Identification and Functional Characterization of Acyl-ACP Thioesterases B (GhFatBs) Responsible for Palmitic Acid Accumulation in Cotton Seeds.

In spite of increasing use in the food industry, high relative levels of palmitic acid (C16:0) in cottonseed oil imposes harmful effects on human health when overconsumed in the diet. The limited understanding of the mechanism in controlling fatty acid composition has become a significant obstacle for breeding novel cotton varieties with high-quality oil. Fatty acyl-acyl carrier protein (ACP) thioesterase B (FatBs) are a group of enzymes which prefer to hydrolyze the thioester bond from saturated acyl-ACPs, thus playing key roles in controlling the accumulation of saturated fatty acids. However, FatB members and their roles in cotton are largely unknown. In this study, a genome-wide characterization of FatB members was performed in allotetraploid upland cotton, aiming to explore the GhFatBs responsible for high accumulations of C16:0 in cotton seeds. A total of 14 GhFatB genes with uneven distribution on chromosomes were identified from an upland cotton genome and grouped into seven subfamilies through phylogenetic analysis. The six key amino acid residues (Ala, Trys, Ile, Met, Arg and Try) responsible for substrate preference were identified in the N-terminal acyl binding pocket of GhFatBs. RNA-seq and qRT-PCR analysis revealed that the expression profiles of GhFatB genes varied in multiple cotton tissues, with eight GhFatBs (GhA/D-FatB3, GhA/D-FatB4, GhA/D-FatB5, and GhA/D-FatB7) having high expression levels in developing seeds. In particular, expression patterns of GhA-FatB3 and GhD-FatB4 were positively correlated with the dynamic accumulation of C16:0 during cotton seed development. Furthermore, heterologous overexpression assay of either GhA-FatB3 or GhD-FatB4 demonstrated that these two GhFatBs had a high substrate preference to 16:0-ACP, thus contributing greatly to the enrichment of palmitic acid in the tested tissues. Taken together, these findings increase our understanding on fatty acid accumulation and regulation mechanisms in plant seeds. GhFatBs, especially GhA-FatB3 and GhD-FatB4, could be molecular targets for genetic modification to reduce palmitic acid content or to optimize fatty acid profiles in cotton and other oil crops required for the sustainable production of healthy edible oil.

Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis.

BACKGROUND: Haematococcus lacustris is an ideal source of astaxanthin (AST), which is stored in oil bodies containing esterified AST (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferases (DGATs) catalyze the last step of acyl-CoA-dependent TAG biosynthesis and are also considered as crucial enzymes involved in EAST biosynthesis in H. lacustris. Previous studies have identified four putative DGAT2-encoding genes in H. lacustris, and only HpDGAT2D allowed the recovery of TAG biosynthesis, but the engineering potential of HpDGAT2s in TAG biosynthesis remains ambiguous. RESULTS: Five putative DGAT2 genes (HpDGAT2A, HpDGAT2B, HpDGAT2C, HpDGAT2D, and HpDGAT2E) were identified in H. lacustris. Transcription analysis showed that the expression levels of the HpDGAT2A, HpDGAT2D, and HpDGAT2E genes markedly increased under high light and nitrogen deficient conditions with distinct patterns, which led to significant TAG and EAST accumulation. Functional complementation demonstrated that HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E had the capacity to restore TAG synthesis in a TAG-deficient yeast strain (H1246) showing a large difference in enzymatic activity. Fatty acid (FA) profile assays revealed that HpDGAT2A, HpDGAT2D, and HpDGAT2E, but not HpDGAT2B, preferred monounsaturated fatty acyl-CoAs (MUFAs) for TAG synthesis in yeast cells, and showed a preference for polyunsaturated fatty acyl-CoAs (PUFAs) based on their feeding strategy. The heterologous expression of HpDGAT2D in Arabidopsis thaliana and Chlamydomonas reinhardtii significantly increased the TAG content and obviously promoted the MUFAs and PUFAs contents. CONCLUSIONS: Our study represents systematic work on the characterization of HpDGAT2s by integrating expression patterns, AST/TAG accumulation, functional complementation, and heterologous expression in yeast, plants, and algae. These results (1) update the gene models of HpDGAT2s, (2) prove the TAG biosynthesis capacity of HpDGAT2s, (3) show the strong preference for MUFAs and PUFAs, and (4) offer target genes to modulate TAG biosynthesis by using genetic engineering methods.

