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Palm oil, widely used in various products, poses environmental and climate change risks. "Yeast oil" produced by Lipomyces starkeyi, an oil-producing yeast, is one of the sustainable alternatives for palm oil and was successfully produced as an edible substitute for palm oil. However, the high cost of the culture medium for oil production remains a challenge for practical applications. Okara is a by-product of tofu and soymilk production. Because yeast extract contributes to the high cost of the culture medium, we considered using okara, a cheap and nitrogen-rich substitute, to reduce costs. In the initial study with okara, the production of yeast oil was confirmed, but its productivity was low due to the high viscosity caused by its insoluble solids. To overcome this, we extracted and concentrated nitrogen components in okara using the membrane concentration process. Using NF (nanofiltration) membrane concentration, oil production increased 1.69 and 1.44 times compared to the unconcentrated extract solution (added 90% (v/v) in the culture medium) and yeast extract (added 5% (w/v) in the culture medium), respectively. These findings indicate the potential for a significant cost reduction in the culture medium and high oil yield in yeast oil production. [ABSTRACT FROM AUTHOR]

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]

ABSTRACT The oleaginous yeast Lipomyces starkeyi is recognized for its remarkable lipid accumulation under nitrogen‐limited conditions. However, precise control of microbial lipid production in L. starkeyi remains challenging due to the complexity of nutrient media. We developed a two‐stage fed‐batch fermentation process using a well‐defined synthetic medium in a 5‐L bioreactor. In the first stage, the specific growth rate was maintained at a designated level by maximizing the cell density through optimizing the feeding rate, molar carbon‐to‐nitrogen (C/N) ratio, and phosphate concentration in feeding media, achieving a high cell density of 213 ± 10 × 107 cells mL−1. In the second stage, we optimized the molar C/N ratio in the feeding medium for lipid production and achieved high biomass (130 ± 5 g L−1), lipid titer (88 ± 6 g L−1), and lipid content (67% ± 2% of dry cellular weight). Our approach yielded a high lipid titer, comparable to the highest reported value of 68 g L−1 achieved in a nutrient medium, by optimizing cultivation conditions with a synthetic medium in L. starkeyi. This highlights the importance of well‐established yet powerful bioprocess approaches for the precise control of microbial cultivation.

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 shift from fossil fuels to renewable energy sources is crucial in addressing environmental challenges. Vegetable oils have been focused on as the main potential source for biodiesel and biolubricant production. However, due to their fatty acid (FA) composition they are characterized by low stability to oxidation and variable viscosity. Single-cell oils (SCOs) from oleaginous microorganisms are a possible alternative to vegetable oils: their composition is more suitable, and it can further be improved by controlling the fermentation's physiological conditions. In the present study, the production of SCOs with targeted technological properties from Lipomyces starkeyi in fermentation under controlled temperatures was assessed. A dairy effluent (scotta) was used as the fermentation substrate to improve the economic sustainability of the process. Batch aerobic fermentations were carried out in a fermenter at two different temperatures (25 °C and 30 °C). The fermentation yields and SCO FA profiles were analyzed. The highest yields of biomass (9.76 g L−1) and microbial oil (1.83 g L−1) were obtained from fermentations carried out at 30 °C. Furthermore, a significantly lower content (46% vs. 55%) of unsaturated FAs and higher content (11% vs. 1.5%) of shorter-chain saturated FAs, with myristic acid almost matching stearic acid, were detected at 30 °C in comparison to 25 °C. Very low peroxide values were also found (0.14 meq O2 kg−1 at 30 °C and 0 meq O2 kg−1 at 25 °C). These results indicate that these SCOs were highly oxidation-resistant, and that a higher fermentation temperature improves their oxidative stability and tribophysical features. The biodiesels' technological properties, calculated from the FA composition, were within the limits of both U.S. standards and E.U. regulations. Then, SCOs produced from L. starkeyi by fermentation of dairy effluents carried out under controlled temperature can be considered a suitable alternative to vegetable oils to produce biodiesel and biolubricants. [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]

