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.

Oleaginous fungi: a promising source of biofuels and nutraceuticals with enhanced lipid production strategies.

Oleaginous fungi have attracted a great deal of interest for their potency to accumulate high amounts of lipids (more than 20% of biomass dry weight) and polyunsaturated fatty acids (PUFAs), which have a variety of industrial and biological applications. Lipids of plant and animal origin are related to some restrictions and thus lead to attention towards oleaginous microorganisms as reliable substitute resources. Lipids are traditionally biosynthesized intra-cellularly and involved in the building structure of a variety of cellular compartments. In oleaginous fungi, under certain conditions of elevated carbon ratio and decreased nitrogen in the growth medium, a change in metabolic pathway occurred by switching the whole central carbon metabolism to fatty acid anabolism, which subsequently resulted in high lipid accumulation. The present review illustrates the bio-lipid structure, fatty acid classes and biosynthesis within oleaginous fungi with certain key enzymes, and the advantages of oleaginous fungi over other lipid bio-sources. Qualitative and quantitative techniques for detecting the lipid accumulation capability of oleaginous microbes including visual, and analytical (convenient and non-convenient) were debated. Factors affecting lipid production, and different approaches followed to enhance the lipid content in oleaginous yeasts and fungi, including optimization, utilization of cost-effective wastes, co-culturing, as well as metabolic and genetic engineering, were discussed. A better understanding of the oleaginous fungi regarding screening, detection, and maximization of lipid content using different strategies could help to discover new potent oleaginous isolates, exploit and recycle low-cost wastes, and improve the efficiency of bio-lipids cumulation with biotechnological significance.

Nitrogen starvation causes lipid remodeling in Rhodotorula toruloides.

BACKGROUND: The oleaginous yeast Rhodotorula toruloides is a promising chassis organism for the biomanufacturing of value-added bioproducts. It can accumulate lipids at a high fraction of biomass. However, metabolic engineering efforts in this organism have progressed at a slower pace than those in more extensively studied yeasts. Few studies have investigated the lipid accumulation phenotype exhibited by R. toruloides under nitrogen limitation conditions. Consequently, there have been only a few studies exploiting the lipid metabolism for higher product titers. RESULTS: We performed a multi-omic investigation of the lipid accumulation phenotype under nitrogen limitation. Specifically, we performed comparative transcriptomic and lipidomic analysis of the oleaginous yeast under nitrogen-sufficient and nitrogen deficient conditions. Clustering analysis of transcriptomic data was used to identify the growth phase where nitrogen-deficient cultures diverged from the baseline conditions. Independently, lipidomic data was used to identify that lipid fractions shifted from mostly phospholipids to mostly storage lipids under the nitrogen-deficient phenotype. Through an integrative lens of transcriptomic and lipidomic analysis, we discovered that R. toruloides undergoes lipid remodeling during nitrogen limitation, wherein the pool of phospholipids gets remodeled to mostly storage lipids. We identify specific mRNAs and pathways that are strongly correlated with an increase in lipid levels, thus identifying putative targets for engineering greater lipid accumulation in R. toruloides. One surprising pathway identified was related to inositol phosphate metabolism, suggesting further inquiry into its role in lipid accumulation. CONCLUSIONS: Integrative analysis identified the specific biosynthetic pathways that are differentially regulated during lipid remodeling. This insight into the mechanisms of lipid accumulation can lead to the success of future metabolic engineering strategies for overproduction of oleochemicals.

Evaluating oleaginous yeasts for enhanced microbial lipid production using sweetwater as a sustainable feedstock.

BACKGROUND: Yeasts exhibit promising potential for the microbial conversion of crude glycerol, owing to their versatility in delivering a wide range of value-added products, particularly lipids. Sweetwater, a methanol-free by-product of the fat splitting process, has emerged as a promising alternative feedstock for the microbial utilization of crude glycerol. To further optimize sweetwater utilization, we compared the growth and lipid production capabilities of 21 oleaginous yeast strains under different conditions with various glycerol concentrations, sweetwater types and pH. RESULTS: We found that nutrient limitation and the unique carbon composition of sweetwater boosted significant lipid accumulation in several strains, in particular Rhodosporidium toruloides NRRL Y-6987. Subsequently, to decipher the underlying mechanism, the transcriptomic changes of R. toruloides NRRL Y-6987 were further analyzed, indicating potential sugars and oligopeptides in sweetwater supporting growth and lipid accumulation as well as exogenous fatty acid uptake leading to the enhanced lipid accumulation. CONCLUSION: Our comparative study successfully demonstrated sweetwater as a cost-effective feedstock while identifying R. toluroides NRRL Y-6987 as a highly promising microbial oil producer. Furthermore, we also suggested potential sweetwater type and strain engineering targets that could potentially enhance microbial lipid production.

