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.

Diversity, distribution, and bioprospecting potentials of carotenogenic yeast from mangrove ecosystem.

Microbial production of carotenoids has gained significant interest for its cost-effectiveness and sustainable nature. This study focuses on 47 red-pigmented yeasts isolated from sediments and plant parts of 13 species of mangrove trees. The relative abundance and distribution of these yeasts varied with plant species and plant parts. The highest number of red yeasts was associated with the mangrove plant Avicennia officinalis (32%). Notably, the leaves harbored the highest percentage (45%) of carotenogenic yeasts, and definite compartmentalization of these yeast species was noticed in mangrove plant parts. All the isolates were molecularly identified and they belonged to the genera of Rhodotorula, Rhodosporidiobolus, and Cryptococcus. The diversity of the pigmented yeasts isolated from A. officinalis was found to be the greatest. Among these strains, Rhodotorula mucilaginosa PV 8 was identified as the most potent producer of carotenoid pigment. Under optimized conditions of physical parameters - 28 °C, pH 5, and 15% salinity led to biomass production of 9.2 ± 0.12 g/L DCW and a pigment yield of 194.78 µg/g. The pigment produced by PV 8 was identified as ß-carotene by thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR). This ß-carotene demonstrated strong antioxidant activity. Moreover, the carotenoid displayed promising antibacterial activity against multidrug-resistant organisms, including Aeromonas sp. and Vibrio sp. In vitro studies revealed the probiotic traits of PV 8. The cytotoxicity of R. mucilaginosa PV 8 was assessed in the invertebrate model Artemia salina and the survival rate showed that it was non-toxic. Furthermore, the ß-carotene from PV 8 demonstrated the ability to transfer its vibrant color to various food products, maintaining color stability even under varied conditions. This research underscores the potential of R. mucilaginosa PV 8, as a versatile and valuable resource for the production of carotenoids.

Glucosylglycerol and proline reverse the effects of glucose on Rhodosporidium toruloides lifespan.

Rhodosporidium toruloides is a novel cell factory used to synthesis carotenoids, biosurfactants, and biofuel feedstocks. However, research on R. toruloides has generally centred on the manufacture of biochemicals, while analyses of its longevity have received scant attention. Understanding of R. toruloides longevity under different nutrient conditions could help to improve its biotechnological significance and metabolite production. Glucosylglycerol (GG) and proline are osmoprotectants that could revert the harmful effects of environmental stress. This study examined how GG and proline affect R. toruloides strain longevity under glucose nutrimental stress. Herein, we provide evidence that GG and proline enhance cell performance and viability. These compatible solutes neutralises the pro-ageing effects of high glucose (10% glucose) on the yeast cell and reverse its cellular stress. GG exhibits the greatest impact on lifespan extension at 100 mM, whereas proline exerts effect at 2 mM. Our data reveal that these compounds significantly affect the culture medium osmolarity. Moreso, GG and proline decreased ROS production and mitohormetic lifespan regulation, respectively. The data indicates that these solutes (proline and GG) support the longevity of R. toruloides at a pro-ageing high glucose culture condition.

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.

Identification of Irpex and Rhodotorula on surveillance bronchoscopy in a pediatric lung transplant recipient: A case report and review of literature of these atypical fungal organisms.

BACKGROUND: Invasive fungal disease (IFD) is a frequent complication in pediatric lung transplant recipients, occurring in up to 12% of patients in the first year. Risk factors for infection include impaired lung defenses and intense immunosuppressive regimens. While most IFD occurs from Aspergillus, other fungal conidia are continuously inhaled, and infections with fungi on a spectrum of human pathogenicity can occur. CASE REPORT: We report a case of a 17-year-old lung transplant recipient in whom Irpex lacteus and Rhodotorula species were identified during surveillance bronchoscopy. She was asymptomatic and deemed to be colonized by Irpex lacteus and Rhodotorula species following transplant. 2 years after transplantation, she developed a fever, respiratory symptoms, abnormal lung imaging, and histological evidence of acute and chronic bronchitis on transbronchial biopsy. After developing symptoms concerning for a pulmonary infection and graft dysfunction, she was treated for a presumed IFD. Unfortunately, further diagnostic testing could not be performed at this time given her tenuous clinical status. Despite the initiation of antifungal therapy, her graft function continued to decline resulting in a second lung transplantation. CONCLUSIONS: This case raises the concern for IFD in lung transplant recipients from Irpex species. Further investigation is needed to understand the pathogenicity of this organism, reduce the incidence and mortality of IFD in lung transplant recipients, and refine the approach to diagnosis and manage the colonization and isolation of rare, atypical fungal pathogens in immunocompromised hosts.

