The proteome analysis of oleaginous yeast Lipomyces starkeyi

Oleaginous yeast Lipomyces starkeyi, a species in the Saccharomycetales order, has the capability to accumulate over 70% of its cell biomass as lipid under defined culture conditions. In this study, analysis of L. starkeyi AS 2.1560 proteome samples from different culture stages during a typical lipid production process was performed using an online multidimensional μRPLC/MS/MS method. Data searching against the proteome database of the yeast Saccharomyces cerevisiae led to the identification of 289 protein hits. Further comparative and semi-quantitative analysis under more stringent criteria revealed 81 proteins with significant expression-level changes. Among them, 52 proteins were upregulated and 29 proteins were downregulated. Gene ontology annotation indicated that global responses occurred when cells were exposed to the nitrogen deficiency environment for lipid production. Protein hits were annotated and largely concerned metabolic processes for alternative nitrogen sources usage or lipid accumulation. Many of the downregulated proteins were related to glycolysis, whereas the majority of the upregulated proteins were involved in proteolysis and peptidolysis, carbohydrate metabolism and lipid metabolism. Insights were provided in terms of cellular responses to nutrient availability as well as the basic biochemistry of lipid accumulation. This work presented potentially valuable information for understanding the biochemical events related to microbial oleaginity and rational engineering of oleaginous yeasts.     

Evaluation of green solvents: Oil extraction from oleaginous yeast Lipomyces starkeyi using cyclopentyl methyl ether (CPME)

Cyclopentyl methyl ether (CPME) was evaluated for extracting oil or triacylglycerol (TAG) from wet cells of the oleaginous yeast Lipomyces starkeyi. CPME is a greener alternative to chloroform as a potential solvent for oil recovery. A monophasic system of CPME and biphasic system of CPME:water (1:0.7) performed poorly having the lowest TAG extraction efficiency and TAG selectivity compared to other monophasic systems of hexane and chloroform and the biphasic Bligh and Dyer method (chloroform:methanol:water). Biphasic systems of CPME:water:alcohol (methanol/ethanol/1‐propanol) were tested and methanol achieved the best oil extraction efficiency compared to ethanol and 1‐propanol. Different biphasic systems of CPME:methanol:water were tested, the best TAG extraction efficiency and TAG selectivity achieved was 9.9 mg/mL and 64.6%, respectively, using a starting ratio of 1:1.7:0.6 and a final ratio of 1:1:0.8 (CPME:methanol:water). Similar results were achieved for the Bligh and Dyer method (TAG extraction efficiency of 10.2 mg/mL and TAG selectivity of 66.0%) indicating that the biphasic CPME system was comparable. The fatty acid profile remained constant across all the solvent systems tested indicating that choice of solvent was not specific for any certain fatty acid. This study was able to demonstrate that CPME could be used as an alternative solvent for the extraction of oil from the wet biomass of oleaginous yeast. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1096–1103, 2017     

Phylogenetic and biochemical characterization of the oil-producing yeast Lipomyces starkeyi

Lipomyces starkeyi is an oleaginous yeast, and has been classified in four distinct groups, i.e., sensu stricto and custers α, β, and γ. Recently, L. starkeyi clusters α, β, and γ were recognized independent species, Lipomyces mesembrius, Lipomyces doorenjongii, and Lipomyces kockii, respectively. In this study, we investigated phylogenetic relationships within L. starkeyi, including 18 Japanese wild strains, and its related species, based on internal transcribed spacer sequences and evaluated biochemical characters which reflected the phylogenetic tree. Phylogenetic analysis showed that most of Japanese wild strains formed one clade and this clade is more closely related to L. starkeyi s.s. clade including one Japanese wild strain than other clades. Only three Japanese wild strains were genetically distinct from L. starkeyi. Lipomyces mesembrius and L. doorenjongii shared one clade, while L. kockii was genetically distinct from the other three species. Strains in L. starkeyi s.s. clade converted six sugars, d-glucose, d-xylose, l-arabinose, d-galactose, d-mannose, and d-cellobiose to produce high total lipid yields. The Japanese wild strains in subclades B, C, and D converted d-glucose, d-galactose, and d-mannose to produce high total lipid yields. Lipomyces mesembrius was divided into two subclades. Lipomyces mesembrius CBS 7737 converted d-xylose, l-arabinose, d-galactose, and d-cellobiose, while the other L. mesembrius strains did not. Lipomyces doorenjongii converted all the sugars except d-cellobiose. In comparison to L. starkeyi, L. mesembrius, and L. doorenjongii, L. kockii produced higher total lipid yields from d-glucose, d-galactose, and d-mannose. The type of sugar converted depended on the subclade classification elucidated in this study.     