Enhancing growth and oil accumulation of a palmitoleic acid-rich Scenedesmus obliquus in mixotrophic cultivation with acetate and its potential for ammonium-containing wastewater purification and biodiesel production.

A palmitoleic acid-rich Scenedesmus obliquus strain SXND-02 was isolated from ammonium-containing wastewater. Biomass and lipid production were examined for this microalgal strain in photoautotrophic, heterotrophic, and mixotrophic cultivations, respectively, in order to extend its application in wastewater purification coupled with production of valued bio-products. Among the tested conditions, the microalga had better growth and higher lipid accumulation in mixotrophy. NH4Cl inhibited the microalgal growth in photoautotrophic cultivation. However, NaAc alleviated this inhibition in both heterotrophy and mixotrophy. Using 7 g L-1 NaAc and 0.5 g L-1 NH4Cl as carbon and nitrogen sources significantly increased the algal biomass and lipid yields under mixotrophic cultivation, with the highest levels up to 1.0 g L-1 and 59.88%, respectively. Fatty acid profiling indicated that palmitoleic acid was 23% in the S. obliquus SXND-02 under mixotrophic condition, which was about 21-fold higher than that in the control S. obliquus. Furthermore, this microalgal strain was tested in the chicken farm wastewater (CFW) containing high ammonium. Compared with other treatments, the S. obliquus SXND-02 cultivated in the 1/2 CFW + NaAc medium produced larger amounts of biomass (2.18 g L-1) and lipids (50.22%), and simultaneously higher removal rates of total nitrogen (TN) (80%), total ammonium nitrogen (TAN) (68%), total phosphate (TP) (82%), biological oxygen demand (BOD) (86%) and chemical oxygen demand (COD) (89%) from wastewater. The present data indicate that this excellent microalga can be used in mixotrophic cultivation for wastewater purification coupled with commercial production of valued biomass and high-quality algal oils.

Advanced treatment of chicken farm flushing wastewater by integrating Fenton oxidation and algal cultivation process for algal growth and nutrients removal.

Algae based wastewater treatment has been considered as the most promising win-win strategy for nutrients removal and biomass accumulation. However, the poor linking between traditional wastewater treatment and algal cultivation limits the achievement of this goal. In this study, a novel combination of Fenton oxidation and algal cultivation (CFOAC) system was investigated for the treatment of chicken farm flushing wastewater (CFFW). Fenton oxidation (FO) was adopted to reduce the excessive ammonia nitrogen, which might inhibit the algal growth. The results showed that single FO pretreatment removed 70.5 %, 96.7 %, 86.1 %, and 96.2 % of TN, TAN, TP, and COD, respectively. The highest biomass (235.8 mg/L/d) and lipid (77.3 mg/L/d) productivities were achieved on optimized CFOAC system after 7 days batch cultivation. Accordingly, the nutrients removal efficiencies increased to almost 100 %. Further fatty acid profile analysis showed that algae grown on optimal CFOAC system accumulated a high level of total lipids (32.8 %) with C16-C18 fatty acid as the most abundant compositions (accounting for over 60.6 %), which were propitious to biodiesel production. In addition, this CFOAC system was magnified from 1 L flask to 50 L horizontal pipe photobioreactor (HPPB) in semi-continuously culture under optimal conditions. The average biomass and lipid productivities were 995.7 mg/L/d and 320.6 mg/L/d, respectively, when cultured at 6 days hydraulic retention time with 1/3 substitution every two days. These findings proved that the novel CFOAC system is efficient in nutrients removal, algal cultivation, and biomass production for advanced treatment of CFFW.

Genome-wide identification and functional characterization of the Camelina sativa WRKY gene family in response to abiotic stress.

