Nitrogen availability is important for preventing catastrophic mitosis in fission yeast.

Mitosis is a critical stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe may demonstrate catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from e.g. mutations in acetyl-CoA/biotin carboxylase (cut6), in fatty acid synthase (fas2/lsd1), or in the transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It was previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride. In this study we demonstrate that mitotic fidelity can be improved by multiple nitrogen sources. Moreover, this improvement is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the partial rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. Our results highlight a novel role for nitrogen availability in mitotic fidelity.

Yeast perilipin Pet10p/Pln1p interacts with Erg6p in ergosterol metabolism.

Lipid droplets (LD) are highly dynamic organelles specialized for the regulation of energy storage and cellular homeostasis. LD consist of a neutral lipid core surrounded by a phospholipid monolayer membrane with embedded proteins, most of which are involved in lipid homeostasis. In this study, we focused on one of the major LD proteins, sterol C24-methyltransferase, encoded by ERG6. We found that the absence of Erg6p resulted in an increased accumulation of yeast perilipin Pet10p in LD, while the disruption of PET10 was accompanied by Erg6p LD over-accumulation. An observed reciprocal enrichment of Erg6p and Pet10p in pet10Δ and erg6Δ mutants in LD, respectively, was related to specific functional changes in the LD and was not due to regulation on the expression level. The involvement of Pet10p in neutral lipid homeostasis was observed in experiments that focused on the dynamics of neutral lipid mobilization as time-dependent changes in the triacylglycerols (TAG) and steryl esters (SE) content. We found that the kinetics of SE hydrolysis was reduced in erg6Δ cells and the mobilization of SE was completely lost in mutants that lacked both Erg6p and Pet10p. In addition, we observed that decreased levels of SE in erg6Δpet10Δ was linked to an overexpression of steryl ester hydrolase Yeh1p. Lipid analysis of erg6Δpet10Δ showed that PET10 deletion altered the composition of ergosterol intermediates which had accumulated in erg6Δ. In conclusion, yeast perilipin Pet10p functionally interacts with Erg6p during the metabolism of ergosterol.

Yarrowia lipolytica as a Platform for Punicic Acid Production.

Punicic acid (PuA) is a polyunsaturated fatty acid with significant medical, biological, and nutraceutical properties. The primary source of punicic acid is the pomegranate seed oil obtained from fruits of trees that are mainly cultivated in subtropical and tropical climates. To establish sustainable production of PuA, various recombinant microorganisms and plants have been explored as platforms with limited efficiencies. In this study, the oleaginous yeast Yarrowia lipolytica was employed as a host for PuA production. First, growth and lipid accumulation of Y. lipolytica were evaluated in medium supplemented with pomegranate seed oil, resulting in the accumulation of lipids up to 31.2%, consisting of 22% PuA esterified in the fraction of glycerolipids. In addition, lipid-engineered Y. lipolytica strains, transformed with the bifunctional fatty acid conjugase/desaturase from Punica granatum (PgFADX), showed the ability to accumulate PuA de novo. PuA was detected in both polar and neutral lipid fractions, especially in phosphatidylcholine and triacylglycerols. Promoter optimization for PgFADX expression resulted in improved accumulation of PuA from 0.9 to 1.8 mg/g of dry cell weight. The best-producing strain expressing PgFADX under the control of a strong erythritol-inducible promoter produced 36.6 mg/L PuA. These results demonstrate that the yeast Y. lipolytica is a promising host for PuA production.

Heterologous Production of Calendic Acid Naturally Found in Calendula officinalis by Recombinant Fission Yeast.

Calendic acid (CA) is a conjugated fatty acid with anti-cancer properties that is widely present in seed oil of Calendula officinalis. Using the co-expression of C. officinalis fatty acid conjugases (CoFADX-1 or CoFADX-2) and Punica granatum fatty acid desaturase (PgFAD2), we metabolically engineered the synthesis of CA in the yeast Schizosaccharomyces pombe without the need for linoleic acid (LA) supplementation. The highest CA titer and achieved accumulation were 4.4 mg/L and 3.7 mg/g of DCW in PgFAD2 + CoFADX-2 recombinant strain cultivated at 16 °C for 72 h, respectively. Further analyses revealed the accumulation of CA in free fatty acids (FFA) and downregulation of the lcf1 gene encoding long-chain fatty acyl-CoA synthetase. The developed recombinant yeast system represents an important tool for the future identification of the essential components of the channeling machinery to produce CA as a high-value conjugated fatty acid at an industrial level.

Yeast Sec14-like lipid transfer proteins Pdr16 and Pdr17 bind and transfer the ergosterol precursor lanosterol in addition to phosphatidylinositol.

Yeast Sec14-like phosphatidylinositol transfer proteins (PITPs) contain a hydrophobic cavity capable of accepting a single molecule of phosphatidylinositol (PI) or another molecule in a mutually exclusive manner. We report here that two yeast Sec14 family PITPs, Pdr16p (Sfh3p) and Pdr17p (Sfh4p), possess high-affinity binding and transfer towards lanosterol. To our knowledge, this is the first identification of lanosterol transfer proteins. In addition, a pdr16Δpdr17Δ double mutant had a significantly increased level of cellular lanosterol compared with the corresponding wild-type. Based on the lipid profiles of wild-type and pdr16Δpdr17Δ cells grown in aerobic and anaerobic conditions, we suggest that PI-lanosterol transfer proteins are important predominantly for the optimal functioning of the post-lanosterol part of sterol biosynthesis.

Correlation between α-synuclein and fatty acid composition in jejunum of rotenone-treated mice is dependent on acyl chain length.

Events associated with the progression of Parkinson´s disease (PD) are closely related to biomembrane dysfunction. The specific role of membrane composition in the conformational stability of alpha synuclein (αS) has already been well documented. Administration of rotenone is one of the best strategies to initiate PD phenotype in animal models. In the present study, daily exposure (14 weeks) of orally administered rotenone (10 mg/kg) was employed in a mouse model. The mitochondrial complex I inhibition resulted in elevated level of αS in whole tissue homogenate of mouse jejunum. In addition, we identified a strong intra-individual correlation between αS level and the specific esterified fatty acids. The observed correlation depends mainly on the acyl chain length. Based on the obtained results, it is suggested that there is a high potential to manipulate fatty acid homeostasis in modulating αS based pathogenesis of PD, at least in experimental conditions.

Defining the Functional Interactome of Spliceosome-Associated G-Patch Protein Gpl1 in the Fission Yeast Schizosaccharomyces pombe.

Pre-mRNA splicing plays a fundamental role in securing protein diversity by generating multiple transcript isoforms from a single gene. Recently, it has been shown that specific G-patch domain-containing proteins are critical cofactors involved in the regulation of splicing processes. In this study, using the knock-out strategy, affinity purification and the yeast-two-hybrid assay, we demonstrated that the spliceosome-associated G-patch protein Gpl1 of the fission yeast S. pombe mediates interactions between putative RNA helicase Gih35 (SPAC20H4.09) and WD repeat protein Wdr83, and ensures their binding to the spliceosome. Furthermore, RT-qPCR analysis of the splicing efficiency of deletion mutants indicated that the absence of any of the components of the Gpl1-Gih35-Wdr83 complex leads to defective splicing of fet5 and pwi1, the reference genes whose unspliced isoforms harboring premature stop codons are targeted for degradation by the nonsense-mediated decay (NMD) pathway. Together, our results shed more light on the functional interactome of G-patch protein Gpl1 and revealed that the Gpl1-Gih35-Wdr83 complex plays an important role in the regulation of pre-mRNA splicing in S. pombe.

Improving the Production of Punicic Acid in Baker's Yeast by Engineering Genes in Acyl Channeling Processes and Adjusting Precursor Supply.

Punicic acid (PuA) is a high-value edible conjugated fatty acid with strong bioactivities and has important potential applications in nutraceutical, pharmaceutical, feeding, and oleochemical industries. Since the production of PuA is severely limited by the fact that its natural source (pomegranate seed oil) is not readily available on a large scale, there is considerable interest in understanding the biosynthesis and accumulation of this plant-based unusual fatty acid in transgenic microorganisms to support the rational design of biotechnological approaches for PuA production via fermentation. Here, we tested the effectiveness of genetic engineering and precursor supply in PuA production in the model yeast strain Saccharomyces cerevisiae. The results revealed that the combination of precursor feeding and co-expression of selected genes in acyl channeling processes created an effective "push-pull" approach to increase PuA content, which could prove valuable in future efforts to produce PuA in industrial yeast and other microorganisms via fermentation.

Sec14 family of lipid transfer proteins in yeasts.

