Computational assessment of lipid production in Rhodosporidium toruloides in two-stage and one-stage batch bioprocesses.

Oleaginous yeasts are promising platforms for microbial lipids production as a renewable and sustainable alternative to vegetable oils in biodiesel production. In this paper, a thorough in silico assessment of lipid production in batch cultivation by Rhodosporidium toruloides was developed. By means of dynamic flux balance analysis, the traditional two-stage bioprocess (TSB) performed by the native strain was contrasted with one-stage bioprocess (OSB) using four designed strains obtained by gene knockout strategies. Lipid titer, yield, content, and productivity were analyzed at different initial C/N ratios as relevant performance indicators used in bioprocesses. By weighting these indicators, a global lipid efficiency metric (GLEM) was defined to consider different scenarios. Under simulated conditions, designed strains for lipid overproduction in OSB outperformed the TSB in terms of lipid title (up to threefold), lipid yield (up to 2.4-fold), lipid content (up to 2.8-fold, with a maximum of 76%), and productivity (up to 1.3-fold), depending on C/N ratios. Using these efficiency parameters and the proposed GLEM, the process of selecting the most suitable candidates for lipid production could be carried out before experimental assays. This methodology holds the potential to be extended to other oleaginous microorganisms and diverse strain design techniques.

ß-Carotene production from sugarcane molasses by a newly isolated Rhodotorula toruloides L/24-26-1.

Production of carotenoids by yeast fermentation is an advantaged technology due to its easy scaling and safety. Nevertheless, carotenoid production needs an economic culture medium and other efficient yeast stains. The study aims to isolate and identify a yeast strain capable of producing carotenoids using a cost-effective substrate. A new strain was identified as Rhodotorula toruloides L/24-26-1, which can produce carotenoids at different pretreated and unpretreated sugarcane molasses concentrations (40 and 80 g/L). The highest biomass concentration (18.6 ± 0.6 g/L) was reached in the culture using 80 g/L of hydrolyzed molasses. On the other hand, the carotenoid accumulation reached the maximum value using pretreated molasses at 40 g/L (715.4 ± 15.1 µg/g d.w). In this case, the ß-carotene was 1.5 times higher than that on the control medium. The yeast growth in molasses was not correlated with carotenoid production. The most outstanding production of The DPPH, ABTS, and FRAP tests demonstrated the antioxidant activity of the obtained carotenogenic extracts. This research demonstrated the R. toruloides L/24-26-1 strain biotechnological potential for carotenoid compounds. The yeast produces carotenoids with antioxidant activity in an inexpensive medium, such as sulfuric acid pretreated and unpretreated molasses.

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.

The dynamic roles of intracellular vacuoles in heavy metal detoxification by Rhodotorula mucilaginosa.

AIMS: Rhodotorula mucilaginosa (Rho) can develop a range of strategies to resist the toxicity of heavy metals. This study aimed to investigate the physiological responses and transcriptomic regulation of the fungus under different heavy metal stresses. METHODS AND RESULTS: This study applied transmission electron microscopy and RNA-seq to investigate the fungal resistance to Pb, Cd, and Cu stresses. Under Pb stress, the activated autophagy-related genes, vesicle-fusing ATPase, and vacuolar ATP synthase improved vacuolar sequestration. This offsets the loss of lipids. However, the metal sequestration by vacuoles was not improved under Cd stress. Vacuolar fusion was also inhibited following the interference of intravacuolar Ca2+ due to their similar ionic radii. Cu2+ showed the maximum toxic effects due to its lowest cellular sorption (as low as 7%) with respect to Pb2+ and Cd2+, although the efflux pumps and divalent metal ion transporters partially contributed to the detoxification. CONCLUSIONS: Divalent cation transporters and vacuolar sequestration are the critical strategies for Rho to resist Pb stress. Superoxide dismutase (SOD) is the main strategy for Cd resistance in Rho. The intracellular Cu level was decreased by efflux pump and divalent metal ion transporters.

Research progress on carotenoid production by Rhodosporidium toruloides.

