Triacylglycerol Production Research Articles

Highly selective isolation and characterization of Lipomyces starkeyi mutants with increased production of triacylglycerol.

The oleaginous yeast Lipomyces starkeyi is an attractive organism for the industrial production of lipids; however, the amount of lipid produced by wild-type L. starkeyi is insufficient. The study aims to obtain L. starkeyi mutants that rapidly accumulate large amounts of triacylglycerol (TAG). Mutagenized yeast cells at the early stages of cultivation were subjected to Percoll density gradient centrifugation; cells with increased production of TAG were expected to be enriched in the resultant upper fraction because of their lower density. Among 120 candidates from the upper fractions, five mutants were isolated that accumulated higher amounts of TAG. Moreover, when omitting cells with mucoid colony morphology, 11 objective mutants from 11 candidates from the upper fraction were effectively (100%) isolated. Of total 16 mutants obtained, detailed characterization of five mutants was performed to reveal that five mutants achieved about 1.5-2.0 times TAG concentration (4.7-6.0g/L) as compared with the wild-type strain (3.6g/L) at day 5. Among these five mutants, strain E15 was the best for industrial use because only strain E15 showed significantly higher TAG concentration as well as significantly higher degree of lipid to glucose and biomass to glucose yields than the wild-type strain. Thus, Percoll density gradient centrifugation is an effective method to isolate mutant cells that rapidly accumulate large amounts of TAG. It is expected that by repeating this procedure as part of a yeast-breeding program, L. starkeyi mutants suitable for industrial lipid production can be easily and effectively obtained.

Fatty acid-binding protein 1 increases steer fat deposition by facilitating the synthesis and secretion of triacylglycerol in liver.

Castration is an important means of improving the beef quality via increasing fat deposition. However, little is known about the molecular mechanism underlying the fat deposition after castration. Here, the intramuscular fat (IMF) content of the steer group was shown to be much higher than the bull group. To understand transcriptional changes in the genes involved in fat deposition following castration, differential expression patterns of mRNAs in liver tissue were investigated in steers and bulls using RNA sequencing. In total, we obtained 58,282,367–54,918,002 uniquely mapped reads, which covered 90.13% of the currently annotated transcripts; 5,864 novel transcripts and optimized 9,088 known genes were determined. These results indicated that castration could change the expression patterns of mRNAs in liver tissue, and 282 differentially expressed genes (DEGs) were detected between steers and bulls. KEGG pathway analysis showed that the DEGs were mostly enriched in PPAR signaling pathway, steroid biosynthesis, steroid hormone biosynthesis, and biosynthesis of fatty acids. Furthermore, eight DEGs were corroborated via quantitative real-time PCR and we found that FABP1 gene knockdown in bovine hepatocytes prominently reduced intracellular triacylglycerol (TAG) synthesis and very low density lipoprotein (VLDL) secretion in culture medium. In summary, these results indicate that FABP1 may promote fat deposition by promoting the production and secretion of TAG and VLDL in steer liver.

Enhancement of Algal Biofeedstocks in a Mixotrophic Batch Culture Supplemented with Exogenous Glycerol