BACKGROUND: WRKY transcription factors are a superfamily of regulators involved in diverse biological processes and stress responses in plants. However, there is limited knowledge about the WRKY family in camelina (Camelina sativa), an important Brassicaceae oil crop with strong tolerance for various stresses. Here, a genome-wide characterization of WRKY proteins is performed to examine their gene structures, phylogenetics, expression, conserved motif organizations, and functional annotation to identify candidate WRKYs that mediate stress resistance regulation in camelinas. RESULTS: A total of 242 CsWRKY proteins encoded by 224 gene loci distributed unevenly over the chromosomes were identified, and they were classified into three groups by phylogenetic analysis according to their WRKY domains and zinc finger motifs. The 15 CsWRKY gene loci generated 33 spliced variants. Orthologous WRKY gene pairs were identified, with 173 pairs in the C. sativa and Arabidopsis genomes as well as 282 pairs in the C. sativa and B. napus genomes, respectively. A total of 137 segmental duplication events were observed, but there was no tandem duplication in the camelina genome. Ten major conserved motifs were examined, with WRKYGQK being the most conserved, and several variants were present in many CsWRKYs. Expression analysis revealed that 50% more CsWRKY genes were expressed constitutively, and a set of them displayed tissue-specific expression. Notably, 11 CsWRKY genes exhibited significant expression changes in seedlings under cold, salt, and drought stresses, showing a preferentially inducible expression pattern in response to the stress. CONCLUSIONS: The present article describes a detailed analysis of the CsWRKY gene family and its expression profiles in 12 tissues and under several stress conditions. Segmental duplication is the major force underlying the broad expansion of this gene family, and a strong purifying pressure occurred for CsWRKY proteins during their evolution. CsWRKY proteins play important roles in plant development, with differential functions in different tissues. Exceptionally, eleven CsWRKYs, particularly five alternative spliced isoforms, were found to be the possible key players in mediating plant responses to various stresses. Overall, our results provide a foundation for understanding the roles of CsWRKYs and the precise mechanism through which CsWRKYs regulate high stress resistance as well as the development of stress tolerance cultivars among Cruciferae crops.

Cloning, expression, and characterization of a novel plant type cryptochrome gene from the green alga Haematococcus pluvialis.

A full-length cDNA sequence of plant type CRY (designated Hae-P-CRY) was cloned from the green alga Haematococcus pluvialis. The cDNA sequence was 3608 base pairs (bp) in length, which contained a 2988-bp open reading frame encoding 995 amino acids with molecular mass of 107.7 kDa and isoelectric point of 6.19. Multiple alignment analysis revealed that the deduced amino acid sequence of Hae-P-CRY shared high identity of 47-66% with corresponding plant type CRYs from other eukaryotes. The catalytic motifs of plant type CRYs were detected in the amino acid sequence of Hae-P-CRY including the typical PHR and CTE domains. Phylogenetic analysis showed that the Hae-P-CRY was grouped together with other plant type CRYs from green algae and higher plants, which distinguished from other distinct groups. The transcriptional level of Hae-P-CRY was strongly decreased after 0-4 h under HL stress. In addition, the Hae-P-CRY gene was heterologously expressed in Escherichia coli BL21 (DE3) and successfully purified. The typical spectroscopic characteristics of plant type CRYs were present in Hae-P-CRY indicated that it may be an active enzyme, which provided valuable clue for further functional investigation in the green alga H. pluvialis. These results lay the foundation for further function and interaction protein identification involved in CRYs mediated signal pathway under HL stress in H. pluvialis.

Analysis of bZIP Transcription Factor Family and Their Expressions under Salt Stress in Chlamydomonas reinhardtii.

The basic leucine-region zipper (bZIP) transcription factors (TFs) act as crucial regulators in various biological processes and stress responses in plants. Currently, bZIP family members and their functions remain elusive in the green unicellular algae Chlamydomonas reinhardtii, an important model organism for molecular investigation with genetic engineering aimed at increasing lipid yields for better biodiesel production. In this study, a total of 17 C. reinhardtii bZIP (CrebZIP) TFs containing typical bZIP structure were identified by a genome-wide analysis. Analysis of the CrebZIP protein physicochemical properties, phylogenetic tree, conserved domain, and secondary structure were conducted. CrebZIP gene structures and their chromosomal assignment were also analyzed. Physiological and photosynthetic characteristics of C. reinhardtii under salt stress were exhibited as lower cell growth and weaker photosynthesis, but increased lipid accumulation. Meanwhile, the expression profiles of six CrebZIP genes were induced to change significantly during salt stress, indicating that certain CrebZIPs may play important roles in mediating photosynthesis and lipid accumulation of microalgae in response to stresses. The present work provided a valuable foundation for functional dissection of CrebZIPs, benefiting the development of better strategies to engineer the regulatory network in microalgae for enhancing biofuel and biomass production.

Splice Variants of the Castor WRI1 Gene Upregulate Fatty Acid and Oil Biosynthesis When Expressed in Tobacco Leaves.