Carotenoids are natural lipophilic pigments, which have been proven to provide significant health benefits to humans, relying on their capacity to efficiently scavenge singlet oxygen and peroxyl radicals as antioxidants. Strains belonging to the genus Rhodosporidium represent a heterogeneous group known for a number of phenotypic traits including accumulation of carotenoids and lipids and tolerance to heavy metals and oxidative stress. As a representative of these yeasts, Rhodosporidium toruloides naturally produces carotenoids with high antioxidant activity and grows on a wide variety of carbon sources. As a result, R. toruloides is a promising host for the efficient production of more value-added lipophilic compound carotenoids, e.g., torulene and torularhodin. This review provides a comprehensive summary of the research progress on carotenoid biosynthesis in R. toruloides, focusing on the understanding of biosynthetic pathways and the regulation of key enzymes and genes involved in the process. Moreover, the relationship between the accumulation of carotenoids and lipid biosynthesis, as well as the stress from diverse abiotic factors, has also been discussed for the first time. Finally, several feasible strategies have been proposed to promote carotenoid production by R. toruloides. It is possible that R. toruloides may become a critical strain in the production of carotenoids or high-value terpenoids by genetic technologies and optimal fermentation processes. KEY POINTS: • Biosynthetic pathway and its regulation of carotenoids in Rhodosporidium toruloides were concluded • Stimulation of abiotic factors for carotenoid biosynthesis in R. toruloides was summarized • Feasible strategies for increasing carotenoid production by R. toruloides were proposed.

Isolation and Identification of Highly Sb-Resistant Rhodotorula glutinis Strain J5 and its Mechanism of Resistance to Sb(III).

Sb-resistant strains can detoxify antimony through metabolic mechanisms such as oxidation and affect the migration, transformation, and ultimate fate of antimony in the environment. In this study, a strain of Sb-resistant fungi, Rhodotorula glutinis sp. Strain J5, was isolated from Xikuangshan mine and its growth characteristics, gene expression differences, and functional annotation under Sb(III) stress were further investigated to reveal the mechanism of resistance to Sb(III). We identified strain J5 as belonging to the Rhodotorula glutinis species optimally growing at pH 5.0 and at 28 °C of temperature. According to gene annotation and differential expression, the resistance mechanism of Strain J5 includes: reducing the endocytosis of antimony by aquaporin AQP8 and transmembrane transporter pst, enhancing the efflux of Sb(III) by the gene expression of acr2, acr3 and ABC, improving the oxidation of Sb(III) by iron-sulfur protein and Superoxide dismutase (SOD), glutathione (GSH) and cysteine (Cys) chelation, methylation of methyltransferase and N-methyltransferase, accelerating cell damage repair and EPS synthesis and other biochemical reaction mechanisms. FT-IR analysis shows that the -OH, -COOH, -NH, -PO, C-O, and other active groups of Strain J5 can be complexed with Sb(III), resulting in chemical adsorption. Strain J5 displays significant resistance to Sb(III) with the MIC of 1300 mg/L, playing a crucial role in the global biochemical transformation of antimony and its potential application in soil microbial remediation.

Corn stover variability drives differences in bisabolene production by engineered Rhodotorula toruloides.

Microbial conversion of lignocellulosic biomass represents an alternative route for production of biofuels and bioproducts. While researchers have mostly focused on engineering strains such as Rhodotorula toruloides for better bisabolene production as a sustainable aviation fuel, less is known about the impact of the feedstock heterogeneity on bisabolene production. Critical material attributes like feedstock composition, nutritional content, and inhibitory compounds can all influence bioconversion. Further, the given feedstocks can have a marked influence on selection of suitable pretreatment and hydrolysis technologies, optimizing the fermentation conditions, and possibly even modifying the microorganism's metabolic pathways, to better utilize the available feedstock. This work aimed to examine and understand how variations in corn stover batches, anatomical fractions, and storage conditions impact the efficiency of bisabolene production by R. toruloides. All of these represent different facets of feedstock heterogeneity. Deacetylation, mechanical refining, and enzymatic hydrolysis of these variable feedstocks served as the basis of this research. The resulting hydrolysates were converted to bisabolene via fermentation, a sustainable aviation fuel precursor, using an engineered R. toruloides strain. This study showed that different sources of feedstock heterogeneity can influence microbial growth and product titer in counterintuitive ways, as revealed through global analysis of protein expression. The maximum bisabolene produced by R. toruloides was on the stalk fraction of corn stover hydrolysate (8.89 ± 0.47 g/L). Further, proteomics analysis comparing the protein expression between the anatomic fractions showed that proteins relating to carbohydrate metabolism, energy production, and conversion as well as inorganic ion transport metabolism were either significantly upregulated or downregulated. Specifically, downregulation of proteins related to the iron-sulfur cluster in stalk fraction suggests a coordinated response by R. toruloides to maintain overall metabolic balance, and this was corroborated by the concentration of iron in the feedstocks. ONE-SENTENCE SUMMARY: This study elucidates the effects of different sources of corn stover on bisabolene production by engineered Rhodotorula toruloides, highlighting the importance of understanding feedstock variability to enhance bioprocess efficiency and economic outcomes.