Palm oil, widely used in various products, poses environmental and climate change risks. “Yeast oil” produced by Lipomyces starkeyi, an oil-producing yeast, is one of the sustainable alternatives for palm oil and was successfully produced as an edible substitute for palm oil. However, the high cost of the culture medium for oil production remains a challenge for practical applications. Okara is a by-product of tofu and soymilk production. Because yeast extract contributes to the high cost of the culture medium, we considered using okara, a cheap and nitrogen-rich substitute, to reduce costs. In the initial study with okara, the production of yeast oil was confirmed, but its productivity was low due to the high viscosity caused by its insoluble solids. To overcome this, we extracted and concentrated nitrogen components in okara using the membrane concentration process. Using NF (nanofiltration) membrane concentration, oil production increased 1.69 and 1.44 times compared to the unconcentrated extract solution (added 90% (v/v) in the culture medium) and yeast extract (added 5% (w/v) in the culture medium), respectively. These findings indicate the potential for a significant cost reduction in the culture medium and high oil yield in yeast oil production.

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.

Sugar-rich waste streams, generated in very high quantities worldwide, constitute an important source of environmental pollution. Their eco-friendly conversions into a plethora of added-value compounds through the use of microbial fermentations is currently a very "hot" scientific topic. The aim of this study, was to assess the potential of single cell oil (SCO), microbial mass and citric acid (CA) production by non-conventional yeast strains growing on expired ("waste") glucose. Six yeast strains (viz. Rhodosporidium toruloides DSM 4444, Rhodotorula glutinis NRRL YB-252, R. toruloides NRRL Y-27012, Yarrowia lipolytica LFMB Y-20, Y. lipolytica ACA-DC 50109 and Lipomyces starkeyi DSM 70296) were initially grown in shake flasks with expired glucose used as substrate under nitrogen limitation, in order to "boost" the cellular metabolism towards the synthesis of SCO and CA, and their growth response was quantitatively evaluated. Initial glucose concentration (Glc0) was adjusted at c. 50 g/L. Besides Y. lipolytica, all other yeast strains produced noticeable SCO quantities [lipid in dry cell weight (DCW) ranging from 25.3% w/w to 55.1% w/w]. Lipids of all yeasts contained significant quantities of oleic acid, being perfect candidates for the synthesis of 2nd generation biodiesel. The highest DCW production (=13.6 g/L) was obtained by L. starkeyi DSM 70296, while both Y. lipolytica strains did not accumulate noticeable lipid quantities, but produced non-negligible CA amounts. The most promising CA-producing strain, namely Y. lipolytica ACA-DC 50109 was further studied in stirred-tank bioreactor systems, while the very promising DCW- and SCO-producing L. starkeyi DSM 70296 was further studied in shake flasks. Both strains were grown on media presenting higher Glc0 concentrations and the same initial nitrogen quantity as previously. Indeed, L. starkeyi grown at Glc0 = 85 g/L, produced DCWmax = 34.0 g/L, that contained lipid =34.1% w/w (thus SCO was =11.6 g/L). The strain ACA-DC 50109 in stirred tank bioreactor with Glc0 ≈ 105 g/L produced CA up to 46 g/L (yield of CA produced on glucose consumed; YCA/Glc ≈ 0.45 g/g). Finally, in fed-batch bioreactor experiment, the significant CA quantity of 82.0 g/L (YCA/Glc = 0.50 g/g) was recorded. Concluding, "waste" glucose proved to be a suitable substrate for a number of non-conventional yeast strains. Y. lipolytica ACA-DC 50109 produced significant quantities of CA while L. starkeyi DSM 70296 was a very interesting DCW- and SCO-producing candidate. These strains can be used as potential cell factories amenable to convert glucose-based residues into the mentioned metabolic compounds, that present high importance for food, chemical and biofuel facilities. [ABSTRACT FROM AUTHOR]

