An integrated transcriptomic and metabolic phenotype analysis to uncover the metabolic characteristics of a genetically engineered Candida utilis strain expressing δ-zein gene

IntroductionCandida utilis (C. utilis) has been extensively utilized as human food or animal feed additives. With its ability to support heterologous gene expression, C. utilis proves to be a valuable platform for the synthesis of proteins and metabolites that possess both high nutritional and economic value. However, there remains a dearth of research focused on the characteristics of C. utilis through genomic, transcriptomic and metabolic approaches.MethodsWith the aim of unraveling the molecular mechanism and genetic basis governing the biological process of C. utilis, we embarked on a de novo sequencing endeavor to acquire comprehensive sequence data. In addition, an integrated transcriptomic and metabolic phenotype analysis was performed to compare the wild-type C. utilis (WT) with a genetically engineered strain of C. utilis that harbors the heterologous δ-zein gene (RCT).Resultsδ-zein is a protein rich in methionine found in the endosperm of maize. The integrated analysis of transcriptomic and metabolic phenotypes uncovered significant metabolic diversity between the WT and RCT C. utilis. A total of 252 differentially expressed genes were identified, primarily associated with ribosome function, peroxisome activity, arginine and proline metabolism, carbon metabolism, and fatty acid degradation. In the experimental setup using PM1, PM2, and PM4 plates, a total of 284 growth conditions were tested. A comparison between the WT and RCT C. utilis demonstrated significant increases in the utilization of certain carbon source substrates by RCT. Gelatin and glycogen were found to be significantly utilized to a greater extent by RCT compared to WT. Additionally, in terms of sulfur source substrates, RCT exhibited significantly increased utilization of O-Phospho-L-Tyrosine and L-Methionine Sulfone when compared to WT.DiscussionThe introduction of δ-zein gene into C. utilis may lead to significant changes in the metabolic substrates and metabolic pathways, but does not weaken the activity of the strain. Our study provides new insights into the transcriptomic and metabolic characteristics of the genetically engineered C. utilis strain harboring δ-zein gene, which has the potential to advance the utilization of C. utilis as an efficient protein feed in agricultural applications

Rapamycin Inhibits Expression of Elongation of Very-long-chain Fatty Acids 1 and Synthesis of Docosahexaenoic Acid in Bovine Mammary Epithelial Cells

Mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth and metabolism and is sufficient to induce specific metabolic processes, including de novo lipid biosynthesis. Elongation of very-long-chain fatty acids 1 (ELOVL1) is a ubiquitously expressed gene and the product of which was thought to be associated with elongation of carbon (C) chain in fatty acids. In the present study, we examined the effects of rapamycin, a specific inhibitor of mTORC1, on ELOVL1 expression and docosahexaenoic acid (DHA, C22:6 n-3) synthesis in bovine mammary epithelial cells (BMECs). We found that rapamycin decreased the relative abundance of ELOVL1 mRNA, ELOVL1 expression and the level of DHA in a time-dependent manner. These data indicate that ELOVL1 expression and DHA synthesis are regulated by mTORC1 in BMECs

Inhibition of Mammalian Target of Rapamycin Complex 1 (mTORC1) Downregulates ELOVL1 Gene Expression and Fatty Acid Synthesis in Goat Fetal Fibroblasts

Elongation of very-long-chain fatty acids 1 (ELOVL1) is a ubiquitously expressed gene that belongs to the ELOVL family and regulates the synthesis of very-long-chain fatty acids (VLCFAs) and sphingolipids, from yeast to mammals. Mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of cell metabolism and is associated with fatty acids synthesis. In this study, we cloned the cDNA that encodes Cashmere goat (Capra hircus) ELOVL1 (GenBank Accession number KF549985) and investigated its expression in 10 tissues. ELOVL1 cDNA was 840 bp, encoding a deduced protein of 279 amino acids, and ELOVL1 mRNA was expressed in a wide range of tissues. Inhibition of mTORC1 by rapamycin decreased ELOVL1 expression and fatty acids synthesis in Cashmere goat fetal fibroblasts. These data show that ELOVL1 expression is regulated by mTORC1 and that mTORC1 has significant function in fatty acids synthesis in Cashmere goat

SQSTM1/p62 interacts with FKBP38 and regulates cell cycle in Cashmere goat foetal fibroblasts

<p>SQSTM1 (sequestosome 1, also known as p62) is a multifunctional scaffold protein implicated in diverse cell physiology processes, such as autophagy, cell signalling, and protein turnover. FKBP38 (FK506-binding protein 38) is a member of FKBPs family and plays a key role in various cellular processes, including signalling transduction, embryonic development and apoptosis. In order to explore the role of SQSTM1/p62 gene in cell-cycle progression and proliferation of goat foetal fibroblast (GFbs), SQSTM1/p62 gene was cloned and characterized. Furthermore, the yeast two-hybrid screening system was used to identify the interaction between SQSTM1/p62 and FKBP38. The results suggested that SQSTM1/p62 directly interacts with FKBP38. Overexpression vector pIRES-EGFP-SQSTM1/p62 and shRNA eukaryotic expression vector pRNAT-U6.1-shSQSTM1/p62 which harboured siRNA targeting the SQSTM1/p62 mRNA were constructed. The overexpression of SQSTM1/p62 gene in GFbs significantly increase the S-phase cells compared with control cells (<i>p </i>< .05). Furthermore, SQSTM1/p62 gene silencing in GFbs leads to a significant decrease of S-phase cells and cell cycle arrest compared with control cells (<i>p </i>< .05). These data indicate that SQSTM1/p62 gene plays an important role in cell-cycle and proliferation of Cashmere GFbs.</p> <p>Abbreviations: Aba: aureobasidin A; EIF1: eukaryotic translation initiation factor 1; FKBP: FK506-binding protein; FKBP38: FK506-binding protein 38; GFbs: goat foetal fibroblast; mTOR: mammalian target of rapamycin; PDLIM7: PDZ and LIM domain 7; SD: standard dropout; SQSTM1/p62: sequestosome 1</p