Two protocols for the detection of oleaginous bacteria using Oil Red O.

The selection of oleaginous bacteria, potentially applicable to biotechnological approaches, is usually carried out by different expensive and time-consuming techniques. In this study, we used Oil Red O (ORO) as an useful dye for staining of neutral lipids (triacylglycerols and wax esters) on thin-layer chromatography plates. ORO could detect minimal quantities of both compounds (detection limit, 0.0025 mg of tripalmitin or 0.005 mg of cetylpalmitate). In addition, we developed a specific, rapid, and inexpensive screening methodology to detect triacylglycerol-accumulating microorganisms grown on the agar plate. This staining methodology detected 9/13 strains with a triacylglycerol content higher than 20% by cellular dry weight. ORO did not stain polyhydroxyalkanoates-producing bacteria. The four oleaginous strains not detected by this screening methodology exhibited a mucoid morphology of their colonies. Apparently, an extracellular polymeric substance produced by these strains hampered the entry of the lipophilic dye into cells. The utilization of the developed screening methodology would allow selecting of oleaginous bacteria in a simpler and faster way than techniques usually used nowadays, based on unspecific staining protocols and spectrophotometric or chromatographic methods. Furthermore, the use of ORO as a staining reagent would easily characterize the neutral lipids accumulated by microorganisms as reserve compounds. KEY POINTS: • Oil Red O staining is specific for triacylglycerols • Oil Red O staining is useful to detect oleaginous bacteria • Fast and inexpensive staining to isolate oleaginous bacteria from the environment.

Identification and characterization of the suppressed lipid accumulation-related gene, SLA1, in the oleaginous yeast Lipomyces starkeyi.

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. SLA1 mutation reduced gene expression in lipid biosynthesis and increased gene expression in ß-oxidation. Our results suggest that SLA1 mutation may leads to decreased lipid productivity. SLA1 deletion also exhibited decreased gene expression in ß-oxidation and increased lipid accumulation, suggesting that SLA1 deletion is a useful tool to improve lipid accumulation in L. starkeyi for industrialization.

Oleaginous Microbial Lipids' Potential in the Prevention and Treatment of Neurological Disorders.

The products of oleaginous microbes, primarily lipids, have gained tremendous attention for their health benefits in food-based applications as supplements. However, this emerging biotechnology also offers a neuroprotective treatment/management potential for various diseases that are seldom discussed. Essential fatty acids, such as DHA, are known to make up the majority of brain phospholipid membranes and are integral to cognitive function, which forms an important defense against Alzheimer's disease. Omega-3 polyunsaturated fatty acids have also been shown to reduce recurrent epilepsy seizures and have been used in brain cancer therapies. The ratio of omega-3 to omega-6 PUFAs is essential in maintaining physiological function. Furthermore, lipids have also been employed as an effective vehicle to deliver drugs for the treatment of diseases. Lipid nanoparticle technology, used in pharmaceuticals and cosmeceuticals, has recently emerged as a biocompatible, biodegradable, low-toxicity, and high-stability means for drug delivery to address the drawbacks associated with traditional medicine delivery methods. This review aims to highlight the dual benefit that lipids offer in maintaining good health for disease prevention and in the treatment of neurological diseases.

Exploring the Fluconazole-Resistance Modifying Activity and Potential Mechanism of Action of Fixed Oil from Caryocar coriaceum Wittm. (Caryocaraceae) against Candida Species.

