Engineering oleaginous red yeasts as versatile chassis for the production of oleochemicals and valuable compounds: Current advances and perspectives.

Enabling the transition towards a future circular bioeconomy based on industrial biomanufacturing necessitates the development of efficient and versatile microbial platforms for sustainable chemical and fuel production. Recently, there has been growing interest in engineering non-model microbes as superior biomanufacturing platforms due to their broad substrate range and high resistance to stress conditions. Among these non-conventional microbes, red yeasts belonging to the genus Rhodotorula have emerged as promising industrial chassis for the production of specialty chemicals such as oleochemicals, organic acids, fatty acid derivatives, terpenoids, and other valuable compounds. Advancements in genetic and metabolic engineering techniques, coupled with systems biology analysis, have significantly enhanced the production capacity of red yeasts. These developments have also expanded the range of substrates and products that can be utilized or synthesized by these yeast species. This review comprehensively examines the current efforts and recent progress made in red yeast research. It encompasses the exploration of available substrates, systems analysis using multi-omics data, establishment of genome-scale models, development of efficient molecular tools, identification of genetic elements, and engineering approaches for the production of various industrially relevant bioproducts. Furthermore, strategies to improve substrate conversion and product formation both with systematic and synthetic biology approaches are discussed, along with future directions and perspectives in improving red yeasts as more versatile biotechnological chassis in contributing to a circular bioeconomy. The review aims to provide insights and directions for further research in this rapidly evolving field. Ultimately, harnessing the capabilities of red yeasts will play a crucial role in paving the way towards next-generation sustainable bioeconomy.

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.

De Novo Synthesis of Resveratrol from Sucrose by Metabolically Engineered Yarrowia lipolytica.

Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis, 4-coumarate CoA ligase from Nicotiana tabacum, and stilbene synthase from Vitis vinifera. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana, malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.

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.

Evaluation of anti-tumor effect of the exopolysaccharide from new cold-adapted yeast, Rhodotorula mucilaginosa sp. GUMS16 on chronic myeloid leukemia K562 cell line.

Recently, the development and application of fungal exopolysaccharides (EPS) as natural biopolymers are on the rise. The present study is based on the investigation of possible antiproliferative and antioxidant activities of EPS from the Rhodotorula mucilaginosa sp. GUMS16 on BCR-ABL positive cells (K562). The cytotoxicity, colony formation assays lactate and dehydrogenase (LDH) activity were performed to assess the possible cancer cell death. To elucidate the underlying antiproliferative mechanism of the EPS, cell cycle analysis following real-time PCR (gene expression assessment) were evaluated. The results indicated that, the EPS with an IC50 dose of 1500 µg/ml, reduced the viability of K562 cells without having toxic effects on normal cells as well as decrease in size and number of colonies in EPS-treated group (p < 0.0001). The increase of LDH was 2.75 times more than the control (p < 0.0001). Gene expression revealed up- and down-regulation of apoptotic and anti-apoptotic genes in EPS group compared with the control. Moreover, the DPPH scavenging activity of the EPS in treated cells was significantly higher than the control group (p < 0.0001). Taken together, we concluded that the EPS from GUMS16 strain is able to inhibit the growth of K562 cells besides having antioxidant activities.

Metabolomics reveals the mechanism of Antarctic yeast Rhodotorula mucliaginosa AN5 to cope with cadmium stress.

Heavy metal pollution in Antarctica has far exceeded expectations. Antarctic yeast is widely present in polar marine environment. The mechanisms of metabolomics effect of heavy metal on polar yeast have not been reported previously. In this study, gas chromatography-mass spectrometry (GC-MS) wascarried out to performed the metabolite profiling analysis of Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5 exposed to different cadmium (Cd) stresses of 5 mM (HM5), 10 mM (HM10) and 20 mM (HM20), respectively. Metabolic profile analysis showed that the composition and contents of cellular metabolites have been altered by cadmium. 93 different metabolites were identified altogether, among which 23, 58 and 81 different metabolites were found in HM5, HM10 and HM20 group respectively. MetaboAnalyst analysis showed that in HM5, HM10 and HM20 groups, 12, 24 and 31 metabolic pathways were involved in the stress of cadmium to R. mucilaginosa, respectively. By contrasting with Kyoto Encyclopedia of Genes and Genomes database, we discovered that exposure of yeast AN5 to Cd stress resulted in profound biochemical changes including amino acids, organic acids and saccharides. These results will supply a nonnegligible basis of studying the adaptive resistance mechanism of Antarctic yeast Rhodotorula mucilaginosa to heavy metal.

Rhodotorula toruloides: an ideal microbial cell factory to produce oleochemicals, carotenoids, and other products.

Requirement of clean energy sources urges us to find substitutes for fossil fuels. Microorganisms provide an option to produce feedstock for biofuel production by utilizing inexpensive, renewable biomass. Rhodotorula toruloides (Rhodosporidium toruloides), a non-conventional oleaginous yeast, can accumulate intracellular lipids (single cell oil, SCO) more than 70% of its cell dry weight. At present, the SCO-based biodiesel is not a price-competitive fuel to the petroleum diesel. Many efforts are made to cut the cost of SCO by strengthening the performance of genetically modified R. toruloides strains and by valorization of low-cost biomass, including crude glycerol, lignocellulosic hydrolysates, food and agro waste, wastewater, and volatile fatty acids. Besides, optimization of fermentation and SCO recovery processes are carefully studied as well. Recently, new R. toruloides strains are developed via metabolic engineering and synthetic biology methods to produce value-added chemicals, such as sesquiterpenes, fatty acid esters, fatty alcohols, carotenoids, and building block chemicals. This review summarizes recent advances in the main aspects of R. toruloides studies, namely, construction of strains with new traits, valorization of low-cost biomass, process detection and optimization, and product recovery. In general, R. toruloides is a promising microbial cell factory for production of biochemicals.