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.

Biotechnological potential of red yeast isolated from birch forests in Poland.

OBJECTIVES: This study aimed to isolate red yeast from sap, bark and slime exudates collected from Polish birch forests and then assessment of their biotechnological potential. RESULTS: 24 strains of red yeast were isolated from the bark, sap and spring slime fluxes of birch (Betula pendula). Strains belonging to Rhodotorula mucilaginosa (6), Rhodosporidiobolus colostri (4), Cystrofilobasidium capitaum (3), Phaffia rhodozyma (3) and Cystobasidium psychroaquaticum (3) were dominant. The highest efficiency of carotenoid biosynthesis (5.04 mg L-1) was obtained by R. mucilaginosa CMIFS 004, while lipids were most efficiently produced by two strains of P. rhodozyma (5.40 and 5.33 g L-1). The highest amount of exopolysaccharides (3.75 g L-1) was produced by the R. glutinis CMIFS 103. Eleven strains showed lipolytic activity, nine amylolytic activity, and only two proteolytic activity. The presence of biosurfactants was not found. The growth of most species of pathogenic moulds was best inhibited by Rhodotorula yeasts. CONCLUSION: Silver birch is a good natural source for the isolation of new strains of red yeast with wide biotechnological potential.

Sustainable trehalose lipid production by Rhodotorula sp.: a promising bio-based alternative.

Global environmental concerns drive research toward the development of new eco-friendly compounds to replace pollutant chemicals. This study focuses on optimizing the production of trehalose lipids (TLs), which are glycolipid biosurfactants (BS) with various applications like antimicrobial or surface tension reduction. New microorganism sources, growth conditions, medium composition, purification conditions, and physicochemical properties of TLs are studied. Addressing a microscale approach, TLs production was successfully achieved using Rhodotorula sp. and Rhodococcus erythropolis to compare, with different media compositions including glucose-based and salt media supplemented with glycerol, glucose, n-hexadecane, n-dodecane. Liquid-liquid extraction using ethyl acetate and methanol was employed for compound extraction, followed by characterization using analytical methods such as Thin layer chromatography (TLC), High performance liquid chromatography (HPLC), and UHPLC. The produced TLs exhibited a minimum surface tension of 47 mN/m and a critical micellar concentration of 4.4 mg/mL. This study also identified Rhodotorula sp. as a new sustainable producer of TLs with improved productivity.

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.

Cultivation of yeasts on liquid digestate to remove organic pollutants and nutrients and for potential application as co-culture with microalgae.

In this study, the growth of yeast and yeast-like fungi in the liquid digestate from vegetable wastes was investigated in order to remove nutrients and organic pollutants, and for their application as co-culture members with green microalgae. The studied yeast strains were characterized for their assimilative and enzymatic profiles as well as temperature requirements. In the first experimental stage, the growth dynamics of each strain were determined, allowing to select the best yeasts for further studies. In the subsequent stage, the ability of selectants to remove organic pollutants was assessed. Different cultivation media containing respectively 1:3, 1:1, 3:1 vol ratio of liquid digestate and the basal minimal medium were used. Among all tested yeast strains, Rhodotorula mucilaginosa DSM 70825 showed the most promising results, demonstrating the highest potential for removing organic substrates and nutrients. Depending on the medium, this strain achieved 50-80% sCOD, 45-60% tVFAs, 21-45% TN, 33-52% PO43- reduction rates. Similar results were obtained for the strain Candida sp. OR687571. The high nutrient and organics removal efficiency by these yeasts could likely be linked to their ability to assimilate xylose (being the main source of carbon in the liquid digestate). In culture media containing liquid digestate, both yeast strains achieved good viability and proliferation potential. In the liquid digestate medium, R. mucilaginosa and Candida sp. showed vitality at the level of 51.5% and 45.0%, respectively. These strains seem to be a good starting material for developing effective digestate treatment strategies involving monocultures and/or consortia with other yeasts or green microalgae.

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.

