Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein.

The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.

Yeasts of the Blastobotrys genus are promising platform for lipid-based fuels and oleochemicals production.

Strains of the yeast genus Blastobotrys (subphylum Saccharomycotina) represent a valuable biotechnological resource for basic biochemistry research, single-cell protein, and heterologous protein production processes. Species of this genus are dimorphic, non-pathogenic, thermotolerant, and can assimilate a variety of hydrophilic and hydrophobic substrates. These can constitute a single-cell oil platform in an emerging bio-based economy as oleaginous traits have been discovered recently. However, the regulatory network of lipogenesis in these yeasts is poorly understood. To keep pace with the growing market demands for lipid-derived products, it is critical to understand the lipid biosynthesis in these unconventional yeasts to pinpoint what governs the preferential channelling of carbon flux into lipids instead of the competing pathways. This review summarizes information relevant to the regulation of lipid metabolic pathways and prospects of metabolic engineering in Blastobotrys yeasts for their application in food, feed, and beyond, particularly for fatty acid-based fuels and oleochemicals. KEY POINTS: • The production of biolipids by heterotrophic yeasts is reviewed. • Summary of information concerning lipid metabolism regulation is highlighted. • Special focus on the importance of diacylglycerol acyltransferases encoding genes in improving lipid production is made.

A repressor activator protein1 homologue from an oleaginous strain of Candida tropicalis increases storage lipid production in Saccharomyces cerevisiae.

The repressor activator protein1 (Rap1) has been studied over the years as a multifunctional regulator in Saccharomyces cerevisiae. However, its role in storage lipid accumulation has not been investigated. This report documents the identification and isolation of a putative transcription factor CtRap1 gene from an oleaginous strain of Candida tropicalis, and establishes the direct effect of its expression on the storage lipid accumulation in S. cerevisiae, usually a non-oleaginous yeast. In silico analysis revealed that the CtRap1 polypeptide binds relatively more strongly to the promoter of fatty acid synthase1 (FAS1) gene of S. cerevisiae than ScRap1. The expression level of CtRap1 transcript in vivo was found to correlate directly with the amount of lipid produced in oleaginous native host C. tropicalis. Heterologous expression of the CtRap1 gene resulted in ∼ 4-fold enhancement of storage lipid content (57.3%) in S. cerevisiae. We also showed that the functionally active CtRap1 upregulates the endogenous ScFAS1 and ScDGAT genes of S. cerevisiae, and this, in turn, might be responsible for the increased lipid production in the transformed yeast. Our findings pave the way for the possible utility of the CtRap1 gene in suitable microorganisms to increase their storage lipid content through transcription factor engineering.