Induction of Lipomyces starkeyi Dextranase

Lipomyces starkeyi ATCC 20825 is a derepressed mutant derived from L. starkeyi ATCC 12659. It requires the presence of an inducer before it produces dextranase. This study was undertaken to determine the most efficient, commercially feasible method for inducing this enzyme. The following compounds induced dextranase synthesis: 1-O-β-methyl-glucopyranoside, 1-O-α-methyl-glucopyranoside, dextran, isomaltopentose, isomaltotetraose, isomaltotriose, and isomaltose. 1-O-β-Methyl-glucopyranoside was found to be a gratuitous inducer. Early in the growth phase, cells produced higher specific levels of enzyme than they did in late log phase. The length of exposure of the yeast cells to the inducer also affected the amount of dextranase produced. The maximum amount of enzyme was produced after 12 h of exposure to the inducer. The saturation concentration was the same for all inducers tested, i.e., approximately 1 mg of inducer for every 2 × 10⁸ cells.     

The purification and characterization of a dextranase from Lipomyces starkeyi

Dextranase produced by Lipomyces starkeyi was purified 43-fold, by carboxymethyl-Sepharose chromatography followed by agarose gel-filtration chromatography. The purified enzyme showed four bands by SDS/polyacrylamide gel electrophoresis with estimated mass 74 kDa, 71 kDa, 68 kDa and 65 kDa. This preparation exhibited multiple isoelectric points between 5.6 and 6.1. All the isoelectric forms were active and catalytically similar. The dextranase contained a carbohydrate moiety (8%). The physical properties of the enzyme were pH and temperature optima of 5.0 and 55 degrees C, respectively. This dextranase was stable between pH 2.5 and 7.0 at temperatures below 40 degrees C. Lipomyces dextranase was a typical endodextranase with the final product of dextran hydrolysis being isomalto-oligosaccharides from glucose to isomaltotetrose.     

Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates

The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.     

Amylolytic enzymes of Lipomyces starkeyi: purification and size-determination

-Amylase accumulated in the growth medium of the yeast Lipomyces starkeyi (NCYC 1436) to a maximum of 12 units ml^–1 after 15 days' batch culture in a soluble starch medium. Extra -amylase activity, about 50% of that released into the medium, could be extracted from the cells by a salt wash. -Amylase was purified by salt precipitation followed by ion exchange chromatography and gel filtration. Native gel electrophoretic analysis showed the presence of three distinct amylolytic enzymes, with molecular weights of approximately 50, 70 and 80 kDa.     

Microbial lipid extraction from Lipomyces starkeyi using irreversible electroporation

The aim of the study was to investigate the feasibility of using irreversible electroporation (EP) as a microbial cell disruption technique to extract intracellular lipid within short time and in an eco‐friendly manner. An EP circuit was designed and fabricated to obtain 4 kV with frequency of 100 Hz of square waves. The yeast cells of Lipomyces starkeyi (L. starkeyi) were treated by EP for 2‐10 min where the distance between electrodes was maintained at 2, 4, and 6 cm. Colony forming units (CFU) were counted to observe the cell viability under the high voltage electric field. The forces of the pulsing electric field caused significant damage to the cell wall of L. starkeyi and the disruption of microbial cells was visualized by field emission scanning electron microscopic (FESEM) image. After breaking the cell wall, lipid was extracted and measured to assess the efficiency of EP over other techniques. The extent of cell inactivation was up to 95% when the electrodes were placed at the distance of 2 cm, which provided high treatment intensity (36.7 kWh m^?3). At this condition, maximum lipid (63 mg g^?1) was extracted when the biomass was treated for 10 min. During the comparison, EP could extract 31.88% lipid while the amount was 11.89% for ultrasonic and 16.8% for Fenton's reagent. The results recommend that the EP is a promising technique for lowering the time and solvent usage for lipid extraction from microbial biomass. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:838–845, 2018     

Lipid Molecular Species of Lipomyces starkeyi

The molecular species of glycerides and phospholipids of the yeast Lipomyces starkeyi IFO 0678 harvested at 60 hr, corresponding to the late exponential phase, were analyzed by gas chromatography-mass spectrometry. The major triglyceride was C_16:0–C_18:1–C_18:1. The major molecular species of phospholipid were 1–C_16:0–2–C_18:1 and 1–C_18:1–2–C_18:2. Although phosphatidylcholine and phosphatidylethanol amine were composed of several kinds of molecular species, 1–C_16:1–2–C_18:1, 1–C_18:1–2–C_18:2 and l-C_16:0–2–C_18:1, phosphatidylserine was composed of almost exclusively 1–C_16:0-2-C_18:1. The lipid and the fatty acid compositions of the yeast harvested at the different growth phases were also investigated.     