The fixed oil from the inner mesocarp of Caryocar coriaceum Wittm. is used in the Chapada do Araripe region of Brazil for the treatment of genitourinary candidiasis. This study aimed to evaluate the chemical composition, antifungal activity, reduction of fungal virulence, and the preliminary toxicity of the fixed oil from the inner mesocarp of C. coriaceum tested against three Candida yeasts. The oil was characterized by gas chromatography (GC-MS and GC-FID). Antifungal activity was assessed using the serial microdilution method. Additionally, the potential of the oil as an enhancer of fluconazole action was tested at sub-inhibitory concentrations (MIC/8). The mechanism of action of C. coriaceum fixed oil was determined by evaluating the inhibition of morphological transition in Candida spp. The chemical composition of the fixed oil of C. coriaceum comprised both unsaturated and saturated fatty acids. Oleic (61 %) and palmitic (33 %) acids were the major constituents. Regarding its anti-Candida activity, the oil inhibited the growth of C. albicans (IC50 : 371 µg/mL) and C. tropicalis (IC50 : 830 µg/mL). Furthermore, the oil reversed the antifungal resistance of C. albicans and C. tropicalis, restoring the susceptibility to fluconazole and reducing their IC50 from 12.33 µg/mL and 362 µg/mL to 0.22 µg/mL and 13.93 µg/mL, respectively. The fixed oil of C. coriaceum completely inhibited the morphological transition of C. albicans and C. tropicalis at a concentration of 512 µg/mL, but exhibited limited low antifungal potential against C. krusei. The observed antifungal activity may be attributed to the overproduction of reactive oxygen species. Additionally, the oil showed no toxic effect on the Drosophila melanogaster in vivo model. The fixed oil from the inner mesocarp of C. coriaceum emerge as a strong candidate for the development of new pharmaceutical formulations to treat infections caused by Candida spp.

Isolation, characterization and optimization of oleaginous Providencia vermicola as a feedstock for biodiesel production using Response Surface Methodology.

Oleaginous organisms accrue more than twenty percent of their biomass as lipids and hence are promising feedstocks for biodiesel production. In this study, lipid accumulating bacteria were isolated from diesel-contaminated soils and screened with Sudan black B stain. The most oleaginous was done using 16s rRNA gene sequencing. Lipid production was initially optimized based on media, nitrogen source, pH and temperature. Response surface methodology (RSM) was then employed for the enhancement of lipid weight and content. Obtained lipid was converted to biodiesel using direct transesterification, and both lipid and biodiesel were characterized using FTIR. A total of thirteen bacteria were isolated and the most prominent lipid producer was identified as Providencia vermicola with lab number BA6. Preliminary optimization studies revealed optimum lipid production when nutrient broth and acetic acid served as carbon source; KNO3 as nitrogen source, pH 7.0 and 30 °C. Optimization using RSM resulted in a 5.1% and 74.1% increase in the biomass and lipid content of BA6 respectively. FTIR analyses confirmed the presence of functional groups characteristic of lipids and biodiesel. P. vermicola is a novel oleaginous organism that represents a promising feedstock for biodiesel production.HIGHLIGHTSThe bacterium designated as BA6 identified as Providencia vermicola has the highest lipid contents of the oleaginous bacteria isolated.It accumulates lipids up to 47.73 % of its biomassThe percentage lipids accumulation increased to about 74 % when RSM was used.Providencia vermicola is being reported as an oleaginous organism for the first time.

Expanding the genetic toolbox for Cutaneotrichosporon oleaginosus employing newly identified promoters and a novel antibiotic resistance marker.

BACKGROUND: Cutaneotrichosporon oleaginosus is an oleaginous yeast that can produce up to 80% lipid per dry weight. Its high capacity for the biosynthesis of single cell oil makes it highly interesting for the production of engineered lipids or oleochemicals for industrial applications. However, the genetic toolbox for metabolic engineering of this non-conventional yeast has not yet been systematically expanded. Only three long endogenous promoter sequences have been used for heterologous gene expression, further three dominant and one auxotrophic marker have been established. RESULTS: In this study, the structure of putative endogenous promoter sequences was analyzed based on more than 280 highly expressed genes. The identified motifs of regulatory elements and translational initiation sites were used to annotate the four endogenous putative promoter sequences D9FADp, UBIp, PPIp, and 60Sp. The promoter sequences were tested in a construct regulating the known dominant marker hygromycin B phosphotransferase. The four newly described promoters and the previously established GAPDHp successfully initiated expression of the resistance gene and PPIp was selected for further marker development. The geneticin G418 resistance (aminoglycoside 3'-phosphotransferase, APH) and the nourseothricin resistance gene N-acetyl transferase (NAT) were tested for applicability in C. oleaginosus. Both markers showed high transformation efficiency, positive rate, and were compatible for combined use in a successive and simultaneous manner. CONCLUSIONS: The implementation of four endogenous promoters and one novel dominant resistance markers for C. oleaginosus opens up new opportunities for genetic engineering and strain development. In combination with recently developed methods for targeted genomic integration, the established toolbox allows a wide spectrum of new strategies for genetic and metabolic engineering of the industrially highly relevant yeast.