The shift from fossil fuels to renewable energy sources is crucial in addressing environmental challenges. Vegetable oils have been focused on as the main potential source for biodiesel and biolubricant production. However, due to their fatty acid (FA) composition they are characterized by low stability to oxidation and variable viscosity. Single-cell oils (SCOs) from oleaginous microorganisms are a possible alternative to vegetable oils: their composition is more suitable, and it can further be improved by controlling the fermentation’s physiological conditions. In the present study, the production of SCOs with targeted technological properties from Lipomyces starkeyi in fermentation under controlled temperatures was assessed. A dairy effluent (scotta) was used as the fermentation substrate to improve the economic sustainability of the process. Batch aerobic fermentations were carried out in a fermenter at two different temperatures (25 °C and 30 °C). The fermentation yields and SCO FA profiles were analyzed. The highest yields of biomass (9.76 g L−1) and microbial oil (1.83 g L−1) were obtained from fermentations carried out at 30 °C. Furthermore, a significantly lower content (46% vs. 55%) of unsaturated FAs and higher content (11% vs. 1.5%) of shorter-chain saturated FAs, with myristic acid almost matching stearic acid, were detected at 30 °C in comparison to 25 °C. Very low peroxide values were also found (0.14 meq O2 kg−1 at 30 °C and 0 meq O2 kg−1 at 25 °C). These results indicate that these SCOs were highly oxidation-resistant, and that a higher fermentation temperature improves their oxidative stability and tribophysical features. The biodiesels’ technological properties, calculated from the FA composition, were within the limits of both U.S. standards and E.U. regulations. Then, SCOs produced from L. starkeyi by fermentation of dairy effluents carried out under controlled temperature can be considered a suitable alternative to vegetable oils to produce biodiesel and biolubricants.

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]

The production of palm oil, which is used in various foods, is associated with environmental destruction and climate change risks; therefore, there is an urgent need for sustainable alternatives. "Yeast oil" produced by Lipomyces starkeyi, an oil-producing yeast, is expected to solve these problems because its fatty acid composition is similar to that of palm oil. To date, we have successfully developed yeast oil as an edible alternative to palm oil. However, conventional processes, including cell collection and lyophilization, are difficult to industrialize in terms of equipment and cost. Therefore, a method for extracting yeast oil from the emulsified liquid generated by crushing the culture was investigated. It is presumed that the emulsified state is stable owing to the components derived from yeast cells and metabolites; thus, solid–liquid filtration separation was attempted before extraction. The extraction recovery ratio of yeast oil was 98.2% when a hexane/ethanol mixture (3:1) was added to the residue after filtration. Furthermore, the energy consumption and processing cost of this new process were estimated to be 26% and 34%, respectively, of that of conventional methods, suggesting that the new process has potential for practical applications. [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 oleaginous yeast Lipomyces starkeyi has a high capacity for starch assimilation, but the genes involved and specific mechanisms in starch degradation remain unclear. This study aimed to identify the critical carbohydrate-active enzyme (CAZyme) genes contributing to starch degradation in L. starkeyi. Comparative transcriptome analysis of cells cultured in glucose and soluble starch medium revealed that 55 CAZymes (including transcript IDs 3772, 1803, and 7314) were highly expressed in soluble starch medium. Protein domain structure and disruption mutant analyses revealed that 3772 encodes the sole secreted α-amylase (LsAmy1p), whereas 1803 and 7314 encode secreted α-glucosidase (LsAgd1p and LsAgd2p, respectively). Triple-gene disruption exhibited severely impaired growth in soluble starch, dextrin, and raw starch media, highlighting their critical role in degrading polysaccharides composed of glucose linked by α-1,4-glucosidic bonds. This study provided insights into the complex starch degradation mechanism in L. starkeyi., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

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]

Abstract 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.

Sugar-rich waste streams, generated in very high quantities worldwide, constitute an important source of environmental pollution. Their eco-friendly conversions into a plethora of added-value compounds through the use of microbial fermentations is currently a very “hot” scientific topic. The aim of this study, was to assess the potential of single cell oil (SCO), microbial mass and citric acid (CA) production by non-conventional yeast strains growing on expired (“waste”) glucose. Six yeast strains (viz. Rhodosporidium toruloides DSM 4444, Rhodotorula glutinis NRRL YB-252, R. toruloides NRRL Y-27012, Yarrowia lipolytica LFMB Y-20, Y. lipolytica ACA-DC 50109 and Lipomyces starkeyi DSM 70296) were initially grown in shake flasks with expired glucose used as substrate under nitrogen limitation, in order to “boost” the cellular metabolism towards the synthesis of SCO and CA, and their growth response was quantitatively evaluated. Initial glucose concentration (Glc0) was adjusted at c. 50 g/L. Besides Y. lipolytica, all other yeast strains produced noticeable SCO quantities [lipid in dry cell weight (DCW) ranging from 25.3% w/w to 55.1% w/w]. Lipids of all yeasts contained significant quantities of oleic acid, being perfect candidates for the synthesis of 2nd generation biodiesel. The highest DCW production (=13.6 g/L) was obtained by L. starkeyi DSM 70296, while both Y. lipolytica strains did not accumulate noticeable lipid quantities, but produced non-negligible CA amounts. The most promising CA-producing strain, namely Y. lipolytica ACA-DC 50109 was further studied in stirred-tank bioreactor systems, while the very promising DCW- and SCO-producing L. starkeyi DSM 70296 was further studied in shake flasks. Both strains were grown on media presenting higher Glc0 concentrations and the same initial nitrogen quantity as previously. Indeed, L. starkeyi grown at Glc0 = 85 g/L, produced DCWmax = 34.0 g/L, that contained lipid =34.1% w/w (thus SCO was =11.6 g/L). The strain ACA-DC 50109 in stirred tank bioreactor with Glc0 ≈ 105 g/L produced CA up to 46 g/L (yield of CA produced on glucose consumed; YCA/Glc ≈ 0.45 g/g). Finally, in fed-batch bioreactor experiment, the significant CA quantity of 82.0 g/L (YCA/Glc = 0.50 g/g) was recorded. Concluding, “waste” glucose proved to be a suitable substrate for a number of non-conventional yeast strains. Y. lipolytica ACA-DC 50109 produced significant quantities of CA while L. starkeyi DSM 70296 was a very interesting DCW- and SCO-producing candidate. These strains can be used as potential cell factories amenable to convert glucose-based residues into the mentioned metabolic compounds, that present high importance for food, chemical and biofuel facilities.