色醇对斯达氏油脂酵母产油能力的影响

研究在发酵培养基中添加色醇对斯达氏油脂酵母发酵的影响。结果表明, 在接种后0 h或12 h时添加色醇, 能够明显抑制菌体生长和油脂积累; 而在培养24 h或36 h添加, 能够明显促进菌体生长, 并增强菌体对底物利用率。与对照组比较, 在36 h添加100 mmol/L色醇, 生物量、油脂量和脂肪系数分别增加7.4%、13.9%和14.2%, 发酵时间缩短13.3%, 明显提高了油脂生产效率。气相色谱分析表明, 添加色醇对菌油脂肪酸组成及其相对含量无显著影响。实验结果有助于建立调控油脂发酵的新策略, 具有重要的理论和工程应用意义。     

Characterization of oil-producing yeast Lipomyces starkeyi on glycerol carbon source based on metabolomics and 13C-labeling

Lipomyces starkeyi is an oil-producing yeast that can produce triacylglycerol (TAG) from glycerol as a carbon source. The TAG was mainly produced after nitrogen depletion alongside reduced cell proliferation. To obtain clues for enhancing the TAG production, cell metabolism during the TAG-producing phase was characterized by metabolomics with ¹³C labeling. The turnover analysis showed that the time constants of intermediates from glycerol to pyruvate (Pyr) were large, whereas those of tricarboxylic acid (TCA) cycle intermediates were much smaller than that of Pyr. Surprisingly, the time constants of intermediates in gluconeogenesis and the pentose phosphate (PP) pathway were large, suggesting that a large amount of the uptaken glycerol was metabolized via the PP pathway. To synthesize fatty acids that make up TAG from acetyl-CoA (AcCoA), 14 molecules of nicotinamide adenine dinucleotide phosphate (NADPH) per C16 fatty acid molecule are required. Because the oxidative PP pathway generates NADPH, this pathway would contribute to supply NADPH for fatty acid synthesis. To confirm that the oxidative PP pathway can supply the NADPH required for TAG production, flux analysis was conducted based on the measured specific rates and mass balances. Flux analysis revealed that the NADPH necessary for TAG production was supplied by metabolizing 48.2% of the uptaken glycerol through gluconeogenesis and the PP pathway. This result was consistent with the result of the ¹³C-labeling experiment. Furthermore, comparison of the actual flux distribution with the ideal flux distribution for TAG production suggested that it is necessary to flow more dihydroxyacetonephosphate (DHAP) through gluconeogenesis to improve TAG yield.

     

Biodegradation of triazine herbicides on polyvinylalcohol gel plates by the soil yeast Lipomyces starkeyi

The soil yeast Lipomyces starkeyi was tested for its ability to degrade triazine herbicides. Polyvinylalcohol (PVA) was employed as a solid medium in culture plates instead of agar. The cell sizes of the control (without nitrogen source) on the PVA gel plate were much smaller than those on the agar gel plate. The difference between the diameters of the sample and control colonies on the PVA gel plate were almost twice those of the colonies on the agar gel plate (1.9 and 1.0 mm, respectively). Thus, the PVA gel plate is much better than the agar plate for evaluating the degree of utilization of a sole nitrogen source. The yeast grew well (more than 4 mm in diameter) with 1,3,5-triazine or cyanuric acid as nitrogen source. In addition, melamine and thiocyanuric acid inhibited growth of the yeast, and the sizes of colonies were smaller than those of the control. All triazine herbicides tested (simazine, atrazine, cyanazine, ametryn, and prometryn) could be degraded and assimilated by L. starkeyi.     

色醇对斯达氏油脂酵母产油能力的影响

研究在发酵培养基中添加色醇对斯达氏油脂酵母发酵的影响.结果表明,在接种后0 h或12 h时添加色醇,能够明显抑制菌体生长和油脂积累;而在培养24 h或36 h添加,能够明显促进菌体生长,并增强菌体对底物利用率.与对照组比较,在36 h添加100 μmol/L色醇,生物量、油脂量和脂肪系数分别增加7.4%、13.9%和14.2%,发酵时间缩短13.3%,明显提高了油脂生产效率.气相色谱分析表明,添加色醇对菌油脂肪酸组成及其相对含量无显著影响.实验结果有助于建立调控油脂发酵的新策略,具有重要的理论和工程应用意义.     

Mechanism of biodegradation of paraquat by Lipomyces starkeyi

The biodegradation of ring-14C- and methyl-14C-labeled paraquat by the soil yeast Lipomyces starkeyi was studied in vitro. It was found that the degradation of paraquat (acting as a sole source of culture nitrogen) resulted in the accumulation in the extracellular medium of radiolabeled acetic acid. The culture also evolved radiolabeled CO2. The results suggest that the degradation of paraquat by L. starkeyi is associated with the integrity of the cell wall and that disruption or removal of the wall results in a complete loss of degradative capability. A mechanism for the degradation of paraquat by this organism is postulated.     

Dextran production by Leuconostoc mesenteroides in the presence of a dextranase producing yeast, Lipomyces starkeyi

Summary A new process for the production of small size dextran is developed in which dextran is produced by cultures of Leuconostoc mesenteroides in the presence of a partially constitutive mutant of Lipomyces starkeyi producing dextranase. Mixed cultures were examined by scanning electron microscopy with ruthenium to show the effects of the mixed culture on low molecular weight dextran (M.W. of 5,000 – 100,000) formation. The presence of the size variation in dextran was confirmed by gel permeation chromatography.     

Structures of Hexadecenoic Acid and Octadecenoic Acid in Lipomyces starkeyi

Taxonomic and some other properties of a yeast strain, Candida sp. 36, which characteristically assimilates n-alkanes, were described. Identification of coenzyme Q, NMR spectroscopy of cell wall polysaccharides, determination of G+C content of DNA and some DNA-DNA hybridization experiments were carried out, in addition to the morphological and physiological observations. All the data were consistent with the suggestion that Candida cloacae Komagata, Nakase and Katsuya and Candida subtropicalis Nakase, Fukazawa and Tsuchiya are the synonyms of Candida maltosa Komagata, Nakase and Katsuya. Candida sp. 36 was identified as C. maltosa, too. The yeast was found to grow most abundantly on n-hexadecane and on n-octadecane in the presence of biotin.     

Mechanism of biodegradation of paraquat by Lipomyces starkeyi.

The biodegradation of ring-14C- and methyl-14C-labeled paraquat by the soil yeast Lipomyces starkeyi was studied in vitro. It was found that the degradation of paraquat (acting as a sole source of culture nitrogen) resulted in the accumulation in the extracellular medium of radiolabeled acetic acid. The culture also evolved radiolabeled CO2. The results suggest that the degradation of paraquat by L. starkeyi is associated with the integrity of the cell wall and that disruption or removal of the wall results in a complete loss of degradative capability. A mechanism for the degradation of paraquat by this organism is postulated.     

Optimization of exopolysaccharide production by probiotic yeast Lipomyces starkeyi VIT-MN03 using response surface methodology and its applications

In the present study, the cultural conditions for exopolysaccharide (EPS) production from probiotic yeast Lipomyces starkeyi VIT-MN03 were optimized using response surface methodology (RSM) to maximize the yield of EPS. Interactions among the various factors viz. sucrose concentration (1–3 g%), NaCl concentration (2–4 g%), pH (3–5), temperature (20–30 °C), and incubation period (20–40 days) during EPS production were studied using Box-Behnken design (BBD). The EPS was purified and characterized using various instrumental analyses. The properties like adhesion, antioxidant, biosurfactant, cholesterol removal, and binding ability to mutagens were also tested for EPS produced. Sixfold increase in EPS production (4.87 g L−1) by L. starkeyi VIT-MN03 was noted under optimized condition. EPS showed a high viscosity (1.8 Pa S−1) and good shear-thinning properties. Instrumental analysis showed that EPS was heteropolysaccharide composed of glucan, mannan, and rhamnan. Lipomyces starkeyi VIT-MN03 exhibited good self-adhesion (95%) and co-aggregation ability (93%). Adhesion efficiency for yeast inoculum containing 5.5 × 107 CFU mL−1 per 9.2 cm2 of Caco-2 cell (colorectal adenocarcinoma) was noted. The probiotic EPS displayed strong antioxidant ability to scavenge hydroxyl radical and DPPH by 58% and 71% respectively. In addition, biosurfactant activity (86%) and cholesterol removal (90%) ability of probiotic EPS was also tested. EPS bound cells of L. starkeyi VIT-MN03 showed good binding ability to mutagens. These results support the effectiveness of using RSM for maximum EPS production. To the best of our knowledge, this is the first report on optimization of EPS production by probiotic yeast.