Deletion of LsSNF1 enhances lipid accumulation in the oleaginous yeast Lipomyces starkeyi.

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.

Automated quantification of lipid contents of Lipomyces starkeyi using deep-learning-based image segmentation.

Intracellular lipid droplets (LDs), subcellular organelles playing a role in long-term carbon storage, have immense potential in biofuel and dietary lipid production. Monitoring the state of LDs in living cells is of utmost importance for quick biomass harvest and screening promising isolates. Here, a deep-learning-based segmentation model was developed for automatic detection and segmentation of LDs using the model yeast species Lipomyces starkeyi, leading to fast and accurate quantification of lipid contents in liquid cultures. The trained model detected the yeast's cell and LDs in light microscopic images with an accuracy of 98% and 92%, respectively. Lipid content prediction using pixel numbers counted in segmented LDs showed high similarity to lipid quantification results obtained with gas chromatography-mass spectrometry. This automated quantification can highly reduce cost and time in real-time monitoring of lipid production, thereby providing an efficient tool in bio-fermentation.

Microbial lipid production from soybean hulls using Lipomyces starkeyi LPB53 in a circular economy.

Soybean hulls are lignocellulosic residuesgeneratedinthe industrial processing of soybean, representing about 5 % of the mass of the whole bean. This by-product isan importantsource of polymers suchas cellulose(34 %) and hemicellulose (11 %),which could bevalorizedvia biotechnology to improvethe economic returnof the oilseed chain. In the present work,soybean hulls were evaluated as a carbon sourcefor biolipid productionbyLipomycesstarkeyi LPB 53. Initially the hulls were treated physicochemically and enzymatically to obtain fermentable sugars. Subsequently, biomass growth was evaluated using different nitrogen sources andthe lipid production was optimized, reaching a maximum cell biomass concentration of 26.5 g/L with 42.5 % of lipids. Around 65 % of the xylose content was consumed.The obtained oil wasmajorlycomposed of oleic, palmitic, palmitoleic, linoleic and stearic fatty acids in a proportion of 54 %, 32 %, 4 %, 3 % and 2 %, respectively.

High hydrostatic pressure-assisted extraction of lipids from Lipomyces starkeyi biomass.

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.

Using gel microdroplets to develop a simple high-throughput screening platform for oleaginous microorganisms.

The oleaginous yeast Lipomyces starkeyi is expected to be a new lipid source since this microorganism is capable of accumulating more than 85% lipid per dry cell weight. For effective utilization of oleaginous yeast, mutants with improved lipid production compared to the wild-type have been screened by methods such as single-cell sorting and Percoll density gradient centrifugation. Because these methods need to reculture all mutated oleaginous yeasts together in a flask, it is difficult to evaluate the growth of each individual mutant. Thus, screening for the slow-growing mutants with high-throughput has never been performed by conventional methods. In this study, we developed a high-throughput method using gel microdroplets (GMD). With this method, the growth and lipid production of L. starkeyi can be evaluated simultaneously. L. starkeyi grew in GMD and the size of these microcolonies was evaluated by scattered light. Finally, a mutant with a 10-fold delay in growth compared to the wild-type was obtained. Analysis of genetic information in this mutant could reveal valuable information about critical genes involved in the growth of these microorganisms, which could then be utilized further.

Growth conditions inducing G1 cell cycle arrest enhance lipid production in the oleaginous yeast Lipomyces starkeyi.

Lipid droplets are cytoplasmic organelles that store lipids for energy and membrane synthesis. The oleaginous yeast Lipomyces starkeyi is one of the most promising lipid producers and has attracted attention as a biofuel source. It is known that the expansion of lipid droplets is enhanced under nutrient-poor conditions. Therefore, we prepared a novel nitrogen-depleted medium (N medium) in which to culture L. starkeyi cells. Lipid accumulation was rapidly induced, and this was reversed by the addition of ammonium. In this condition, cell proliferation stopped, and cells with giant lipid droplets were arrested in G1 phase. We investigated whether cell cycle arrest at a specific phase is required for lipid accumulation. Lipid accumulation was repressed in hydroxyurea-synchronized S phase cells and was increased in nocodazole-arrested G2/M phase cells. Moreover, the enrichment of G1 phase cells seen upon rapamycin treatment induced massive lipid accumulation. From these results, we conclude that L. starkeyi cells store lipids from G2/M phase and then arrest cell proliferation in the subsequent G1 phase, where lipid accumulation is enhanced. Cell cycle control is an attractive approach for biofuel production.

System analysis of Lipomyces starkeyi during growth on various plant-based sugars.

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 ß-glucosidases for extracellular hydrolysis of cellobiose.

Enhancing microbial lipids yield for biodiesel production by oleaginous yeast Lipomyces starkeyi fermentation: A review.