Rhodotorula mucilaginosa A8, a potential helper strain in a vitamin C microbial fermentation process.

In the vitamin C microbial fermentation system, oxidative stress limits the growth and 2-keto-l-gulonic acid (2-KLG, the precursor of vitamin C) production of Ketogulonicigenium vulgare. Most Bacillus strains, as helper strains, have been reported to release key biomolecules to reduce oxidative stress and promote the growth and 2-KLG production of K. vulgare. To understand the specific mechanism by which the helper strain and K. vulgare interact to reduce oxidative stress, a novel helper strain, Rhodotorula mucilaginosa A8, was used to construct a consortium in the co-culture fermentation system. Based on the activities of the antioxidant enzymes and quantitative polymerase chain reaction (qPCR) analysis, R. mucilaginosa A8 could reduce oxidative stress and increase 2-KLG production in K. vulgare by upregulating antioxidant enzyme activities and related gene-expression levels. In addition, the carotenoids of R. mucilaginosa promoted 2-KLG production in K. vulgare. Coculture of R. mucilaginosa with K. vulgare increased the yield of carotenoids. This study suggested that helper strains with the ability to reduce oxidative stress in K. vulgare would likely act as potential helper strains for facilitating 2-KLG biosynthesis. This work could provide a theoretical basis for the search for potential helper strains for vitamin C microbial fermentation and for the construction of synthetic microbial communities to produce valuable products.

Production optimization of food functional factor ergothioneine in wild-type red yeast Rhodotorula mucilaginosa DL-X01.

BACKGROUND: Ergothioneine (EGT) is a high-value food functional factor that cannot be synthesized by humans and other vertebrates, and the low yield limits its application. RESULTS: In this study, the optimal fermentation temperature, fermentation time, initial pH, inoculum age, and inoculation ratio on EGT biosynthesis of Rhodotorula mucilaginosa DL-X01 were optimized. In addition, the effects of three key precursor substances - histidine, methionine, and cysteine - on fungal EGT synthesis were verified. The optimal conditions were further obtained by response surface optimization. The EGT yield of R. mucilaginosa DL-X01 under optimal fermentation conditions reached 64.48 ± 2.30 mg L-1 at shake flask fermentation level. Finally, the yield was increased to 339.08 ± 3.31 mg L-1 (intracellular) by fed-batch fermentation in a 5 L bioreactor. CONCLUSION: To the best of our knowledge, this is the highest EGT yield ever reported in non-recombinant strains. The fermentation strategy described in this study will promote the efficient biosynthesis of EGT in red yeast and its sustainable production in the food industry. © 2024 Society of Chemical Industry.

Genomic analysis of the marine yeast Rhodotorula sphaerocarpa ETNP2018 reveals adaptation to the open ocean.

BACKGROUND: Despite a rising interest in the diversity and ecology of fungi in marine environments, there are few published genomes of fungi isolated from the ocean. The basidiomycetous yeast (unicellular fungus) genus Rhodotorula are prevalent and abundant in the open ocean, and they have been isolated from a wide range of other environments. Many of these environments are nutrient poor, such as the Antarctica and the Atacama deserts, raising the question as to how Rhodotorula yeasts may have adapted their metabolic strategies to optimize survival under low nutrient conditions. In order to understand their adaptive strategies in the ocean, the genome of R. sphaerocarpa ETNP2018 was compared to that of fourteen representative Rhodotorula yeasts, isolated from a variety of environments. RESULTS: Rhodotorula sphaerocarpa ETNP2018, a strain isolated from the oligotrophic part of the eastern tropical North Pacific (ETNP) oxygen minimum zone (OMZ), hosts the smallest of the fifteen genomes and yet the number of protein-coding genes it possesses is on par with the other strains. Its genome exhibits a distinct reduction in genes dedicated to Major Facilitator Superfamily transporters as well as biosynthetic enzymes. However, its core metabolic pathways are fully conserved. Our research indicates that the selective pressures of the ETNP OMZ favor a streamlined genome with reduced overall biosynthetic potential balanced by a stable set of core metabolisms and an expansion of mechanisms for nutrient acquisition. CONCLUSIONS: In summary, this study offers insights into the adaptation of fungi to the oligotrophic ocean and provides valuable information for understanding the ecological roles of fungi in the ocean.