Oleaginous yeasts for biochemicals, biofuels and food from lignocellulose-hydrolysate and crude glycerol.

Microbial lipids produced from lignocellulose and crude glycerol (CG) can serve as sustainable alternatives to vegetable oils, whose production is, in many cases, accompanied by monocultures, land use changes or rain forest clearings. Our projects aim to understand the physiology of microbial lipid production by oleaginous yeasts, optimise the production and establish novel applications of microbial lipid compounds. We have established methods for fermentation and intracellular lipid quantification. Following the kinetics of lipid accumulation in different strains, we found high variability in lipid formation even between very closely related oleaginous yeast strains on both, wheat straw hydrolysate and CG. For example, on complete wheat straw hydrolysate, we saw that one Rhodotorula glutinis strain, when starting assimilating D-xylosealso assimilated the accumulated lipids, while a Rhodotorula babjevae strain could accumulate lipids on D-xylose. Two strains (Rhodotorula toruloides CBS 14 and R. glutinis CBS 3044) were found to be the best out of 27 tested to accumulate lipids on CG. Interestingly, the presence of hemicellulose hydrolysate stimulated glycerol assimilation in both strains. Apart from microbial oil, R. toruloides also produces carotenoids. The first attempts of extraction using the classical acetone-based method showed that ß-carotene is the major carotenoid. However, there are indications that there are also substantial amounts of torulene and torularhodin, which have a very high potential as antioxidants.

Phytochemical screening and antioxidative property evaluation of lipid-producing fungi.

The increased demands for natural bioactive compounds have induced the search for unusual sources. Microorganisms, especially fungi are a potent source of secondary metabolites, which could act mainly as antioxidant compounds to prevent oxidative stress. In the present study three soil-isolated fungi Aspergillus niger, Aspergillus heteromorphus and Aspergillus fumigatus, were screened for their oleaginous property as well as their potential for the production of bioactive compounds. Fungal biomasses were freeze dried and extracted with methanol using a cold percolation process for the production of intracellular metabolites and the fungal culture media after fermentation were examined for extracellular metabolites. Intracellular and extracellular extracts of the isolated fungi along with the single-cell oils extracted from those fungi were screened for phytochemicals, which showed the presence of alkaloids, flavonoides, glycosides, phenols, saponins and terpenoids. All strains showed potent antioxidant activity, determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) activity. Extracellular extract and single-cell oil of A. heteromorphus showed the highest antioxidant activity with maximum ABTS radical scavenging activity and reducing potential. Highest content of phenolic and flavonoid compounds within the isolated fungi was found to be 37.58 mg gallic acid equivalent (GAE)/g and 62.07 mg catechine equivalent (CE)/g, respectively. Chromatographic analysis of the intracellular and extracellular extracts of the fungi showed the presence of gallic acid, di-hydroxy benzoic acid, ferulic acid, quercetin, epigerin, kampferol, trans cinnamic acid, chlorogenic acid and rutin, which made them biologically important and beneficial for human health.

Hannaella oleicumulans sp. nov. and Hannaella higashiohmiensis sp. nov., two novel oleaginous basidiomycetous yeast species.

Three strains of novel oleaginous yeast species were isolated from soil samples collected in Shiga Prefecture, Japan. The sequences of the internal transcribed spacer (ITS) region and the D1/D2 region of the large subunit (LSU) of the rRNA genes indicated that these novel yeast species are members of the genus Hannaella. The results of molecular phylogenetic analysis indicated that strains 38-3 and 8s1 were closely related to Hannaella oryzae. They differed by 10 nucleotide substitutions and one gap (1.77 %) in the D1/D2 region of the LSU of the rRNA genes and by 17-18 nucleotide substitutions and 10-11 gaps (5.45-5.85 %) in the ITS region. Strain 51-4 differed from the type strain of the most closely related species, Hannaella pagnoccae, by 26 nucleotide substitutions (4.46 %) in the D1/D2 region of the LSU of the rRNA genes and by 20 nucleotide substitutions and six gaps (5.42 %) in the ITS region. The names proposed for these previously undescribed species are Hannaella oleicumulans sp. nov. and Hannaella higashiohmiensis sp. nov.