Chlorella vulgaris is a fast growing, unicellular microalgae. With its limited nutritional requirements and ability to grow in adverse environments, it is a promising biodiesel feedstock that will not compete with agricultural resources.1 Published literature has shown C. vulgaris can produce up to 42% lipids per dry mass, the most desired of which are triacylglycerols (TAGs).2 TAGs consist of three fatty acids esterified to a glycerol backbone and are the most reduced, energy‐dense biomolecules. To generate drop‐in ready biodiesel, TAGs are transesterified to yield methyl‐ester alkyls and a glycerol byproduct.To realistically produce fuel on an industrial scale, microalgal TAG production must be increased beyond current levels. Previous work published by the Kolling Lab increased lipid accumulation in C. vulgaris by using an exogenous carbon source to form a heteromixotrophic culture, in which the cells are energetically supplemented by both exogenous carbon as well as photosynthetic light reactions.3 This project seeks to further increase TAG production by supplying cells with exogenous glycerol, thus providing a use for the transesterification byproduct while reducing the cellular energy investment for TAG production.While published studies have found 10 g/L glycerol to be optimal for lipid production in C. vulgaris, this was completed heterotrophically.4 While growing mixotrophically, we have increased lipid accumulation beyond previously published glycerol studies and have increased overall lipid productivity in glycerol concentrations of 31.5 and 63 g/L. We also monitored photosynthetic performance before and after glycerol addition and observed an increased reliance on cyclic electron transport (a mode of photosynthesis that does not result in the production of NADPH), indicating that the ATP need is greater than that of NADPH for TAG formation under photomixotrophic conditions. Finally, using isotopically labeled glycerol, we have used GC‐MS to determine the rate of glycerol uptake and that exogenously provided glycerol is directly used for the TAG backbone and not metabolized for the generation of fatty acids.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952.Amanda Smythers acknowledges a fellowship from the NASA West Virginia Space Grant Consortium, Grant #NNX15AK74A and Training Grant # NNX15AI01H.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Triacylglycerol Production in the Snow Algae Chlamydomonas nivalis under Different Nutrient Conditions.

Eukaryotic microalgae have been known for high competency in the accumulation of triacylglycerol (TAG), a representative class of storage lipid. The snow algal species, Chlamydomonas nivalis, is a unique green eukaryotic microalga that can grow and survive in a wide range of temperatures. Although a few metabolomic studies of C. nivalis were conducted, no study has reported on TAG accumulation in C. nivalis. Herein, the present work aimed to investigate TAG production in C. nivalis under nutrient-starved conditions at 22 °C. Compared to phosphorus starvation, C. nivalis under nitrogen starvation showed a less severe growth defect, greater capacity for TAG production, and simple acyl composition in TAG enriched with 18:1. These features suggest that C. nivalis may be a significant model species to investigate glycerolipid metabolism for basic and applied research.

The Light Regime Effect on Triacylglycerol Accumulation of Isochrysis zhangjiangensis

Stress state of microalgal cells is caused under unfavorable conditions such as disordered light regime and depleted nitrogen. The stress state can impair photosynthetic efficiency, inhibit cell growth and result in the accumulation of triacylglycerol (TAG) from protective mechanisms. Continuous light or nitrogen starvation was applied on microalgae and performed effectively on inducing TAG production. To evaluate the light regime effect on inducing TAG production, the effect of different light regimes on nitrogen-starved Isochrysis zhangjiangensis was investigated in this work. The continuous light and nitrogen starvation elevated TAG content of biomass by 73% and 193%, respectively. Furthermore, the TAG accumulation of I. zhangjiangensis cell under nitrogen starvation decreased under aggravated stress from continuous illumination. Our results demonstrated that culturing the cells with 14L: 10D light regime under nitrogen starvation is the optimal mode to achieve maximal accumulation of TAG. A recovery in light regime was necessary for I. zhangjiangensis cultivation.

Effect of light absorption rate and nitrate concentration on TAG accumulation and productivity of Parachlorella kessleri cultures grown in chemostat mode

This study aims to elucidate the role of light absorption rate and nitrate concentration on triacylglycerol (TAG) productivity and cellular TAG accumulation in nitrogen limited Parachlorella kessleri cultures grown in chemostat mode. In batch operated nitrogen starved cultures the cells accumulate large amounts of TAGs at the expense of biomass growth leading to severe decrease in cell division rate prompting eventual cell death. In PBRs operated in continuous mode cell multiplication can persist in nitrogen limited conditions with simultaneous TAG accumulation. P. kessleri cultures were grown in a flat-plate PBR with constant dilution rate of 0.01 1/h and feed-medium nitrate concentration ranging from 1 to 16 mM. The steady-state biomass, pigment, carbohydrate, protein, total lipid, and TAG concentrations as well as the spectral absorption and scattering cross-sections of the microalgae were measured for each culture. These were used to estimate the specific mean rate of photon absorption (MRPA) in the PBR. Maximum areal TAG productivity of 2.6 g/m2⋅day was obtained for the culture grown with nitrate concentration equal to 3.65 mM and a mean rate of photon absorption equal to 17,700 μmolhν/kgXA⋅s. TAG productivity and cellular TAG accumulation varied with the nitrate concentration in the feed medium and MRPA. Finally, our results show that TAG productivity can be further increased by optimizing the mean rate of photon absorption through adjusting culture dilution rate and feed-medium nitrate concentration.

