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

Introduction: Candida 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. Methods: With 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). Results: δ-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. Discussion: The 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.

Cellular Uptake, Metabolism and Sensing of Long-Chain Fatty Acids.

Fatty acids (FAs) are critical nutrients that regulate an organism's health and development in mammal. Long-chain fatty acids (LCFAs) can be divided into saturated and unsaturated fatty acids, depending on whether the carbon chain contains at least 1 double bond. The fatty acids that are required for humans and animals are obtained primarily from dietary sources, and LCFAs are absorbed from outside of cells in mammals. LCFAs enter cells through several mechanisms, including passive diffusion and protein-mediated translocation across the plasma membrane, the latter in which FA translocase (FAT/CD36), plasma membrane FA-binding protein (FABPpm), FA transport protein (FATP), and caveolin-1 are believed to have important functions. The LCFAs that are taken up by cells bind to FA-binding proteins (FABPs) and are transported to the specific organelles, where they are activated into acyl-CoA to target specific metabolic pathways. LCFA-CoAs can be esterified to phospholipids, triacylglycerol, cholesteryl ester, and other specialized lipids. Non-esterified free fatty acids are preferentially stored as triacylglycerol molecules. The main pathway by which fatty acids are catabolized is ß-oxidation, which occurs in mitochondria and peroxisomes. stearoyl-CoA desaturase (SCD)-dependent and Fatty acid desaturases (FADS)-dependent fatty acid desaturation pathways coexist in cells and provide metabolic plasticity. The process of fatty acid elongation occurs by cycling through condensation, reduction, dehydration, and reduction. Extracellular LCFA can be mediated by membrane protein G protein-coupled receptor 40 (GPR40) or G protein-coupled receptor 120 (GPR120) to activate mammalian target of rapamycin complex 1 (mTORC1) signaling, and intracellular LCFA's sensor remains to be determined. The crystal structures of a phosphatidic acid phosphatase and a membrane-bound fatty acid elongase-condensing enzyme and other LCFA-related proteins provide important insights into the mechanism of utilization, increasing our understanding of the cellular uptake, metabolism and sensing of LCFAs.

mTORC1 mediates peptidoglycan induced inflammatory cytokines expression and NF-κB activation in macrophages.

Peptidoglycan (PGN) is the major structural component of the bacterial cell wall, especially gram positive bacteria, which induces inflammatory responses. Mammalian target of rapamycin (mTOR) regulates the production of inflammatory cytokines induced by antigens, while the function of mTORC1 in peptidoglycan induced inflammatory response is unknown. This study aims to examine the role and the regulatory mechanism of mTOR signaling pathway in peptidoglycan induced cytokine expression in mouse macrophages. We observed that peptidoglycan upregulated the secretion of proinflammatory cytokines IL-6, TNF-α and anti-inflammatory cytokine IL-10 in a dose- and time-dependent manner. mTORC1 positively regulates IL-6 and TNF-α, but negatively regulates IL-10 secretion. mTORC1 regulates NF-κB p65 activation by degrading IκB-α in response to peptidoglycan. mTOR, NF-κB and STAT3 signaling pathways are involved in peptidoglycan induced inflammatory cytokines expression via a TLR1/TLR2-dependent mechanism in macrophages. Thus, mTORC1 pathway regulates the innate immune response to bacterial peptidoglycan.

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.

Current models of mammalian target of rapamycin complex 1 (mTORC1) activation by growth factors and amino acids.

Mammalian target of rapamycin (mTOR), which is now referred to as mechanistic target of rapamycin, integrates many signals, including those from growth factors, energy status, stress, and amino acids, to regulate cell growth and proliferation, protein synthesis, protein degradation, and other physiological and biochemical processes. The mTOR-Rheb-TSC-TBC complex co-localizes to the lysosome and the phosphorylation of TSC-TBC effects the dissociation of the complex from the lysosome and activates Rheb. GTP-bound Rheb potentiates the catalytic activity of mTORC1. Under conditions with growth factors and amino acids, v-ATPase, Ragulator, Rag GTPase, Rheb, hVps34, PLD1, and PA have important but disparate effects on mTORC1 activation. In this review, we introduce five models of mTORC1 activation by growth factors and amino acids to provide a comprehensive theoretical foundation for future research.