The hydrophobicity of lipids prevents their free movement across the cytoplasm. To achieve highly heterogeneous and precisely regulated lipid distribution in different cellular membranes, lipids are transported by lipid transfer proteins (LTPs) in addition to their transport by vesicles. Sec14 family is one of the most extensively studied groups of LTPs. Here we provide an overview of Sec14 family of LTPs in the most studied yeast Saccharomyces cerevisiae as well as in other selected non-Saccharomyces yeasts-Schizosaccharomyces pombe, Kluyveromyces lactis, Candida albicans, Candida glabrata, Cryptococcus neoformans, and Yarrowia lipolytica. Discussed are specificities of Sec14-domain LTPs in various yeasts, their mode of action, subcellular localization, and physiological function. In addition, quite few Sec14 family LTPs are target of antifungal drugs, serve as modifiers of drug resistance or influence virulence of pathologic yeasts. Thus, they represent an important object of study from the perspective of human health.

Metabolism of Storage Lipids and the Role of Lipid Droplets in the Yeast Schizosaccharomyces pombe.

Storage lipids, triacylglycerols (TAG), and steryl esters (SE), are predominant constituents of lipid droplets (LD) in fungi. In several yeast species, metabolism of TAG and SE is linked to various cellular processes, including cell division, sporulation, apoptosis, response to stress, and lipotoxicity. In addition, TAG are an important source for the generation of value-added lipids for industrial and biomedical applications. The fission yeast Schizosaccharomyces pombe is a widely used unicellular eukaryotic model organism. It is a powerful tractable system used to study various aspects of eukaryotic cellular and molecular biology. However, the knowledge of S. pombe neutral lipids metabolism is quite limited. In this review, we summarize and discuss the current knowledge of the homeostasis of storage lipids and of the role of LD in the fission yeast S. pombe with the aim to stimulate research of lipid metabolism and its connection with other essential cellular processes. We also discuss the advantages and disadvantages of fission yeast in lipid biotechnology and recent achievements in the use of S. pombe in the biotechnological production of valuable lipid compounds.

Metabolism of phospholipids in the yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an important model organism for the study of fundamental questions in eukaryotic cell and molecular biology. A plethora of cellular processes are membrane associated and/or dependent on the proper functioning of cellular membranes. Phospholipids are not only the basic building blocks of cellular membranes; they also serve as precursors to numerous signaling molecules. In this review, we describe the biosynthetic pathways leading to major S. pombe phospholipids, how these pathways are regulated, and what is known about degradation and turnover of fission yeast phospholipids. This review also addresses the synthesis, regulation and the role of water-soluble phospholipid precursors. The last chapter of the review is devoted to the use of S. pombe for the biotechnological production of value-added lipid molecules.

Yeast phosphatidylinositol transfer protein Pdr17 does not require high affinity phosphatidylinositol binding for its cellular function.

Yeast phosphatidylinositol transfer protein (PITP) Pdr17 is an essential component of the complex required for decarboxylation of phosphatidylserine (PS) to phosphatidylethanolamine (PE) at a non-mitochondrial location. According to current understanding, this process involves the transfer of PS from the endoplasmic reticulum to the Golgi/endosomes. We generated a Pdr17E237A, K269A mutant protein to better understand the mechanism by which Pdr17p participates in the processes connected to the decarboxylation of PS to PE. We show that the Pdr17E237A, K269A mutant protein is not capable of binding phosphatidylinositol (PI) using permeabilized human cells, but still retains the ability to transfer PI between two membrane compartments in vitro. We provide data together with molecular models showing that the mutations E237A and K269A changed only the lipid binding cavity of Pdr17p and not its surface properties. In contrast to Pdr16p, a close homologue, the ability of Pdr17p to bind PI is not required for its major cellular function in the inter-membrane transfer of PS. We hypothesize that these two closely related yeast PITPs, Pdr16p and Pdr17p, have evolved from a common ancestor. Pdr16p fulfills those role(s) in which the ability to bind and transfer PI is required, while Pdr17p appears to have adapted to a different role which does not require the high affinity binding of PI, although the protein retains the capacity to transfer PI. Our results indicate that PITPs function in complex ways in vivo and underscore the need to consider multiple PITP parameters when studying these proteins in vitro.

Engineering Arabidopsis long-chain acyl-CoA synthetase 9 variants with enhanced enzyme activity.