Increased triacylglycerol production in oleaginous microalga Neochloris oleoabundans by overexpression of plastidial lysophosphatidic acid acyltransferase

BackgroundMicroalgae are promising sources of lipid triacylglycerol (TAG) for sustainable production of natural edible oils and biofuels. Nevertheless, products derived from microalgal TAG are not yet economically feasible; increasing TAG content via targeted genetic engineering of genes in TAG biosynthesis pathway are important to achieve economic viability. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with the endogenous enzyme lysophosphatidic acid acyltransferase (NeoLPAAT1) responsible for plastidial TAG biosynthesisResultsNeoLPAAT1 was found to contain all canonical motifs attributed to LPAAT proteins, two hypothetical membrane-spanning domains and a putative chloroplast transit peptide, indicating as a member of plastidial LPAAT type 1 subfamily. The NeoLPAAT1-expression cassette integrated in N. oleoabundans transformant was confirmed by PCR. The neutral lipid content in the transformant detected by Nile red staining was 1.6-fold higher than in wild type. The NeoLPAAT1 transcript was twofold higher in the transformant than wild type. Considerably higher lipid quantity was found in the transformant than wild type: total lipid content increased 1.8- to 1.9-fold up to 78.99 ± 1.75% dry cell weight (DCW) and total lipid productivity increased 1.8- to 2.4-fold up to 16.06 ± 2.68 mg/L/day; while TAG content increased 2.1- to 2.2-fold up to 55.40 ± 5.56% DCW and TAG productivity increased 1.9- to 2.8-fold up to 10.67 ± 2.37 mg/L/day. A slightly altered fatty acid composition was detected in the transformant compared to wild type; polyunsaturated fatty acid (C18:2) increased to 19% from 11%. NeoLPAAT1-overexpression stability was observed in the transformant continuously maintained in solid medium over 150 generations in a period of about 6 years.ConclusionsOur results demonstrate the considerably increased TAG content and productivity in N. oleoabundans by overexpression of plastidial NeoLPAAT1 that are important for products derived from microalgal TAG to achieve economic viability. Plastidial LPAAT1 can be a candidate for target genetic manipulation to increase TAG content in other microalgal species with desired characteristics for production of natural edible oils and biofuels.

Exploring the metabolic potential of oleaginous actinomycetes in biodiesel production from cassava wastewater

The depletion in fossil fuel reserves has instigated the search for renewable sources such as biofuels. Biofuel production provides a sustainable alternative to fossil fuels. However, the progression of biofuel industry has been largely affected by several uncertainties including the sustainability of production processes. Finding an economically viable process and the right substrates have been a source of constant debate. Biodiesel production is one such area which is gaining momentum in the last few decades. We are currently investigating cassava wastewater as a substrate and also as a source of oleaginous bacteria. The production of tri-acyl glycerol from mycolic acid containing actinomycetes predominantly Rhodococcus, has been identified as a potential source. These bacteria which are largely ubiquitous provide a significant amount of triglyceride when cultivated under low cost waste. The growth of oleaginous bacteria for the accumulation of triglycerides on low cost and abundant, cassava waste still remains unexplored, especially in Nigeria which is the largest producer of cassava in the world. We started our initial investigations on the cassava waste water sample following culture dependent and independent approach. We prognosticate that the identification of microflora isolated from different stages in the sample will provide a basis for understanding the nature of the substrate and the potential for the synthesis of biodiesel from cassava waste water.