Challenges and prospects of yeast-based microbial oil production within a biorefinery concept.

Biodiesel, unlike to its fossil-based homologue (diesel), is renewable. Its use contributes to greater sustainability in the energy sector, mainly by reducing greenhouse gas emissions. Current biodiesel production relies on plant- and animal-related feedstocks, resulting in high final costs to the prices of those raw materials. In addition, the production of those materials competes for arable land and has provoked a heated debate involving their use food vs. fuel. As an alternative, single-cell oils (SCOs) obtained from oleaginous microorganisms are attractive sources as a biofuel precursor due to their high lipid content, and composition similar to vegetable oils and animal fats. To make SCOs competitive from an economic point of view, the use of readily available low-cost substrates becomes essential. This work reviews the most recent advances in microbial oil production from non-synthetic sugar-rich media, particularly sugars from lignocellulosic wastes, highlighting the main challenges and prospects for deploying this technology fully in the framework of a Biorefinery concept.

Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector.

BACKGROUND: Sustainable production of triglycerides for various applications is a major focus of microbial factories. Oleaginous yeast species have been targeted for commercial production of microbial oils. Among all the oleaginous yeasts examined in a previous comparative study, Cutaneotrichosporon oleaginosus showed the highest lipid productivity. Moreover, a new lipid production process for C. oleaginosus with minimal waste generation and energy consumption resulted in the highest lipid productivity in the history of oleaginous yeasts. However, productivity and product diversity are restricted because of the genetic intractability of this yeast. To date, successful targeted genetic engineering of C. oleaginosus has not yet been reported. RESULTS: The targeted gene editing was successfully carried out in C. oleaginosus using CRISPR/Cas system. A tailored enzyme system isolated to degrade the C. oleaginosus cell wall enabled the isolation of viable spheroplasts that are amenable to in-cell delivery of nucleic acids and proteins. The employment of both Cas9 protein and Cas mRNA was effective in obtaining strains with URA5 knockout that did not exhibit growth in the absence of uracil. Subsequently, we successfully created several strains with enhanced lipid yield (54% increase compared to that in wild type) or modified fatty acid profiles comparable with those of cocoa butter or sunflower oil compositions. CONCLUSION: This study establishes the first targeted engineering technique for C. oleaginosus using the CRISPR/Cas system. The current study creates the foundation for flexible and targeted strain optimizations towards building a robust platform for sustainable microbial lipid production. Moreover, the genetic transformation of eukaryotic microbial cells using Cas9 mRNA was successfully achieved.

The monitoring of oil production process by deep learning based on morphology in oleaginous yeasts.

BACKGROUND: Monitoring jar fermenter-cultured microorganisms in real time is important for controlling productivity of bioproducts in large-scale cultivation settings. Morphological data is used to understand the growth and fermentation states of these microorganisms during monitoring. Oleaginous yeasts are used for their high productivity of single-cell oils but the relationship between lipid productivity and morphology has not been elucidated in these organisms. RESULTS: In this study, we investigated the relationship between the morphology of oleaginous yeasts (Lipomyces starkeyi and Rhodosporidium toruloides were used) and their cultivation state in a large-scale cultivation setting using a real-time monitoring system. We combined this with deep learning by feeding a large amount of high-definition cell images obtained from the monitoring system to a deep learning algorithm. Our results showed that the cell images could be grouped into 7 distinct groups and that a strong correlation existed between each group and its biochemical activity (growth and oil-productivity). CONCLUSIONS: This is the first report describing the morphological variations of oleaginous yeasts in a large-scale cultivation, and describes a promising new avenue for improving productivity of microorganisms in large-scale cultivation through the use of a real-time monitoring system combined with deep learning. KEY POINTS: • A real-time monitoring system followed the morphological change of oleaginous yeasts. • Deep learning grouped them into 7 distinct groups based on their morphology. • A correlation between the cultivation state and the shape of the yeast was observed.

LsSpt23p is a regulator of triacylglycerol synthesis in the oleaginous yeast Lipomyces starkeyi.

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 LsSPT23M1108T overexpression mutant showed fivefold higher TAG production than control.

Nile red-based lipid fluorometry protocol and its use for statistical optimization of lipids in oleaginous yeasts.

