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The oleaginous yeast Lipomyces starkeyi is an attractive industrial yeast that can accumulate high amounts of intracellular lipids. Identification of genes involved in lipid accumulation contributes not only to elucidating the lipid accumulation mechanism but also to breeding industrially useful high lipid-producing strains. In this study, the suppressed lipid accumulation-related gene (SLA1) was identified as the causative gene of the sr22 mutant with decreased lipid productivity. Suppressed lipid accumulation-related gene mutation reduced gene expression in lipid biosynthesis and increased gene expression in β-oxidation. Our results suggest that SLA1 mutation may leads to decreased lipid productivity. Suppressed lipid accumulation-related gene deletion also exhibited decreased gene expression in β-oxidation and increased lipid accumulation, suggesting that SLA1 deletion is a useful tool to improve lipid accumulation in L. starkeyi for industrialization. [ABSTRACT FROM AUTHOR]

Only a few reports available about the assimilation of hydrophobic or oil-based feedstock as carbon sources by Lipomyces starkeyi. In this study, the ability of L. starkeyi to efficiently utilize free fatty acids (FFAs) and real biomass like palm acid oil (PAO) as well as crude palm kernel oil (CPKO) for growth and lipid production was investigated. PAO, CPKO, and FFAs were evaluated as sole carbon sources or in the mixed medium containing glucose. L. starkeyi was able to grow on the medium supplemented with PAO and FFAs, which contained long-chain length FAs and accumulated lipids up to 35% (w/w) of its dry cell weight. The highest lipid content and lipid concentration were achieved at 50% (w/w) and 10.1 g/L, respectively, when L. starkeyi was cultured in nitrogen-limited mineral medium (-NMM) supplemented with PAO emulsion. Hydrophobic substrate like PAO could be served as promising carbon source for L. starkeyi. • Lipomyces starkeyi could grow on specific types of hydrocarbon or plant-oil-based feedstocks and accumulate lipid. • Palm acid oil (PAO) and long-chain length fatty acids such as C16:0 and C18:1 could be efficiently utilized by L. starkeyi. • L. starkeyi was not able to use carbon sources consisting of C12:0 such as CPKO and sodium laurate. • Addition of Tween 20 as a surfactant to emulsify PAO increases the availability of PAO in the culture medium. [ABSTRACT FROM AUTHOR]

The oleaginous yeast, Lipomyces starkeyi can have diverse industrial applications due to its remarkable capacity to use various carbon sources for the biosynthesis intracellular triacylglycerides (TAGs). In L. starkeyi , TAG synthesis is enhanced through upregulation of genes involved in citrate-mediated acyl-CoA synthesis and Kennedy pathways through the transcriptional regulator LsSpt23p. High expression of LsSPT23 can considerably enhance TAG production. Altering the regulatory factors associated with lipid production can substantially augment lipid productivity. In this study, we identified and examined the L. starkeyi homolog sucrose nonfermenting 1 SNF1 (LsSNF1) of YlSNF1 , which encodes a negative regulator of lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica. The deletion of LsSNF1 enhanced TAG productivity in L. starkeyi , suggesting that LsSnf1p is a negative regulator in TAG production. The enhancement of TAG production following deletion of LsSNF1 can primarily be attributed to the upregulation of genes in the citrate-mediated acyl-CoA synthesis and Kennedy pathways, pivotal routes in TAG biosynthesis. The overexpression of LsSPT23 enhanced lipid productivity; strain overexpressing LsSPT23 and without LsSNF1 exhibited increased TAG production capacity per cell. LsSnf1p also has a significant role in the utilization of carbon sources, including xylose or glycerol, in L. starkeyi. Our study results elucidated the role of LsSnf1p in the negative regulation of TAG synthesis in L. starkeyi , which has not previously been reported. • Lipomyces starkeyi SNF1 homolog (LsSNF1) was identified and the LsSNF1 deletion mutant was constructed. • LsSnf1p had important roles in lipid production and carbon-source utilization in L. starkeyi. • The overexpression of LsSpt23p and without LsSnf1p exhibited increased TAG production capacity per cell. [ABSTRACT FROM AUTHOR]

Recently, microbial oil has become one of the promising next-generation feedstocks for producing biodiesel. While microbial oil can be extracted from different sources, there is only limited work on microbial production from fruits and vegetables. In this work, biodiesel was extracted through a two-step process: microbial conversion of vegetable waste into microbial oil using Lipomyces starkeyi, followed by transesterification of microbial oil into biodiesel. The lipid accumulation, composition of microbial oil, and the fuel properties of biodiesel were evaluated. The microbial oil consisted mainly of C16:0, C18:0 and C18:1, which were close to the properties of palm oil. The fuel properties of biodiesel comply with the EN14214:2012 standard. Thus, the vegetable waste can be a good biodiesel feedstock. Three blends (MOB10, MOB20 and MOB30 with 10, 20, and 30% of biodiesel) were tested for engine performance and emission characteristics in a 3.5 kW VCR research engine. At full load, MOB20 reduced the pollutant emissions of CO and HC by 47.8% and 33.2% with the penalty of increased NOx by 3.9%, while BTE reduced by 0.8% with the increased BSFC by 5.2%. Thus, the addition of vegetable waste biodiesel blends reduced the emissions of CO and HC significantly with slight reduction of brake thermal efficiency. [ABSTRACT FROM AUTHOR]

The clean energy demand and limited fossil fuel reserves require an alternate source that is sustainable and eco-friendly. This demand for clean energy steered the introduction of biofuels such as bioethanol and biodiesel. The third-generation biodiesel is promising as it surpasses the difficulties associated with food security and land usage. The third-generation biodiesel comprises biodiesel derived from oil produced by oleaginous microbes. The term oleaginous refers to microbes with the ability to accumulate lipids to about 20% of the biomass and is found in the form of triacylglycerols. Yeasts can be grown easily on a commercial scale and are amenable to modifications to increase single-cell oil (SCO) productivity. The oleaginous yeast L. starkeyi is a potential lipid producer that can accumulate up to 70% of SCO of its cell dry weight under optimum conditions. Compared to other oleaginous organisms, it can be grown on a wide range of feedstock and a good part of the lipid produced can be converted to biodiesel. This review presents the recent advances in single-cell oil production from L starkeyi and strategies to increase lipid production are analyzed. [ABSTRACT FROM AUTHOR]

Recently, microbial oil has become one of the promising next-generation feedstocks for producing biodiesel. While microbial oil can be extracted from different sources, there is only limited work on microbial production from fruits and vegetables. In this work, biodiesel was extracted through a two-step process: microbial conversion of vegetable waste into microbial oil using Lipomyces starkeyi, followed by transesterification of microbial oil into biodiesel. The lipid accumulation, composition of microbial oil, and the fuel properties of biodiesel were evaluated. The microbial oil consisted mainly of C16:0, C18:0 and C18:1, which were close to the properties of palm oil. The fuel properties of biodiesel comply with the EN14214:2012 standard. Thus, the vegetable waste can be a good biodiesel feedstock. Three blends (MOB10, MOB20 and MOB30 with 10, 20, and 30% of biodiesel) were tested for engine performance and emission characteristics in a 3.5 kW VCR research engine. At full load, MOB20 reduced the pollutant emissions of CO and HC by 47.8% and 33.2% with the penalty of increased NOx by 3.9%, while BTE reduced by 0.8% with the increased BSFC by 5.2%. Thus, the addition of vegetable waste biodiesel blends reduced the emissions of CO and HC significantly with slight reduction of brake thermal efficiency. [ABSTRACT FROM AUTHOR]

Pine needles are relatively less explored lignocellulosic forest waste which can be saccharified to fermentable sugars and be used in subsequent conversion to biofuels. Microbial lipids were produced from pine needles hydrolysate and recovered lipid liquor. Ultrasound assisted pretreatment increased the cellulose content by 20.679%, 27.17%, 32.21%, and 35.61% when treated for 60 min at varying NaOH concentrations (1–4%). The 4-feed batch sachharification was performed to investigate the effect of increasing substrate loadings and fixed initial concentration (5% w/v) and yielded maximum increase of 70.23% in reducing sugars. The pine needle hydrolysate yielded 19.581 g/L total lipids, 35.888 g/L biomass concentration and lipid concentration at 67.46% g/g. Simultaneously, at equimolar ratios of HCl and liquor resulted in highest yields for total lipids (6.92 g/L) and traces of inhibitors. The fatty acids distribution and biodiesel properties revealed a similar biodiesel quality to that of palm oil. Findings from this study facilitate the complete utilisation of pine needles for microbial lipid production to resolve forest fires, and in keeping view of climate, fuels and inorganic resources towards developing a circular bioeconomy. [Display omitted] [ABSTRACT FROM AUTHOR]

The oleaginous yeast Lipomyces starkeyi is a promising triacylglycerol (TAG) producer for biodiesel fuel. However, it is necessary to further improve TAG productivity in L. starkeyi from a mixed sugar of glucose and xylose. This study aimed to construct an L. starkeyi mutant with increased TAG productivity from glucose/xylose mixed-sugar and to elucidate the causes underlying increased lipid productivity. Ultra-violet (UV) mutagenesis combined with enrichment culture with ethanol and H 2 O 2 and selection of low-density cells was applied to L. starkeyi to obtain the L. starkeyi mutant strain UMP47, which exhibited higher TAG production from glucose/xylose. Transcriptome analysis revealed high expression of genes involved in transporter activity and carbohydrate metabolism, whereas genes involved in DNA replication exhibited lower expression in the mutant strain UMP47 than in the wild-type strain. Altogether, the lipid productivity of L. starkeyi was successfully improved by UV mutagenesis. Transcriptome analysis suggested the importance of previously unidentified genes in TAG production. This study provides information on potential target genes for improving TAG production through the genetic modification of oleaginous yeast. • Lipomyces starkeyi is a promising triacylglycerol producer for biodiesel fuel. • UV mutagenesis successfully improved the lipid productivity of L. starkeyi. • UMP47 strain showed increased TAG productivity from glucose/xylose mixed-sugar. • Transport and carbohydrate metabolism exhibited higher expression in the mutant. • DNA replication exhibited lower expression in the mutant. [ABSTRACT FROM AUTHOR]

Microorganisms have a high potential as biofuel sources. Co-culture of microalgae and yeasts can result in high lipid production as a modification treatment. The goal of this study was to see how the co-culture of the Glagah consortium (diversity of associated microalgae and bacteria from Glagah Lagoon, Yogyakarta) and Lipomyces starkeyi affected the production of biomass, lipids, proteins, and carbohydrates. The culture was performed under airtight conditions on a shaker at 127 rpm, with a light intensity of 27.75 mol/m²/s and a temperature of 30°C. The culture was subjected to a dark: light (6:18) treatment. Biomass was measured by dry weight, lipids by the Bligh and Dyer method, proteins by the Bradford method and carbohydrates by the phenol- sulfuric acid method. On day 3, L. starkey culture produced the most biomass, yielding 2.21 g/L with a productivity of 0.49 g/ L/day. On day 4, the highest lipids produced from co-culture treatment yielded 1.03 g/g with a productivity of 0.21 g/L/day. The highest protein yield was obtained from L. starkeyi culture treatment on day 4, yielding 0.60 g/g with a productivity of 0.12 g/L/ day. On day 6, co-culture produced the total carbohydrates, yielding 4.78 g/g with a productivity of 0.68 g/L/day. The co-culture treatment produced the highest lipids and carbohydrates production (1.03 g/g and 4.78 g/g) and productivity (0.21 g/L/day and 0.68 g/L/day), while L. starkeyi culture produced the highest total biomass and protein production (2.21 g/L and 0.6 g/g) and productivity (0.49 g\L\day and 0.12 g/L/day). In microalgae culture, CO2 generally given directly through the aeration process. In this study, the source of CO2 was yeast, whereas yeast also obtained O2 from microalgae in the consortium for their metabolic process. This mutualism symbiosis will help in providing benefits in reducing the costs for the cultivation process, especially in optimizing the production of biomass an lipids. [ABSTRACT FROM AUTHOR]

The oleaginous yeast Lipomyces starkeyi has considerable potential in industrial application, since it can accumulate a large amount of triacylglycerol (TAG), which is produced from sugars under nitrogen limitation condition. However, the regulation of lipogenesis in L. starkeyi has not been investigated in depth. In this study, we compared the genome sequences of wild-type and mutants with increased TAG productivity, and identified a regulatory protein, LsSpt23p, which contributes to the regulation of TAG synthesis in L. starkeyi. L. starkeyi mutants overexpressing LsSPT23 had increased TAG productivity compared with the wild-type strain. Quantitative real-time PCR analysis showed that LsSpt23p upregulated the expression of GPD1, which encodes glycerol 3-phosphate dehydrogenase; the Kennedy pathway genes SCT1, SLC1, PAH1, DGA1, and DGA2; the citrate-mediated acyl-CoA synthesis pathway-related genes ACL1, ACL2, ACC1, FAS1, and FAS2; and OLE1, which encodes ∆9 fatty acid desaturase. Chromatin immunoprecipitation-quantitative PCR assays indicated that LsSpt23p acts as a direct regulator of SLC1 and PAH1, all the citrate-mediated acyl-CoA synthesis pathway–related genes, and OLE1. These results indicate that LsSpt23p regulates TAG synthesis. Phosphatidic acid is a common substrate of phosphatidic acid phosphohydrolase, which is used for TAG synthesis, and phosphatidate cytidylyltransferase 1 for phospholipid synthesis in the Kennedy pathway. LsSpt23p directly regulated PAH1 but did not affect the expression of CDS1, suggesting that the preferred route of carbon is the Pah1p-mediated TAG synthesis pathway under nitrogen limitation condition. The present study contributes to understanding the regulation of TAG synthesis, and will be valuable in future improvement of TAG productivity in oleaginous yeasts. Key points: LsSpt23p was identified as a positive regulator of TAG biosynthesis LsSPT23 overexpression enhanced TAG biosynthesis gene expression and TAG production LsSPT23M1108Toverexpression mutant showed fivefold higher TAG production than control [ABSTRACT FROM AUTHOR]

The purpose of this study is to evaluate the effect of high hydrostatic pressure (HHP) as a novel approach for yeast cell disruption and lipid extraction from Lipomyces starkeyi DSM 70295 grown in glucose medium (40 g/L and C/N:55/1) at initial pH of 5.0, 25°C, and 130 rpm for 8 days. HHP extraction conditions including pressure, time, and temperature were optimized by response surface methodology. The high speed homogenizer‐assisted extraction (HSH) was also used for comparison. The biomass subjected to HHP was examined under scanning electron microscopy and light microscope. A maximal lipid yield of 45.8 ± 2.1% in dry cell basis (w/w) was achieved at 200 MPa, 40°C, and 15 min, while a minimum yield of 15.2 ± 0.9% was observed at 300 MPa, 40°C, and 10 min (p < 0.05). The lipid yield decreased with increasing pressure. It was demonstrated that low pressure (200 MPa) collapsed the cells, while high pressure (400 MPa) created protrusions on the cell wall and cell fragments spread in the environment. This study favors HHP as a promising method for Lipomyces oil extraction. Practical Application: Single‐cell oils are considered future alternatives to plant‐based oils as food additives and dietary supplements. Oleaginous microorganisms accumulate oils in their cell plasma, which makes extraction essential. One of the main obstacles with existing methods is the utilization of strong acids to destroy cell walls. This study aims to demonstrate high hydrostatic pressure as a rapid method for lipid extraction from oleaginous yeast Lipomyces starkeyi. [ABSTRACT FROM AUTHOR]

Oleaginous yeasts have received significant attention due to their substantial lipid storage capability. The accumulated lipids can be utilized directly or processed into various bioproducts and biofuels. Lipomyces starkeyi is an oleaginous yeast capable of using multiple plant-based sugars, such as glucose, xylose, and cellobiose. It is, however, a relatively unexplored yeast due to limited knowledge about its physiology. In this study, we have evaluated the growth of L. starkeyi on different sugars and performed transcriptomic and metabolomic analyses to understand the underlying mechanisms of sugar metabolism. Principal component analysis showed clear differences resulting from growth on different sugars. We have further reported various metabolic pathways activated during growth on these sugars. We also observed non-specific regulation in L. starkeyi and have updated the gene annotations for the NRRL Y-11557 strain. This analysis provides a foundation for understanding the metabolism of these plant-based sugars and potentially valuable information to guide the metabolic engineering of L. starkeyi to produce bioproducts and biofuels. Key points: • L. starkeyi metabolism reprograms for consumption of different plant-based sugars. • Non-specific regulation was observed during growth on cellobiose. • L. starkeyi secretes β-glucosidases for extracellular hydrolysis of cellobiose. [ABSTRACT FROM AUTHOR]

Co-cultures of different microorganisms are considered promising inocula for treating palm oil mill effluents (POME) and producing value-added bio-products (e.g., biofuels and fatty acid-derived materials). However, the efficiency of yeast-bacteria co-culture for microbial lipid production through bioremediation of wastewater remains a bottleneck. In this study, the performance of a co-culture for lipid accumulation through POME bioremediation was investigated using a yeast (Lipomyces starkeyi) and a bacterium (Bacillus cereus). A maximum biomass of 8.89 ± 0.33 g/L and lipid production of 2.27 ± 0.10 g/L were achieved by the co-culture inoculum, which were substantially higher than those of the monocultures. Besides, the co-culture inoculum attained a maximum chemical oxygen demand (COD) removal of 83.66 ± 1.9%, while the individual cultures of B. cereus and L. starkeyi obtained 74.35 ± 1.7% and 69.01 ± 2.3%, respectively. The bioremediation efficiency was confirmed by the seed germination index (GI) of Vigna radiata (Mung bean). It was observed that the co-culture inoculum had a higher GI compared to the untreated POME and even the monoculture-treated POME. We argue that the symbiotic association of a yeast-bacteria co-culture in POME could be an attractive approach for achieving maximum biomass as well as lipid production and simultaneous bioremediation of POME. [Display omitted] [ABSTRACT FROM AUTHOR]

The efficient valorization of lignocellulosic biomass into valuable chemicals represents a cornerstone for advancing sustainable bioeconomy practices. This study focuses on the innovative conversion of oil palm empty fruit bunch (OPEFB) into lignin monomeric alcohols and lipids through a novel two-step process combining alkaline nitrobenzene depolymerization and bioconversion by Lipomyces starkeyi. Initially, OPEFB undergoes alkaline nitrobenzene depolymerization to break down complex lignin structures into monomeric aldehydes. The resulting derivates serve as substrates for the bioconversion by L. starkeyi , which is competent at metabolizing lignin-derived compounds into higher-value lignin monomeric alcohols (syringyl, vanillyl, and 4-hydroxybenzyl alcohol) and lipids, simultaneously. The strategy delineates the optimization of depolymerization conditions, sodium hydroxide concentration, nitrobenzene effect, and reaction time to maximize the yield of depolymerized lignin suitable for bioconversion. Subsequently, the bioconversion process toward cell growth, lipid production, and fatty acid profiles in various fermentations by L. starkeyi was explored. The highest syringaldehyde and vanillin obtained reached up to 4.81 mM and 0.64 mM, respectively. Then, it successfully produced 3.57 mM and 0.40 mM of syringyl alcohol and vanillyl alcohol, respectively, with 5.86 g L−1 of the lipid titer. The study demonstrates the potential of this integrated approach to convert OPEFB into valuable lignin-derived biochemicals and lipids, thereby contributing to the development of alternative strategies for biomass utilization efficiently. [Display omitted] • Lignin monomeric alcohols and lipids were produced from OPEFB. • The two-step utilization process of OPEFB lignin valorization was demonstrated. • Syringaldehyde, vanillin, and 4-hydroxybenzaldehyde were extracted from OPEFB. • Lignin alcohol production and lipid biosynthesis via L. starkeyi were evaluated. • Bioconversion achieved significant amounts of lipid and lignin alcohol production. [ABSTRACT FROM AUTHOR]

Background: Lipomyces starkeyi has been widely regarded as a promising oleaginous yeast with broad industrial application prospects because of its wide substrate spectrum, good adaption to fermentation inhibitors, excellent fatty acid composition for high-quality biodiesel, and negligible lipid remobilization. However, the currently low experimental lipid yield of L. starkeyi prohibits its commercial success. Metabolic model is extremely valuable to comprehend the complex biochemical processes and provide great guidance for strain modification to facilitate the lipid biosynthesis. Results: A small-scale metabolic model of L. starkeyi NRRL Y-11557 was constructed based on the genome annotation information. The theoretical lipid yields of glucose, cellobiose, xylose, glycerol, and acetic acid were calculated according to the flux balance analysis (FBA). The optimal flux distribution of the lipid synthesis showed that pentose phosphate pathway (PPP) independently met the necessity of NADPH for lipid synthesis, resulting in the relatively low lipid yields. Several targets (NADP-dependent oxidoreductases) beneficial for oleaginicity of L. starkeyi with significantly higher theoretical lipid yields were compared and elucidated. The combined utilization of acetic acid and other carbon sources and a hypothetical reverse β-oxidation (RBO) pathway showed outstanding potential for improving the theoretical lipid yield. Conclusions: The lipid biosynthesis potential of L. starkeyi can be significantly improved through appropriate modification of metabolic network, as well as combined utilization of carbon sources according to the metabolic model. The prediction and analysis provide valuable guidance to improve lipid production from various low-cost substrates. [ABSTRACT FROM AUTHOR]