Long-chain acyl-CoA synthetase (LACS, EC 6.2.1.3) catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which, in turn, serves as the major acyl donor for various lipid metabolic pathways. Increasing the size of acyl-CoA pool by enhancing LACS activity appears to be a useful approach to improve the production and modify the composition of fatty acid-derived compounds, such as triacylglycerol. In the present study, we aimed to improve the enzyme activity of Arabidopsis thaliana LACS9 (AtLACS9) by introducing random mutations into its cDNA using error-prone PCR. Two AtLACS9 variants containing multiple amino acid residue substitutions were identified with enhanced enzyme activity. To explore the effect of each amino acid residue substitution, single-site mutants were generated and the amino acid substitutions C207F and D238E were found to be primarily responsible for the increased activity of the two variants. Furthermore, evolutionary analysis revealed that the beneficial amino acid site C207 is conserved among LACS9 from plant eudicots, whereas the other beneficial amino acid site D238 might be under positive selection. Together, our results provide valuable information for the production of LACS variants for applications in the metabolic engineering of lipid biosynthesis in oleaginous organisms.

Substrate preferences of long-chain acyl-CoA synthetase and diacylglycerol acyltransferase contribute to enrichment of flax seed oil with α-linolenic acid.

Seed oil from flax (Linum usitatissimum) is enriched in α-linolenic acid (ALA; 18:3Δ9cis,12cis,15cis ), but the biochemical processes underlying the enrichment of flax seed oil with this polyunsaturated fatty acid are not fully elucidated. Here, a potential process involving the catalytic actions of long-chain acyl-CoA synthetase (LACS) and diacylglycerol acyltransferase (DGAT) is proposed for ALA enrichment in triacylglycerol (TAG). LACS catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which in turn may serve as an acyl-donor in the DGAT-catalyzed reaction leading to TAG. To test this hypothesis, flax LACS and DGAT cDNAs were functionally expressed in Saccharomyces cerevisiae strains to probe their possible involvement in the enrichment of TAG with ALA. Among the identified flax LACSs, LuLACS8A exhibited significantly enhanced specificity for ALA over oleic acid (18:1Δ9cis ) or linoleic acid (18:2Δ9cis,12cis ). Enhanced α-linolenoyl-CoA specificity was also observed in the enzymatic assay of flax DGAT2 (LuDGAT2-3), which displayed ∼20 times increased preference toward α-linolenoyl-CoA over oleoyl-CoA. Moreover, when LuLACS8A and LuDGAT2-3 were co-expressed in yeast, both in vitro and in vivo experiments indicated that the ALA-containing TAG enrichment process was operative between LuLACS8A- and LuDGAT2-3-catalyzed reactions. Overall, the results support the hypothesis that the cooperation between the reactions catalyzed by LACS8 and DGAT2 may represent a route to enrich ALA production in the flax seed oil.

Bioactivity and biotechnological production of punicic acid.

Punicic acid (PuA; 18: 3Δ 9cis,11trans,13cis ) is an unusual 18-carbon fatty acid bearing three conjugated double bonds. It has been shown to exhibit a myriad of beneficial bioactivities including anti-cancer, anti-diabetes, anti-obesity, antioxidant, and anti-inflammatory properties. Pomegranate (Punica granatum) seed oil contains approximately 80% PuA and is currently the major natural source of this remarkable fatty acid. While both PuA and pomegranate seed oil have been used as functional ingredients in foods and cosmetics for some time, their value in pharmaceutical/medical and industrial applications are presently under further exploration. Unfortunately, the availability of PuA is severely limited by the low yield and unstable supply of pomegranate seeds. In addition, efforts to produce PuA in transgenic crops have been limited by a relatively low content of PuA in the resulting seed oil. The production of PuA in engineered microorganisms with modern fermentation technology is therefore a promising and emerging method with the potential to resolve this predicament. In this paper, we provide a comprehensive review of this unusual fatty acid, covering topics ranging from its natural sources, biosynthesis, extraction and analysis, bioactivity, health benefits, and industrial applications, to recent efforts and future perspectives on the production of PuA in engineered plants and microorganisms.

Metabolic engineering of Schizosaccharomyces pombe to produce punicic acid, a conjugated fatty acid with nutraceutic properties.

Punicic acid (PuA) is a conjugated linolenic acid (C18:3Δ9c,11t,13c) with a wide range of nutraceutic effects with the potential to reduce the incidence of a number of health disorders including diabetes, obesity, and cancer. It is the main component of seed oil from Punica granatum and Trichosanthes kirilowii. Previously, production of relatively high levels of this unusual fatty acid in the seed oil of transgenic Arabidopsis thaliana plant was accomplished by the use of A. thaliana fad3/fae1 mutant high in linoleic acid (18:2∆9c,12c) and by co-expression of P. granatum FATTY ACID CONJUGASE (PgFADX) with Δ12-DESATURASE (FAD2). In the current study, P. granatum cDNAs governing PuA production were introduced into the yeast Schizosaccharomyces pombe. Expression of PgFADX alone resulted in production of PuA at the level of 19.6% of total fatty acids. Co-expression PgFADX with PgFAD2, however, further enhanced PuA content to 25.1% of total fatty acids, the highest level reported to date for heterologous expression. Therefore, microbial systems can be considered as a potential alternative to plant sources for a source of PuA for nutraceutic applications.