The enhanced production of microbial lipids suitable for manufacturing biodiesel from oleaginous yeast Lipomyces starkeyi is critically reviewed. Recent advances in several aspects involving the biosynthetic pathways of lipids, current conversion efficiencies using various carbon sources, intensification strategies for improving lipid yield and productivity in L. starkeyi fermentation, and lipid extraction approaches are analyzed from about 100 papers for the past decade. Key findings on strategies are summarized, including (1) optimization of parameters, (2) cascading two-stage systems, (3) metabolic engineering strategies, (4) mutagenesis followed by selection, and (5) co-cultivation of yeast and algae. The current technical limitations are analyzed. Research suggestions like examination of more gene targets via metabolic engineering are proposed. This is the first comprehensive review on the latest technical advances in strategies from the perspective of process and metabolic engineering to further increase the lipid yield and productivity from L. starkeyi fermentation.

Strains and approaches for genetic crosses in the oleaginous yeast Lipomyces starkeyi.

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.

Identification and analysis of sugar transporters capable of co-transporting glucose and xylose simultaneously.

Simultaneous co-fermentation of glucose and xylose is a key desired trait of engineered Saccharomyces cerevisiae for efficient and rapid production of biofuels and chemicals. However, glucose strongly inhibits xylose transport by endogenous hexose transporters of S. cerevisiae. We identified structurally distant sugar transporters (Lipomyces starkeyi LST1_205437 and Arabidopsis thaliana AtSWEET7) capable of co-transporting glucose and xylose from previously unexplored oleaginous yeasts and plants. Kinetic analysis showed that LST1_205437 had lenient glucose inhibition on xylose transport and AtSWEET7 transported glucose and xylose simultaneously with no inhibition. Modelling studies of LST1_205437 revealed that Ala335 residue at sugar binding site can accommodates both glucose and xylose. Docking studies with AtSWEET7 revealed that Trp59, Trp183, Asn145, and Asn179 residues stabilized the interactions with sugars, allowing both xylose and glucose to be co-transported. In addition, we altered sugar preference of LST1_205437 by single amino acid mutation at Asn365. Our findings provide a new mechanistic insight on glucose and xylose transport mechanism of sugar transporters and the identified sugar transporters can be employed to develop engineered yeast strains for producing cellulosic biofuels and chemicals.

Mitochondrial dynamics and degradation in the oleaginous yeast Lipomyces starkeyi.

Emerging evidence implicates the vital role of mitochondria in lipid consumption and storage, highlighting the intimate link between energy production and saving. Although formation of giant lipid droplets, which is the key hallmark of the oleaginous yeast Lipomyces starkeyi, appears to be regulated in response to changes in mitochondrial shape and metabolism, technical limitations of genetic manipulation have become an obstacle to uncover the mitochondrial behavior in this nonconventional yeast. Here, we established an L. starkeyi strain stably expressing a fluorescent marker for monitoring mitochondrial morphology and degradation and found that mitochondria are mostly fragmented in L. starkeyi cells under fermentable, nonfermentable, and nitrogen depletion conditions. Notably, a fraction of mitochondria-specific fluorescent signals was localized to the vacuole, a lytic organelle in yeast, indicating degradation of mitochondria in those cells. This possible catabolic event was more predominant in cells under nutrient-poor conditions than that in cells under nutrient-rich conditions, concomitantly with lipid droplet formation. Collectively, our studies provide a new tool to investigate mitochondrial dynamics in L. starkeyi and decipher the potential role of mitochondrial degradation in lipid metabolism.

Genetically Engineered Oleaginous Yeast Lipomyces starkeyi for Sesquiterpene α-Zingiberene Production.

Oleaginous yeast, such as Lipomyces starkeyi, are logical organisms for production of higher energy density molecules like lipids and terpenes. We demonstrate that transgenic L. starkeyi strains expressing an α-zingiberene synthase gene from lemon basil or Hall's panicgrass can produce up to 17 mg/L α-zingiberene in yeast extract peptone dextrose (YPD) medium containing 4% glucose. The transgenic strain was further examined in 8% glucose media with C/N ratios of 20 or 100, and YPD. YPD medium resulted in 59 mg/L α-zingiberene accumulation. Overexpression of selected genes from the mevalonate pathway achieved 145% improvement in α-zingiberene synthesis. Optimization of the growth medium for α-zingiberene production led to 15% higher titer than YPD medium. The final transgenic strain produced 700 mg/L α-zingiberene in fed-batch bioreactor culture. This study opens a new synthetic route to produce α-zingiberene or other terpenoids in L. starkeyi and establishes this yeast as a platform for jet fuel biosynthesis.

Carotenoid Production in Oleaginous Yeasts.

Oleaginous yeasts, Yarrowia lipolytica and Lipomyces starkeyi, can synthesize more than 20% of lipids per dry cell weight from a wide variety of substrates. This feature is attractive for cost-efficient production of industrial biodiesel fuel. These yeasts are also very promising hosts for the efficient production of more value-added lipophilic compound carotenoids, e.g., lycopene and astaxanthin, although they cannot naturally biosynthesize carotenoids. Here, we review recent progress in researches on carotenoid production by oleaginous yeasts, which include red yeasts that naturally produce carotenoids, e.g., Rhodotorula glutinis and Xanthophyllomyces dendrorhous. Our new results on pathway engineering of L. starkeyi for lycopene production are also revealed in the present review.