The oleaginous yeast Lipomyces starkeyi is an intriguing lipid producer that can produce triacylglycerol (TAG), a feedstock for biodiesel production. We previously reported that the L. starkeyi mutant E15 with high levels of TAG production compared with the wild-type was efficiently obtained using Percoll density gradient centrifugation. However, considering its use for biodiesel production, it is necessary to further improve the lipid productivity of the mutant. In this study, we aimed to obtain mutants with better lipid productivity than E15, evaluate its lipid productivity, and analyze lipid synthesis-related gene expression in the wild-type and mutant strains. The mutants E15-11, E15-15, and E15-25 exhibiting higher lipid productivity than E15 were efficiently isolated from cells exposed to ultraviolet light using Percoll density gradient centrifugation. They exhibited approximately 4.5-fold higher lipid productivity than the wild-type on day 3. The obtained mutants did not exhibit significantly different fatty acid profiles than the wild-type and E15 mutant strains. E15-11, E15-15, and E15-25 exhibited higher expression of acyl-CoA synthesis- and Kennedy pathway-related genes than the wild-type and E15 mutant strains. Activation of the pentose phosphate pathway, which supplies NADPH, was also observed. These results suggested that the increased expression of acyl-CoA synthesis- and Kennedy pathway-related genes plays a vital role in lipid productivity in the oleaginous yeast L. starkeyi. [ABSTRACT FROM AUTHOR]

Fatty acid desaturases, especially Δ12 fatty acid desaturases, are key enzymes for the production of unsaturated fatty acids in oleaginous yeasts. In this study, we identified and characterized a gene encoding Δ12 fatty acid desaturase of Pseudozyma antarctica named PaFAD2. Almost all oleic acid (C18:1) was converted to linoleic acid by the heterologous expression of the PaFAD2 gene in Saccharomyces cerevisiae and Lipomyces starkeyi oleaginous yeast. Notably, PaFad2 converted not only oleic acid to linoleic acid, but also palmitoleic acid (C16:1) to 9,12-hexadecadienoic acid (C16:2). These results indicated that the PaFAD2 gene was very useful for the production of polyunsaturated fatty acids in yeast, including oleaginous yeast. [ABSTRACT FROM AUTHOR]

The oleaginous yeast Lipomyces starkeyi is an excellent sustainable lipid producer, which can convert industrial wastes into lipids and accumulate triacylglycerols (TAG) by > 70% of its dry cell weight. Recent studies using omics technologies applied in L. starkeyi have aided in obtaining greater understanding of the important mechanisms of lipid metabolism in L. starkeyi. Therefore, the development of genetic engineering tools for L. starkeyi has led to accelerated efforts for a highly efficient production of lipids. This review focuses on the aspects of TAG and fatty acid synthesis pathways in L. starkeyi. We also present a quite effective strategy to obtain L. starkeyi mutants accumulating a larger amount of lipids and having a higher lipid production rate than the wild-type strain. The analysis of these mutants exhibiting high lipid production has led to the identification of important genes for achieving highly effective lipid production and thus advanced improvement in lipid production. Herein, our aim was to provide useful information to advance the development of L. starkeyi as a cost-effective TAG feedstock. Key Points •Oleaginous yeast Lipomyces starkeyi is an excellent sustainable lipid producer. •Efficient isolation of lipid-enriched L. starkeyi mutants depends on the low density of lipids. •Increased acyl-CoA synthesis pathway is important for improving lipid productivity. [ABSTRACT FROM AUTHOR]

The oleaginous yeast Lipomyces starkeyi is a potential cost-effective source for the production of microbial lipids. Fatty acid elongases have vital roles in the syntheses of long-chain fatty acids. In this study, two genes encoding fatty acid elongases of L. starkeyi, LsELO1, and LsELO2 were identified and characterized. Heterologous expression of these genes in Saccharomyces cerevisiae revealed that LsElo1 is involved in the production of saturated long-chain fatty acids with 24 carbon atoms (C24:0) and that LsElo2 is involved in the conversion of C16 fatty acids to C18 fatty acids. In addition, both LsElo1 and LsElo2 were able to elongate polyunsaturated fatty acids. LsElo1 elongated linoleic acid (C18:2) to eicosadienoic acid (C20:2), and LsElo2 elongated α-linolenic acid (C18:3) to eicosatrienoic acid (C20:3). Overexpression of LsElo2 in L. starkeyi caused a reduction in C16 fatty acids, such as palmitic and palmitoleic acids, and an accumulation of C18 fatty acids such as oleic and linoleic acids. Our findings have the potential to contribute to the remodeling of fatty acid composition and the production of polyunsaturated long-chain fatty acids in oleaginous yeasts. [ABSTRACT FROM AUTHOR]

Studies on endo -inulinases from yeast are scarce, compared to those from other microbial sources. In this study, a novel endo -inulinase from Lipomyces starkeyi NRRL Y-11557 was identified, expressed in its soluble form, and characterized its physicochemically properties, together with its enzymatic activity and production of fructooligosaccharides (FOSs). A putative endo -inulinase gene inu3 was identified through rational genome mining. Through enzymatic activity and SDS-PAGE analysis, the endo -inulinase putative function of the protein encoded by inu3B gene (INU3B) was confirmed, and its soluble expression was achieved with pET22b (+) in Escherichia coli. INU3B showed effective catalytic activity and high thermostability. To our knowledge, the specific activity of INU3B against inulin reported in this study, 2262.8 ± 82.3 U·mg−1, at 70 °C and pH 5.0–6.0, is the highest reported to date. When the enzyme catalyzed FOSs production, the main products were DP3, DP4 and DP5. Overall, this report describes a novel yeast-derived endo -inulinase with optimal enzymatic properties, and thus, the reported enzyme has great potential for industrial production of FOSs. [ABSTRACT FROM AUTHOR]

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 (4.7–6.0 g/L) as compared with the wild-type strain (3.6 g/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. [ABSTRACT FROM AUTHOR]

The ability of oleaginous yeast Lipomyces starkeyi to efficiently produce lipids when cultivated on sap extracted from felled oil palm trunk (OPT) as a novel inexpensive renewable carbon source was evaluated. OPT sap was found to contain approximately 98 g/L glucose and 32 g/L fructose. Batch fermentations were performed using three different OPT sap medium conditions: regular sap, enriched sap, and enriched sap at pH 5.0. Under all sap medium conditions, the cell biomass and lipid production achieved were approximately 30 g/L and 60% (w/w), respectively. L. starkeyi tolerated acidified medium (initial pH ≈ 3) and produced considerable amounts of ethanol as well as xylitol as by-products. The fatty acid profile of L. starkeyi was remarkably similar to that of palm oil, one of the most common vegetable oil feedstock used in biodiesel production with oleic acid as the major fatty acid followed by palmitic, stearic and linoleic acids. Image 1 [ABSTRACT FROM AUTHOR]

