity of these microor ganisms has high potential for the development of more sustainable and efficient industrial applications (e.g., for the production of biodiesel, the degradation of plastic substances or the manufacture of bio-based ingre dients). Our research teams are working with rumen digesta of sheep. We use high throughput screening to search for new enzymes related to lipid metabolism (hydratases and dehydrogenases). Potential applications in clude contribution to biotransformation, for example, by converting fatty acids from various waste products, such as used frying oils, into higher value-added products (hydroxy- and keto-acids

Thermostable in vitro transcription-translation compatible with microfluidic droplets

Background: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Objectives: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. Results: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Conclusions: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughputThis work has received funding from the European Union\u2019s Research and Innovation Framework programs FP7 and Horizon 2020 under Grant Agreement numbers 324439, 635595, 685474, 695669 and 10100560 and from the Spanish Ministry of Economy and Competitiveness under grant number BIO-2013-44963-R. The CBM is funded by \u201CCentre of Excellence Severo Ochoa\u201D Grant CEX2021-001154-S from MICIU/AEI / https://doi.org/10.13039/501100011033 and receives institutional support by Fundaci\u00F3n Ram\u00F3n Arece

A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity

In the last two decades, several PET-degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high-throughput screening protocols for PET-hydrolyzing activity that avoid the use of surrogate substrates. Herein, a microplate fluorescence screening assay was described. It was based on the coupled activity of ketoreductases (KREDs) and diaphorase to release resorufin in the presence of the products of PET degradation. Six KREDs were identified in a commercial panel that were able to use the PET building block, ethylene glycol, as substrate. The most efficient KRED, KRED61, was combined with the diaphorase from Clostridium kluyveri to monitor the PET degradation reaction catalyzed by the thermostable variant of the cutinase-type polyesterase from Saccharomonospora viridis AHK190. The PET degradation products were measured both fluorimetrically and by HPLC, with excellent correlation between both methodsts This work was supported by a grant from the Universidad Autónoma de Madrid (Project for Young Researchers, Ref. SI1/PJI/ 2019-00394). D. M. M. was supported by a Research Talent Attraction contract from the Community of Madrid (Ref. 2016-T2/ BIO-1960). We thank Carmen Ortega (Center of Molecular Biology “Severo Ochoa”, UAM) for preliminary tests with KREDs. We are also grateful to Jose Luis González Alfonso (Institute of Catalysis and Petrochemistry, CSIC) and David Rodrigo Frutos (Analiza Calidad Madrid S.L.) for technical support on HPLC analyse

Thermostability engineering of a class ii pyruvate aldolase from Escherichia coli by in vivo folding interference

[Image: see text] The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better “evolvability” of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (T(m)) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T(m) compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T(m) 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters