Activation of diacylglycerol acyltransferase expressed in Saccharomyces cerevisiae: overexpression of Dga1p lacking the N-terminal region in the ∆snf2 disruptant produces a significant increase in its enzyme activity

We previously found that overexpression of DGA1 encoding diacylglycerol acyltransferase (DGAT) in the ∆snf2 disruptant of Saccharomyces cerevisiae caused a significant increase in lipid accumulation and DGAT activity. The present study was conducted to investigate how Dga1p is activated in the ∆snf2 disruptant. To analyze the expression of Dga1p in wild type and the ∆snf2 disruptant, we overexpressed Dga1p with a 6x His tag at the N-terminus and a FLAG tag at the C-terminus. Immunoblotting using anti-6x His and anti-FLAG antibodies revealed that, in addition to full-length protein, Dga1p lacking the N-terminus was produced only in the ∆snf2 disruptant. Full-length Dga1p and N-terminally truncated Dga1p were separated and purified from the lipid body fraction by using anti-FLAG M2 agarose and TALON metal affinity resin. Major DGAT activity was recovered in the purified fraction of N-terminally truncated Dga1p, indicating that proteolytic cleavage at the N-terminal region is involved in DGAT activation in the ∆snf2 disruptant. Analysis of the cleavage site of N-terminally truncated Dga1p revealed a major site between Lys-29 and Ser-30. We then overexpressed truncated Dga1p variants that lacked different N-terminal amino acids and had a FLAG tag at the C-terminus. The homogenate and lipid body fraction of the ∆snf2 disruptant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1∆N2p) had higher DGAT activity than that overexpressing Dga1p, indicating that Dga1∆N2p is activated Dga1p. Dga1∆N2p-FLAG(C-terminus) was purified to near homogeneity by anti-FLAG M2 agarose chromatography and maintained significant DGAT activity. These results provide a new strategy to engineer expression of DGAT.     

Identification of triacylglycerol and steryl ester synthases of the methylotrophic yeast Pichia pastoris

In yeast like in many other eukaryotes, fatty acids are stored in the biologically inert form of triacylglycerols (TG) and steryl esters (SE) as energy reserve and/or as membrane building blocks. In the present study, we identified gene products catalyzing formation of TG and SE in the methylotrophic yeast Pichia pastoris. Based on sequence homologies to Saccharomyces cerevisiae, the two diacylglycerol acyltransferases Dga1p and Lro1p and one acyl CoA:sterol acyltransferase Are2p from P. pastoris were identified. Mutants bearing single and multiple deletions of the respective genes were analyzed for their growth phenotype, lipid composition and the ability to form lipid droplets. Our results indicate that the above mentioned gene products are most likely responsible for the entire TG and SE synthesis in P. pastoris. Lro1p which has low fatty acid substrate specificity in vivo is the major TG synthase in this yeast, whereas Dga1p contributes less to TG synthesis although with some preference to utilize polyunsaturated fatty acids as substrates. In contrast to S. cerevisiae, Are2p is the only SE synthase in P. pastoris. Also this enzyme exhibits some preference for certain fatty acids as judged from the fatty acid profile of SE compared to bulk lipids. Most interestingly, TG formation in P. pastoris is indispensable for lipid droplet biogenesis. The small amount of SE synthesized by Are2p in a dga1?lro1? double deletion mutant is insufficient to initiate the formation of the storage organelle. In summary, our data provide a first insight into the molecular machinery of non-polar lipid synthesis and storage in P. pastoris and demonstrate specific features of this machinery in comparison to other eukaryotic cells, especially S. cerevisiae.     

Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica

Although there are numerous oleochemical applications for ricinoleic acid (RA) and its derivatives, their production is limited and subject to various safety legislations. In an effort to produce RA from alternative sources, we constructed a genetically modified strain of the oleaginous yeast Yarrowia lipolytica. This strain is unable to perform β-oxidation and is invalidated for the native triacylglycerol (TAG) acyltransferases (Dga1p, Dga2p, and Lro1p) and the ?12 desaturase (Fad2p). We also expressed the Ricinus communis ?12 hydroxylase (RcFAH12) under the control of the TEF constitutive promoter in this strain. However, RA constituted only 7 % of the total lipids produced by this modified strain. By contrast, expression of the Claviceps purpurea hydroxylase CpFAH12 in this background resulted in a strain able to accumulate RA to 29 % of total lipids, and expression of an additional copy of CpFAH12 drove RA accumulation up to 35 % of total lipids. The co-expression of the C. purpurea or R. communis type II diacylglycerol acyltransferase (RcDGAT2 or CpDGAT2) had negative effects on RA accumulation in this yeast, with RA levels dropping to below 14 % of total lipids. Overexpression of the native Y. lipolytica PDAT acyltransferase (Lro1p) restored both TAG accumulation and RA levels. Thus, we describe the consequences of rerouting lipid metabolism in this yeast so as to develop a cell factory for RA production. The engineered strain is capable of accumulating RA to 43 % of its total lipids and over 60 mg/g of cell dry weight; this is the most efficient production of RA described to date.     

Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds

Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24 kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06 % higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.     

Lipid productivity and fatty acid composition in Chlorella and Scenepdesmus strains grown in nitrogen-stressed conditions

Thirty Chlorella and 30 Scenedesmus strains grown in nitrogen-stressed conditions (70 mg L^?1 N) were analyzed for biomass accumulation, lipid productivity, protein, and fatty acid (FA) composition. Scenedesmus strains produced more biomass (4.02?±?0.73 g L^?1) after 14 days in culture compared to Chlorella strains (2.57?±?0.12 g L^?1). Protein content decreased and lipid content increased from days 8 to 14 with an increase in triacylglycerol (TAG) accumulation in most strains. By day 14, Scenedesmus strains generally had higher lipid productivity (53.5?±?3.7 mg lipid L^?1 day^?1) than Chlorella strains (35.1?±?2.8 mg lipid L^?1 day^?1) with the lipids consisting mainly of C16–18 TAGs. Scenedesmus strains generally had a more suitable FA profile with higher amounts of saturated fatty acids and monounsaturated fatty acids (MUFAs) and a smaller polyunsaturated fatty acid (PUFA) component. Chlorella strains had a larger PUFA component and smaller MUFA component. The general trend in the FA composition of Chlorella strains was oleic > palmitic > α-linolenic = linoleic > eicosenoic > heptadecenoic > stearic acid. For Scenedesmus strains, the general trend was oleic > palmitic > linoleic > α-linolenic > stearic > eicosenoic > palmitoleic > heptadecenoic acid. The most promising strains with the highest lipid productivity and most suitable FA profiles were Scenedesmus sp. MACC 401, Scenedesmus soli MACC 721, and Scenedesmus ecornis MACC 714. Although Chlorella sp. MACC 519 had lower lipid productivity, the FA profile was good with a lower PUFA component compared to the other Chlorella strains analyzed and a low linolenic acid concentration.     

Effects of Initial Phosphorus Concentration and Light Intensity on Biomass Yield per Phosphorus and Lipid Accumulation of Scenedesmus sp. LX1

Phosphorus has been considered as one of the most important limiting resources of large-scale production of microalgal biofuel. The approaches to increase biomass yield per phosphorus, along with the lipid accumulation properties of Scenedesmus sp. LX1, were investigated in this study. It was found that practical biomass yield per phosphorous was reduced with the increase of initial phosphorus (P) concentration, but increased with light intensity. The highest biomass yield per P of 4,500 kg-biomass/kg-P was achieved at initial phosphorus concentration of 0.05 mg?·?L^?1 under the light intensity of 320 μmol photon?·?m^?2?·?s^?1. Furthermore, the lipid content per biomass and triacylglycerols (TAGs) content per lipid were found to be positively correlated to biomass yield per P. With the biomass yield increased from 2,800 kg-biomass/kg-P to 4,500 kg-biomass/kg-P, the lipid content per microalgal biomass and TAG content per lipid increased from 18.7 % to 35.0 % and from 69.5 % to 83.0 %. These results suggested a possible approach to achieve high biomass production and high lipid content simultaneously.     

At high temperature lipid production in Ettlia oleoabundans occurs before nitrate depletion

Temperature and light intensity effects on biomass and lipid production were investigated in Ettlia oleoabundans to better understand some fundamental properties of this potentially useful but poorly studied microalgal species. E. oleoabundans entered dormant state at 5 °C, showed growth at 10 °C, and when exposed to light at 70 μmol photons per square meter per second at 10 °C, cells reached a biomass concentration of >2.0 g?L^?1 with fatty acid methyl esters of 11.5 mg?L^?1. Highest biomass productivity was at 15 °C and 25 °C regardless of light intensity, and accumulation of intracellular lipids was stimulated by nitrate depletion under these conditions. Although growth was inhibited at 35 °C, at 130 μmol photons per square meter per second lipid content reached 10.37 mg?L^?1 with fatty acid content more favorable to biodiesel dominating; this occurred without nitrate depletion. In a two-phase temperature shift experiment at two nitrate levels, cells were shifted after 21 days at 15 °C to 35 °C for 8 days. Although after the shift growth continued, lipid productivity per cell was less than that in the 35 °C cultures, again without nitrate depletion. This study showed that E. oleoabundans grows well at low temperature and light intensity, and high temperature can be a useful trigger for lipid accumulation independent of nitrate depletion. This will prove useful for improving our knowledge about lipid production in this and other oleaginous algae for modifying yield and quality of algal lipids being considered for biodiesel production.     

Reactive oxygen species generation and antioxidant defense system in hydroponically grown wheat (Triticum aestivum) upon β-pinene exposure: an early time course assessment

We investigated the effect of β-pinene on reactive oxygen species (ROS: lipid peroxidation, membrane integrity, hydrogen peroxide and superoxide ions) generation and activity of antioxidant defense system during early hours of treatment (4, 8, 16 and 24 h) in hydroponically grown Triticum aestivum (wheat). β-Pinene reduced the root and shoot growth of the hydroponically grown wheat. However, the reduction was more pronounced in root length than in shoot length. β-Pinene enhanced ROS generation as indicated by increased levels of malondialdehyde (20–87 %), hydrogen peroxide (9–45 %) and superoxide ion (23–179 %) content, thereby suggesting lipid peroxidation and induction of oxidative stress in a time- and concentration-dependent manner. The oxidative damage was more pronounced at ≥10 µM β-pinene and at ≥8 h after exposure. β-Pinene caused a severe electrolyte leakage from wheat roots indicating membrane disruption and loss of integrity. Enhanced lipid peroxidation and loss of membrane integrity were confirmed by in situ histochemical studies. β-Pinene provoked increase in the activity of lipoxygenase and upregulation in the activities of antioxidant enzymes: catalases, superoxide dismutases, ascorbate peroxidases, guaiacol peroxidases and glutathione reductases. The enhanced activity of lipoxygenases evoked by β-pinene paralleled higher accumulation of MDA, thereby suggesting that antioxidant defense mechanism was not able to prevent β-pinene-induced lipid peroxidation.     

Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.     

Effects of different nitrogen, phosphorus, and iron concentrations and salinity on lipid production in newly isolated strain of the tropical green microalga, Scenedesmus dimorphus KMITL

The fatty acid composition, the effect of different concentrations of nitrogen (16.5-344 mg ?L^?1), phosphorus (9–45 mg? L^?1), iron (9–45 mg? L^?1) and salinity levels (0–20 psu) on lipid production in the green microalga Scenedesmus dimorphus KMITL, a new strain isolated from a tropical country, Thailand, were studied. The alga was isolated from a freshwater fish pond, and cultured in Chlorella medium by varying one parameter at a time. The main fatty acid composition of this strain was C16–C18 (97.52 %) fatty acids. A high lipid content was observed in conditions of 16.5 mg? L^?1-N, or 22 mg ?L^?1-P, or 45 mg ?L^?1-Fe, or 5 psu salinity, which accumulated lipids to 20.3?±?0.4, 19.4?±?0.2, 24.7?±?0.5, and 14.3?±?0.2 % of algal biomass, respectively. Increasing lipid content and lipid productivity was noted when the alga was cultured under high iron concentration and high salinity, as well as under reduced phosphorus conditions, whereas nitrogen limitation only resulted in an increased lipid content.     

Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin

Milbemycins A3/A4 are important 16-membered macrolides which have been commercialized and widely used as pesticide and veterinary medicine. However, similar to other milbemycin producers, the production of milbemycins A3/A4 in Streptomyces bingchenggensis is usually accompanied with undesired by-products such as C5-O-methylmilbemycins B2/B3 (α-class) and β1/β2 (β-class) together with nanchangmycin. In order to obtain high yield milbemycins A3/A4-producing strains that produce milbemycins A3/A4 as main components, milD, a putative C5-O-methyltransferase gene of S. bingchenggensis, was biofunctionally investigated by heterologous expression in Escherichia coli. Enzymatic analysis indicated that MilD can catalyze both α-class (A3/A4) and β-class milbemycins (β11) into C5-O-methylmilbemycins B2/B3 and β1, respectively, suggesting little effect of furan ring formed between C6 and C8a on the C5-O-methylation catalyzed by MilD. Deletion of milD gene resulted in the elimination of C5-O-methylmilbemycins B2/B3 and β1/β2 together with an increased yield of milbemycins A3/A4 in disruption strain BCJ13. Further disruption of the gene nanLD encoding loading module of polyketide synthase responsible for the biosynthesis of nanchangmycin led to strain BCJ36 that abolished the production of nanchangmycin. Importantly, mutant strain BCJ36 (?milD?nanLD) produced milbemycins A3/A4 as main secondary metabolites with a yield of 2312?±?47 μg/ml, which was approximately 74 % higher than that of the initial strain S. bingchenggensis BC-109-6 (1326?±?37 μg/ml).     

Effects of photoperiod,salinity and pH on cell growth and lipid content of Pavlova lutheri

The aim of the present work was to study the effects of photoperiod, salinity and pH on growth and lipid content of Pavlova lutheri microalgae for biodiesel production in small-scale and large-scale open-pond tanks. In a 250-mL flask, the cultures grew well under 24 h illumination with maximum specific growth rate, μ _max , of 0.12 day^?1 and lipid content of 35 % as compared to 0.1 day^?1 and 15 % lipid content in the dark. The salinity was optimum for the cell growth at 30–35 ppt, but the lipid content of 34–36 % was higher at 35–40 ppt. Algal growth and lipid accumulation was optimum at pH 8–9. Large-scale cultivation in 5-L and 30-L tanks achieved μ _max of 0.13–0.14 day^?1 as compared to 0.12 day^?1 in small-scale and 300L cultures.     

pH effects on growth and lipid accumulation of the biofuel microalgae Nannochloropsis salina and invading organisms

Biofuels derived from non-crop sources, such as microalgae, offer their own advantages and limitations. Despite high growth rates and lipid accumulation, microalgae cultivation still requires more energy than it produces. Furthermore, invading organisms can lower efficiency of algae production. Simple environmental changes might be able to increase algae productivity while minimizing undesired organisms like competitive algae or predatory algae grazers. Microalgae are susceptible to pH changes. In many production systems, pH is kept below 8 by CO₂ addition. Here, we uncouple the effects of pH and CO₂ input, by using chemical pH buffers and investigate how pH influences Nannochloropsis salina growth and lipid accumulation as well as invading organisms. We used a wide range of pH levels (5, 6, 7, 8, 9, and 10). N. salina showed highest growth rates at pH 8 and 9 (0.19?±?0.008 and 0.19?±?0.011, respectively; mean ± SD). Maximum cell densities in these treatments were reached around 21 days into the experiment (95.6?×?10⁶?±?9?×?10⁶ cells mL^?1 for pH 8 and 92.8?×?10⁶?±?24?×?10⁶ cells mL^?1 for pH 9). Lipid accumulation of unbuffered controls were 21.8?±?5.8 % fatty acid methyl esters content by mass, and we were unable to trigger additional significant lipid accumulation by manipulating pH levels at the beginning of stationary phase. Ciliates (grazing predators) occurred in significant higher densities at pH 6 (56.9?±?39.6?×?10⁴ organisms mL^?1) than higher pH treatments (0.1–6.8?×?10⁴ organisms mL^?1). Furthermore, the addition of buffers themselves seemed to negatively impact diatoms (algal competitors). They were more abundant in an unbuffered control (12.7?±?5.1?×?10⁴ organisms mL^?1) than any of the pH treatments (3.6–4.7?×?10⁴ organisms mL^?1). In general, pH values of 8 to 9 might be most conducive to increasing algae production and minimizing invading organisms. CO₂ addition seems more valuable to algae as an inorganic carbon source and not as an essential mechanism to reduce pH.     