The objective of this study was to disrupt the non-homologous end-joining (NHEJ) pathway gene (Lsku70Δ) and evaluate the effects of selected gene deletions related to glycogen synthesis (LsGSY1) and lipid degradation (LsMFE1, LsPEX10, and LsTGL4) on lipid production in the oleaginous yeast Lipomyces starkeyi. Disruption of the NHEJ pathway to reduce the rate of non-homologous recombination is a common approach used to overcome low-efficiency targeted deletion or insertion in various organisms. Here, the homologue of the LsKU70 gene was identified and disrupted in L. starkeyi NRRL Y-11558. The LsGSY1, LsMFE1, LsPEX10, LsTGL4, and LsURA3 genes were then replaced with a resistance marker in the Lsku70Δ strain and several site-specific insertions were assessed for targeted over-expression of selected genes. The targeted disruption efficiency of five selected genes (LsGSY1, LsMFE1, LsPEX10, LsTGL4, and LsURA3) was increased from 0 to 10% in the parent to 50-100% of transformants screened in the Lsku70Δ strain with 0.8-1.4 kb homologous flanking sequences, while the efficiency of site-specific gene insertion with the β-glucuronidase reporter gene was 100% in the locus near the 3′-end coding (LsKU70) and non-coding (LsGSY1, LsMFE1, and LsPEX10) regions. Disruption of LsKU70 in isolation and in conjunction with LsGSY1, LsMFE1, LsPEX10, or LsTGL4 did not affect lipid production in L. starkeyi. Furthermore, β-glucuronidase reporter gene activity was similar in strains containing site-specific targeted insertions. Therefore, over-expression of genes related to lipid synthesis at targeted loci can be further examined for improvement of total lipid production in L. starkeyi. [ABSTRACT FROM AUTHOR]

The main objective of this study was to investigate the effects of a low cost minimal medium, developed by the UL Bioprocessing Lab, on the cultivation of Lipomyces starkeyi NRRL Y-11557 using repeated fed-batch and continuous fermentation strategies. The highest cell and lipid concentrations obtained were 22.7 g L-1 and 11.67 g L-1 under repeated fed-batch cultivation, respectively. Continuous cultivation with the dilution rate of 0.06 h-1 presented the highest cell (0.401 g g-1) and lipid yields (0.177g g-1). Exopolysaccharide production was observed when L. starkeyi was cultivated in the minimal media supplemented with 90 g L-1 glucose under repeated fed-batch fermentation. The produced exopolysaccharide is likely composed of 4-5 repeating sugar units, incorporating mannose and galactose and their respective uronic acids. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Oleaginous microbes can convert substrates such as carbon dioxide, sugars, and organic acids to single-cell oils (SCOs). Among the oleaginous microorganisms, Lipomyces starkeyi is a particularly well-suited host given its impressive native abilities, including the capability to utilize a wide variety of carbon sources. In this work, the potential of L. starkeyi NBRC10381 to produce SCOs in a synthetically nitrogen-limited mineral medium (-NMM) was investigated by differing the inoculum size using glucose and/or xylose as a carbon source. Fermentation using glucose and xylose as mixed carbon sources generated the highest production of biomass at 40.8 g/L, and achieved a lipid content of 84.9% (w/w). When either glucose or xylose was used separately, the totals for achieved lipid content were 79.6% (w/w) and 85.1% (w/w), respectively. However, biomass production was higher for glucose than for xylose (30.3 vs. 28.7 g/L, respectively). This study describes the first simultaneous achievement of higher levels of cell mass and lipid production using glucose and/or xylose as the carbon sources in different inoculum sizes. [ABSTRACT FROM AUTHOR]

Lignin carries great potential as an attractive alternative to petroleum-based products. In particular, biological lignin conversion to various value-added products has recently gained attention owing to its promising benefits in supporting the future bioeconomy. The oleaginous yeast Lipomyces starkeyi is a promising organism due to its impressive native abilities to tolerate and metabolize various lignin monomer derivatives and its ability to synthesize single-cell oil (lipids) inside the cells. Herein, we investigated the coupling abilities of lignin monomer metabolism and lipid biosynthesis via L. starkeyi. Three representatives of lignin derivative aldehydes (4-hydroxybenzaldehyde (H-lignin), vanillin (G-lignin), and syringaldehyde (S-lignin)) were converted into their alcohol forms (4-hydroxybenzyl alcohol, vanillyl alcohol, and syringyl alcohol, respectively) without the formation of undesirable byproducts, and each was maintained in its alcohol form for the remaining cultivation. In addition, a double concentration of syringaldehyde in individual lignin fermentation promoted cell growth (17.01 g L−1) and lipid accumulation (30.72 % (w w−1)). However, mixed fermentation of all lignin aldehydes resulted in heavy inhibition of the cells. Hence, combination fermentation strategies of high initial inoculum size using a medium with a high C:N ratio were beneficial to improving the fermentation time and lipid titer increment up to 2.6-fold. Overall, this study describes the first prospective of lignin alcohol and lipid production from lignin derivative aldehydes via L. starkeyi. [Display omitted] • Lignin derivative alcohols were produced from lignin aldehydes by L. starkeyi. • Lignin derivative alcohols metabolism via L. starkeyi was evaluated. • L. starkeyi lipid biosynthesis while fermented under lignin derivative aldehydes. • High inoculum size and high C:N ratio improved fermentation time and lipid titer. [ABSTRACT FROM AUTHOR]

The discharge of palm oil mill effluent (POME) on arable land causes large amounts of environmental distress due to its high concentration of phenolic compounds and chemical oxygen demand (COD). The approach of simultaneous microbial oil production and wastewater treatment is an attractive option to combine renewable energy production and environmental resilience. This study aims to produce cost effective microbial lipids using the oleaginous yeast Lipomyces starkeyi through the bioremediation of POME. A moderately dilute solution (50%) of POME showed higher microbial growth and lipid accumulation and offered a significantly higher degree of bioremediation. A lipid content of 21.32% was achieved with 50% POME, whereas the value was 15.14% for 25% POME. Three different techniques including ultrasonic treatment, Fenton's reagent and Fenton's + ultrasonic were employed to extract lipids from microbial biomass, and the maximum lipid concentration was obtained using the Fenton's + ultrasonic treatment. The degree of bioremediation was evaluated by the calculating seed germination index (GI) values. Higher GI values were observed for the 25% and 50% dilutions compared to undiluted (100%) POME. This combined approach can be a potential alternative technology that integrates bioremediation of POME with microbial lipid production. [ABSTRACT FROM AUTHOR]

The oleaginous yeast Lipomyces starkeyi was engineered for the production of long-chain fatty alcohols by expressing a fatty acyl-CoA reductase, mFAR1, from Mus musculus. The optimal conditions for production of fatty alcohols by this strain were investigated. Increased carbon-to-nitrogen ratios led to efficient C16 and C18 fatty alcohol production from glucose, xylose and glycerol. Batch cultivation resulted in a titer of 1.7 g/L fatty alcohol from glucose which represents a yield of 28 mg of fatty alcohols per gram of glucose. This relatively high level of production with minimal genetic modification indicates that L. starkeyi may be an excellent host for the bioconversion of carbon-rich waste streams, particularly lignocellulosic waste, to C16 and C18 fatty alcohols. [ABSTRACT FROM AUTHOR]

Enhanced single cell oil (SCO) production by the oleaginous yeast Lipomyces starkeyi DSM 70296, immobilised on delignified porous cellulose, is reported. Pure glucose media were initially used. The effects of substrate pH and treatment temperature were evaluated, showing that 30 °C and pH 5.0 were the optimum conditions for SCO production by the immobilised yeast. The immobilisation technique led to increased lipid accumulation and cell growth by 44% and 8%, respectively, in the glucose media, compared to free cells in suspension . This positive effect was also shown when low concentration mixed agro-industrial waste suspensions were used as substrates, leading to 85% enhanced SCO production in comparison with free cells. Higher fatty acid (HFA) analysis showed that yeast immobilisation led to increased formation of unsaturated HFAs (6%) and reduced saturated HFAs (5%) compared to free cells. [ABSTRACT FROM AUTHOR]

