Sustainable Production of Microbial Isoprenoid Derived Advanced Biojet Fuels Using Different Generation Feedstocks: A Review

As the fastest mode of transport, the aircraft is a major driver for globalization and economic growth. The development of alternative advanced liquid fuels is critical to sustainable development within the sector. Such fuels should be compatible with existing infrastructure and derived from second generation feedstocks to avoid competition with food markets. With properties similar to petroleum based fuels, isoprenoid derived compounds such as limonene, bisabolane, farnesane, and pinene dimers are of increasing interest as “drop-in” replacement jet fuels. In this review potential isoprenoid derived jet fuels and progress toward their microbial production was discussed in detail. Although substantial advancements have been achieved, the use of first generation feedstocks remains ubiquitous. Lignocellulosic biomass is the most abundant raw material available for biofuel production, however, technological constraints associated with its pretreatment and saccharification hinder its economic feasibility for low-value commodity production. Non-conventional microbes with novel characteristics including cellulolytic bacteria and fungi capable of highly efficient lignocellulose degradation and xylose fermenting oleaginous yeast with enhanced lignin-associated inhibitor tolerance were investigated as alternatives to traditional model hosts. Finally, innovative bioprocessing methods including consolidated bioprocessing and sequential bioreactor approaches, with potential to capitalize on such unique natural capabilities were considered

Redox metabolism for improving whole-cell P450-catalysed terpenoid biosynthesis

The growing preference for producing cytochrome P450-mediated natural products in microbial systems stems from the challenging nature of the organic chemistry approaches. The P450 enzymes are redox-dependent proteins, through which they source electrons from reducing cofactors to drive their activities. Widely researched in biochemistry, most of the previous studies have extensively utilised expensive cell-free assays to reveal mechanistic insights into P450 functionalities in presence of commercial redox partners. However, in the context of microbial bioproduction, the synergic activity of P450- reductase proteins in microbial systems have not been largely investigated. This is mainly due to limited knowledge about their mutual interactions in the context of complex systems. Hence, manipulating the redox potential for natural product synthesis in microbial chassis has been limited. As the potential of redox state as crucial regulator of P450 biocatalysis has been greatly underestimated by the scientific community, in this review, we re-emphasize their pivotal role in modulating the in vivo P450 activity through affecting the product profile and yield. Particularly, we discuss the applications of widely used in vivo redox engineering methodologies for natural product synthesis to provide further suggestions for patterning on P450-based terpenoids production in microbial platforms

Multiplex Genome Engineering Methods for Yeast Cell Factory Development

As biotechnological applications of synthetic biology tools including multiplex genome engineering are expanding rapidly, the construction of strategically designed yeast cell factories becomes increasingly possible. This is largely due to recent advancements in genome editing methods like CRISPR/Cas tech and high-throughput omics tools. The model organism, baker’s yeast (Saccharomyces cerevisiae) is an important synthetic biology chassis for high-value metabolite production. Multiplex genome engineering approaches can expedite the construction and fine tuning of effective heterologous pathways in yeast cell factories. Numerous multiplex genome editing techniques have emerged to capitalize on this recently. This review focuses on recent advancements in such tools, such as delta integration and rDNA cluster integration coupled with CRISPR-Cas tools to greatly enhance multi-integration efficiency. Examples of pre-placed gate systems which are an innovative alternative approach for multi-copy gene integration were also reviewed. In addition to multiple integration studies, multiplexing of alternative genome editing methods are also discussed. Finally, multiplex genome editing studies involving non-conventional yeasts and the importance of automation for efficient cell factory design and construction are considered. Coupling the CRISPR/Cas system with traditional yeast multiplex genome integration or donor DNA delivery methods expedites strain development through increased efficiency and accuracy. Novel approaches such as pre-placing synthetic sequences in the genome along with improved bioinformatics tools and automation technologies have the potential to further streamline the strain development process. In addition, the techniques discussed to engineer S. cerevisiae, can be adapted for use in other industrially important yeast species for cell factory development

Rational Design of CRISPR/Cas12a-RPA Based One-Pot COVID-19 Detection with Design of Experiments

Simple and effective molecular diagnostic methods have gained importance due to the devastating effects of the COVID-19 pandemic. Various isothermal one-pot COVID-19 detection methods have been proposed as favorable alternatives to standard RT-qPCR methods as they do not require sophisticated and/or expensive devices. However, as one-pot reactions are highly complex with a large number of variables, determining the optimum conditions to maximize sensitivity while minimizing diagnostic cost can be cumbersome. Here, statistical design of experiments (DoE) was employed to accelerate the development and optimization of a CRISPR/Cas12a-RPA-based one-pot detection method for the first time. Using a definitive screening design, factors with a significant effect on performance were elucidated and optimized, facilitating the detection of two copies/μL of full-length SARS-CoV-2 (COVID-19) genome using simple instrumentation. The screening revealed that the addition of a reverse transcription buffer and an RNase inhibitor, components generally omitted in one-pot reactions, improved performance significantly, and optimization of reverse transcription had a critical impact on the method's sensitivity. This strategic method was also applied in a second approach involving a DNA sequence of the N gene from the COVID-19 genome. The slight differences in optimal conditions for the methods using RNA and DNA templates highlight the importance of reaction-specific optimization in ensuring robust and efficient diagnostic performance. The proposed detection method is automation-compatible, rendering it suitable for high-throughput testing. This study demonstrated the benefits of DoE for the optimization of complex one-pot molecular diagnostics methods to increase detection sensitivity

