New CRISPR Technology for Creating Cell Models of Lipoprotein Assembly and Secretion.

PURPOSE OF REVIEW: This review is aimed at providing an overview of new developments in gene editing technology, including examples of how this technology has been used to develop cell models for studying the effects of gene ablation or missense mutations on lipoprotein assembly and secretion. RECENT FINDINGS: CRISPR/Cas9-mediated gene editing is superior to other technologies because of its ease, sensitivity, and low off-target effects. This technology has been used to study the importance of microsomal triglyceride transfer protein in the assembly and secretion of apolipoprotein B-containing lipoproteins, as well as to establish causal effects of APOB gene missense mutations on lipoprotein assembly and secretion. CRISPR/Cas9 technology is anticipated to provide unprecedented flexibility in studying protein structure and function in cells and animals and to yield mechanistic insights into variants in the human genome.

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.

Lipid production by oleaginous yeasts.

Microbial lipid production has been studied extensively for years; however, lipid metabolic engineering in many of the extraordinarily high lipid-accumulating yeasts was impeded by inadequate understanding of the metabolic pathways including regulatory mechanisms defining their oleaginicity and the limited genetic tools available. The aim of this review is to highlight the prominent oleaginous yeast genera, emphasizing their oleaginous characteristics, in conjunction with diverse other features such as cheap carbon source utilization, withstanding the effect of inhibitory compounds, commercially favorable fatty acid composition-all supporting their future development as economically viable lipid feedstock. The unique aspects of metabolism attributing to their oleaginicity are accentuated in the pretext of outlining the various strategies successfully implemented to improve the production of lipid and lipid-derived metabolites. A large number of in silico data generated on the lipid accumulation in certain oleaginous yeasts have been carefully curated, as suggestive evidences in line with the exceptional oleaginicity of these organisms. The different genetic elements developed in these yeasts to execute such strategies have been scrupulously inspected, underlining the major types of newly-found and synthetically constructed promoters, transcription terminators, and selection markers. Additionally, there is a plethora of advanced genetic toolboxes and techniques described, which have been successfully used in oleaginous yeasts in the recent years, promoting homologous recombination, genome editing, DNA assembly, and transformation at remarkable efficiencies. They can accelerate and effectively guide the rational designing of system-wide metabolic engineering approaches pinpointing the key targets for developing industrially suitable yeast strains.

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.

Recent advances in lipid metabolic engineering of oleaginous yeasts.

With the increasing demand to develop a renewable and sustainable biolipid feedstock, several species of non-conventional oleaginous yeasts are being explored. Apart from the platform oleaginous yeast Yarrowia lipolytica, the understanding of metabolic pathway and, therefore, exploiting the engineering prospects of most of the oleaginous species are still in infancy. However, in the past few years, enormous efforts have been invested in Rhodotorula, Rhodosporidium, Lipomyces, Trichosporon, and Candida genera of yeasts among others, with the rapid advancement of engineering strategies, significant improvement in genetic tools and techniques, generation of extensive bioinformatics and omics data. In this review, we have collated these recent progresses to make a detailed and insightful summary of the major developments in metabolic engineering of the prominent oleaginous yeast species. Such a comprehensive overview would be a useful resource for future strain improvement and metabolic engineering studies for enhanced production of lipid and lipid-derived chemicals in oleaginous yeasts.

Characterization of two sugar transporters responsible for efficient xylose uptake in an oleaginous yeast Candida tropicalis SY005.

