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.     

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.     

Cloning and characterization of a novel diacylglycerol acyltransferase from the diatom Phaeodactylum tricornutum

In this study, a cDNA encoding a novel acyl-CoA:diacylglycerol acyltransferase (DGAT)-like protein is identified and isolated from the diatom microalga Phaeodactylum tricornutum (PtDGAT3). Analysis of the sequence reveals that ptDGAT3 cDNA encodes a protein of 504 amino acids with a molecular mass of 64.5 KDa. The putative ptDGAT3 protein has two catalytic domains: a wax ester synthase-like acyl-CoA acyltransferase domain and a bacteria-specific acyltransferase domain, which shows higher similarity to the DGAT3 of Acinetobacter calcoaceticus than reported DGAT1 or DGAT2 from high plants or algae. Its activity was confirmed by heterologous expression of PtDGAT3 in a neutral lipid-deficient quadruple mutant yeast Saccharomyces cerevisiae H1246. The recombinant yeast restored the formation of a lipid body and displayed a preference to the incorporation of unsaturated C₁₈ fatty acids into triacyglycerol (TAG). This is the first characterized algal DGAT3 gene, giving further evidence to the occurrence of a DGAT3-mediated TAG biosynthesis pathway.     

Synthesis of Novel Lipids in Saccharomyces cerevisiae by Heterologous Expression of an Unspecific Bacterial Acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri

In order to identify novel genes encoding enzymes involved in the terminal step of triacylglycerol (TAG) formation, a database search was carried out in the genome of the unicellular photoautotrophic green alga Ostreococcus tauri. The search led to the identification of three putative type 2 acyl-CoA:diacylglycerol acyltransferase-like sequences (DGAT; EC 2.3.1.20), and revealed the absence of any homolog to type 1 or type 3 DGAT sequence in the genome of O. tauri. For two of the cDNA sequences (OtDGAT2A and B) enzyme activity was detected by heterologous expression in Saccharomyces cerevisiae mutant strains with impaired TAG metabolism. However, activity of OtDGAT2A was too low for further analysis. Analysis of their amino acid sequences showed that they share limited identity with other DGAT2 from different plant species, such as Ricinus communis and Vernicia fordii with ～25 to 30% identity. Lipid analysis of the mutant yeast cells revealed that OtDGAT2B showed broad substrate specificity accepting saturated as well as mono- and poly-unsaturated acyl-CoAs as substrates.     

Identification and characterization of a novel diacylglycerol acyltransferase gene from <Emphasis Type="Italic">Mortierella alpina</Emphasis>

Objectives

To clone and express a diacylglycerol acyltransferase (DGAT) gene from Mortierella alpina in Saccharomyces cerevisiae and characterize oil production and fatty acid composition of the resulting recombinant

Results

A new, full-length cDNA, putatively encoding a DGAT, was cloned from M. alpina. We subsequently cloned the gene, except the transmembrane-encoding region, termed MaDGAT, its molecular mass was 31.3 kDa. MaDGAT shares 75% identity with a DGAT from Mortierella verticillata NRRL 6337. A recombinant vector expressing MaDGAT, pYES2-DGAT, was constructed and transformed into S. cerevisiae H1246, a neutral, lipid-deficient quadruple mutant. TLC analysis showed that the recombinant vector restored triacylglycerol biosynthesis and its content in the recombinant strain was 3.9%.

Conclusion

MaDGAT is a novel DGAT gene and could increase TAG biosynthesis in M. alpina or other filamentous fungi, thereby promoting the synthesis of polyunsaturated fatty acids.

     

Directed evolution of acyl-CoA:diacylglycerol acyltransferase: Development and characterization of Brassica napus DGAT1 mutagenized libraries

Metabolic flux to triacylglycerol (TAG) may be limited by the level of acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) activity. In some species, this enzyme also appears to play a role in the channeling of specific fatty acyl moieties into TAG. The objective of this work is to implement a directed evolution approach to enhance the catalytic efficiency of type-1 DGAT from Brassica napus (BnDGAT1). We generated randomly mutagenized libraries of BnDGAT1 in a yeast expression vector using error-prone PCR. The mutagenized libraries were used to transform a Saccharomyces cerevisiae strain devoid of neutral lipid biosynthesis and analyzed using a high-throughput screening (HTS) system. The HTS, recently developed for this purpose, consisted of a positive selection of clones expressing active DGAT mutants followed by quantification of DGAT activity by fluorescence detection of TAG in yeast cells. The initial results indicated that the positive selection system efficiently eliminated DGAT mutants lacking enzyme activity. Screening of 1528 selected mutants revealed that some DGAT clones had enhanced ability to synthesize TAG in yeast. This was confirmed by analysis of individual clones that could carry mutations resulting in an increased catalytic efficiency. The directed evolution approach could lead to the development of an improved plant DGAT1 for increasing seed oil content in oleaginous crops.     

