A structural model of EFL teachers' physical activity, emotion regulation, and competence for online teaching.

BACKGROUND: The COVID-19 pandemic has prompted a rapid shift to online teaching, placing unprecedented demands on educators' physical and mental well-being. However, the relationship between English as a Foreign Language (EFL) teachers' physical activity, emotion regulation, and competence for online teaching remains underexplored. OBJECTIVES: This study aimed to investigate the interplay between EFL teachers' physical activity, emotion regulation strategies, and competence for online teaching. RESULTS: Structural equation modeling revealed significant direct and indirect effects, indicating that physical activity positively influences emotion regulation, which, in turn, enhances teachers' competence for online instruction. Furthermore, emotion regulation was found to mediate the relationship between physical activity and online teaching competence. CONCLUSIONS: These findings underscore the importance of promoting physical activity among EFL teachers as a means to enhance their emotion regulation skills and competence for online teaching, particularly in the context of the COVID-19 pandemic. IMPLICATIONS: The study highlights the need for targeted interventions aimed at supporting EFL teachers' well-being and professional development, with implications for educational policies, teacher training programs, and institutional support structures in the digital learning landscape.

High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components.

BACKGROUND: Lupeol exhibits novel physiological and pharmacological activities, such as anticancer and immunity-enhancing activities. However, cytotoxicity remains a challenge for triterpenoid overproduction in microbial cell factories. As lipophilic and relatively small molecular compounds, triterpenes are generally secreted into the extracellular space. The effect of increasing triterpene efflux on the synthesis capacity remains unknown. RESULTS: In this study, we developed a strategy to enhance triterpene efflux through manipulation of lipid components in Y. lipolytica by overexpressing the enzyme Δ9-fatty acid desaturase (OLE1) and disturbing phosphatidic acid phosphatase (PAH1) and diacylglycerol kinase (DGK1). By this strategy combined with two-phase fermentation, the highest lupeol production reported to date was achieved, where the titer in the organic phase reached 381.67 mg/L and the total production was 411.72 mg/L in shake flasks, exhibiting a 33.20-fold improvement over the initial strain. Lipid manipulation led to a twofold increase in the unsaturated fatty acid (UFA) content, up to 61-73%, and an exceptionally elongated cell morphology, which might have been caused by enhanced membrane phospholipid biosynthesis flux. Both phenotypes accelerated the export of toxic products to the extracellular space and ultimately stimulated the capacity for triterpenoid synthesis, which was proven by the 5.11-fold higher ratio of extra/intracellular lupeol concentrations, 2.79-fold higher biomass accumulation and 2.56-fold higher lupeol productivity per unit OD in the modified strains. This strategy was also highly efficient for the biosynthesis of other triterpenes and sesquiterpenes, including α-amyrin, ß-amyrin, longifolene, longipinene and longicyclene. CONCLUSIONS: In conclusion, we successfully created a high-yield lupeol-producing strain via lipid manipulation. We demonstrated that the enhancement of lupeol efflux and synthesis capacity was induced by the increased UFA content and elongated cell morphology. Our study provides a novel strategy to promote the biosynthesis of valuable but toxic products in microbial cell factories.

Gene repression via multiplex gRNA strategy in Y. lipolytica.

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest. RESULTS: In this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized. CONCLUSION: Taken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.