Microbial conversion of lignocellulosic feedstock to the target bioproduct requires efficient assimilation of its constituent sugars, a large part of which comprises of glucose and xylose. This study aims to identify and characterize sugar transporters capable of xylose uptake in an oleaginous strain of the industrially relevant yeast Candida tropicalis. In silico database mining resulted in two sugar transporter proteins- CtStp1 and CtStp2, containing conserved amino acid residues and motifs that have been previously reported to be involved in xylose transport in other organisms. Several softwares predicted the likelihood of 10-12 transmembrane (TM) helices to be present in both the Stps, while molecular modelling showed 12 TM helices that were organized into a typical structure found in the major facilitator superfamily of transporters. Docking with different sugars also predicted favorable interactions. Heterologous expression in a Saccharomyces cerevisiae strain harboring functional xylose metabolic genes validated the broad substrate specificity of the two Stps. Each transporter supported prominent growth of recombinant S. cerevisiae strains on six sugars including xylose at various concentrations. Expression of CtSTP1 and CtSTP2 along with the xylose metabolic genes in yeast transformants grown in presence of xylose was confirmed by transcript detection. Growth curve and sugar consumption profiles revealed uptake of both glucose and xylose simultaneously by the recombinant yeast strains, though CtStp1 showed relatively less effect of glucose repression in mixed sugars and was a better transporter of xylose than CtStp2. Such glucose-xylose utilizing efficient transporters can be effective tools for developing co-fermenting yeasts through genetic engineering in future, with noteworthy applications in renewable biomass utilization.

Engineering an oleaginous yeast Candida tropicalis SY005 for enhanced lipid production.

Candida tropicalis has recently emerged as a valuable yeast species with respect to lipid metabolism, not only for its oleaginous characteristics but also for its ability to utilize diverse range of substrates. Hence, it can be explored as an ideal host for lipid metabolic engineering, although inadequate genetic transformation system for developing the stable transformant has limited this scope. To resolve this existing constraint, we have come up with a novel strategy of a genomic integrating system in the oleaginous strain SY005 of C. tropicalis. Employing this system, comprising of host-specific regulatable promoter, transcription terminator, and integration locus, we have first examined the expression of a reporter gene, and then ectopically expressed a transcription factor CtRAP1 encoding the repressor activator protein 1 of C. tropicalis SY005. A maximum lipid content of 0.37 g/g dry cell weight was achieved in the engineered strain upon galactose induction, leading to 60% (w/w) increase relative to the wild type strain SY005. This work demonstrates the use of a markerless integrative transformation system to promote lipid accumulation in the diploid yeast without applying nutrient stress and hampering cell growth. The findings of this study will augment the research on lipid metabolic engineering and exploit the enormous potential of C. tropicalis as an industrial lipid feedstock. Key points •A transformation system was established in oleaginous yeast Candida tropicalis SY005 •Activity of host-specific molecular elements was verified by reporter gene expression •SY005 was engineered to ectopically express a transcriptional regulator gene CtRAP1 •The engineered strain exhibited 60% increase in lipid content on galactose induction •The increase in lipid content was correlated with the induced expression of CtRAP1.

Identification and functional characterization of a lipid droplet protein CtLDP1 from an oleaginous yeast Candida tropicalis SY005.

Proteins residing in lipid droplets (LDs) of organisms exhibit diverse physiological roles. Since the LD proteins of yeasts are largely unexplored, we have identified a putative LD protein gene, CtLDP1 in the oleaginous yeast Candida tropicalis SY005 and characterized its function. The increased lipid accumulation in SY005 could be correlated with enhanced (~2.67-fold) expression of the CtLDP1 after low-nitrogen stress. The N-terminal transmembrane domain similar to perilipin proteins and the amphipathic α-helices predicted in silico, presumably aid in targeting the CtLDP1 to LD membranes. Heterologous expression of CtLDP1-mCherry fusion in Saccharomyces cerevisiae revealed localization in LDs, yet the expression of CtLDP1 did not show significant effect on LD formation in transformed cells. Molecular docking showed favourable interactions of the protein with sterol class of molecules, but not with triacylglycerol (TAG); and this was further experimentally verified by co-localization of the mCherry-tagged protein in TAG-deficient (but steryl ester containing) LDs. While oleic acid supplementation caused coalescence of LDs into supersized ones (average diameter = 1.19 ± 0.12 µm; n = 160), this effect was suppressed due to CtLDP1 expression, and the cells mostly exhibited numerous smaller LDs (average diameter = 0.46 ± 0.05 µm; n = 160). Moreover, CtLDP1 expression in pet10Δ knockout strain of S. cerevisiae restored multiple LD formation, indicating functional complementation of the protein. Overall, this study documents functional characterization of an LD-stabilizing protein from an oleaginous strain of Candida genus for the first time, and provides insights on the characteristics of LD proteins in oleaginous yeasts for future metabolic engineering.