Rhodotorula sp. as a cell factory for production of valuable biomolecules.

Rhodotorula sp. are well-known for their ability to biosynthesize a diverse range of valuable biomolecules, including carotenoids, lipids, enzymes, and polysaccharides. Despite the high number of studies conducted using Rhodotorula sp. at the laboratory scale, most of these do not address all processual aspects necessary for scaling up these processes for industrial applications. This chapter explores the potential of Rhodotorula sp. as a cell factory for the production of distinct biomolecules, with a particular emphasis on exploring their use from a biorefinery perspective. Through in-depth discussions of the latest research and insights into non-conventional applications, we aim to provide a comprehensive understanding of Rhodotorula sp.'s ability to produce biofuels, bioplastics, pharmaceuticals, and other valuable biochemicals. This book chapter also examines the fundamentals and challenges associated with the optimizing upstream and downstream processing of Rhodotorula sp-based processes. We believe that through this chapter, readers with different levels of expertise will gain insights into strategies for enhancing the sustainability, efficiency, and effectiveness of producing biomolecules using Rhodotorula sp.

Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation.

BACKGROUND: The non-conventional yeast Rhodotorula toruloides is an emerging host organism in biotechnology by merit of its natural capacity to accumulate high levels of carotenoids and intracellular storage lipids from a variety of carbon sources. While the number of genetic engineering strategies that employ R. toruloides is increasing, the lack of genetic tools available for modification of this yeast is still limiting strain development. For instance, several strong, constitutive R. toruloides promoters have been characterized, but to date, only five inducible promoters have been identified. Although nitrogen-limited cultivation conditions are commonly used to induce lipid accumulation in this yeast, no promoters regulated by nitrogen starvation have been described for R. toruloides. RESULTS: In this study, we used a combination of genomics and transcriptomics methods to identify novel R. toruloides promoter sequences that are either inducible or repressible by nitrogen starvation. RNA sequencing was used to assess gene expression in the recently isolated strain R. toruloides BOT-A2 during exponential growth and during nitrogen starvation, when cultivated with either glucose or xylose as the carbon source. The genome of BOT-A2 was sequenced using a combination of long- and short-read sequencing and annotated with support of the RNAseq data. Differential expression analysis was used to identify genes with a |log2 fold change|≥ 2 when comparing their expression during nitrogen depletion to that during exponential growth. The promoter regions from 16 of these genes were evaluated for their ability to drive the expression of a fluorescent reporter gene. Three promoters that were clearly upregulated under nitrogen starvation and three that were downregulated were selected and further characterized. One promoter, derived from gene RTBOTA2_003877, was found to function like an on-off switch, as it was only upregulated under full nitrogen depletion and downregulated in the presence of the nitrogen source. CONCLUSIONS: Six new R. toruloides promoters that were either upregulated or downregulated under nitrogen-starvation were identified. These substantially contribute to the available promoters when engineering this organism and are foreseen to be particularly useful for future engineering strategies requiring specific regulation of target genes in accordance with nitrogen availability.

Enhancement of novel Endo-polygalacturonase expression in Rhodotorula mucilaginosa PY18: insights from mutagenesis and molecular docking.

Pectinase is a particular type of enzyme that can break down pectin compounds and is extensively utilised in the agricultural field. In this study, twenty yeast isolates were isolated and assayed for pectinase activity. Molecular identification by PCR amplification and sequencing of internal transcribed spacer (ITS) regions of isolate no. 18 had the highest pectinase activity of 46.35 U/mg, was identified as Rhodotorula mucilaginosa PY18, and was submitted under accession no. (OM275426) in NCBI. Rhodotorula mucilaginosa PY18 was further enhanced through sequential mutagenesis, resulting in a mutant designated as Rhodotorula mucilaginosa E54 with a specific activity of 114.2 U/mg. Using Response Surface Methodology (RSM), the best culture conditions for the pectinase-producing yeast mutant Rhodotorula mucilaginosa E54 were pH 5, 72-h incubation, 2.5% xylose, and 2.5% malt extract, with a pectinase-specific activity of 156.55 U/mg. Then, the obtained sequences of the endo-polygalacturonase PGI gene from Rhodotorula mucilaginosa PY18 and mutant Rhodotorula mucilaginosa E54 were isolated for the first time, sequenced, and submitted to NCBI accession numbers OQ283005 and OQ283006, respectively. The modelled 3D structure of the endo-PGI enzyme (485 residues) was validated using Ramachandran's plot, which showed 87.71, 85.56, and 91.57% in the most favourable region for template Rhodotorula mucilaginosa KR, strain Rhodotorula mucilaginosa PY18, and mutant Rhodotorula mucilaginosa E54, respectively. In molecular docking studies, the results of template Rhodotorula mucilaginosa KR endo-PG1 showed an interaction with an affinity score of - 6.0, - 5.9, and - 5.6 kcal/mol for active sites 1, 2, and 3, respectively. Rhodotorula mucilaginosa PY18 endo-PG1 showed an interaction affinity with a score of - 5.8, - 6.0, and - 5.0 kcal/mol for active sites 1, 2, and 3, respectively. Mutant Rhodotorula mucilaginosa E54 endo-PG1 showed an interaction affinity of - 5.6, - 5.5, - 5.5 and - 5.4 kcal/mol for active sites 1, 2, and 3, respectively. The endo-PGI genes of both the yeast strain Rhodotorula mucilaginosa PY18 and mutant Rhodotorula mucilaginosa E54 were successfully cloned and expressed in E. coli DH5α, showing significantly higher endo-PG1 activity, which recorded 94.57 and 153.10 U/mg for recombinant Rhodotorula mucilaginosa pGEM-PGI-PY18 and recombinant mutant Rhotorula pGEM-PGI-E54, respectively.