Exploring optimal Taxol® CYP725A4 activity in Saccharomyces cerevisiae

Background: CYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae. / Results: Investigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses. / Conclusions: This study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains

Relación entre el establecimiento de la dirección escolar y la comunicación efectiva de los directivos de la Unidad Educativa Eugenio Espejo, 2018

El presente trabajo de investigación se titula “Relación entre el establecimiento de la dirección escolar y la comunicación efectiva de los directivos de la unidad educativa Eugenio Espejo, 2018” y está vinculada a la gestión escolar con la pretensión de aportar al desarrollo de la calidad educativa en las escuelas y centros de formación de educación superior, por ende se fijó como objetivo general determinar la relación existe entre establecimiento de dirección escolar y comunicación efectiva de los directivos de la unidad educativa Eugenio Espejo, 2018. Para realizar dicha investigación se ha considerado una muestra de 35 docentes de la unidad educativa Eugenio Espejo. El diseño es de tipo correlacional y de corte transversal. Se ha consideraron dos instrumentos de investigación para evaluar ambas variables, un cuestionario de Establecimiento de la dirección escolar y otro cuestionario de comunicación efectiva, ambos validados por el alfa de Cronbach, de 0,902 y 0,988, respectivamente; y para la prueba de hipótesis general como de las específicas, se usaron el coeficiente de correlación de Pearson y el coeficiente de determinación R2 En cuanto a los resultados, con los estadísticos mencionados se ha obtenido en la tabla N° 04, un coeficiente de correlación de Pearson de 0,815, considerada como directa fuerte entre las variables de estudio establecimiento de la dirección escolar y comunicación efectiva, con lo cual se puede concluir que el tratamiento adecuado del establecimiento de la dirección escolar conduce a la mejora de la comunicación efectiva, pudiendo también considerarse que el tratamiento adecuado de la comunicación efectiva conduce a la mejora del establecimiento de la dirección escolar; y por lo cual, se recomienda investigaciones de corte experimental con el diseño de programas experimentales que consideren estrategias y técnicas adecuadas

Niveles de ansiedad y satisfacción de los pacientes de Bioholística Clínica, Lima, 2021

El presente trabajo de investigación titulado “Niveles de ansiedad y satisfacción de los pacientes de Bioholística Clínica, Lima, 2021”, presentó como objetivo principal determinar la relación entre los niveles de ansiedad y satisfacción de los pacientes de Bioholística Clínica, Lima, 2021. Para ello, se empleó una metodología investigación de enfoque cuantitativo, de método hipotético- deductivo, de nivel descriptivo, y de diseño no experimental, transversal – correlacional. Se empleó como muestra 105 pacientes, que respondieron los instrumentos que fueron dos encuestas (SERVQUAL modificada y Test de Beck modificado). Para el análisis de datos se empleó una prueba de normalidad Kolmogórov-Smirnov, que determinó una distribución anormal, por lo que se empleó la prueba no paramétrica de Rho de Spearman para el contraste de las hipótesis. Se obtuvo como principales resultados descriptivos que, la mayoría de pacientes que fueron 31 (29.5%) presentan muy baja ansiedad, seguido de 42 (40%) que presentan baja ansiedad, así mismo 55 (52.4%) se encuentran satisfechos y 40 (38.1%) totalmente satisfechos. De los resultados inferenciales se obtuvo que, la correlación entre la ansiedad y satisfacción presentó un p>0.05 (p=0.214) mientras que el Rho fue de -0.122. Por lo tanto, se concluye que no existe una relación significativa entre ambas variables

Biophysical characterization of the inactivation of E. coli transketolase by aqueous co-solvents

Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic, and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo- and holo-TK activity and structure to a range of polar organic co-solvents: acetonitrile (AcCN), n-butanol (nBuOH), ethyl acetate (EtOAc), isopropanol (iPrOH), and tetrahydrofuran (THF). The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not simply correlated to global conformational stability. The co-solvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts. Small amounts of aggregate formed in some cases, but this was not sufficient to explain the enzyme inactivation. TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) AcCN, or 6% (v/v) nBuOH over 3 h. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops