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1.
High‐performance variants of plant diacylglycerol acyltransferase 1 generated by directed evolution provide insights into structure function
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Guanqun Chen Yang Xu Rodrigo M. P. Siloto Kristian Mark P. Caldo Thomas Vanhercke Anna El Tahchy Nathalie Niesner Yongyan Chen Elzbieta Mietkiewska Randall J. Weselake 《The Plant journal : for cell and molecular biology》2017,92(2):167-177
Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the acyl‐CoA‐dependent biosynthesis of triacylglycerol, the predominant component of seed oil. In some oil crops, including Brassica napus, the level of DGAT1 activity can have a substantial effect on triacylglycerol production. Structure–function insights into DGAT1, however, remain limited because of the lack of a three‐dimensional detailed structure for this membrane‐bound enzyme. In this study, the amino acid residues governing B. napus DGAT1 (BnaDGAT1) activity were investigated via directed evolution, targeted mutagenesis, in vitro enzymatic assay, topological analysis, and transient expression of cDNA encoding selected enzyme variants in Nicotiana benthamiana. Directed evolution revealed that numerous amino acid residues were associated with increased BnaDGAT1 activity, and 67% of these residues were conserved among plant DGAT1s. The identified amino acid residue substitution sites occur throughout the BnaDGAT1 polypeptide, with 89% of the substitutions located outside the putative substrate binding or active sites. In addition, cDNAs encoding variants I447F or L441P were transiently overexpressed in N. benthamiana leaves, resulting in 33.2 or 70.5% higher triacylglycerol content, respectively, compared with native BnaDGAT1. Overall, the results provide novel insights into amino acid residues underlying plant DGAT1 function and performance‐enhanced BnaDGAT1 variants for increasing vegetable oil production. 相似文献
2.
Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content 总被引:4,自引:0,他引:4
Weselake RJ Shah S Tang M Quant PA Snyder CL Furukawa-Stoffer TL Zhu W Taylor DC Zou J Kumar A Hall L Laroche A Rakow G Raney P Moloney MM Harwood JL 《Journal of experimental botany》2008,59(13):3543-3549
Top-down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content. 相似文献
3.
Rodrigo M.P. Siloto Martin Truksa Disa Brownfield Allen G. Good Randall J. Weselake 《Plant Physiology and Biochemistry》2009,47(6):456-461
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. 相似文献
4.
Cloning and functional analysis of two type 1 diacylglycerol acyltransferases from Vernonia galamensis 总被引:2,自引:0,他引:2
Vernonia galamensis accumulates vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid) as the major fatty acid in its seed oil. Such epoxy fatty acids are useful in a number of industrial applications. Successful genetic engineering of commercial oilseed crops to produce high levels of vernolic acid depends on a better understanding of the source plant enzymes for vernolic acid accumulation. Developing V. galamensis seed microsome assays demonstrate that diacylglycerol acyltransferase (DGAT), an enzyme for the final step of triacylglycerol synthesis, has a strong substrate preference for vernolic acid bearing substrates including acyl-CoA and diacylglycerol. There are two classes of DGATs known as DGAT1 and DGAT2. Here we report on the isolation, characterization, and functional analysis of two DGAT1 cDNAs from V. galamensis (VgDGAT1a and VgDGAT1b). VgDGAT1a and VgDGAT1b are expressed in all plant tissues examined with highest expression in developing seeds. Enzymatic assays using isolated microsomes from transformed yeast show that VgDGAT1a and VgDGAT1b have the same DGAT activity levels and substrate specificities. Oleoyl-CoA and sn-1,2-dioleoylglycerol are preferred substrates over vernoloyl-CoA and sn-1,2-divernoloylglycerol. This data indicates that the two VgDGAT1s are functional, but not likely to be responsible for the selective accumulation of vernolic acid in V. galamensis seed oil. 相似文献
5.
Pamela J. McFie Sandra L. Stone Shanna L. Banman Scot J. Stone 《The Journal of biological chemistry》2010,285(48):37377-37387
Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols. Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of synthesizing diacylglycerol, retinyl, and wax esters in addition to triacylglycerol. Here, we report the membrane topology for murine DGAT1 using protease protections assays and indirect immunofluorescence in conjunction with selective permeabilization of cellular membranes. Topology models based on prediction algorithms suggested that DGAT1 had eight transmembrane domains. In contrast, our data indicate that DGAT1 has three transmembrane domains with the N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which accounts for ∼50% of the protein, is present in the endoplasmic reticulum lumen and contains a highly conserved histidine residue (His-426) that may be part of the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1 to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is not required for the catalytic activity of DGAT1 but, instead, may be involved in regulating enzyme activity and dimer/tetramer formation. 相似文献
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7.
植物二酰甘油酰基转移酶基因(DGAT)研究进展 总被引:2,自引:0,他引:2
三酰甘油(TAG)是油料作物最主要的储藏脂类,二酰甘油酰基转移酶(DGAT,EC2.3.1.20)是TAG合成途径的限速酶,其主要作用是催化二酰甘油加上酰基脂肪酸形成三酰甘油.在植物中已发现了3种不同类型的DGAT基因,分别为DGAT1、DGAT2和DGAT3.该文对近年来国内外有关植物DGAT相关基因及其蛋白分类、定位、结构及其在脂肪酸合成、种子发育与萌发、幼苗发育、叶片新陈代谢等过程中的作用等研究进展进行综述.为提高油料作物种子油含量以及特定脂肪酸积累提供理论参考. 相似文献
8.
Xu J Francis T Mietkiewska E Giblin EM Barton DL Zhang Y Zhang M Taylor DC 《Plant biotechnology journal》2008,6(8):799-818
SUMMARY: A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content. 相似文献
9.
Orland MD Anwar K Cromley D Chu CH Chen L Billheimer JT Hussain MM Cheng D 《Biochimica et biophysica acta》2005,1737(1):76-82
We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K(m) values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9+/-2.1 microM and 13.9+/-0.3 microM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits approximately 85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl-oleate formation without influencing the retinyl-palmitate formation. Using this inhibitor, we estimate that approximately 64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells. 相似文献
10.
11.
Acyl-coenzyme A:monoacylglycerol acyltransferase 3 (MGAT3) is a member of the MGAT family of enzymes that catalyze the synthesis of diacylglycerol (DAG) from monoacylglycerol (MAG), a committed step in dietary fat absorption. Although named after the initial identification of its MGAT activity, MGAT3 shares higher sequence homology with acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2) than with other MGAT enzymes, suggesting that MGAT3 may also possess significant DGAT activity. This study compared the catalytic properties of MGAT3 with those of MGAT1 and MGAT2 enzymes using both MAG and DAG as substrates. Our results showed that in addition to the expected MGAT activity, the recombinant MGAT3 enzyme expressed in Sf-9 insect cells displayed a strong DGAT activity relative to that of MGAT1 and MGAT2 enzymes in the order MGAT3 > MGAT1 > MGAT2. In contrast, none of the three MGAT enzymes recognized biotinylated acyl-CoA or MAG as a substrate. Although MGAT3 possesses full DGAT activity, it differs from DGAT1 in catalytic properties and subcellular localization. The MGAT3 activity was sensitive to inhibition by the presence of 1% CHAPS, whereas DGAT1 activity was stimulated by the detergent. Consistent with high sequence homology with DGAT2, the MGAT3 enzyme demonstrated a similar subcellular distribution pattern to that of DGAT2, but not DGAT1, when expressed in COS-7 cells. Our data suggest that MGAT3 functions as a novel triacylglycerol (TAG) synthase that catalyzes efficiently the two consecutive acylation steps in TAG synthesis. 相似文献
12.
DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes 总被引:1,自引:0,他引:1
Harris CA Haas JT Streeper RS Stone SJ Kumari M Yang K Han X Brownell N Gross RW Zechner R Farese RV 《Journal of lipid research》2011,52(4):657-667
The total contribution of the acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, to mammalian triacylglycerol (TG) synthesis has not been determined. Similarly, whether DGAT enzymes are required for lipid droplet (LD) formation is unknown. In this study, we examined the requirement for DGAT enzymes in TG synthesis and LDs in differentiated adipocytes with genetic deletions of DGAT1 and DGAT2. Adipocytes with a single deletion of either enzyme were capable of TG synthesis and LD formation. In contrast, adipocytes with deletions of both DGATs were severely lacking in TG and did not have LDs, indicating that DGAT1 and DGAT2 account for nearly all TG synthesis in adipocytes and appear to be required for LD formation during adipogenesis. DGAT enzymes were not absolutely required for LD formation in mammalian cells, however; macrophages deficient in both DGAT enzymes were able to form LDs when incubated with cholesterol-rich lipoproteins. Although adipocytes lacking both DGATs had no TG or LDs, they were fully differentiated by multiple criteria. Our findings show that DGAT1 and DGAT2 account for the vast majority of TG synthesis in mice, and DGAT function is required for LDs in adipocytes, but not in all cell types. 相似文献
13.
Lozeman FJ Middleton CK Deng J Kazala EC Verhaege C Mir PS Laroche A Bailey DR Weselake RJ 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2001,130(1):105-115
The activity of the triacylglycerol bioassembly enzyme, diacylglycerol acyltransferase (DGAT), was characterized in microsomal fractions prepared from bovine subcutaneous (SC) adipose, intramuscular (IM) adipose, and muscle (pars costalis diaphragmatis) tissue. The activity of DGAT was generally higher from SC adipose tissue than from IM adipose or muscle tissue. The characteristics of DGAT activity from the three bovine tissues resembled the activity characteristics observed in previous studies from various other organisms and tissues; the pH optimum was near neutrality, the activity was almost completely inhibited by pre-incubation with N-ethylmaleimide (NEM), and the enzyme accepted a broad range of acyl-CoAs and sn-1,2-diacylglycerols. In some aspects, the SC adipose tissue DGAT activity was different from the DGAT activity from the other two tissues. The SC adipose tissue DGAT activity was not as susceptible to inhibition by NEM as the enzymes from the two other tissue sources, and it exhibited increased specificity for substrates containing oleoyl moieties. The differences in DGAT properties between the three bovine tissues may account to some extent for the differences in the relative fatty acid composition and the positional distribution of fatty acids in triacylglycerol between bovine tissues. The observed differences in enzymatic properties also support recent biochemical and molecular genetic observations that imply the existence of multiple DGAT genes and/or isoforms. 相似文献
14.