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species. [ABSTRACT FROM AUTHOR]

Microbial lipids are sustainable feedstock for the production of oleochemicals and biodiesel. Oleaginous yeasts have recently been proposed as alternative lipid producers to plants and animals to promote sustainability in the chemical and fuel industries. The oleaginous yeast Lipomyces starkeyi has great industrial potential as an excellent lipid producer. However, improvement of its lipid productivity is essential for the cost-effective production of oleochemicals and fuels. Genetic and metabolic engineering of L. starkeyi via gene manipulation techniques may result in improvements in lipid production and our understanding of the mechanisms behind lipid biosynthesis pathways. We previously described an integrative transformation system using a drug-resistant marker for L. starkeyi. However, gene-targeting frequencies were very low because non-homologous recombination is probably predominant in L. starkeyi. Genetic engineering tools for L. starkeyi have not been sufficiently developed. In this study, we describe a new genetic tool and its application in L. starkeyi. To develop a highly efficient gene-targeting system for L. starkeyi, we constructed a series of mutants by disrupting genes for LsKu70p, LsKu80p, and/or LsLig4p, which share homology with other yeasts Ku70p, Ku80p, and Lig4p, respectively, being involved in non-homologous end-joining pathway. Deletion of the LsLIG4 gene dramatically improved the homologous recombination efficiency (80.0%) at the LsURA3 locus compared with that in the wild-type strain (1.4%), when 2000-bp homologous flanking regions were used. The homologous recombination efficiencies of the double mutant ∆ l sku70∆ lslig4 and the triple mutant ∆ lsku70∆ lsku80∆ lslig4 were also markedly enhanced. Therefore, the L. starkeyi ∆ lslig4 background strains have promise as efficient recipient strains for genetic and metabolic engineering approaches in this yeast. [ABSTRACT FROM AUTHOR]

Background: Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. Results: To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Conclusions: Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. The effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well. [ABSTRACT FROM AUTHOR]

The capability of using crude glycerol as the sole carbon source for microbial oil production by Lipomyces starkeyi AS 2.1560 was investigated. The optimal crude glycerol concentration, nitrogen source, C/N ratio, inoculum concentration, culture temperature, and pH for lipid production were 70 g/L, yeast extract + peptone, 60, 10.0%, 30 °C, and 6.0, respectively. Under the optimal condition, the maximum biomass, lipid content, lipid yield, and lipid coefficient were 21.1 g/L, 35.7%, 7.5 g/L, and 17.3%, respectively. Methanol present in the crude glycerol had minor inhibition on the lipid production. Addition of 0.05 g/L PEG-200, 0.1 g/L potassium oleate or sodium stearate into the medium enhanced the lipid production by 6.4%, 7.7% and 10.4%, respectively. Lipid fermentation was further performed in a 5 L fermentor and the biomass, lipid content, and lipid yield after 10 days’ fermentation were 29.2 g/L, 42.9%, and 12.5 g/L, respectively. The corresponding kinetic models for the cell growth, lipid synthesis, and glycerol consumption were built. The maximum specific growth rate and the correlation coefficient of lipid synthesis to cell growth were 0.40 and 0.56, respectively. This study shows that L. starkeyi AS 2.1560 is a promising strain for lipid production on crude glycerol. [ABSTRACT FROM AUTHOR]

The aim of this study was to produce single cell oil (SCO) from Lipomyces starkeyi ATCC 56304 using a biphasic fed-batch fermentation strategy. Fed-batch fermentations were performed to compare oil production using glucose, xylose, mixed-sugars (1:1 ratio of glucose:xylose), and a biphasic system supplying glucose for cell growth and xylose for oil production. The fermentation using xylose as the sole carbon source generated an oil yield of 0.16 g g −1 and a productivity of 0.12 g L −1 h −1 , two-times the oil yield (0.08 g g −1 ) and productivity (0.06 g L −1 h −1 ) achieved with glucose. At 120 h, the biphasic fermentation produced a greater oil productivity (0.13 g L −1 h −1 ) compared to the mixed-sugar fermentation (0.09 g L −1 h −1 ) but similar oil productivities (0.14 g L −1 h −1 ) were achieved for both at longer fermentation times. The biphasic fermentation generated a final oil yield of 0.17 g g −1 and an oil content of 60.1% (w w −1 ), but oil productivity showed minor improvement at 0.14 g L −1 h −1 after 255 h. Oleic acid accounted for at least 65% of the fatty acid content, suggesting that oil from L. starkeyi has potential for application in the biofuel and oleochemical industries. [ABSTRACT FROM AUTHOR]

Olive mill wastewater (OMW) contains a variety of assimilable carbon sources and therefore it can be regarded as fermentation medium for the production of high-added value products rather than as a waste material. In this study, OMW enriched with low cost carbon sources was used as culture medium for selected yeast strains cultivated in flasks and bioreactors. Lipomyces starkeyi NRRL Y- 11557 and Yarrowia lipolytica strains showed a noteworthy ability to accumulate lipids (15–25%, w/w) cultivated on OMW based media. Oleic acid was the major fatty acid in the lipids produced by the above mentioned yeasts followed by palmitic acid. On OMW media enriched with glycerol Y. lipolytica A6 strain produced also mannitol in considerable amounts (i.e. 13.4 g/L), while Y. lipolytica LGAM S (7) strain produced high quantities of citric acid (i.e. 30.3 g/L). Candida tropicalis LFMB 16 and Saccharomyces cerevisiae MAK-1, cultivated under non-aseptic conditions on OMW media enriched with glucose produced 21.9 and 31.3 g/L of ethanol, respectively. Remarkable phenolic removal was performed by strains of Y. lipolytica , C. tropicalis and S. cerevisiae , while color removal was only observed in trials performed with C. tropicalis and S. cerevisiae . The present study provides a new perspective for the production of high-added value metabolites having biotechnological interest from OMW based media along with OMW management. [ABSTRACT FROM AUTHOR]

Background: Current biological pathways to produce biofuel intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels are not cost effective. Previously, oleaginous yeasts have been investigated primarily for lipid production. However, yeasts store neutral lipids intracellularly making recovery difficult and expensive. In addition, once recovered from the cells, lipids are difficult to blend directly with the existing fuels without upgrading. We have, therefore, begun to investigate secreted fatty acid-derived products which can be easily recovered and upgraded to fuels. Results: In this study, we successfully demonstrate the production of fatty alcohols by the oleaginous yeasts, Yarrowia lipolytica and Lipomyces starkeyi, through expression of the fatty acyl-CoA reductase gene from Marinobactor aquaeolei VT8. This strategy resulted in the production of 167 and 770 mg/L of fatty alcohols in shake flask from Y. lipolytica and L starkeyi, respectively. When using a dodecane overlay during fermentation, 92 and 99% of total fatty alcohols produced by Y. lipolytica and L. starkeyi, respectively, were extracted into the dodecane phase, which compares favorably to the 3 and 50% recovered, respectively, without the dodecane layer. In both oleaginous yeasts, long chain length, saturated fatty alcohols, i.e., hexadecanol (C16:0) and octadecanol (C18:0), were predominant and accounted for more than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Conclusion: This work demonstrates that the oleaginous yeasts, Y. lipolytica and L. starkeyi, can serve as platform organisms for the production of fatty acid-derived biofuels and bioproducts. [ABSTRACT FROM AUTHOR]