Efficient xylose utilization leads to highest lipid productivity in Candida tropicalis SY005 among six yeast strains grown in mixed sugar medium.

Six local isolates of yeasts were screened for cell mass and lipid production in mixed glucose and xylose medium. Candida tropicalis SY005 and Trichosporon (Apiotrichum) loubieri SY006 showed significant lipid accumulation of 24.6% and 32% (dry cell weight), respectively when grown in medium containing equal mass of both the sugars. SY005 produced relatively higher cell mass of 9.66 gL-1 due to higher rate of sugar consumption, which raised the lipid productivity of the organism to 0.792 gL-1day-1 as compared to 0.446 gL-1day-1 in SY006. When grown with each sugar separately, the xylose consumption rate of SY005 was found to be 0.55 gL-1 h-1 after 4 days as compared to 0.52 gL-1 h-1 for SY006. Transcript expression of the high affinity xylose transporter (Cthaxt), xylose reductase (Ctxyl1), and xylitol dehydrogenase (Ctxyl2) of SY005 was monitored to unravel such high rate of sugar consumption. Expression of all the three genes was observed to vary in mixed sugars with Cthaxt exhibiting the highest expression in presence of only xylose. Expression levels of both Ctxyl1 and Ctxyl2, involved in xylose catabolism, were maximum during 24-48 h of growth, indicating that xylose utilization started in the presence of glucose, which was depleted in the medium after 96 h. Together, the present study documents that C. tropicalis SY005 consumes xylose concomitant to glucose during early period of growth, and it is a promising yeast strain for viable production of storage lipid or other high-value oleochemicals utilizing lignocellulose hydrolysate.

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.

Enhancement of lipid productivity in oleaginous Colletotrichum fungus through genetic transformation using the yeast CtDGAT2b gene under model-optimized growth condition.

Oleaginous fungi are of special interest among microorganisms for the production of lipid feedstocks as they can be cultured on a variety of substrates, particularly waste lingocellulosic materials, and few fungal strains are reported to accumulate inherently higher neutral lipid than bacteria or microalgae. Previously, we have characterized an endophytic filamentous fungus Colletotrichum sp. DM06 that can produce total lipid ranging from 34% to 49% of its dry cell weight (DCW) upon growing with various carbon sources and nutrient-stress conditions. In the present study, we report on the genetic transformation of this fungal strain with the CtDGAT2b gene, which encodes for a catalytically efficient isozyme of type-2 diacylglycerol acyltransferase (DGAT) from oleaginous yeast Candida troplicalis SY005. Besides the increase in size of lipid bodies, total lipid titer by the transformed Colletotrichum (lipid content ∼73% DCW) was found to be ∼1.7-fold more than the wild type (lipid content ∼38% DCW) due to functional activity of the CtDGAT2b transgene when grown under standard condition of growth without imposition of any nutrient-stress. Analysis of lipid fractionation revealed that the neutral lipid titer in transformants increased up to 1.8-, 1.6- and 1.5-fold compared to the wild type when grown under standard, nitrogen stress and phosphorus stress conditions, respectively. Lipid titer of transformed cells was further increased to 1.7-fold following model-based optimization of culture conditions. Taken together, ∼2.9-fold higher lipid titer was achieved in Colletotrichum fungus due to overexpression of a rate-limiting crucial enzyme of lipid biosynthesis coupled with prediction-based bioprocess optimization.