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]

Extracellular production of long chain fatty acid-based products, such as fatty alcohols, are being investigated as intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels. We previously demonstrated that the oleaginous yeast, Lipomyces starkeyi expressing the fatty acyl-CoA reductase (FAR) gene from Marinobacter aquaeolei VT8, produced up to 770 mg/L of fatty alcohols. The FAR genes expressed in L. starkeyi transformants exhibited different fatty alcohol production levels. To better understand fatty alcohol production and potentially further increase fatty alcohol titer and yields, we investigated the expression levels, copy numbers, and enzymatic activities of the expressed FAR genes. Our results showed that fatty alcohol production is dependent on the above factors for higher titers of fatty alcohols. Fatty alcohol titer was further increased in the highest producing transformant by testing different basic process configurations (batch and fed-batch) with different initial nitrogen concentrations and feed rates. In fed-batch, a maximum of 4.2 g/L fatty alcohols were produced in 5-L bioreactor. The oleaginous yeast L. starkeyi can produce high titers of fatty alcohols particularly when periodically fed glucose and nitrogen.

Abstract Background Lipids from oleaginous yeasts emerged as a sustainable alternative to vegetable oils and animal fat to produce biodiesel, the biodegradable and environmentally friendly counterpart of petro-diesel fuel. To develop economically viable microbial processes, the use of residual feedstocks as growth and production substrates is required. Results In this work we investigated sugar beet pulp (SBP) and molasses, the main residues of sugar beet processing, as sustainable substrates for the growth and lipid accumulation by the oleaginous yeast Lipomyces starkeyi. We observed that in hydrolysed SBP the yeast cultures reached a limited biomass, cellular lipid content, lipid production and yield (2.5 g/L, 19.2%, 0.5 g/L and 0.08 g/g, respectively). To increase the initial sugar availability, cells were grown in SBP blended with molasses. Under batch cultivation, the cellular lipid content was more than doubled (47.2%) in the presence of 6% molasses. Under pulsed-feeding cultivation, final biomass, cellular lipid content, lipid production and lipid yield were further improved, reaching respectively 20.5 g/L, 49.2%, 9.7 g/L and 0.178 g/g. Finally, we observed that SBP can be used instead of ammonium sulphate to fulfil yeasts nitrogen requirement in molasses-based media for microbial oil production. Conclusions This study demonstrates for the first time that SBP and molasses can be blended to create a feedstock for the sustainable production of lipids by L. starkeyi. The data obtained pave the way to further improve lipid production by designing a fed-batch process in bioreactor. Graphical abstract