Developmental Profile of Diacylglycerol Acyltransferase in Maturing Seeds of Oilseed Rape and Safflower and Microspore-Derived Cultures of Oilseed Rape 总被引:12,自引:3,他引:9
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Weselake RJ Pomeroy MK Furukawa TL Golden JL Little DB Laroche A 《Plant physiology》1993,102(2):565-571
Diacylglycerol acyltransferase (EC 2.3.1.20) activity was assayed during the maturation of seeds of oilseed rape (Brassica napus L.) and safflower (Carthamus tinctorius L.). Developmental studies were also conducted with microspore-derived embryos of oilseed rape (B. napus L. cv Topas) and an embryogenic microspore-derived cell-suspension culture of winter oilseed rape (B. napus L. cv Jet Neuf). In the maturing seeds, diacylglycerol acyltransferase activity increased to a maximum during rapid accumulation of lipid and declined, thereafter, with seed maturity. In microspore-derived embryos of oilseed rape (cv Topas), high levels of diacylglycerol acyltransferase activity were found throughout the early torpedo to late cotyledonary developmental stages with maximum enzyme specific activity associated with the mid-cotyledonary developmental stage. The cell-suspension culture of winter oilseed rape (cv Jet Neuf) contained 3 to 4% triacylglycerol on a dry weight basis and represented about half of the total lipid. The fatty acid profile of total lipid and triacylglycerol in the cell-suspension culture was similar in samples taken during a 1-year period. The Jet Neuf culture contained diacylglycerol acyltransferase with specific activity similar to that of Topas microspore-derived embryos. Jet Neuf diacylglycerol acyltransferase also displayed an enhanced specificity for erucoyl-CoA over oleoyl-CoA when assayed with 14 [mu]M acyl-coenzyme A in the reaction mixture. The specific activity of diacylglycerol acyltransferase in homogenates prepared from the Jet Neuf culture ranged from 5 to 15 pmol of triacylglycerol min-1 mg-1 of protein when assayed at intervals during a period of 1 year. Thus, the cell-suspension culture may represent an attractive tissue source for purification and characterization of triacyl-glycerol biosynthetic enzymes. 相似文献
15.
16.
Engineering Camelina sativa (L.) Crantz for enhanced oil and seed yields by combining diacylglycerol acyltransferase1 and glycerol‐3‐phosphate dehydrogenase expression
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Sudesh Chhikara Hesham M. Abdullah Parisa Akbari Danny Schnell Om Parkash Dhankher 《Plant biotechnology journal》2018,16(5):1034-1045
Plant seed oil‐based liquid transportation fuels (i.e., biodiesel and green diesel) have tremendous potential as environmentally, economically and technologically feasible alternatives to petroleum‐derived fuels. Due to their nutritional and industrial importance, one of the major objectives is to increase the seed yield and oil production of oilseed crops via biotechnological approaches. Camelina sativa, an emerging oilseed crop, has been proposed as an ideal crop for biodiesel and bioproduct applications. Further increase in seed oil yield by increasing the flux of carbon from increased photosynthesis into triacylglycerol (TAG) synthesis will make this crop more profitable. To increase the oil yield, we engineered Camelina by co‐expressing the Arabidopsis thaliana (L.) Heynh. diacylglycerol acyltransferase1 (DGAT1) and a yeast cytosolic glycerol‐3‐phosphate dehydrogenase (GPD1) genes under the control of seed‐specific promoters. Plants co‐expressing DGAT1 and GPD1 exhibited up to 13% higher seed oil content and up to 52% increase in seed mass compared to wild‐type plants. Further, DGAT1‐ and GDP1‐co‐expressing lines showed significantly higher seed and oil yields on a dry weight basis than the wild‐type controls or plants expressing DGAT1 and GPD1 alone. The oil harvest index (g oil per g total dry matter) for DGTA1‐ and GPD1‐co‐expressing lines was almost twofold higher as compared to wild type and the lines expressing DGAT1 and GPD1 alone. Therefore, combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield. 相似文献
17.
Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1
Michael D. Orland Kamran Anwar Debra Cromley Ching-Hsuen Chu Luping Chen Jeffrey T. Billheimer M. Mahmood Hussain Dong Cheng 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2005,1737(1):76-82
We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent Km values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ~85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ~64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells. 相似文献
18.
Heping Cao Jay M. Shockey K. Thomas Klasson Dorselyn C. Chapital Catherine B. Mason Brian E. Scheffler 《PloS one》2013,8(10)
Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms. 相似文献
19.
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. 相似文献