Attenuating the triacylglycerol catabolism enhanced lipid production of Rhodotorula strain U13N3.

Enhancing the lipid production of oleaginous yeasts is conducive to cutting the cost of feedstock for biodiesel. To increase the lipid productivity of Rhodotorula sp. U13N3, genes involving lipid degradation were knocked out and fermentation conditions were investigated. Results of transcription analysis demonstrated that genes encoding the ATG15-like lipase (ATG15) and peroxisomal acyl-CoA oxidase (ACOX2) were upregulated significantly at the lipogenesis stage. When ATG15 and ACOX2 were knocked out separately from the genome by the CRISPR/Cas9 method, both ΔATG15 and ΔACOX2 mutants showed better lipid production ability than the parent strain. Flow cytometry and confocal microscopic analyses indicated that simultaneous the knockout of ATG15 and ACOX2 did not impact the cell viability, whereas the lipid production was enhanced markedly as the lipid yield increased by 67.03% in shake flasks. Afterward, the ΔATG15ΔACOX2 transformant (TO2) was cultivated in shake flasks in the fed-batch mode; the highest biomass and lipid yield reached 45.76 g/L and 27.14 g/L at 216 h, respectively. Better performance was achieved when TO2 was cultivated in the 1-L bioreactor. At the end of fermentation (180 h), lipid content, yield, yield coefficient, and productivity reached 65.53%, 27.35 g/L, 0.277 g/g glycerol, and 0.152 g/L/h, respectively. These values were at the high level in comparison with Rhodotorula strains cultivated in glycerol media. Besides, fermentation modes did not affect the fatty acid composition of TO2 significantly. In conclusion, blocking the lipid degradation was an applicable strategy to increase the lipid production of Rhodotorula strains without compromising their cell viability. KEY POINTS: • ATG15-like lipase and acyl-CoA oxidase (ACOX2) participated in lipid degradation. • Knockout of ATG15 and ACOX2 increased lipid productivity, and lipid yield coefficient. • Cell viability maintained at high level in the knockout mutants during fermentation.

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.

Responses of Rhodotorula mucilaginosa under Pb(II) stress: carotenoid production and budding.

Rhodotorula mucilaginosa resists heavy metal (HM) stress because of its abundant extracellular polymeric substances and functional vesicles. In this study, we provided new insights into its survival strategies at both biochemical and genetic levels. After lead exposure, carotenoid biosynthesis was initiated within 24 h incubation and then increased to the maximum after 96 h of incubation. Raman analysis confirmed that carotenoids (primarily ß-carotene) were the major identifiable chemical substances on the cell surface. Moreover, the increased carotenoid production was accompanied by a rising budding rate, ~40% higher than that in the cultures without Pb. During the 96 h of incubation, the driving force for Pb accumulation was assigned to this elevated budding rate. After 96 h, biosorption was primarily attributed to the enhanced antioxidant ability of the single cells during carotenoid production. Furthermore, the yeast budding cells demonstrated an evidently heterogeneous biosorption of Pb, i.e., the rejuvenated daughters had a relatively lower Pb level than the mother cells. This resulted in the protection of the buds from Pb stress. After investigating phosphorus uptake and the RNA sequencing data, we finally confirmed two tightly correlated pathways that resist HM stress, i.e., biochemical (carotenoid production) and reproductive (healthy buds) pathways.