Transcriptional Regulation of Acyl-CoA:Glycerol-sn-3-Phosphate Acyltransferases.

Acyl-CoA:glycerol-sn-3-phosphate acyltransferase (GPAT) is an enzyme responsible for the rate-limiting step in the synthesis of glycerophospholipids and triacylglycerol (TAG). The enzymes of mammalian species are classified into four isoforms; GPAT1 and GPAT2 are localized in the mitochondrial outer membrane, whereas GPAT3 and GPAT4 are localized in the endoplasmic reticulum membrane. The activity of each enzyme expressed is associated with physiological and pathological functions. The transcriptional regulation is well known, particularly in GPAT1. GPAT1 mRNA expression is mainly regulated by the binding of the transcriptional factor SREBP-1c to the specific element (the sterol regulatory element) flanking the GPAT1 promoter. The TAG level is controlled by the insulin-induced transcriptional expression of GPAT1, which occupies most of the GPAT activity in the liver. The transcriptional regulation of the other three GPAT isoforms remains undetermined in detail. It is predicted that retinoic acid serves as a transcription factor in the GPAT2 promoter. PPARγ (peroxisome proliferator-activated receptor γ) increases the mRNA expression of GPAT3, which is associated with TAG synthesis in adipose tissues. Although GPAT has been considered to be a key enzyme in the production of TAG, unexpected functions have recently been reported, particularly in GPAT2. It is likely that GPAT2 is associated with tumorigenesis and normal spermatogenesis. In this review, the physiological and pathophysiological roles of the four GPAT isoforms are described, alongside the transcriptional regulation of these enzymes.

Eliciting the impacts of cellular noise on metabolic trade-offs by quantitative mass imaging

Optimal metabolic trade-offs between growth and productivity are key constraints in strain optimization by metabolic engineering; however, how cellular noise impacts these trade-offs and drives the emergence of subpopulations with distinct resource allocation strategies, remains largely unknown. Here, we introduce a single-cell strategy for quantifying the trade-offs between triacylglycerol production and growth in the oleaginous microorganism Yarrowia lipolytica. The strategy relies on high-throughput quantitative-phase imaging and, enabled by nanoscale secondary ion mass spectrometry analyses and dedicated image processing, allows us to image how resources are partitioned between growth and productivity. Enhanced precision over population-averaging biotechnologies and conventional microscopy demonstrates how cellular noise impacts growth and productivity differently. As such, subpopulations with distinct metabolic trade-offs emerge, with notable impacts on strain performance and robustness. By quantifying the self-degradation of cytosolic macromolecules under nutrient-limiting conditions, we discover the cell-to-cell heterogeneity in protein and fatty-acid recycling, unmasking a potential bet-hedging strategy under starvation.

Characterization of type I and type II diacylglycerol acyltransferases from the emerging model alga Chlorella zofingiensis reveals their functional complementarity and engineering potential

