The Yarrowia lipolytica PAH1 homologue contributes but is not required for triacylglycerol biosynthesis during growth on glucose (original) (raw)
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Yeast
The PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the Mg 2+-dependent dephosphorylation of phosphatidate to produce diacylglycerol, which can be acylated to form triacylglycerol (TAG). In the model oleaginous yeast Yarrowia lipolytica, TAG is the major lipid produced, and its biosynthesis requires a continuous supply of diacylglycerol, which can be provided by the PAP reaction. However, the regulation of Pah1 has not been studied in detail in Y. lipolytica, and thus its contribution to the biosynthesis of TAG in this yeast is not well understood. In this work, we examined the regulation of the PAH1-mediated PAP activity and Pah1 abundance and localization in cells growing on glucose. We found that Pah1 abundance and localization were regulated in a growth-dependent manner, yet the loss of Pah1 did not have a major effect on PAP activity. We also examined the effects of the Y. lipolytica pah1Δ mutation on cell physiology and lipid biosynthesis. The lack of Pah1 in the pah1Δ mutant resulted in a moderate decrease in TAG levels and an increase in phospholipid levels. These results showed that Pah1 contributed to TAG biosynthesis in Y. lipolytica but also suggested the presence of other activities in the pah1Δ mutant that compensate for the loss of Pah1. Also, the levels of linoleic acid were elevated in pah1Δ cells with a concomitant decrease in the oleic acid levels suggesting that the pah1Δ mutation affected the biosynthesis of fatty acids.
Yeast (Chichester, England), 2018
Phosphatidate phosphatase (PA phosphatase) dephosphorylates the membrane phospholipid phosphatidate (PA) to diacylglycerol (DAG) that can be used for the synthesis of the storage lipid triacylglycerol (TAG). In Y. lipolytica, TAG biosynthesis is induced during the lipogenic phase which results in the accumulation of this lipid in cells. The accumulation of TAG during lipogenesis requires the supply of DAG, but the source of this DAG is not known in Y. lipolytica. In this study, the regulation of PA phosphatase during lipogenesis and its contribution to TAG biosynthesis was examined in Y. lipolytica. Lipogenesis was triggered by growing cells in high-glucose media, while control cultures were grown in low-glucose media. PA phosphatase activity increased in a time-dependent manner as high-glucose cells progressed in the lipogenic phase. In contrast, the activity decreased in low-glucose cells that did not accumulate lipids. An analysis of the subcellular localization of the PA phospha...
Metabolic Engineering, 2011
The oleaginous yeast Yarrowia lipolytica is able to accumulate lipids up to 38% of its dry weight (DW). Factors involved into lipid and particularly triglycerides accumulation are not well identified. Using different mutants of the glycerol-3-phosphate (G3P) shuttle (∆gut2 or overexpressing GPD1, affecting the anabolic and catabolic dehydrogenases, respectively), we were able to modulate G3P concentration. We show that an increase in G3P concentration leads to a higher triacylglyceride (TAG) accumulation associated to a decrease in glycerol concentration, suggesting that Y. lipolytica does not possess a glycerol-3-phosphatase activity and presents a modified and original glycerol metabolism, which could contribute to its oleaginous character. Moreover, coupling the G3P shuttle disorders to a deficient β-oxidation pathway increased overall lipid accumulation and TAG storage. Finally we obtained strains accumulating up to 65-75% of lipid (W/W dry). Transcriptional analysis in these strains, revealed that high lipids levels resulted from the over-expression of genes involved into TAG synthesis (SCT1 and DGA1, encoding an sn-1 acyltransferase and an acylCoA:diacylglycerol acyltransferase, respectively) and the repression of genes involved in the TAG degradation (TGL3 and TGL4, encoding triacylglycerol lipases). These findings, demonstrate that the expression of genes involved in lipid metabolism during TAG homeostasis is regulated by the G3P shuttle and the β-oxidation pathway. Moreover, the synergistic contributions of acyltransferase gene expression to G3P synthesis are required for high TAG synthesis and lipid accumulation in Y. lipolytica.
Applied Biochemistry and Biotechnology, 2020
Yarrowia lipolytica is a well-known oleaginous yeast that naturally accumulates lipids to more than 20% of their dry cell weight. Due to its brief doubling time and Generally Recognized as Safe (GRAS) properties, Y. lipolytica has been exploited for the production of commercially valuable lipids. Among the genes related to the lipid synthesis, the gene YALI0E16797g (LRO1) encoding a major triacylglycerol synthase of Y. lipolytica shows a significant impact during the acylation process. Thus, in the present work, we explore the contributions of hp4d or TEFintron promoters to the response of LRO1 expression on lipid accumulation by molecular cloning technology. Results showed that over-expression of LRO1 led to higher lipid content as well as lipid yield. The one with the hp4d promoter showed the highest lipid content of 12% wt. However, such an enhancement also caused a growth defect of cells. On the other hand, the lipid content of the cells over-expressing LRO1 with TEFintron promoter revealed only a modest increase in lipid content, but it promoted cell growth. Therefore, all things considered the one with the TEFintron promoter showed the highest lipid yield.
The metabolism and genetic regulation of lipids in the oleaginous yeast Yarrowia lipolytica
Brazilian Journal of Microbiology, 2018
The biotechnological potential of Yarrowia lipolytica, as a single cell oil-producing microorganism, is presented in this review. Although initially this yeast species was considered as a lipid-degrading, recently, it was reclassified as a lipid-producing microorganism, since it has been reported to be capable of accumulating diverse desirable fatty acids after metabolic pathway engineering. In the first part of the present document, a general revision of the oil metabolic pathways and the capacity of oil production in Y. lipolytica is presented. The single cell oil produced by these metabolic engineering strategies has been designed by optimization, introduction, or suppression of new pathways to increase yield on lipid production. Later on, the genetic regulation systems and the lipid composition generated by this yeast for industrial purposes are discussed. These lipids could be safely used in the chemical food and biofuel industries, due to their high proportion of oleic acid. This document emphasizes in the overviewing at Y. lipolytica as an ideal oil cell factory, and as an excellent model to produce single cell oil.
Control of Lipid Accumulation in the Yeast Yarrowia lipolytica
Applied and Environmental Microbiology, 2008
A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This ⌬gut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to -oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal -oxidation. Additional deletion of the POX1 to POX6 genes in the ⌬gut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.
Overexpression of DGAT inYarrowia lipolyticaaffects lipid body size, number, and distribution
FEMS Yeast Research, 2016
In the oleaginous yeast Yarrowia lipolytica, the diacylglycerol acyltransferases (DGATs) are major factors for triacylglycerol (TAG) synthesis. The Q4 strain, in which the four acyltransferases have been deleted, is unable to accumulate lipids and to form lipid bodies (LBs). However, the expression of a single acyltransferase in this strain restores TAG accumulation and LB formation. Using this system, it becomes possible to characterize the activity and specificity of an individual DGAT. Here, we examined the effects of DGAT overexpression on lipid accumulation and LB formation in Y. lipolytica. Specifically, we evaluated the consequences of introducing one or two copies of the Y. lipolytica DGAT genes YlDGA1 and YlDGA2. Overall, multi-copy DGAT overexpression increased the lipid content of yeast cells. However, the size and distribution of LBs depended on the specific DGAT overexpressed. YlDGA2 overexpression caused the formation of large LBs, while YlDGA1 overexpression generated smaller but more numerous LBs. This phenotype was accentuated through the addition of a second copy of the overexpressed gene and might be linked to the distinct subcellular localization of each DGAT, i.e. YlDga1 being localized in LBs, while YlDga2 being localized in a structure strongly resembling the endoplasmic reticulum.
Leucine Biosynthesis Is Involved in Regulating High Lipid Accumulation in Yarrowia lipolytica
mBio, 2017
The yeast Yarrowia lipolytica is a potent accumulator of lipids, and lipogenesis in this organism can be influenced by a variety of factors, such as genetics and environmental conditions. Using a multifactorial study, we elucidated the effects of both genetic and environmental factors on regulation of lipogenesis in Y. lipolytica and identified how two opposite regulatory states both result in lipid accumulation. This study involved comparison of a strain overexpressing diacylglycerol acyltransferase (DGA1) with a control strain grown under either nitrogen or carbon limitation conditions. A strong correlation was observed between the responses on the transcript and protein levels. Combination of DGA1 overexpression with nitrogen limitation resulted in a high level of lipid accumulation accompanied by downregulation of several amino acid biosynthetic pathways, including that of leucine in particular, and these changes were further correlated with a decrease in metabolic fluxes. This ...
PLOS ONE, 2015
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 longchain 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 mediumchain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.