The oleaginous yeast Lipomyces starkeyi is a powerful lipid producer with great industrial potential. Recent studies have reported the isolation of mutant L. starkeyi cells with higher lipid producing capacity. Although genetic engineering strategies have been applied to L. starkeyi, classical genetic approaches are lacking. The development of tools that facilitate genetic crosses in L. starkeyi would not only make it possible to build improved lipid‐producing strains but also facilitate molecular biological analysis of this species. In this study, I report a set of strains and approaches useful for performing genetic crosses with L. starkeyi. The homothallic L. starkeyi reportedly forms an ascus containing two to 20 spores. These spores were resistant to glusulase and could be dissected using a micromanipulator, suggesting that random spore and tetrad (spore dissection) analysis can be adapted for L. starkeyi. Additionally, to isolate a pair of heterothallic strains useful for genetic crosses, the homothallic strain was exposed to UV irradiation, and 10 self‐sterile strains were crossed with one another. One of these combinations, Ls75 and Ls100, sporulated stably. Moreover, to detect genetic recombination, I introduced a different drug resistance marker into each strain and crossed them. The resulting progeny exhibited Mendelian segregation of the resistance markers. Altogether, the work reported here provides a powerful resource for genetic analysis in L. starkeyi. [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]

Abstract Background Lipid extraction for quantification of fat content in oleaginous yeasts often requires strong acids and harmful organic solvents; it is laborious and time-consuming. Therefore, in most cases just endpoint measurements of lipid accumulation are performed and kinetics of intracellular lipid accumulation is difficult to follow. To address this, we created a prediction model using Fourier-transform near-infrared (FT-NIR) spectroscopy. This method allows to measure lipid content in yeast. Methods The FT-NIR calibration sets were constructed from spectra of freeze-dried cells of the oleaginous yeasts Rhodotorula toruloides CBS 14, Lipomyces starkeyi CBS 1807 and Yarrowia lipolytica CBS 6114. The yeast cells were obtained from different cultivation conditions. Freeze-dried cell pellets were scanned using FT-NIR in the Multi Purpose Analyser (MPA) from Bruker. The obtained spectra were assigned corresponding to total fat content, obtained from lipid extraction using a modified Folch method. Quantification models using partial least squares (PLS) regression were built, and the calibration sets were validated on independently cultivated samples. The R. toruloides model was additionally tested on Rhodotorula babjevae DBVPG 8058 and Rhodotorula glutinis CBS 2387. Results The R 2 of the FT-NIR model for R. toruloides was 98%, and the root mean square error of cross-validation (RMSECV) was 1.53. The model was validated using a separate set of R. toruloides samples with a root mean square error of prediction (RMSEP) of 3.21. The R 2 of the Lipomyces model was 96%, with RMSECV 2.4 and RMSEP 3.8. The R 2 of the mixed model, including all tested yeast strains, was 90.5%, with RMSECV 2.76 and RMSEP 3.22, respectively. The models were verified by predicting the total fat content in newly cultivated and freeze-dried samples. Additionally, the kinetics of lipid accumulation of a culture were followed and compared with standard lipid extraction methods. Conclusions Using FT-NIR spectroscopy, we have developed a faster, less laborious and non-destructive quantification of yeast intracellular lipid content compared to methods using lipid extraction.

Abstract Background Efficient and economically viable production of biofuels from lignocellulosic biomass is dependent on mechanical and chemical pretreatment and enzymatic hydrolysis of plant material. These processing steps yield simple sugars as well as plant-derived and process-added organic acids, sugar-derived dehydration products, aldehydes, phenolics and other compounds that inhibit the growth of many microorganisms. Lipomyces starkeyi is an oleaginous yeast capable of robust growth on a variety of sugars and lipid accumulation on pretreated lignocellulosic substrates making it attractive as an industrial producer of biofuels. Here, we examined gene expression during batch growth and lipid accumulation in a 20-L bioreactor with either a blend of pure glucose and xylose or pretreated corn stover (PCS) that had been enzymatically hydrolyzed as the carbon sources. Results We monitored sugar and ammonium utilization as well as biomass accumulation and found that growth of L. starkeyi is inhibited with PCS hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to L. starkeyi in PCS hydrolysate. We quantified gene expression at seven time-points for each carbon source during batch growth and found that gene expression is similar at physiologically equivalent points. Analysis of promoter regions revealed that gene expression during the transition to lipid accumulation is regulated by carbon and nitrogen catabolite repression, regardless of carbon source and is associated with decreased expression of the translation machinery and suppression of the cell cycle. We identified 73 differentially expressed genes during growth phase in the bioreactor that may be involved in detoxification of corn stover hydrolysate. Conclusions Growth of L. starkeyi is inhibited by compounds present in PCS hydrolysate. Here, we monitored key metabolites to establish physiologically equivalent comparisons during a batch bioreactor run comparing PCS hydrolysate and purified sugars. L. starkeyi’s response to PCS hydrolysate is primarily at the beginning of the run during growth phase when inhibitory compounds are presumably at their highest concentration and inducing the general detoxification response by L. starkeyi. Differentially expressed genes identified herein during growth phase will aid in the improvement of industrial strains capable of robust growth on substrates containing various growth inhibitory compounds.