BackgroundThe green alga Chlorella zofingiensis has been recognized as an industrially relevant strain because of its robust growth under multiple trophic conditions and the potential for simultaneous production of triacylglycerol (TAG) and the high-value keto-carotenoid astaxanthin. Nevertheless, the mechanism of TAG synthesis remains poorly understood in C. zofingiensis. Diacylglycerol acyltransferase (DGAT) is thought to catalyze the committed step of TAG assembly in the Kennedy pathway. C. zofingiensis genome is predicted to possess eleven putative DGAT-encoding genes, the greatest number ever found in green algae, pointing to the complexity of TAG assembly in the alga.ResultsThe transcription start site of C. zofingiensis DGATs was determined by 5′-rapid amplification of cDNA ends (RACE), and their coding sequences were cloned and verified by sequencing, which identified ten DGAT genes (two type I DGATs designated as CzDGAT1A and CzDGAT1B, and eight type II DGATs designated as CzDGTT1 through CzDGTT8) and revealed that the previous gene models of seven DGATs were incorrect. Function complementation in the TAG-deficient yeast strain confirmed the functionality of most DGATs, with CzDGAT1A and CzDGTT5 having the highest activity. In vitro DGAT assay revealed that CzDGAT1A and CzDGTT5 preferred eukaryotic and prokaryotic diacylglycerols (DAGs), respectively, and had overlapping yet distinctive substrate specificity for acyl-CoAs. Subcellular co-localization experiment in tobacco leaves indicated that both CzDGAT1A and CzDGTT5 were localized at endoplasmic reticulum (ER). Upon nitrogen deprivation, TAG was drastically induced in C. zofingiensis, accompanied by a considerable up-regulation of CzDGAT1A and CzDGTT5. These two genes were probably regulated by the transcription factors (TFs) bZIP3 and MYB1, as suggested by the yeast one-hybrid assay and expression correlation. Moreover, heterologous expression of CzDGAT1A and CzDGTT5 promoted TAG accumulation and TAG yield in different hosts including yeast and oleaginous alga.ConclusionsOur study represents a pioneering work on the characterization of both type I and type II C. zofingiensis DGATs by systematically integrating functional complementation, in vitro enzymatic assay, subcellular localization, yeast one-hybrid assay and overexpression in yeast and oleaginous alga. These results (1) update the gene models of C. zofingiensis DGATs, (2) shed light on the mechanism of oleaginousness in which CzDGAT1A and CzDGTT5, have functional complementarity and probably work in collaboration at ER contributing to the abundance and complexity of TAG, and (3) provide engineering targets for future trait improvement via rational manipulation of this alga as well as other industrially relevant ones.

Investigation of Liquid Culture Conditions for Triacylglycerol Production from Waste Peach by Lipomyces Wild-type Strain.

In this study, waste peach (WP) liquid culture conditions for the maintenance of high triacylglycerol (TG)-accumulation ability in Lipomyces wild-type strain, obtained from WP plate medium were investigated. As the concentration of WP juice was high, the medium viscosity became high, and TG accumulation ability was suppressed. In a 5-L jar fermenter, the negative influence of viscosity on TG-accumulation ability was significantly improved by an agitation speed of 150 rpm (0.4 vvm). Where a bench scale pilot plant (90-L jar fermenter) was operated at 40 rpm, TG-accumulation ability reached 6.8 mg/108cells. This ability was 85% of that obtained with WP plate medium.

Tissue-specific differences in metabolites and transcripts contribute to the heterogeneity of ricinoleic acid accumulation in Ricinus communis L. (castor) seeds.

Castor (Ricinus communis L.) seeds are valued for their production of oils which can comprise up to 90% hydroxy-fatty acids (ricinoleic acid). Castor oil contains mono-, di- and tri- ricinoleic acid containing triacylglycerols (TAGs). Although the enzymatic synthesis of ricinoleic acid is well described, the differential compartmentalization of these TAG molecular species has remained undefined. To examine the distribution of hydroxy fatty acid accumulation within the endosperm and embryo tissues of castor seeds. Matrix assisted laser desorption/ionization mass spectrometry imaging was used to map the distribution of triacylglycerols in tissue sections of castor seeds. In addition, the endosperm and embryo (cotyledons and embryonic axis) tissues were dissected and extracted for quantitative lipidomics analysis and Illumina-based RNA deep sequencing. This study revealed an unexpected heterogeneous tissue distribution of mono-, di- and tri- hydroxy-triacylglycerols in the embryo and endosperm tissues of castor seeds. Pathway analysis based on transcript abundance suggested that distinct embryo- and endosperm-specific mechanisms may exist for the shuttling of ricinoleic acid away from phosphatidylcholine (PC) and into hydroxy TAG production. The embryo-biased mechanism appears to favor removal of ricinoleic acid from PC through phophatidylcholine: diacylglycerol acyltransferase while the endosperm pathway appears to remove ricinoleic acid from the PC pool by preferences of phospholipase A (PLA2α) and/or phosphatidylcholine: diacylglycerol cholinephosphotransferase. Collectively, a combination of lipidomics and transcriptomics analyses revealed previously undefined spatial aspects of hydroxy fatty acid metabolism in castor seeds. These studies underscore a need for tissue-specific studies as a means to better understand the regulation of triacylglycerol accumulation in oilseeds.