Squalene is lipotoxic to yeast cells defective in lipid droplet biogenesis.

The toxic effect of overloaded lipids on cell physiology and viability was described in various organisms. In this study we focused on the potential lipotoxicity of squalene, a linear triterpene synthesized in eukaryotic cells as an intermediate in sterol biosynthesis. Squalene toxicity was studied in the yeast Saccharomyces cerevisiae, a model unicellular eukaryote established in lipotoxicity studies. Squalene levels in yeast are typically low but its accumulation can be induced under specific conditions, e.g. by inhibition of squalene monooxygenase with the antimycotic terbinafine. At higher levels squalene is stored in lipid droplets. We demonstrated that low doses of terbinafine caused severe impairment of growth and loss of viability of the yeast mutant dga1Δ lro1Δ are1Δ are2Δ unable to form lipid droplets and that these defects were linked to squalene accumulation. The hypersensitivity of the lipid droplet-less mutant to terbinafine was alleviated by decreasing squalene accumulation with low doses of squalene synthase inhibitor zaragozic acid. Our results proved that accumulated squalene is lipotoxic to yeast cells if it cannot be efficiently sequestered in lipid droplets. This supports the hypothesis about the role of squalene in the fungicidal activity of terbinafine. Squalene toxicity may represent also a limiting factor for production of this high-value lipid in yeast.

Baker's Yeast Deficient in Storage Lipid Synthesis Uses cis-Vaccenic Acid to Reduce Unsaturated Fatty Acid Toxicity.

The role of cis-vaccenic acid (18:1n-7) in the reduction of unsaturated fatty acids toxicity was investigated in baker's yeast Saccharomyces cerevisiae. The quadruple mutant (QM, dga1Δ lro1Δ are1Δ are2Δ) deficient in enzymes responsible for triacylglycerol and steryl ester synthesis has been previously shown to be highly sensitive to exogenous unsaturated fatty acids. We have found that cis-vaccenic acid accumulated during cultivation in the QM cells but not in the corresponding wild type strain. This accumulation was accompanied by a reduction in palmitoleic acid (16:1n-7) content in the QM cells that is consistent with the proposed formation of cis-vaccenic acid by elongation of palmitoleic acid. Fatty acid analysis of individual lipid classes from the QM strain revealed that cis-vaccenic acid was highly enriched in the free fatty acid pool. Furthermore, production of cis-vaccenic acid was arrested if the mechanism of fatty acids release to the medium was activated. We also showed that exogenous cis-vaccenic acid did not affect viability of the QM strain at concentrations toxic for palmitoleic or oleic acids. Moreover, addition of cis-vaccenic acid to the growth medium provided partial protection against the lipotoxic effects of exogenous oleic acid. Transformation of palmitoleic acid to cis-vaccenic acid is thus a rescue mechanism enabling S. cerevisiae cells to survive in the absence of triacylglycerol synthesis as the major mechanism for unsaturated fatty acid detoxification.

Phosphatidylinositol binding of Saccharomyces cerevisiae Pdr16p represents an essential feature of this lipid transfer protein to provide protection against azole antifungals.

Pdr16p is considered a factor of clinical azole resistance in fungal pathogens. The most distinct phenotype of yeast cells lacking Pdr16p is their increased susceptibility to azole and morpholine antifungals. Pdr16p (also known as Sfh3p) of Saccharomyces cerevisiae belongs to the Sec14 family of phosphatidylinositol transfer proteins. It facilitates transfer of phosphatidylinositol (PI) between membrane compartments in in vitro systems. We generated Pdr16p(E235A, K267A) mutant defective in PI binding. This PI binding deficient mutant is not able to fulfill the role of Pdr16p in protection against azole and morpholine antifungals, providing evidence that PI binding is critical for Pdr16 function in modulation of sterol metabolism in response to these two types of antifungal drugs. A novel feature of Pdr16p, and especially of Pdr16p(E235A, K267A) mutant, to bind sterol molecules, is observed.