Inorganic carbon and pH effect on growth and lipid productivity of Tetraselmis suecica and Chlorella sp (Chlorophyta) grown outdoors in bag photobioreactors

There has been considerable interest in cultivation of green microalgae (Chlorophyta) as a source of lipid that can alternatively be converted to biodiesel. However, almost all mass cultures of algae are carbon-limited. Therefore, to reach a high biomass and oil productivities, the ideal selected microalgae will most likely need a source of inorganic carbon. Here, growth and lipid productivities of Tetraselmis suecica CS-187 and Chlorella sp were tested under various ranges of pH and different sources of inorganic carbon (untreated flue gas from coal-fired power plant, pure industrial CO₂, pH-adjusted using HCl and sodium bicarbonate). Biomass and lipid productivities were highest at pH 7.5 (320?±?29.9 mg biomass L^?1 day^?1and 92?±?13.1 mg lipid L^?1 day^?1) and pH 7 (407?±?5.5 mg biomass L^?1 day^?1 and 99?±?17.2 mg lipid L^?1 day^?1) for T. suecica CS-187 and Chlorella sp, respectively. In general, biomass and lipid productivities were pH 7.5?>?pH 7?>?pH 8?>?pH 6.5 and pH 7?>?pH 7.5?=?pH 8?>?pH 6.5?>?pH 6?>?pH 5.5 for T. suecica CS-187 and Chlorella sp, respectively. The effect of various inorganic carbon on growth and productivities of T. suecica (regulated at pH?=?7.5) and Chlorella sp (regulated at pH?=?7) grown in bag photobioreactors was also examined outdoor at the International Power Hazelwood, Gippsland, Victoria, Australia. The highest biomass and lipid productivities of T. suecica (51.45?±?2.67 mg biomass L^?1 day^?1 and 14.8?±?2.46 mg lipid L^?1 day^?1) and Chlorella sp (60.00?±?2.4 mg biomass L^?1 day^?1 and 13.70?±?1.35 mg lipid L^?1 day^?1) were achieved when grown using CO₂ as inorganic carbon source. No significant differences were found between CO₂ and flue gas biomass and lipid productivities. While grown using CO₂ and flue gas, biomass productivities were 10, 13 and 18 %, and 7, 14 and 19 % higher than NaHCO₃, HCl and unregulated pH for T. suecica and Chlorella sp, respectively. Addition of inorganic carbon increased specific growth rate and lipid content but reduced biomass yield and cell weight of T. suecica. Addition of inorganic carbon increased yield but did not change specific growth rate, cell weight or content of the cell weight of Chlorella sp. Both strains showed significantly higher maximum quantum yield (F_v/F_m) when grown under optimum pH.     

Reduction of furan derivatives by overexpressing NADH-dependent Adh1 improves ethanol fermentation using xylose as sole carbon source with Saccharomyces cerevisiae harboring XR–XDH pathway

Several alcohol dehydrogenase (ADH)-related genes have been identified as enzymes for reducing levels of toxic compounds, such as, furfural and/or 5-hydroxymethylfurfural (5-HMF), in hydrolysates of pretreated lignocelluloses. To date, overexpression of these ADH genes in yeast cells have aided ethanol production from glucose or glucose/xylose mixture in the presence of furfural or 5-HMF. However, the effects of these ADH isozymes on ethanol production from xylose as a sole carbon source remain uncertain. We showed that overexpression of mutant NADH-dependent ADH1 derived from TMB3000 strain in the recombinant Saccharomyces cerevisiae, into which xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway of Pichia stipitis has been introduced, improved ethanol production from xylose as a sole carbon source in the presence of 5-HMF. Enhanced furan-reducing activity is able to regenerate NAD⁺ to relieve redox imbalance, resulting in increased ethanol yield arising from decreased xylitol accumulation. In addition, we found that overexpression of wild-type ADH1 prevented the more severe inhibitory effects of furfural in xylose fermentation as well as overexpression of TMB3000-derived mutant. After 120 h of fermentation, the recombinant strains overexpressing wild-type and mutant ADH1 completely consumed 50 g/L xylose in the presence of 40 mM furfural and most efficiently produced ethanol (15.70 g/L and 15.24 g/L) when compared with any other test conditions. This is the first report describing the improvement of ethanol production from xylose as the sole carbon source in the presence of furan derivatives with xylose-utilizing recombinant yeast strains via the overexpression of ADH-related genes.     