Isolation and characterization of Lipomyces starkeyi mutants with greatly increased lipid productivity following UV irradiation.

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.

Obtaining hemicellulosic hydrolysate from sugarcane bagasse for microbial oil production by Lipomyces starkeyi.

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.

Integrated cascade biorefinery processes for the production of single cell oil by Lipomyces starkeyi from Arundo donax L. hydrolysates.

Giant reed (Arundo donax L.) is a promising source of carbohydrates that can be converted into single cell oil (SCO) by oleaginous yeasts. Microbial conversion of both hemicellulose and cellulose fractions represents the key step for increasing the economic sustainability for SCO production. Lipomyces starkeyi DSM 70,296 was cultivated in two xylose-rich hydrolysates, obtained by the microwave-assisted hydrolysis of hemicellulose catalysed by FeCl3 or Amberlyst-70, and in two glucose-rich hydrolysates obtained by the enzymatic hydrolysis of cellulose. L. starkeyi grew on both undetoxified and partially-detoxified hydrolysates, achieving the lipid content of 30 wt% and yield values in the range 15-24 wt%. For both integrated cascade processes the final production of about 8 g SCO from 100 g biomass was achieved. SCO production through integrated hydrolysis cascade processes represents a promising solution for the effective exploitation of lignocellulosic feedstock from perennial grasses towards new generation biodiesel and other valuable bio-based products.

Optimizing lime pretreatment of rice straw for biolipid production using oleaginous microorganisms.

This work focuses on the lime pretreatment of rice straw, agricultural waste in northeast Vietnam, for microbial lipid (bio-lipid) production. A response surface methodology approach was performed to optimize parameters for the pretreatment process. These parameters are the concentration of Ca(OH)2, hydrolysis temperature, and maintained time; the effect is considered bio-lipid production yield. The lipid yield was estimated through consolidated bioprocessing of the pretreated rice straw as a substrate and using the fungus strain (Aspergillus oryzae 32) and the yeast strain (Lipomyces starkeyi 22). As a result, the optimal pretreatment conditions for maximum lipid yield were obtained at Ca(OH)2 concentration of 12 g/L and hydrolysis temperature of 110 °C within 60 min. The accumulated lipid in fermentation using these oleaginous microorganisms was 8.5g/100g oven-dry weight of rice straw (10.9g/100g for pretreated rice straw). The biolipid consisted of 42.6% saturated, 21.1% monounsaturated (MUFA), and 35.1% polyunsaturated (PUFA) fatty acids.

Engineering xylose metabolism in yeasts to produce biofuels and chemicals.

Xylose is the second most abundant sugar in lignocellulosic biomass. Efficient and rapid xylose utilization is essential for the economic bioconversion of lignocellulosic biomass into value-added products. Building on previous pathway engineering efforts to enable xylose fermentation in Saccharomyces cerevisiae, recent work has focused on reprogramming regulatory networks to enhance xylose utilization by engineered S. cerevisiae. Also, potential benefits of using xylose for the production of various value-added products have been demonstrated. With increasing needs of lipid-derived bioproducts, activation and enhancement of xylose metabolism in oleaginous yeasts have been attempted. This review highlights recent progress of metabolic engineering to achieve efficient and rapid xylose utilization by S. cerevisiae and oleaginous yeasts, such as Yarrowia lipolytica, Rhodosporidium toruloides, and Lipomyces starkeyi.

Conversion of sugar beet residues into lipids by Lipomyces starkeyi for biodiesel production.

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.

Improved production of an acidic exopolysaccharide, the efficient flocculant, by Lipomyces starkeyi U9 overexpressing UDP-glucose dehydrogenase gene.

In order to increase content of glucuronic acid in the exopolysaccharide (EPS) and its flocculating activity, an UDP-glucose dehydrogenase gene was overexpressed in Lipomyces starkeyi V19. The obtained U9 strain could produce 62.1 ± 1.2 g/l EPS while the V19 strain only produced 53.5 ± 1.3 g/l EPS. The compositions of monosaccharides (mannose, glucuronic acid and galactose) in the purified EPS (U9-EPS) from the U9 strain contained 3.79:1:5.52 while those in the purified EPS (V19-EPS) were 3.94:1:6.29. The flocculation rate of the U9-EPS on kaolin clay reached 87.9%, which was significantly higher than that (74.7%) of the V19-EPS while the decolorization rate of Congo Red (CR) by the U9-EPS reached 94.3%, which was significantly higher than that of CR by the V19-EPS (86.23%). The results showed that the purified bioflocculant U9-EPS had effective flocculation of kaolin clay. The U9-EPS also had high ability to flocculate the polluted river water and decolorize Congo red.