Lipid accumulation by the yeast Lipomyces starkeyi grown on various carbon (C) sources (glucose, xylose, hydrolysate of oil palm empty fruit bunch (OPEFB)) is reported. In media with glucose as the main C-source, a glucose concentration of 60–80 g L−1 was optimal for producing microbial biomass and lipids. Glucose at 60 g L−1 afforded a final dry biomass concentration of 21.2 g L−1 with a 35.8% lipid content. The biomass concentration was reduced to 18.7 g L−1 if the glucose was 80 g L−1 and the lipids were reduced to 11.8%. With pure xylose (80 g L−1), the final biomass concentration was 14.0 g L−1 and the lipid content of the biomass was 42.1%. A 4:1 by mass mixture of glucose and xylose was used to simulate a typical hydrolysate of OPEFB. With this mixture (glucose = 64 g L−1, xylose = 16 g L−1), the maximum biomass concentration was 17.9 g L−1 and the lipids in the biomass exceeded 37.9%. A hydrolysate of OPEFB was prepared by enzymatic hydrolysis of the alkali-pretreated OPEFB. With this hydrolysate as the main C-source, the final yeast biomass concentration was 14.9 g L−1, but the lipid content in the biomass was only 6%. The lipid content of the yeast biomass could be raised to >40% by using a hydrolysate diluted with distilled water. Apparently, dilution alleviated the effects of inhibitory compounds that formed during hydrolysis of lignocellulosic biomass with the commercial enzymes. The lipid yield on C-sources was 0.162 g g−1 in a medium with a C:N mole ratio of 71.7:1. [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]

Lignocellulosic biomass is an attractive raw material for the sustainable production of chemicals and materials using microbial cell factories. Most of the existing bioprocesses focus on second-generation ethanol production using genetically modified Saccharomyces cerevisiae, however, this microorganism is naturally unable to consume xylose. Moreover, extensive metabolic engineering has to be carried out to achieve high production levels of industrially relevant building blocks. Hence, the use of non-Saccharomyces species, or non-conventional yeasts, bearing native metabolic routes, allows conversion of a wide range of substrates into different products, and higher tolerance to inhibitors improves the efficiency of biorefineries. In this study, nine non-conventional yeast strains were selected and screened on a diluted hemicellulosic hydrolysate from Birch. Kluyveromyces marxianus CBS 6556, Scheffersomyces stipitis CBS 5773, Lipomyces starkeyi DSM 70295, and Rhodotorula toruloides CCT 7815 were selected for further characterization, where their growth and substrate consumption patterns were analyzed under industrially relevant substrate concentrations and controlled environmental conditions in bioreactors. K. marxianus CBS 6556 performed poorly under higher hydrolysate concentrations, although this yeast was determined among the fastest-growing yeasts on diluted hydrolysate. S. stipitis CBS 5773 demonstrated a low growth and biomass production while consuming glucose, while during the xylose-phase, the specific growth and sugar co-consumption rates were among the highest of this study (0.17 h–1 and 0.37 g/gdw*h, respectively). L. starkeyi DSM 70295 and R. toruloides CCT 7815 were the fastest to consume the provided sugars at high hydrolysate conditions, finishing them within 54 and 30 h, respectively. R. toruloides CCT 7815 performed the best of all four studied strains and tested conditions, showing the highest specific growth (0.23 h–1), substrate co-consumption (0.73 ± 0.02 g/gdw*h), and xylose consumption (0.22 g/gdw*h) rates. Furthermore, R. toruloides CCT 7815 was able to produce 10.95 ± 1.37 gL–1 and 1.72 ± 0.04 mgL–1 of lipids and carotenoids, respectively, under non-optimized cultivation conditions. The study provides novel information on selecting suitable host strains for biorefinery processes, provides detailed information on substrate consumption patterns, and pinpoints to bottlenecks possible to address using metabolic engineering or adaptive evolution experiments.