The plant-specific WRINKLED1 (WRI1) is a member of the AP2/EREBP class of transcription factors that positively regulate oil biosynthesis in plant tissues. Limited information is available for the role of WRI1 in oil biosynthesis in castor bean (Ricinus connunis L.), an important industrial oil crop. Here, we report the identification of two alternatively spliced transcripts of RcWRI1, designated as RcWRI1-A and RcWRI1-B. The open reading frames of RcWRI1-A (1341 bp) and RcWRI1-B (1332 bp) differ by a stretch of 9 bp, such that the predicted RcWRI1-B lacks the three amino acid residues "VYL" that are present in RcWRI1-A. The RcWRI1-A transcript is present in flowers, leaves, pericarps and developing seeds, while the RcWRI1-B mRNA is only detectable in developing seeds. When the two isoforms were individually introduced into an Arabidopsiswri1-1 loss-of-function mutant, total fatty acid content was almost restored to the wild-type level, and the percentage of the wrinkled seeds was largely reduced in the transgenic lines relative to the wri1-1 mutant line. Transient expression of each RcWRI1 splice isoform in N. benthamiana leaves upregulated the expression of the WRI1 target genes, and consequently increased the oil content by 4.3-4.9 fold when compared with the controls, and RcWRI1-B appeared to be more active than RcWRI1-A. Both RcWRI1-A and RcWRI1-B can be used as a key transcriptional regulator to enhance fatty acid and oil biosynthesis in leafy biomass.

Systematic characterization of CsbZIP transcription factors in Camelina sativa and functional analysis of CsbZIP-A12 mediating regulation of unsaturated fatty acid-enriched oil biosynthesis.

The basic leucine zipper (bZIP) transcription factors (TFs) function importantly in numerous life processes in plants. However, bZIP members and their biological roles remain unknown in Camelina sativa, a worldwide promising oil crop. Here, 220 CsbZIP proteins were identified in camelina and classified into thirteen groups. Two and 347 pairs of tandem and segmental duplication genes were detected to be underwent purification selection, with segmental duplication as the main driven-force of CsbZIP gene family expansion. Most CsbZIP genes displayed a tissue-specific expression pattern. Particularly, CsbZIP-A12 significantly positively correlated with many FA/oil biosynthesis-related genes, indicating CsbZIP-A12 may regulate lipid biosynthesis. Notably, yeast one-hybrid (Y1H), ß-Glucuronidase (GUS), dual-luciferase (LUC) and EMSA assays evidenced that CsbZIP-A12 located in nucleus interacted with the promoters of CsSAD2-3 and CsFAD3-3 genes responsible for unsaturated fatty acid (UFA) synthesis, thus activating their transcriptions. Overexpression of CsbZIP-A12 led to an increase of total lipid by 3.275 % compared to the control, followed with oleic and α-linolenic acid levels enhanced by 3.4 % and 5.195 %, and up-regulated the expressions of CsSAD2-3, CsFAD3-3 and CsPDAT2-3 in camelina seeds. Furthermore, heterogeneous expression of CsbZIP-A12 significantly up-regulated the expressions of NtSAD2, NtFAD3 and NtPDAT genes in tobacco plants, thereby improving the levels of total lipids and UFAs in both leaves and seeds without negative effects on other agronomic traits. Together, our findings suggest that CsbZIP-A12 upregulates FA/oil biosynthesis by activating CsSAD2-3 and CsFAD3-3 as well as possible other related genes. These data lay a foundation for further functional analyses of CsbZIPs, providing new insights into the TF-based lipid metabolic engineering to increase vegetable oil yield and health-beneficial quality in oilseeds.

Expression of yeast acyl-CoA-∆9 desaturase leads to accumulation of unusual monounsaturated fatty acids in soybean seeds.

An acyl-CoA-Δ9 desaturase from Saccharomyces cerevisiae was expressed by subcellular-targeting in soybean (Glycine max) seeds with the goal of increasing palmitoleic acid (16:1Δ9), a high-valued fatty acid (FA), and simultaneously decreasing saturated FA in oil. The expression resulted in the conversion of palmitic acid (16:0) to 16:1Δ9 in soybean seeds. 16:1Δ9 and its elongation product cis-vaccenic acid (18:1Δ11) were increased to 17 % of the total fatty acids by plastid-targeted expression of the enzyme. Other lipid changes include the decrease of polyunsaturated FA and saturated FA, suggesting that a mechanism exists downstream in oil biosynthesis to compensate the FA alternation. This is the first time a cytosolic acyl-CoA-∆9 desaturase is functionally expressed in plastid and stronger activity was achieved than its cytosolic expression. The present study provides a new strategy for converting 16:0 to 16:1Δ9 by engineering acyl-CoA-Δ9 desaturase in commercialized oilseeds.

Optimization of hydrolysis conditions of crop straw and its effect in Chlorella sorokiniana culture.