CUT&RUN identifies centromeric DNA regions of Rhodotorula toruloides IFO0880.

Rhodotorula toruloides has been increasingly explored as a host for bioproduction of lipids, fatty acid derivatives and terpenoids. Various genetic tools have been developed, but neither a centromere nor an autonomously replicating sequence (ARS), both necessary elements for stable episomal plasmid maintenance, has yet been reported. In this study, cleavage under targets and release using nuclease (CUT&RUN), a method used for genome-wide mapping of DNA-protein interactions, was used to identify R. toruloides IFO0880 genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and four were tested for, but did not show, ARS activity. These centromeric sequences contained below average GC content, corresponded to transcriptional cold spots, were primarily nonrepetitive and shared some vestigial transposon-related sequences but otherwise did not show significant sequence conservation. Future efforts to identify an ARS in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative ARS elements.

Metabolic engineering of Rhodotorula toruloides for resveratrol production.

BACKGROUND: Resveratrol is a plant-derived phenylpropanoid with diverse biological activities and pharmacological applications. Plant-based extraction could not satisfy ever-increasing market demand, while chemical synthesis is impeded by the existence of toxic impurities. Microbial production of resveratrol offers a promising alternative to plant- and chemical-based processes. The non-conventional oleaginous yeast Rhodotorula toruloides is a potential workhorse for the production of resveratrol that endowed with an efficient and intrinsic bifunctional phenylalanine/tyrosine ammonia-lyase (RtPAL) and malonyl-CoA pool, which may facilitate the resveratrol synthesis when properly rewired. RESULTS: Resveratrol showed substantial stability and would not affect the R. toruloides growth during the yeast cultivation in flasks. The heterologus resveratrol biosynthesis pathway was established by introducing the 4-coumaroyl-CoA ligase (At4CL), and the stilbene synthase (VlSTS) from Arabidopsis thaliana and Vitis labrusca, respectively. Next, The resveratrol production was increased by 634% through employing the cinnamate-4-hydroxylase from A. thaliana (AtC4H), the fused protein At4CL::VlSTS, the cytochrome P450 reductase 2 from A. thaliana (AtATR2) and the endogenous cytochrome B5 of R. toruloides (RtCYB5). Then, the related endogenous pathways were optimized to affect a further 60% increase. Finally, the engineered strain produced a maximum titer of 125.2 mg/L resveratrol in YPD medium. CONCLUSION: The non-conventional oleaginous yeast R. toruloides was engineered for the first time to produce resveratrol. Protein fusion, co-factor channeling, and ARO4 and ARO7 overexpression were efficient for improving resveratrol production. The results demonstrated the potential of R. toruloides for resveratrol and other phenylpropanoids production.

Metabolic engineering of Rhodotorula toruloides IFO0880 improves C16 and C18 fatty alcohol production from synthetic media.

BACKGROUND: The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. RESULTS: Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides, while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. CONCLUSIONS: The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

Altering the fatty acid profile of Yarrowia lipolytica to mimic cocoa butter by genetic engineering of desaturases.

BACKGROUND: Demand for Cocoa butter is steadily increasing, but the supply of cocoa beans is naturally limited and under threat from global warming. One route to meeting the future demand for cocoa butter equivalent (CBE) could be to utilize microbial cell factories such as the oleaginous yeast Yarrowia lipolytica. RESULTS: The main goal was to achieve triacyl-glycerol (TAG) storage lipids in Y. lipolytica mimicking cocoa butter. This was accomplished by replacing the native Δ9 fatty acid desaturase (Ole1p) with homologs from other species and changing the expression of both Ole1p and the Δ12 fatty acid desaturase (Fad2p). We thereby abolished the palmitoleic acid and reduced the linoleic acid content in TAG, while the oleic acid content was reduced to approximately 40 percent of the total fatty acids. The proportion of fatty acids in TAG changed dramatically over time during growth, and the fatty acid composition of TAG, free fatty acids and phospholipids was found to be very different. CONCLUSIONS: We show that the fatty acid profile in the TAG of Y. lipolytica can be altered to mimic cocoa butter. We also demonstrate that a wide range of fatty acid profiles can be achieved while maintaining good growth and high lipid accumulation, which, together with the ability of Y. lipolytica to utilize a wide variety of carbon sources, opens up the path toward sustainable production of CBE and other food oils.