Bioproduction of yeast single cell oil with acute oral toxicity study intended for edible oil application.

Human nutrition and health rely on edible oils. Global demand for edible oils is expanding, necessitating the discovery of new natural oil sources subjected to adequate quality and safety evaluation. However, in contrast to other agricultural products, India's edible oil supply is surprisingly dependent on imports. The microbial oil is generated by fermentation of oleaginous yeast Rhodotorula mucilaginosa IIPL32 MTCC 25056 using biodiesel plant byproduct crude glycerol as a fermentable carbon source. Enriched with monounsaturated fatty acid, nutritional indices mapping based on the fatty acid composition of the yeast SCO, suggested its plausible use as an edible oil blend. In the present study, acute toxicity evaluation of the yeast SCO in C57BL/6 mice has been performed by randomly dividing the animals into 5 groups with 50, 300, 2000, and 5000 mg/Kg yeast SCO dosage, respectively, and predicted the median lethal dose (LD50). Detailed blood biochemistry and kidney and liver histopathology analyses were also reported. The functions of the liver enzymes were also evaluated to check and confirm the anticipated toxicity. To determine cell viability and in vitro biocompatibility, the 3T3-L1 cell line and haemolysis tests were performed. The results suggested the plausible use of yeast SCO as an edible oil blend due to its non-toxic nature in mice models.

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.

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.

A Tripartite Interaction among the Basidiomycete Rhodotorula mucilaginosa, N2-Fixing Endobacteria, and Rice Improves Plant Nitrogen Nutrition.

Nitrogen (N) limits crop yield, and improvement of N nutrition remains a key goal for crop research; one approach to improve N nutrition is identifying plant-interacting, N2-fixing microbes. Rhodotorula mucilaginosa JGTA-S1 is a basidiomycetous yeast endophyte of narrowleaf cattail (Typha angustifolia). JGTA-S1 could not convert nitrate or nitrite to ammonium but harbors diazotrophic (N2-fixing) endobacteria (Pseudomonas stutzeri) that allow JGTA-S1 to fix N2 and grow in a N-free environment; moreover, P. stutzeri dinitrogen reductase was transcribed in JGTA-S1 even under adequate N. Endobacteria-deficient JGTA-S1 had reduced fitness, which was restored by reintroducing P. stutzeri JGTA-S1 colonizes rice (Oryza sativa), significantly improving its growth, N content, and relative N-use efficiency. Endofungal P. stutzeri plays a significant role in increasing the biomass and ammonium content of rice treated with JGTA-S1; also, JGTA-S1 has better N2-fixing ability than free-living P. stutzeri and provides fixed N to the plant. Genes involved in N metabolism, N transporters, and NODULE INCEPTION-like transcription factors were upregulated in rice roots within 24 h of JGTA-S1 treatment. In association with rice, JGTA-S1 has a filamentous phase and P. stutzeri only penetrated filamentous JGTA-S1. Together, these results demonstrate an interkingdom interaction that improves rice N nutrition.

Sustainable production of single-cell oil and protein from wastepaper hydrolysate: identification and optimization of a Rhodotorula mucilaginosa strain as a promising yeast.

This study investigated the potential of wastepaper hydrolysate as a sustainable and low-cost carbon source for single-cell oil and protein production, attending to the growing need for alternative feedstocks and waste management strategies. Wastepaper, characterized by its high carbohydrate content, was subjected to enzymatic and chemo-enzymatic treatments for carbohydrate release. The chemo-enzymatic treatment performed better, yielding 65.3 g l-1 of fermentable sugars. A total of 62 yeast strains were screened for single-cell oil accumulation, identifying Rhodotorula mucilaginosa M1K4 as the most advantageous oleaginous yeast. M1K4 lipid production was optimized in liquid culture, and its fatty acid profile was analyzed, showing a high content of industrially valuable fatty acids, particularly palmitic (28%) and oleic (51%). Batch-culture of M1K4 in a 3-l reactor demonstrated the strain's ability to utilize wastepaper hydrolysate as a carbon source, with dry cell weight, total lipid and protein production of 17.7 g l-1, 4.5 g l-1, and 2.1 g l-1, respectively. Wastepaper as a substrate provides a sustainable solution for waste management and bioproduction. This research highlights the potential of R. mucilaginosa for lipid and protein production from wastepaper hydrolysate.

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.