Screening for Hydrolytic Enzymes Reveals Ayr1p as a Novel Triacylglycerol Lipase in Saccharomyces cerevisiae

Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317–23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301–37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.     

The Endoplasmic Reticulum Enzyme DGAT2 Is Found in Mitochondria-associated Membranes and Has a Mitochondrial Targeting Signal That Promotes Its Association with Mitochondria

The synthesis and storage of neutral lipids in lipid droplets is a fundamental property of eukaryotic cells, but the spatial organization of this process is poorly understood. Here we examined the intracellular localization of acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), an enzyme that catalyzes the final step of triacylglycerol (TG) synthesis in eukaryotes. We found that DGAT2 expressed in cultured cells localizes to the endoplasmic reticulum (ER) under basal conditions. After providing oleate to drive TG synthesis, DGAT2 also localized to near the surface of lipid droplets, where it co-localized with mitochondria. Biochemical fractionation revealed that DGAT2 is present in mitochondria-associated membranes, specialized domains of the ER that are highly enriched in lipid synthetic enzymes and interact tightly with mitochondria. The interaction of DGAT2 with mitochondria depended on 67 N-terminal amino acids of DGAT2, which are not conserved in family members that have different catalytic functions. This targeting signal was sufficient to localize a red fluorescent protein to mitochondria. A highly conserved, positively charged, putative mitochondrial targeting signal was identified in murine DGAT2 between amino acids 61 and 66. Thus, DGAT2, an ER-resident transmembrane domain-containing enzyme, is also found in mitochondria-associated membranes, where its N terminus may promote its association with mitochondria.Most eukaryotic cells can synthesize neutral lipids, such as triacylglycerols (TGs)² and sterol esters, and store them in cytosolic lipid droplets. Yet, a molecular understanding of this process and how it is spatially organized is lacking. For example, lipid substrates for TG synthesis (fatty acids and glycerolipid precursors) are found in the cytoplasm and membranes, energy for activating fatty acids (by converting to fatty acyl-CoA) comes from mitochondria, and the enzymes that catalyze TG formation are primarily found in the mitochondria and endoplasmic reticulum (ER). How the cell orchestrates this complex anabolic process to maximize lipid synthesis and storage during times of substrate excess is poorly understood.In most cells, TG synthesis occurs via the glycerol 3-phosphate (Kennedy) pathway and involves multiple enzymatic reactions in different subcellular compartments (1). The fatty acids for TG synthesis must first be “activated” by acyl-CoA synthases, a family of enzymes that localize to membranes of different compartments, including the ER, mitochondria, and plasma membrane (2), and utilize ATP to ligate CoA to the fatty acyl chain. Next, these fatty acids enter the Kennedy pathway of glycerolipid synthesis, in which the first two reactions occur in both the ER and mitochondria. In the first reaction, glycerol 3-phosphate and a fatty acyl-CoA are combined to yield lysophosphatidic acid through the actions of glycerol-3-phosphate acyltransferase enzymes (1, 3). In the second reaction, 1-acylglycerol-3-phosphate O-acyltransferase enzymes catalyze the esterification of lysophosphatidic acid with fatty acyl-CoA to form phosphatidic acid (1, 4). Next, phosphatidic acid is dephosphorylated at membrane surfaces by phosphatidate phosphatase to yield diacylglycerol (1, 5, 6). All these steps are highly organized spatially, which is likely to be important for the efficiency of the pathway.The final reaction of TG synthesis is catalyzed by acyl-CoA: diacylglycerol acyltransferase (DGAT) enzymes (7-9). The two mammalian DGATs, DGAT1 and DGAT2 (10, 11), which are encoded by genes of different families, have distinct roles in TG synthesis (12). DGAT2 is the major TG biosynthetic enzyme in eukaryotes. Dgat2-deficient mice die shortly after birth and are almost completely devoid of TG (13), indicating an essential requirement for DGAT2. Catalysis of TG synthesis is conserved in the DGAT2 gene family, with functional orthologs in many species, including Dga1p in Saccharomyces cerevisiae, which contributes to a major portion of TG synthesis (14-16).Little is known about the intracellular localization of DGAT enzymes. DGAT activity is present in microsomes (7, 17, 18), but in vitro assays do not distinguish between DGAT1 and DGAT2. A DGAT2-green fluorescent fusion protein expressed in HeLa cells localized to the ER (19), and Dga1p activity in S. cerevisiae localizes to the ER and lipid droplets (16). DGAT1 and DGAT2 expressed in COS-7 cells localized primarily to the ER (20). A recent study of the subcellular localizations of tung tree DGAT1 and DGAT2 in tobacco BY-2 cells revealed that the enzymes are located in distinct, non-overlapping regions of the ER (21). Most recently, DGAT2 was reported to co-localize with lipid droplets in cultured adipocytes (22). As a step toward a better understanding of the cellular organization of processes that contribute to TG synthesis and storage, we determined the subcellular localization of murine DGAT2 in mammalian cells.     