Background: Microbial lipids can represent a valuable alternative feedstock for biodiesel production in the context of a viable bio-based economy. This production can be driven by cultivating some oleaginous microorganisms on crude-glycerol, a 10 % (w/w) by-product produced during the transesterification process from oils into biodiesel. Despite attractive, the perspective is still economically unsustainable, mainly because impurities in crude glycerol can negatively affect microbial performances. In this view, the selection of the best cell factory, together with the development of a robust and effective production process are primary requirements. Results: The present work compared crude versus pure glycerol as carbon sources for lipid production by three different oleaginous yeasts: Rhodosporidium toruloides (DSM 4444), Lipomyces starkeyi (DSM 70295) and Cryptococcus curvatus (DSM 70022). An efficient yet simple feeding strategy for avoiding the lag phase caused by growth on crude glycerol was developed, leading to high biomass and lipid production for all the tested yeasts. Flow-cytometry and fourier transform infrared (FTIR) microspectroscopy, supported by principal component analysis (PCA), were used as non-invasive and quick techniques to monitor, compare and analyze the lipid production over time. Gas chromatography (GC) analysis completed the quali-quantitative description. Under these operative conditions, the highest lipid content (up to 60.9 % wt/wt) was measured in R. toruloides, while L. starkeyi showed the fastest glycerol consumption rate (1.05 g L-1 h-1). Being productivity the most industrially relevant feature to be pursued, under the presented optimized conditions R. toruloides showed the best lipid productivity (0.13 and 0.15 g L-1 h-1 on pure and crude glycerol, respectively). Conclusions: Here we demonstrated that the development of an efficient feeding strategy is sufficient in preventing the inhibitory effect of crude glycerol, and robust enough to ensure high lipid accumulation by three different oleaginous yeasts. Single cell and in situ analyses allowed depicting and comparing the transition between growth and lipid accumulation occurring differently for the three different yeasts. These data provide novel information that can be exploited for screening the best cell factory, moving towards a sustainable microbial biodiesel production. [ABSTRACT FROM AUTHOR]

Single cell oils (SCOs) are considered potential raw material for the production of biodiesel. Rhodosporidium sp. and Lipomyces sp. are good candidates for SCO production. Lipid extractability differs according to yeast species and literature on the most suitable method for each oleaginous yeast species is scarce. This work aimed to investigate the efficiency of the most cited strategies for extracting lipids from intact and pretreated cells of Rhodosporidium toruloides and Lipomyces starkeyi. Lipid extractions were conducted using hexane or combinations of chloroform and methanol. The Folch method resulted in the highest lipid yields for both yeasts (42% for R. toruloides and 48% for L. starkeyi). Also, this method eliminates the cell pretreatment step. The Bligh and Dyer method underestimated the lipid content in the tested strains (25% for R. toruloides and 34% for L. starkeyi). Lipid extractability increased after acid pretreatment for the Pedersen, hexane, and Bligh and Dyer methods. For R. toruloides unexpected fatty acid methyl esters (FAME) composition were found for some lipid extraction strategies tested. Therefore, this work provides useful information for analytical and process development aiming at biodiesel production from the SCO of these two yeast species. [ABSTRACT FROM AUTHOR]

Aims To screen yeasts in relation to the potential to produce single cell oil ( SCO) from biodiesel-derived glycerol and to enhance SCO production in Lipomyces starkeyi and Rhodosporidium toruloides yeasts. Methods and Results Yarrowia lipolytica, Cryptococcus curvatus, R. toruloides and L. starkeyi were grown in nitrogen-limited flask cultures. Yarrowia lipolytica strains produced citric acid and mannitol. Lipomyces starkeyi DSM 70296 and R. toruloides NRRL Y-27012 showed potential for SCO production, and were cultivated at increasing the initial glycerol concentrations with the initial nitrogen concentration remaining constant. Significant biomass and SCO production were reported even in cultures with high initial glycerol concentrations (i.e. 180 g l−1). Lipid quantities of c. 12 g l−1 (lipid in dry cell weight 35-40%) were obtained for both L. starkeyi and R. toruloides, quite high values compared with literature values for oleaginous micro-organisms growing on glycerol. However, these strains exhibited different kinetic profiles in the synthesis of intracellular polysaccharides. Lipomyces starkeyi produced a significant quantity of polysaccharides ( c. 7 g l−1). The yeast lipids contained mainly oleic and palmitic and to a lesser extent linoleic and stearic acids. Conclusions Lipomyces starkeyi and R. toruloides are potential SCO producers from crude glycerol. Significance and Impact of the Study Very scarce numbers of reports have indicated the production of SCO by L. starkeyi and R. toruloides growing on glycerol. We report here that these yeasts are able efficiently to convert raw glycerol into SCO, while L. starkeyi also synthesizes intracellular polysaccharides in marked quantities. [ABSTRACT FROM AUTHOR]

[Display omitted] • Combination of SSF of corn stover with CBP of cassava starch was firstly developed. • The combination strategy showed synergistic effect for improving lipid production. • Rapid hydrolysis of starch into fermentable sugars eliminated a pre-hydrolysis step. • The enzyme cost could be significantly decreased via the combination strategy. • Biodiesel from the combination strategy showed high-quality fuel properties. Highly integrated processes are crucial for the commercial success of microbial lipid production from low-cost substrates. Here, combination of simultaneous saccharification and fermentation (SSF) of corn stover with consolidated bioprocessing (CBP) of cassava starch by Lipomyces starkeyi was firstly developed as a novel strategy for lipid production. Starch was quickly hydrolyzed within 24 h by the amylolytic enzymes secreted by L. starkeyi to provide adequate fermentable sugars at the initial stage of culture, which eliminated the pre-hydrolysis step. More interestingly, synergistic effect for achieving higher lipid production by combined utilization of corn stover and cassava starch at relatively low enzyme dosage was realized, in comparison with the separate utilization of these two substrates. The fatty acid profiles indicated that lipid prepared by the combination strategy was suitable precursor for biodiesel production. The combined SSF&CBP strategy offers a simplified, highly-efficient, and economical route for co-valorization of low-cost substrates into lipids. [ABSTRACT FROM AUTHOR]

The article discusses a study related to integration and expression of heterologous genes in the oleaginous yeast, Lipomyces starkeyi. Topics discussed include development of a system for targeted recombinant DNA integration of multiple copies of a gene into Lipomyces starkeyi genome using spheroplast-polyethylene glycol transformation, importance of Lipomyces starkeyi as an excellent lipid producer and need for the establishment of methods for molecular breeding for Lipomyces starkeyi.