As lipogenic yeasts are becoming increasingly harnessed as biofactories of oleochemicals, the availability of efficient protocols for the determination and optimization of lipid titers in these organisms is necessary. In this study, we optimized a quick, reliable, and high-throughput Nile red-based lipid fluorometry protocol adapted for oleaginous yeasts and validated it using different approaches, the most important of which is using gas chromatography coupled to flame ionization detection and mass spectrometry. This protocol was applied in the optimization of the concentrations of ammonium chloride and glycerol for attaining highest lipid titers in Rhodotorula toruloides NRRL Y-6987 and Yarrowia lipolytica W29 using response surface central composite design (CCD). Results of this optimization showed that the optimal concentration of ammonium chloride and glycerol is 4 and 123 g/L achieving a C/N ratio of 57 for R. toruloides, whereas for Y. lipolytica, concentrations are 4 and 139 g/L with a C/N ratio of 61 for Y. lipolytica. Outside the C/N of 33 to 74 and 45 to 75, respectively, for R. toruloides and Y. lipolytica, lipid productions decrease by more than 10%. The developed regression models and response surface plots show the importance of the careful selection of C/N ratio to attain maximal lipid production. KEY POINTS: • Nile red (NR)-based lipid fluorometry is efficient, rapid, cheap, high-throughput. • NR-based lipid fluorometry can be well used for large-scale experiments like DoE. • Optimal molar C/N ratio for maximum lipid production in lipogenic yeasts is ~60.

Effect of carbon and nitrogen concentrations on lipid accumulation and regulation of acetyl-CoA carboxylase in Papiliotrema laurentii.

Biodiesel is an interesting alternative to petroleum diesel as it is renewable, biodegradable, and has a low pollutant content. Yeast oils can be used for biodiesel production instead of edible oils, mitigating the use of arable land and water for biodiesel production. Maximum lipid accumulation is reached at 48 h of cultivation by the oleaginous yeast Papiliotrema laurentii UFV-1. Nevertheless, the effects of carbon and nitrogen concentrations on lipid accumulation, as well as the regulation of lipid metabolism in this yeast are still not well-characterised. Therefore, this work evaluated the effects of carbon and nitrogen concentrations on the lipid accumulation in P. laurentti, the expression of the ACC gene, and the activity of the enzyme acetyl-CoA carboxylase (ACCase) in different carbon:nitrogen ratios (C:N) and glucose concentrations. The variation of ammonium sulfate concentration did not affect the growth and lipid accumulation in P. laurentii UFV-1. On the other hand, glucose concentration remarkably influenced biomass and lipid production by this yeast. Therefore, the carbon concentration is more important than the nitrogen concentration for lipid production by P. laurentii UFV-1. Importantly, the levels of both ACC gene expression and ACCase activity were maximum during the late-exponential growth phase and decreased after reaching the highest lipid contents, which was easier evidenced during the accumulation and maximum lipid levels. As such, the reduction of ACCase enzyme activity seems to be related to the decrease in the expression level of the ACC gene.

Determining mating type and ploidy in Rhodotorula toruloides and its effect on growth on sugars from lignocellulosic biomass.

Rhodotorula toruloides is being developed for the use in industrial biotechnology processes because of its favorable physiology. This includes its ability to produce and store large amounts of lipids in the form of intracellular lipid bodies. Nineteen strains were characterized for mating type, ploidy, robustness for growth, and accumulation of lipids on inhibitory switchgrass hydrolysate (SGH). Mating type was determined using a novel polymerase chain reaction (PCR)-based assay, which was validated using the classical microscopic test. Three of the strains were heterozygous for mating type (A1/A2). Ploidy analysis revealed a complex pattern. Two strains were triploid, eight haploid, and eight either diploid or aneuploid. Two of the A1/A2 strains were compared to their parents for growth on 75%v/v concentrated SGH. The A1/A2 strains were much more robust than the parental strains, which either did not grow or had extended lag times. The entire set was evaluated in 60%v/v SGH batch cultures for growth kinetics and biomass and lipid production. Lipid titers were 2.33-9.40 g/L with a median of 6.12 g/L, excluding the two strains that did not grow. Lipid yields were 0.032-0.131 (g/g) and lipid contents were 13.5-53.7% (g/g). Four strains had significantly higher lipid yields and contents. One of these strains, which had among the highest lipid yield in this study (0.131 ± 0.007 g/g), has not been previously described in the literature. SUMMARY: The yeast Rhodotorula toruloides was used to produce oil using sugars extracted from a bioenergy grass.