Growth Retardation, Impaired Triacylglycerol Catabolism, Hepatic Steatosis, and Lethal Skin Barrier Defect in Mice Lacking Comparative Gene Identification-58 (CGI-58)

Comparative gene identification-58 (CGI-58), also designated as α/β-hydrolase domain containing-5 (ABHD-5), is a lipid droplet-associated protein that activates adipose triglyceride lipase (ATGL) and acylates lysophosphatidic acid. Activation of ATGL initiates the hydrolytic catabolism of cellular triacylglycerol (TG) stores to glycerol and nonesterified fatty acids. Mutations in both ATGL and CGI-58 cause “neutral lipid storage disease” characterized by massive accumulation of TG in various tissues. The analysis of CGI-58-deficient (Cgi-58^−/−) mice, presented in this study, reveals a dual function of CGI-58 in lipid metabolism. First, systemic TG accumulation and severe hepatic steatosis in newborn Cgi-58^−/− mice establish a limiting role for CGI-58 in ATGL-mediated TG hydrolysis and supply of nonesterified fatty acids as energy substrate. Second, a severe skin permeability barrier defect uncovers an essential ATGL-independent role of CGI-58 in skin lipid metabolism. The neonatal lethal skin barrier defect is linked to an impaired hydrolysis of epidermal TG. As a consequence, sequestration of fatty acids in TG prevents the synthesis of acylceramides, which are essential lipid precursors for the formation of a functional skin permeability barrier. This mechanism may also underlie the pathogenesis of ichthyosis in neutral lipid storage disease patients lacking functional CGI-58.     

高内涵脂滴三维影像定量分析研究细胞内的脂质储存

摘要 目的：研究细胞内脂滴含量的变化对肥胖、糖尿病等代谢性疾病发生发展的影响。方法：建立高内涵脂滴三维成像和定量分析系统,获得脂滴三维动态表型参数,例如细胞内脂滴的总体积量、脂滴平均体积、单一细胞内脂滴平均数量等指标。选择HeLa、AML-12、COS-7和3T3-L1四种细胞系进行油酸、基因沉默、酶活性抑制剂的处理,量化处理后四种细胞内的脂滴数量与大小的表型差异。结果：在加入油酸情况下,细胞随油酸浓度增加而生成更多、更大的脂滴,但AML-12细胞只有展现增加脂滴数量的变化表型;在HeLa细胞中进行19种中性脂合成通路上关键基因的转录表达沉默,发现需要同时双敲降两种甘油三酯合成酶DGAT1和DGAT2才能显着降低细胞内脂滴总体积储存量,但在COS-7细胞中只需要单敲降DGAT1即可降低脂滴存量;进一步使用了DGAT1/2抑制剂处理四种细胞后,发现对抑制剂响应可区分为两类细胞分组（HeLa、AML-12与COS-7、3T3-L1）的脂滴存量表型差异,其原因是DGAT1和DGAT2的转录表达谱在这两类细胞分组中的不同。结论：建立了高内涵脂滴三维成像和定量分析系统,量化了四种细胞系的脂滴数量与大小的表型差异,揭示了细胞的脂滴脂储存方式与蛋白酶表达谱的关系。     

Membrane topology and identification of key functional amino acid residues of murine acyl-CoA:diacylglycerol acyltransferase-2

Triacylglycerols are the predominant molecules of energy storage in eukaryotes. However, excessive accumulation of triacylglycerols in adipose tissue leads to obesity and, in nonadipose tissues, is associated with tissue dysfunction. Hence, it is of great importance to have a better understanding of the molecular mechanisms of triacylglycerol synthesis. The final step in triacylglycerol synthesis is catalyzed by the acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although recent studies have shed light on metabolic functions of these enzymes, little is known about the molecular aspects of their structures or functions. Here we report the topology for murine DGAT2 and the identification of key amino acids that likely contribute to enzymatic function. Our data indicate that DGAT2 is an integral membrane protein with both the N and C termini oriented toward the cytosol. A long hydrophobic region spanning amino acids 66-115 likely comprises two transmembrane domains or, alternatively, a single domain that is embedded in the membrane bilayer. The bulk of the protein lies distal to the transmembrane domains. This region shares the highest degree of homology with other enzymes of the DGAT2 family and contains a sequence HPHG that is conserved in all family members. Mutagenesis of this sequence in DGAT2 demonstrated that it is required for full enzymatic function. Additionally, a neutral lipid-binding domain that is located in the putative first transmembrane domain was also required for full enzymatic function. Our findings provide the first insights into the topography and molecular aspects of DGAT2 and related enzymes.     

Gene Expression in Plant Lipid Metabolism in Arabidopsis Seedlings

Events in plant lipid metabolism are important during seedling establishment. As it has not been experimentally verified whether lipid metabolism in 2- and 5-day-old Arabidopsis thaliana seedlings is diurnally-controlled, quantitative real-time PCR analysis was used to investigate the expression of target genes in acyl-lipid transfer, β-oxidation and triacylglycerol (TAG) synthesis and hydrolysis in wild-type Arabidopsis WS and Col-0. In both WS and Col-0, ACYL-COA-BINDING PROTEIN3 (ACBP3), DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and DGAT3 showed diurnal control in 2- and 5-day-old seedlings. Also, COMATOSE (CTS) was diurnally regulated in 2-day-old seedlings and LONG-CHAIN ACYL-COA SYNTHETASE6 (LACS6) in 5-day-old seedlings in both WS and Col-0. Subsequently, the effect of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) from the core clock system was examined using the cca1lhy mutant and CCA1-overexpressing (CCA1-OX) lines versus wild-type WS and Col-0, respectively. Results revealed differential gene expression in lipid metabolism between 2- and 5-day-old mutant and wild-type WS seedlings, as well as between CCA1-OX and wild-type Col-0. Of the ACBPs, ACBP3 displayed the most significant changes between cca1lhy and WS and between CCA1-OX and Col-0, consistent with previous reports that ACBP3 is greatly affected by light/dark cycling. Evidence of oil body retention in 4- and 5-day-old seedlings of the cca1lhy mutant in comparison to WS indicated the effect of cca1lhy on storage lipid reserve mobilization. Lipid profiling revealed differences in primary lipid metabolism, namely in TAG, fatty acid methyl ester and acyl-CoA contents amongst cca1lhy, CCA1-OX, and wild-type seedlings. Taken together, this study demonstrates that lipid metabolism is subject to diurnal regulation in the early stages of seedling development in Arabidopsis.     

Characterization of the interaction of diacylglycerol acyltransferase-2 with the endoplasmic reticulum and lipid droplets

Acyl CoA:diacylglycerol acyltransferase-2 (DGAT2) is an integral membrane protein that catalyzes the synthesis of triacylglycerol (TG). DGAT2 is present in the endoplasmic reticulum (ER) and also localizes to lipid droplets when cells are stimulated with oleate. Previous studies have shown that DGAT2 can interact with membranes and lipid droplets independently of its two transmembrane domains, suggesting the presence of an additional membrane binding domain. In order to identify additional membrane binding regions, we confirmed that DGAT2 has only two transmembrane domains and demonstrated that the loop connecting them is present in the ER lumen. Increasing the length of this short loop from 5 to 27 amino acids impaired the ability of DGAT2 to localize to lipid droplets. Using a mutagenesis approach, we were able to identify a stretch of amino acids that appears to have a role in binding DGAT2 to the ER membrane. Our results confirm that murine DGAT2 has only two transmembrane domains but also can interact with membranes via a previously unidentified helical domain containing its active site.     