Identification of a chloroplast fatty acid exporter protein, CmFAX1, and triacylglycerol accumulation by its overexpression in the unicellular red alga Cyanidioschyzon merolae

Triacylglycerols (TAGs) produced by microalgae are a promising biofuel resource, but low productivity is still a problem for practical production. Understanding the underlying molecular mechanisms of TAG production is essential to improve productivity, which remains mostly in a primitive stage. Recently, a nuclear gene encoding a chloroplast inner membrane protein, AtFAX1, which is involved in the export of fatty acids (FAs) from the chloroplast, was identified and analyzed in Arabidopsis thaliana. In this study, we identified and analyzed a nuclear-encoded homolog of AtFAX1, named CmFAX1, from the unicellular red alga Cyanidioschyzon merolae. CmFAX1 was localized around the chloroplast envelope membrane in C. merolae, and heterologous expression in budding yeast suggested a role in FA traffic through membranes. Overexpression of CmFAX1 in C. merolae cells resulted in a 0.4-fold decrease in intracellular free FAs and a 2.4-fold increase in intracellular TAGs under normal growth conditions, suggesting that export of the free FAs produced in the chloroplast was enhanced and they were consumed for TAG synthesis in the cytosol. The accumulated TAGs in the CmFAX1 overexpression strain included short chain FAs, 14:0 and 14:1, which were not observed in the TAGs of the control strain and are suitable for biojet/biodiesel production because of their low cloud points. Overproduction of CmFAX1 resulted in higher cell yields after exponential growth phase culture. This was probably because of decreased photosynthetic pigments (chlorophyll a) and increased light permeability into the highly dense cell culture, which are beneficial characteristics for increased biomass production. These results indicate that CmFAX1 could be a useful target protein for improvement of algal biofuel production.

Interaction of DGAT1 and PDAT1 to enhance TAG assembly in Arabidopsis

ABSTRACTSeeds contain a large quantity of oils, which are mainly constituted of triacylglycerol (TAG), a fundamental source of carbon and energy. TAG biosynthesis is catalyzed by a series of multiple enzymes. In particular, two key enzymes catalyzing the last acylation step for TAG production, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1), are rate-limiting enzymes that determine TAG accumulation in Arabidopsis seeds. We recently showed that the two enzymes are transcriptionally coordinated by the R2R3-type MYB96 transcription factor to promote TAG assembly during seed maturation in Arabidopsis. Here, we further found that DGAT1 and PDAT1 physically associate, possibly to enhance the efficiency of TAG production. Overall, our findings suggest that TAG biosynthesis is intricately regulated at multiple levels, and these molecular strategies can potentially be used for metabolic engineering in plants.

Daily changes in phytoplankton lipidomes reveal mechanisms of energy storage in the open ocean

Sunlight is the dominant control on phytoplankton biosynthetic activity, and darkness deprives them of their primary external energy source. Changes in the biochemical composition of phytoplankton communities over diel light cycles and attendant consequences for carbon and energy flux in environments remain poorly elucidated. Here we use lipidomic data from the North Pacific subtropical gyre to show that biosynthesis of energy-rich triacylglycerols (TAGs) by eukaryotic nanophytoplankton during the day and their subsequent consumption at night drives a large and previously uncharacterized daily carbon cycle. Diel oscillations in TAG concentration comprise 23 ± 11% of primary production by eukaryotic nanophytoplankton representing a global flux of about 2.4 Pg C yr−1. Metatranscriptomic analyses of genes required for TAG biosynthesis indicate that haptophytes and dinoflagellates are active members in TAG production. Estimates suggest that these organisms could contain as much as 40% more calories at sunset than at sunrise due to TAG production.