Taking straws of corn, wheat, and millet as raw materials, we pretreated them with alkaline hydrogen peroxide, and then hydrolyzed by cellulase and xylanase. We selected the total sugar content in the hydrolysate as the indicator to evaluate the hydrolysis of the straws from three crop species, and further optimized the conditions. Then, the hydrolysates of three types of crop straws were used as carbon source for Chlorella sorokiniana culture to assess their effects on microalgal cultivation. The results showed that the optimal hydrolysis conditions for the three crop straws were identified as solid-liquid ratio of 1:15, temperature of 30 ℃, and treatment time of 12 h. Under such optimal condition, the total sugar contents increased up to 1.677, 1.412, and 1.211 g·L-1 in the corn, millet and wheat straw hydrolysate, respectively. The hydrolysates from the three crop straw could significantly increase both algal biomass and lipid content of C. sorokiniana. Corn straw hydrolysate had the best effect, with high levels of algal biomass (1.801 g·L-1) and lipid content (30.1%). Therefore, we concluded that crop straw hydrolysates as carbon source could significantly promote microalgal biomass and lipid enrichment. The results could lay the foundation for the efficient conversion and utilization of straw lignocellulose raw materials, provide new knowledge for the resource utilization of agricultural wastes, as well as the theoretical basis for the efficient cultivation of microalgae using crop straw hydrolysates.

[Characterization the response of Chlamydomonas reinhardtii serine/threonine protein kinase mutant to blue light].

In order to investigate the molecular mechanism of silk/threonine protein kinase (STK)-mediated blue light response in the algal Chlamydomonas reinhardtii, phenotype identification and transcriptome analysis were conducted for C. reinhardtii STK mutant strain crstk11 (with an AphvIII box reverse insertion in stk11 gene coding region) under blue light stress. Phenotypic examination showed that under normal light (white light), there was a slight difference in growth and pigment contents between the wild-type strain CC5325 and the mutant strain crstk11. Blue light inhibited the growth and chlorophyll synthesis in crstk11 cells, but significantly promoted the accumulation of carotenoids in crstk11. Transcriptome analysis showed that 860 differential expression genes (DEG) (559 up-regulated and 301 down-regulated) were detected in mutant (STK4) vs. wild type (WT4) upon treatment under high intensity blue light for 4 days. After being treated under high intensity blue light for 8 days, a total of 1 088 DEGs (468 upregulated and 620 downregulated) were obtained in STK8 vs. WT8. KEGG enrichment analysis revealed that compared to CC5325, the crstk11 blue light responsive genes were mainly involved in catalytic activity of intracellular photosynthesis, carbon metabolism, and pigment synthesis. Among them, upregulated genes included psaA, psaB, and psaC, psbA, psbB, psbC, psbD, psbH, and L, petA, petB, and petD, as well as genes encoding ATP synthase α, ß and c subunits. Downregulated genes included petF and petJ. The present study uncovered that the protein kinase CrSTK11 of C. reinhardtii may participate in the blue light response of algal cells by mediating photosynthesis as well as pigment and carbon metabolism, providing new knowledge for in-depth analysis of the mechanism of light stress resistance in the algae.

Critical metabolic pathways and genes cooperate for epoxy fatty acid-enriched oil production in developing seeds of Vernonia galamensis, an industrial oleaginous plant.

BACKGROUND: Vernonia galamensis native to Africa is an annual oleaginous plant of Asteraceae family. As a newly established industrial oil crop, this plant produces high level (> 70%) of vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid), which is an unusual epoxy fatty acid (EFA) with multiple industrial applications. Here, transcriptome analysis and fatty acid profiling from developing V. galamensis seeds were integrated to uncover the critical metabolic pathways responsible for high EFA accumulation, aiming to identify the target genes that could be used in the biotechnological production of high-value oils. RESULTS: Based on oil accumulation dynamics of V. galamensis seeds, we harvested seed samples from three stages (17, 38, and 45 days after pollination, DAP) representing the initial, fast and final EFA accumulation phases, and one mixed sample from different tissues for RNA-sequencing, with three biological replicates for each sample. Using Illumina platform, we have generated a total of 265 million raw cDNA reads. After filtering process, de novo assembly of clean reads yielded 67,114 unigenes with an N50 length of 1316 nt. Functional annotation resulted in the identification of almost all genes involved in diverse lipid-metabolic pathways, including the novel fatty acid desaturase/epoxygenase, diacylglycerol acyltransferases, and phospholipid:diacylglycerol acyltransferases. Expression profiling revealed that various genes associated with acyl editing, fatty acid ß-oxidation, triacylglycerol assembly and oil-body formation had greater expression levels at middle developmental stage (38 DAP), which were consistent with the fast accumulation of EFA in V. galamensis developing seed, these genes were detected to play fundamental roles in EFA production. In addition, we isolated some transcription factors (such as WRI1, FUS3 and ABI4), which putatively regulated the production of V. galamensis seed oils. The transient expression of the selected genes resulted in a synergistic increase of EFA-enriched TAG accumulation in tobacco leaves. Transcriptome data were further confirmed by quantitative real-time PCR for twelve key genes in EFA biosynthesis. Finally, a comprehensive network for high EFA accumulation in V. galamensis seed was established. CONCLUSIONS: Our findings provide new insights into molecular mechanisms underlying the natural epoxy oil production in V. galamensis. A set of genes identified here could be used as the targets to develop other oilseeds highly accumulating valued epoxy oils for commercial production.