DGAT1 from the arachidonic-acid-producing microalga <Emphasis Type="Italic">Lobosphaera incisa</Emphasis> shows late gene expression under nitrogen starvation and substrate promiscuity in a heterologous system

We report the identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1)-encoding gene from the green oleaginous microalga Lobosphaera incisa (SAG 2468), a prolific photosynthetic producer of the n-6 very long chain polyunsaturated fatty acid (VLC-PUFA), arachidonic acid. The gene expression pattern of LiDGAT1 in L. incisa cells showed a weak increase in mRNA abundance in the course of nitrogen starvation under low light; however, LiDGAT1 expression was significantly upregulated with the progression of N-starvation under high light. Heterologous expression of LiDGAT1 in the neutral lipid-deficient mutant H1246 of Saccharomyces cerevisiae complemented the mutant phenotype and demonstrated an excelling TAG production compared to the yeast endogenous DGAT gene (DGA1). The TAG that formed in the LiDGAT1-expressing H1246 cells contained higher proportions of C16:0 and C18:0 fatty acids, suggesting that at least in a heterologous system, lacking PUFA biosynthesis, the enzyme seems to favor saturated over monounsaturated fatty acids. LiDGAT1 expression prompted an incorporation of several tested exogenous C18 PUFA and C20 VLC-PUFA into TAG. LiDGAT1-driven activity mediated the incorporation of either n-3 or n-6 VLC-PUFA, supplied as substrates for the TAG assembly; however, somewhat of a preference for 18:3n-3 over 20:4n-6 was demonstrated by lipidomics analysis. A structure-functional analysis of LiDGAT1 revealed that the N-terminal Pleckstrin homology (PH) domain is important but not essential for TAG generation in the yeast expression system. Deletion of the PH domain led to decreased TAG formation and ARA incorporation into TAG in yeast. Remarkably, we found the PH domain to be present in the DGAT1 of a number of chlorophytes, in a charophyceaen multicellular alga, in two diatoms and in the liverwort Marchantia polymorpha, but absent from those of red algae, higher plants and animals. Our findings indicate the promiscuity of LiDGAT1 for VLC-PUFA and suggest a specific role for this enzyme in the neutral lipid metabolism of L. incisa that needs to be further investigated by molecular engineering approaches.     

Saccharomyces cerevisiae and Candida tropicalis as starter cultures for the alcoholic fermentation of tchapalo, a traditional sorghum beer

Starter cultures of Candida tropicalis and Saccharomyces cerevisiae isolated from tchapalo were tested in pure culture and co-culture of four ratios [2:1, 25:4, 1:4, 2:3 (cells/cells)] for their ability to ferment sorghum wort to produce tchapalo. All the starters showed means growth rate between 0.043 and 0.101 h^?1. Only C. tropicalis in pure culture showed growth rate lower than that of S. cerevisiae in single culture. During fermentation, according to total soluble solids depletion, yeast starters could be grouped in four different profiles. But in the beer produced, total soluble solids contents were statistically identical. The lowest values were obtained with co-culture C. tropicalis + S. cerevisiae in the ratios of 2:1 and 2:3. Starter cultures with large ratio of C. tropicalis produced a higher organic acids and 2-butanone than S. cerevisiae in pure culture. However, co-culture C. tropicalis + S. cerevisiae (2:1) was the alone starter which produced higher ethanol than S. cerevisiae in pure culture. The beers produced with C. tropicalis + S. cerevisiae (25:4), C. tropicalis + S. cerevisiae (1:4) and C. tropicalis were widely different from those produced with the others starter cultures.     

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.     

Defects in triacylglycerol lipolysis affect synthesis of triacylglycerols and steryl esters in the yeast

Tgl3p, Tgl4p and Tgl5p are the major triacylglycerol lipases of the yeast Saccharomyces cerevisiae catalyzing degradation of triacylglycerols stored in lipid droplets. Previous results from our laboratory (Athenstaedt and Daum, 2005, J. Biol. Chem. 280, 37301–37309) demonstrated that a yeast strain lacking all three triacylglycerol lipases accumulates not only triacylglycerols at high amount, but also steryl esters. Here we show a metabolic link between synthesis and mobilization of non-polar lipids. In particular, we demonstrate that a block in tri-acylglycerol degradation in a tgl3?tgl4?tgl5? triple mutant lacking all major triacylglycerol lipases causes marked changes in non-polar lipid synthesis. Under these conditions formation of triacylglycerols is reduced, whereas steryl ester synthesis is enhanced as shown by quantification of non-polar lipids, in vivo labeling of lipids using [¹⁴C]oleic acid and [¹⁴C]acetic acid as precursors, and enzyme analyses in vitro. In summary, this study demonstrates that triacylglycerol metabolism and steryl ester metabolism are linked processes. The importance of balanced storage and degradation of these components for lipid homeostasis in the yeast is highlighted.