Comparative transcriptome and metabolome analysis suggests bottlenecks that limit seed and oil yields in transgenic Camelina sativa expressing diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase

BackgroundCamelina sativa has attracted much interest as alternative renewable resources for biodiesel, other oil-based industrial products and a source for edible oils. Its unique oil attributes attract research to engineering new varieties of improved oil quantity and quality. The overexpression of enzymes catalyzing the synthesis of the glycerol backbone and the sequential conjugation of fatty acids into this backbone is a promising approach for increasing the levels of triacylglycerol (TAG). In a previous study, we co-expressed the diacylglycerol acyltransferase (DGAT1) and glycerol-3-phosphate dehydrogenase (GPD1), involved in TAG metabolism, in Camelina seeds. Transgenic plants exhibited a higher-percentage seed oil content, a greater seed mass, and overall improved seed and oil yields relative to wild-type plants. To further increase seed oil content in Camelina, we utilized metabolite profiling, in conjunction with transcriptome profiling during seed development to examine potential rate-limiting step(s) in the production of building blocks for TAG biosynthesis.ResultsTranscriptomic analysis revealed approximately 2518 and 3136 transcripts differentially regulated at significant levels in DGAT1 and GPD1 transgenics, respectively. These transcripts were found to be involved in various functional categories, including alternative metabolic routes in fatty acid synthesis, TAG assembly, and TAG degradation. We quantified the relative contents of over 240 metabolites. Our results indicate major metabolic switches in transgenic seeds associated with significant changes in the levels of glycerolipids, amino acids, sugars, and organic acids, especially the TCA cycle and glycolysis intermediates.ConclusionsFrom the transcriptomic and metabolomic analysis of DGAT1, GPD1 and DGAT1 + GPD1 expressing lines of C. sativa, we conclude that TAG production is limited by (1) utilization of fixed carbon from the source tissues supported by the increase in glycolysis pathway metabolites and decreased transcripts levels of transcription factors controlling fatty acids synthesis; (2) TAG accumulation is limited by the activity of lipases/hydrolases that hydrolyze TAG pool supported by the increase in free fatty acids and monoacylglycerols. This comparative transcriptomics and metabolomics approach is useful in understanding the regulation of TAG biosynthesis, identifying bottlenecks, and the corresponding genes controlling these pathways identified as limitations, for generating Camelina varieties with improved seed and oil yields.

The type 2 acyl-CoA:diacylglycerol acyltransferase family of the oleaginous microalga Lobosphaera incisa

BackgroundOleaginous microalgae are promising sources of energy-rich triacylglycerols (TAGs) for direct use for food, feed and industrial applications. Lobosphaera incisa is a fresh water unicellular alga, which in response to nutrient stress accumulates a high amount of TAGs with a high proportion of arachidonic acid (ARA). The final committed step of de novo TAG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferases (DGATs), which add a fatty acid (FA) to the final sn-3 position of diacylglycerol (DAG).ResultsGenome analysis revealed the presence of five putative DGAT isoforms in L. incisa, including one DGAT of type 1, three DGATs of type 2 and a single isoform of a type 3 DGAT. For LiDGAT1, LiDGAT2.1, LiDGAT2.2 and LiDGAT2.3 enzyme activity was confirmed by expressing them in the TAG-deficient yeast strain H1246. Feeding experiments of yeast transformants with fatty acids suggest a broad substrate specificity spectrum for LiDGAT1. A significant TAG production in response to exogenous ARA was found for LiDGAT2.2. Cellular localization of the four type 1 and type 2 DGATs expressed in yeast revealed that they all localize to distinct ER domains. A prominent association of LiDGAT1 with ER domains in close proximity to forming lipid droplets (LDs) was also observed.ConclusionsThe data revealed a distinct molecular, functional and cellular nature of type 1 and type 2 DGATs from L. incisa, with LiDGAT1 being a major contributor to the TAG pool. LiDGATs of type 2 might be in turn involved in the incorporation of unusual fatty acids into TAG and thus regulate the composition of TAG. This report provides a valuable resource for the further research of microalgae DGATs oriented towards production of fresh-water strains with higher oil content of valuable composition, not only for oil industry but also for human and animal nutrition.

Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene

Saccharomyces cerevisiae is an efficient host for natural-compound production and preferentially employed in academic studies and bioindustries. However, S. cerevisiae exhibits limited production capacity for lipophilic natural products, especially compounds that accumulate intracellularly, such as polyketides and carotenoids, with some engineered compounds displaying cytotoxicity. In this study, we used a nature-inspired strategy to establish an effective platform to improve lipid oil–triacylglycerol (TAG) metabolism and enable increased lycopene accumulation. Through systematic traditional engineering methods, we achieved relatively high-level production at 56.2 mg lycopene/g cell dry weight (cdw). To focus on TAG metabolism in order to increase lycopene accumulation, we overexpressed key genes associated with fatty acid synthesis and TAG production, followed by modulation of TAG fatty acyl composition by overexpressing a fatty acid desaturase (OLE1) and deletion of Seipin (FLD1), which regulates lipid-droplet size. Results showed that the engineered strain produced 70.5 mg lycopene/g cdw, a 25% increase relative to the original high-yield strain, with lycopene production reaching 2.37 g/L and 73.3 mg/g cdw in fed-batch fermentation and representing the highest lycopene yield in S. cerevisiae reported to date. These findings offer an effective strategy for extended systematic metabolic engineering through lipid engineering.

Dual expression of plastidial GPAT1 and LPAT1 regulates triacylglycerol production and the fatty acid profile in Phaeodactylum tricornutum

BackgroundMetabolic engineering has emerged as a potential strategy for improving microalgal lipid content through targeted changes to lipid metabolic networks. However, the intricate nature of lipogenesis has impeded metabolic engineering. Therefore, it is very important to identify the crucial metabolic nodes and develop strategies to exploit multiple genes for transgenesis. In an attempt to unravel the microalgal triacylglycerol (TAG) pathway, we overexpressed two key lipogenic genes, glycerol-3-phosphate acyltransferase (GPAT1) and lysophosphatidic acid acyltransferase (LPAT1), in oleaginous Phaeodactylum tricornutum and determined their roles in microalgal lipogenesis.ResultsEngineered P. tricornutum strains showed enhanced growth and photosynthetic efficiency compared with that of the wild-type during the growth phase of the cultivation period. However, both the cell types reached stationary phase on day 7. Overexpression of GPAT1 and LPAT1 increased the TAG content by 2.3-fold under nitrogen-replete conditions without compromising cell growth, and they also orchestrated the expression of other key genes involved in TAG synthesis. The transgenic expression of GPAT1 and LPAT1 influenced the expression of malic enzyme and glucose 6-phosphate dehydrogenase, which enhanced the levels of lipogenic NADPH in the transgenic lines. In addition, GPAT1 and LPAT1 preferred C16 over C18 at the sn-2 position of the glycerol backbone.ConclusionOverexpression of GPAT1 together with LPAT1 significantly enhanced lipid content without affecting growth and photosynthetic efficiency, and they orchestrated the expression of other key photosynthetic and lipogenic genes. The lipid profile for elevated fatty acid content (C16-CoA) demonstrated the involvement of the prokaryotic TAG pathway in marine diatoms. The results suggested that engineering dual metabolic nodes should be possible in microalgal lipid metabolism. This study also provides the first demonstration of the role of the prokaryotic TAG biosynthetic pathway in lipid overproduction and indicates that the fatty acid profile can be tailored to improve lipid production.