Characterisation of two novel genes encoding Δ9 fatty acid desaturases (CeSADs) for oleic acid accumulation in the oil-rich tuber of Cyperus esculentus.

Cyperus esculentus is considered one of the most promising oil crops due to its oil-rich tuber, wide adaptability and large biomass production. Preferable triacylglycerol (TAG) composition, especially high oleic acid content, makes tuber oil suitable for human consumption and biodiesel production. However, the mechanism underlying oleic acid enrichment in the tuber remains unknown. Plastidial stearoyl-ACP desaturase (SAD) catalyses the formation of monounsaturated fatty acids (MUFAs), which may function crucially for high accumulation of oleic acid in C. esculentus tubers. In this study, two full-length cDNAs encoding SAD were isolated from the developing tubers of C. esculentus, namely, CeSAD1 and CeSAD2, with ORFs of 1194 bp and 1161 bp, respectively. Quantitative RT-PCR analysis showed that CeSAD genes were highly expressed in tubers. The expression pattern during tuber formation was also significantly correlated with fatty acid and oil accumulation dynamics. Overexpression of each CeSAD gene could restore the normal growth of the defective yeast BY4389, indicating that both CeSADs had fatty acid desaturase activity to catalyse MUFA biosynthesis. A tobacco genetic transformation assay demonstrated that both CeSAD enzymes had high enzyme activity. Exogenous addition of exogenous fatty acids to feed yeast revealed that CeSAD1 has a more substantial substrate preference ratio for C18:0 than CeSAD2 did. Moreover, the overexpression of CeSAD1 significantly increased host tolerance against low-temperature stress. Our data add new insights into the deep elucidation of oleic acid-enriched oils in Cyperus esculentus tubers, showing CeSAD, especially CeSAD1, as the target gene in genetic modification to increase oil and oleic yields in oil crops as well as stress tolerance.

Astaxanthin From Haematococcus pluvialis Prevents High-Fat Diet-Induced Hepatic Steatosis and Oxidative Stress in Mice by Gut-Liver Axis Modulating Properties.

Scope: Evidence is mounting that astaxanthin (ATX), a xanthophyll carotenoid, used as a nutritional supplement to prevent chronic metabolic diseases. The present study aims to identify the potential function of ATX supplementation in preventing steatohepatitis and hepatic oxidative stress in diet-induced obese mice. Methods and Results: In this study, ATX as dose of 0.25, 0.5, and 0.75% have orally administered to mice along with a high-fat diet (HFD) to investigate the role of ATX in regulating liver lipid metabolism and gut microbiota. The study showed that ATX dose-dependently reduces body weight, lipid droplet formation, hepatic triglycerides and ameliorated hepatic steatosis and oxidative stress. 0.75% ATX altered the levels of 34 lipid metabolites related to hepatic cholesterol and fatty acid metabolism which might be associated with downregulation of lipogenesis-related genes and upregulation of bile acid biosynthesis-related genes. The result also revealed that ATX alleviates HFD-induced gut microbiota dysbiosis by significantly inhibiting the growth of obesity-related Parabacteroides and Desulfovibrio while promoting the growth of Allobaculum and Akkermansia. Conclusion: The study results suggested that dietary ATX may prevent the development of hepatic steatosis and oxidative stress with the risk of metabolic disease by gut-liver axis modulating properties.

Functional Characterization of Soybean Diacylglycerol Acyltransferase 3 in Yeast and Soybean.