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]

Objective: The extraction of the hemicellulose fraction of sugarcane bagasse (SCB) by acid hydrolysis was evaluated in an autoclave and a Parr reactor aiming the application of the hydrolysate as a carbon source for lipid production by Lipomyces starkeyi. Results: The hydrolysis that resulted in the highest sugar concentration was obtained by treatment in the Parr reactor (HHR) at 1.5% (m/v) H2SO4 and 120 °C for 20 min, reaching a hemicellulose conversion of approximately 82%. The adaptation of the yeast to the hydrolysate provided good fermentability and no lag phase. The fermentation of hemicellulose-derived sugars (HHR) by L. starkeyi resulted in a 27.8% (w/w) lipid content and YP/S of 0.16 g/l.h. Increasing the inoculum size increased the lipid content by approximately 61%, reaching 44.8% (w/w). Conclusion: The hemicellulose hydrolysate from SCB is a potential substrate for L. starkeyi to produce lipids for biodiesel synthesis based on the biorefinery concept. [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.

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]

Abstract 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.

The present study aimed to evaluate the rheological and interfacial behaviors of a novel microbial exopolysaccharide fermented by L. starkeyi (LSEP). The structure of LSEP was measured by LC-MS, 1H and 13C NMR spectra, and FT-IR. Results showed that the monosaccharide composition of LSEP was D-mannose (8.53%), D-glucose (79.25%), D-galactose (7.15%), and L-arabinose (5.07%); there existed the anomeric proton of α-configuration and the anomeric carbon of α- and β-configuration; there appeared the characteristic absorption peak of the phosphate ester bond. The molecular weight of LSEP was 401.8 kDa. The water holding capacity (WHC, 2.10 g/g) and oil holding capacity (OHC, 12.89 g/g) were also evaluated. The results of rheological properties showed that the aqueous solution of LSEP was a non-Newtonian fluid, exhibiting the shear-thinning characteristics. The adsorption of LSEP can reduce the interfacial tension (11.64 mN/m) well and form an elastic interface layer at the MCT–water interface. Such functional properties make LSEP a good candidate for use as thickener, gelling agent, and emulsifier to form long-term emulsions for food, pharmaceutical, and cosmetic products.

The oleaginous yeast Lipomyces starkeyi is an excellent producer of triacylglycerol (TAG) as a feedstock for biodiesel production. To understand the regulation of TAG synthesis, we attempted to isolate mutants with decreased lipid productivity and analyze the expression of TAG synthesis-related genes in this study. A mutant with greatly decreased lipid productivity, sr22, was obtained by an effective screening method using Percoll density gradient centrifugation. The expression of citrate-mediated acyl-CoA synthesis-related genes (ACL1, ACL2, ACC1, FAS1, and FAS2) was decreased in the sr22 mutant compared with that of the wild-type strain. Together with a notion that L. starkeyi mutants with increased lipid productivities had increased gene expression, there was a correlation between the expression of these genes and TAG synthesis. To clarify the importance of citrate-mediated acyl-CoA synthesis pathway on TAG synthesis, we also constructed a strain with no ATP-citrate lyase responsible for the first reaction of citrate-mediated acyl-CoA synthesis and investigated the importance of ATP-citrate lyase on TAG synthesis. The ATP-citrate lyase was required for the promotion of cell growth and TAG synthesis in a glucose medium. This study may provide opportunities for the development of an efficient TAG synthesis for biodiesel production.

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]

It is inevitably for cellobiose to be co-generated during enzymatic hydrolysis of cellulose, especially when the cellulase is lack of β-glucosidase activity. In the present study, cellobiose was found superior to glucose for cell growth by L. starkeyi, regardless of the sugar concentrations. Glucose was assimilated preferentially when cellobiose and glucose were co-fermented. Deficiency of β-glucosidase was observed to be beneficial for the simultaneous saccharification and lipid production (SSLP). High lipid titer and cellulose conversion of 9.1 g/L and 92.4%, respectively, were achieved when cellulase with low β-glucosidase activity was supplemented. The SSLP achieved higher lipid titer of 9.5 g/L when a pre-hydrolysis process was introduced. The glucosidase generated by L. starkeyi was primarily cell-bound, which contributed significantly to the cellobiose utilization and the high lipid production. These results provided a novel scheme for enhanced lipid production from lignocellulosic biomass with reduced enzyme usage, which is believed to facilitate the design of a more cost-effective lignocellulose-to-lipid route. [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]