Candida zeylanoides as a new yeast model for lipid metabolism studies: effect of nitrogen sources on fatty acid accumulation

Lipid homeostasis is well-known in oleaginous yeasts, but there are few non-oleaginous yeast models apart from Saccharomyces cerevisiae. We are proposing the non-oleaginous yeast Candida zeylanoides QU 33 as model. The aim of this study was to investigate the influence of the carbon/nitrogen ratio and the type of nitrogen source upon oil accumulation by this yeast grown on shake flask cultures. The maximum biomass was obtained in yeast extract (2.39?±?0.19 g/l), followed by peptone (2.24?±?0.05 g/l), while the highest content of microbial oil (0.35?±?0.01 g/l) and the maximum lipid yield (15.63 %) were achieved with peptone. Oleic acid was the predominant cellular fatty acid in all culture media (>32.23 %), followed by linoleic (>15.79 %) and palmitic acids (>13.47 %). The highest lipid yield using glucose and peptone was obtained at the C/N ratio of 200:1.     

Metabolic engineering of lipid pathways in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and staged bioprocess for enhanced lipid production and cellular physiology

Microbially produced lipids have attracted attention for their environmental benefits and commercial value. We have combined lipid pathway engineering in Saccharomyces cerevisiae yeast with bioprocess design to improve productivity and explore barriers to enhanced lipid production. Initially, individual gene expression was tested for impact on yeast growth and lipid production. Then, two base strains were prepared for enhanced lipid accumulation and stabilization steps by combining DGAT1, ΔTgl3 with or without Atclo1, which increased lipid content ~?1.8-fold but reduced cell viability. Next, fatty acid (FA) biosynthesis genes Ald6-SEACS^L641P alone or with ACC1** were co-expressed in base strains, which significantly improved lipid content (8.0% DCW, 2.6-fold than control), but severely reduced yeast growth and cell viability. Finally, a designed two-stage process convincingly ameliorated the negative effects, resulting in normal cell growth, very high lipid productivity (307 mg/L, 4.6-fold above control) and improved cell viability.     

Effect of Lipid Particle Biogenesis on the Subcellular Distribution of Squalene in the Yeast Saccharomyces cerevisiae

Squalene belongs to the group of isoprenoids and is a precursor for the synthesis of sterols, steroids, and ubiquinons. In the yeast Saccharomyces cerevisiae, the amount of squalene can be increased by variation of growth conditions or by genetic manipulation. In this report, we show that a hem1Δ mutant accumulated a large amount of squalene, which was stored almost exclusively in cytoplasmic lipid particles/droplets. Interestingly, a strain bearing a hem1Δ deletion in a dga1Δlro1Δare1Δare2Δ quadruple mutant background (QMhem1Δ), which is devoid of the classical storage lipids, triacylglycerols and steryl esters, and lacks lipid particles, accumulated squalene at similar amounts as the hem1Δ mutant in a wild type background. In QMhem1Δ, however, increased amounts of squalene were found in cellular membranes, especially in microsomes. The fact that QMhem1Δ did not form lipid particles indicated that accumulation of squalene solely was not sufficient to initiate proliferation of lipid particles. Most importantly, these results also demonstrated that (i) squalene was not lipotoxic under the conditions tested, and (ii) organelle membranes in yeast can accommodate relatively large quantities of this non-polar lipid without compromising cellular functions. In summary, localization of squalene as described here can be regarded as an unconventional example of non-polar lipid storage in cellular membranes.