Diacylglycerol acyltransferases (DGAT) function as the key rate-limiting enzymes in de novo biosynthesis of triacylglycerol (TAG) by transferring an acyl group from acyl-CoA to sn-3 of diacylglycerol (DAG) to form TAG. Here, two members of the type 3 DGAT gene family, GmDGAT3-1 and GmDGAT3-2, were identified from the soybean (Glycine max) genome. Both of them were predicted to encode soluble cytosolic proteins containing the typical thioredoxin-like ferredoxin domain. Quantitative PCR analysis revealed that GmDGAT3-2 expression was much higher than GmDGAT3-1's in various soybean tissues such as leaves, flowers, and seeds. Functional complementation assay using TAG-deficient yeast (Saccharomyces cerevisiae) mutant H1246 demonstrated that GmDGAT3-2 fully restored TAG biosynthesis in the yeast and preferentially incorporated monounsaturated fatty acids (MUFAs), especially oleic acid (C18:1) into TAGs. This substrate specificity was further verified by fatty-acid feeding assays and in vitro enzyme activity characterization. Notably, transgenic tobacco (Nicotiana benthamiana) data showed that heterogeneous expression of GmDGAT3-2 resulted in a significant increase in seed oil and C18:1 levels but little change in contents of protein and starch compared to the EV-transformed tobacco plants. Taken together, GmDGAT3-2 displayed a strong enzymatic activity to catalyze TAG assembly with high substrate specificity for MUFAs, particularly C18:1, playing an important role in the cytosolic pathway of TAG synthesis in soybean. The present findings provide a scientific reference for improving oil yield and FA composition in soybean through gene modification, further expanding our knowledge of TAG biosynthesis and its regulatory mechanism in oilseeds.

[Overexpression of NtAGPase small subunit gene increases leaf starch content and tobacco biomass].

Production of biofuels such as ethanol from non-grain crops may contribute to alleviating the global energy crisis and reducing the potential threat to food security. Tobacco (Nicotiana tabacum) is a commercial crop with high biomass yield. Breeding of starch-rich tobacco plants may provide alternative raw materials for the production of fuel ethanol. We cloned the small subunit gene NtSSU of ADP-glucose pyrophosphorylase (NtAGPase), which controls starch biosynthesis in tobacco, and constructed a plant expression vector pCAMBIA1303-NtSSU. The NtSSU gene was overexpressed in tobacco upon Agrobacterium-mediated leaf disc transformation. Phenotypic analysis showed that overexpression of NtSSU gene promoted the accumulation of starch in tobacco leaves, and the content of starch in tobacco leaves increased from 17.5% to 41.7%. The growth rate and biomass yield of the transgenic tobacco with NtSSU gene were also significantly increased. The results revealed that overexpression of NtSSU gene could effectively redirect more photosynthesis carbon flux into starch biosynthesis pathway, which led to an increased biomass yield but did not generate negative effects on other agronomic traits. Therefore, NtSSU gene can be used as an excellent target gene in plant breeding to enrich starch accumulation in vegetative organs to develop new germplasm dedicated to fuel ethanol production.

Characterization of a Haematococcus pluvialis Diacylglycerol Acyltransferase 1 and Its Potential in Unsaturated Fatty Acid-Rich Triacylglycerol Production.

The unicellular green alga Haematococcus pluvialis has been recognized as an industry strain to produce simultaneously esterified astaxanthin (EAST) and triacylglycerol (TAG) under stress induction. It is necessary to identify the key enzymes involving in synergistic accumulation of EAST and TAG in H. pluvialis. In this study, a novel diacylglycerol acyltransferase 1 was systematically characterized by in vivo and in silico assays. The upregulated expression of HpDGAT1 gene was positively associated with the significant increase of TAG and EAST contents under stress conditions. Functional complementation by overexpressing HpDGAT1 in a TAG-deficient yeast strain H1246 revealed that HpDGAT1 could restore TAG biosynthesis and exhibited a high substrate preference for monounsaturated fatty acyl-CoAs (MUFAs) and polyunsaturated fatty acyl-CoAs (PUFAs). Notably, heterogeneous expression of HpDGAT1 in Chlamydomonas reinhardtii and Arabidopsis thaliana resulted in a significant enhancement of total oils and concurrently a high accumulation of MUFAs- and PUFAs-rich TAGs. Furthermore, molecular docking analysis indicated that HpDGAT1 contained AST-binding sites. These findings evidence a possible dual-function role for HpDGAT1 involving in TAG and EAST synthesis, demonstrating that it is a potential target gene to enrich AST accumulation in this alga and to design oil production in both commercial algae and oil crops.