The ability of the oleaginous yeast Lipomyces starkeyi to efficiently produce lipids when cultivated on saccharified sweet sorghum stalks juice was evaluated. Initially the production of lipids using synthetic media mimicking sweet sorghum stalks has been studied and optimized concerning the nitrogen source and the C:N ratio. Under optimum conditions (yeast extract as nitrogen source and C:N ratio of 190) the lipid production reached 5.81 g/L with a lipid content of 47.3% (w/w) from a mixture of sucrose, glucose and fructose, mimicking the sugar composition of sorghum. When cultivated on sweet sorghum stalks juice, it was observed that no external nitrogen addition was necessary which could result in substantial decrease of the initial C:N ratio. Moreover a distinct saccharification process prior to yeast cultivation improved the lipid production yield as it resulted in an increase of the C:N ratio. The highest lipid production, which was 6.40 g/L with a lipid content of 29.5% (w/w), was obtained when juice from saccharified sweet sorghum stalks at an initial sorghum content of 12% (w/w) was used as feedstock. [ABSTRACT FROM AUTHOR]

Flour-rich waste (FRW) and by-product streams generated by bakery, confectionery and wheat milling plants could be employed as the sole raw materials for generic fermentation media production, suitable for microbial oil synthesis. Wheat milling by-products were used in solid state fermentations (SSF) of Aspergillus awamori for the production of crude enzymes, mainly glucoamylase and protease. Enzyme-rich SSF solids were subsequently employed for hydrolysis of FRW streams into nutrient-rich fermentation media. Batch hydrolytic experiments using FRW concentrations up to 205 g/L resulted in higher than 90% (w/w) starch to glucose conversion yields and 40% (w/w) total Kjeldahl nitrogen to free amino nitrogen conversion yields. Starch to glucose conversion yields of 98.2, 86.1 and 73.4% (w/w) were achieved when initial FRW concentrations of 235, 300 and 350 g/L were employed in fed-batch hydrolytic experiments, respectively. Crude hydrolysates were used as fermentation media in shake flask cultures with the oleaginous yeast Lipomyces starkeyi DSM 70296 reaching a total dry weight of 30.5 g/L with a microbial oil content of 40.4% (w/w), higher than that achieved in synthetic media. Fed-batch bioreactor cultures led to a total dry weight of 109.8 g/L with a microbial oil content of 57.8% (w/w) and productivity of 0.4 g/L/h. [ABSTRACT FROM AUTHOR]

Producing microbial oils via oleaginous yeast fermentation has drawn broad attention in the biodiesel industry. The oleaginous yeast Lipomyces starkeyi utilizing diverse carbon sources including glucose, xylose, glycerol, and willow wood sawdust (WWS) hydrolysate for the biosynthesis of oils in its cell growth were investigated in this study. High carbon/nitrogen ratios within the glucose media significantly increased the lipid content of Lipomyces starkeyi cells and modified the fatty acid composition of lipids, promoting the accumulation of C16:0 fatty acids and saturated fatty acids (C16:0 and C18:0). The accumulation of C18 fatty acids (C18:0, C18:1, and C18:2) and unsaturated fatty acids (C16:1, C18:1, and C18:2) was restricted. When crude glycerol and WWS hydrolysate were used as the sole carbon sources for L. starkeyi fermentation, the dry cell weight, lipid content, and lipid productivity were 9.1 g/L, 46.2%, and 4.2 g/L, respectively, for glycerol, and 8.2 g/L, 42.7%, and 3.5 g/L, respectively, for the hydrolysate solution. This study provides useful information for producing oils with L. starkeyi fermentation using glycerol and WWS hydrolysate as the primary or secondary carbon substrates. [ABSTRACT FROM AUTHOR]

We report the development of an efficient genetic transformation system for Lipomyces starkeyi based on a modified lithium acetate transformation protocol. L. starkeyi is a highly lipogenic yeast that grows on a wide range of substrates. The initial transformation rate for this species was extremely low, and required very high concentrations of DNA. A systematic approach for optimizing the protocol resulted in an increase in the transformation efficiency by four orders of magnitude. Important parameters included cell density, the duration of incubation and recovery periods, the heat shock temperature, and the concentration of lithium acetate and carrier DNA within the transformation mixture. We have achieved efficiencies in excess of 8,000 transformants/µg DNA, which now make it possible to screen libraries in the metabolic engineering of this yeast. Metabolic engineering based on this transformation system could improve lipogenesis and enable formation of higher value products. [ABSTRACT FROM AUTHOR]

Highlights: [•] Simultaneous utilization of glucose and mannose for lipid production by L. starkeyi. [•] Glucose and mannose as major sugars present in spent cell mass of R. toruloides. [•] Efficient lipid production on the hydrolysates of spent yeast cell mass. [•] The hydrolysates of spent yeast cell mass as both carbon and nitrogen sources. [ABSTRACT FROM AUTHOR]

In this paper, potato starch wastewater as culture medium was treated by the oleaginous yeast Lipomyces starkeyi to biosynthesize microbial lipid. The result indicated that carbon source types, carbon source concentration, nitrogen source types, nitrogen source concentration, inoculum size, end cultivation time all had a significant effect on cell growth and microbial lipid accumulation in batch cultures. A measure of 120 g/L of glucose concentration, 3.0 g/L of (NH4)2SO4 concentration, 10% inoculum size, and incubation time 96 h cultivated in a shaking flask at 30 °C were found to be the optimal conditions not only for cell growth but also for lipid synthesis. Under this condition, the cellular biomass and lipid content could reach 2.59 g/L and 8.88%, respectively. This work provides a new method for effective utilization of potato starch wastewater, which has particular social and economic benefits for yeast treatment technology. [ABSTRACT FROM AUTHOR]

Abstract: Hydrolysates of lignocellulosic biomass contain glucose, xylose, arabinose, cellobiose, among other sugars. Effective utilization of these sugars remains challenging for microbial conversion, because most microorganisms consume such sugars sequentially with a strong preference for glucose. In the present study, the oleaginous yeast, Lipomyces starkeyi, was shown to consume cellobiose and xylose simultaneously and to produce intracellular lipids from cellobiose, xylose and glucose. In flask cultures with glucose, cellobiose or a mixture of cellobiose/xylose as carbon sources, overall substrate consumption rates were close to 0.6g/L/h, and lipid coefficients were 0.19g lipid/g sugar, respectively. This cellobiose/xylose co-fermentation strategy provides an opportunity to efficiently utilize lignocellulosic biomass for microbial lipid production, which is important for biorefinery and biofuel production. [Copyright &y& Elsevier]

Abstract: The monosodium glutamate (MSG) wastewater as a medium was treated by Lipomyces starkeyi to produce microbial lipid in the study. The effect of related factors (initial glucose concentration, inoculation concentration, initial culture pH, and cultivation time) on biomass, lipid production and lipid content was discussed, respectively. According to the experiments, the optimal fermentation conditions were determined: addition of 80g/L glucose, 10% inoculation concentration, initial pH about 5.0, incubation time 96h. Under this condition, the biomass production reached up to 4.61g/L, lipid production and lipid content was 1.14g/L and 24.73%, respectively. Simultaneously, protein and COD removal rate was 78.60% and 74.96%, respectively. The main composition of fatty acid in the resultant lipid was analyzed by gas chromatography–mass spectrometry, which showed: oleic acid (C18:1) 35.85%, palmitic acid (C16:0) 19.91%, palmitoleic acid (C16:1) 17.65%, and myristic acid (C14:0) 16.03%. [Copyright &y& Elsevier]