Functional characterization of an novel acyl-CoA:diacylglycerol acyltransferase 3-3 (CsDGAT3-3) gene from Camelina sativa.

Diacylglycerol acyltransferases (DGAT) catalyze the final committed step of de novo biosynthesis of triacylglycerol (TAG) in plant seeds. This study was to functionally characterize DGAT3 genes in Camelina sativa, an important oil crops accumulating high levels of unsaturated fatty acids (UFAs) in seeds. Three camelina DGAT3 genes (CsDGAT3-1, CsDGAT3-2 and CsDGAT3-3) were identified, and the encoded proteins were predicted to be cytosolic-soluble proteins present as a homodimer containing the 2Fe-2S domain. They had divergent expression patterns in various tissues, suggesting that they may function in tissue-specific manner with CsDGAT3-1 in roots, CsDGAT3-2 in flowers and young seedlings, and CsDGAT3-3 in developing seeds. Functional complementation assay in yeast demonstrated that CsDGAT3-3 restored TAG synthesis. TAG content and UFAs, particularly eicosenoic acid (EA, 20:1n-9) were largely increased by adding exogenous UFAs in the yeast medium. Further heterogeneously transient expression in N. benthamiana leaves and seed-specific expression in tobacco seeds indicated that CsDGAT3-3 significantly enhanced oil and UFA accumulation with much higher level of EA. Overall, CsDGAT3-3 exhibited a strong abilty catalyzing TAG synthesis and high substrate preference for UFAs, especially for 20:1n-9. The present data provide new insights for further understanding oil biosynthesis mechanism in camelina seeds, indicating that CsDGAT3-3 may have practical applications for increasing both oil yield and quality.

Ectopic overexpression of a type-II DGAT (CeDGAT2-2) derived from oil-rich tuber of Cyperus esculentus enhances accumulation of oil and oleic acid in tobacco leaves.

BACKGROUND: Engineering triacylglycerol (TAG) accumulation in vegetative tissues of non-food crops has become a promising way to meet our increasing demand for plant oils, especially the renewable production of biofuels. The most important target modified in this regard is diacylglycerol acyltransferase (DGAT) enzyme responsible for the final rate-limiting step in TAG biosynthesis. Cyperus esculentus is a unique plant largely accumulating oleic acid-enriched oil in its underground tubers. We speculated that DGAT derived from such oil-rich tubers could function more efficiently than that from oleaginous seeds in enhancing oil storage in vegetative tissues of tobacco, a high-yielding biomass crops. RESULTS: Three CeDGAT genes namely CeDGAT1, CeDGAT2-1 and CeDGAT2-2 were identified in C. esculentus by mining transcriptome of developing tubers. These CeDGATs were expressed in tissues tested, with CeDGAT1 highly in roots, CeDGAT2-1 abundantly in leaves, and CeDGAT2-2 predominantly in tubers. Notably, CeDGAT2-2 expression pattern was in accordance with oil dynamic accumulation during tuber development. Overexpression of CeDGAT2-2 functionally restored TAG biosynthesis in TAG-deficient yeast mutant H1246. Oleic acid level was significantly increased in CeDGAT2-2 transgenic yeast compared to the wild-type yeast and ScDGA1-expressed control under culture with and without feeding of exogenous fatty acids. Overexpressing CeDGAT2-2 in tobacco led to dramatic enhancements of leafy oil by 7.15- and 1.7-fold more compared to the wild-type control and plants expressing Arabidopsis seed-derived AtDGAT1. A substantial change in fatty acid composition was detected in leaves, with increase of oleic acid from 5.1% in the wild type to 31.33% in CeDGAT2-2-expressed tobacco and accompanied reduction of saturated fatty acids. Moreover, the elevated accumulation of oleic acid-enriched TAG in transgenic tobacco exhibited no significantly negative impact on other agronomic traits such as photosynthesis, growth rates and seed germination except for small decline of starch content. CONCLUSIONS: The present data indicate that CeDGAT2-2 has a high enzyme activity to catalyze formation of TAG and a strong specificity for oleic acid-containing substrates, providing new insights into understanding oil biosynthesis mechanism in plant vegetative tissues. Overexpression of CeDGAT2-2 alone can significantly increase oleic acid-enriched oil accumulation in tobacco leaves without negative impact on other agronomy traits, showing CeDGAT2-2 as the desirable target gene in metabolic engineering to enrich oil and value-added lipids in high-biomass plants for commercial production of biofuel oils.