Transcriptomic and Metabolomic Analyses Provide Insights into the Enhancement of Torulene and Torularhodin Production in Rhodotorula glutinis ZHK under Moderate Salt Conditions.

Carotenoids are a group of tetraterpene pigments widely used in the food, pharmaceutical, and cosmetic industries. Torulene, torularhodin, and ß-carotene, three principal carotenoids synthesized by Rhodotorula glutinis ZHK, possess strong health-promoting properties such as antioxidant, provitamin A, and antitumor. Here, the effect of different salt conditions on carotenoids production of R. glutinisZHK was investigated. The results showed that the total carotenoids were significantly enhanced in 0.5 M (3.91 mg/L) and 0.75 M (5.41 mg/L) NaCl treatments than that in 1.0 M (0.35 mg/L) and control (1.42 mg/L) after 120 h of cultivation. Of which, the increase in torulene and torularhodin production acts as the main contributor to the enhancement of total carotenoids. Transcriptome profiling revealed that salt stress efficiently promotes the gene expression of crtI, which could explain the molecular mechanisms of the enhanced torulene and torularhodin production under salt stress. Further experiments indicated that torulene and torularhodin play an important role in quenching excrescent reactive oxygen species induced by salt stress. Together, the present study reports an effective strategy for simultaneously improving torulene and torularhodin production in R. glutinis ZHK.

Near-perfect kinetic resolution of o-methylphenyl glycidyl ether by RpEH, a novel epoxide hydrolase from Rhodotorula paludigena JNU001 with high stereoselectivity.

In order to provide more alternative epoxide hydrolases for industrial production, a novel cDNA gene Rpeh-encoding epoxide hydrolase (RpEH) of Rhodotorula paludigena JNU001 identified by 26S rDNA sequence analysis was amplified by RT-PCR. The open-reading frame (ORF) of Rpeh was 1236 bp encoding RpEH of 411 amino acids and was heterologously expressed in Escherichia coli BL21(DE3). The substrate spectrum of expressed RpEH showed that the transformant E. coli/Rpeh had excellent enantioselectivity to 2a, 3a, and 5a-10a, among which E. coli/Rpeh had the highest activity (2473 U/g wet cells) and wonderful enantioselectivity (E = 101) for 8a, and its regioselectivity coefficients, αR and ßS, toward (R)- and (S)-8a were 99.7 and 83.2%, respectively. Using only 10 mg wet cells/mL of E. coli/Rpeh, the near-perfect kinetic resolution of rac-8a at a high concentration (1000 mM) was achieved within 2.5 h, giving (R)-8a with more than 99% enantiomeric excess (ees) and 46.7% yield and producing (S)-8b with 93.2% eep and 51.4% yield with high space-time yield (STY) for (R)-8a and (S)-8b were 30.6 and 37.3 g/L/h.

Genomics- and Metabolomics-Based Investigation of the Deep-Sea Sediment-Derived Yeast, Rhodotorula mucilaginosa 50-3-19/20B.

Red yeasts of the genus Rhodotorula are of great interest to the biotechnological industry due to their ability to produce valuable natural products, such as lipids and carotenoids with potential applications as surfactants, food additives, and pharmaceuticals. Herein, we explored the biosynthetic potential of R. mucilaginosa 50-3-19/20B collected from the Mid-Atlantic Ridge using modern genomics and untargeted metabolomics tools. R. mucilaginosa 50-3-19/20B exhibited anticancer activity when grown on PDA medium, while antimicrobial activity was observed when cultured on WSP-30 medium. Applying the bioactive molecular networking approach, the anticancer activity was linked to glycolipids, namely polyol esters of fatty acid (PEFA) derivatives. We purified four PEFAs (1-4) and the known methyl-2-hydroxy-3-(1H-indol-2-yl)propanoate (5). Their structures were deduced from NMR and HR-MS/MS spectra, but 1-5 showed no anticancer activity in their pure form. Illumina-based genome sequencing, de novo assembly and standard biosynthetic gene cluster (BGC) analyses were used to illustrate key components of the PEFA biosynthetic pathway. The fatty acid producing BGC3 was identified to be capable of producing precursors of PEFAs. Some Rhodotorula strains are able to convert inulin into high-yielding PEFA and cell lipid using a native exo-inulinase enzyme. The genomic locus for an exo-inulinase enzyme (g1629.t1), which plays an instrumental role in the PEFA production via the mannitol biosynthesis pathway was identified. This is the first untargeted metabolomics study on R. mucilaginosa providing new genomic insights into PEFA biosynthesis.

Molecular identification and antifungal susceptibility testing of Pucciniomycotina red yeast clinical isolates from Rio de Janeiro, Brazil.

Infections caused by Rhodotorula spp. are increasing worldwide. This study identified, through the light of the new taxonomic advances on the subphylum Pucciniomycotina, 16 isolates from blood cultures and compared their antifungal susceptibility on microdilution and gradient diffusion methods. Internal transcriber spacer sequencing identified Rhodotorula mucilaginosa (n = 12), Rhodotorula toruloides (n = 2), Rhodotorula dairenensis (n = 1), and Cystobasidium minutum (n = 1). Amphotericin B was the most effective drug. A good essential agreement was observed on MIC values of amphotericin B and voriconazole determined by the two methods. Therefore, the gradient method is useful for susceptibility tests of R. mucilaginosa against these drugs.

Improvement of selenium enrichment in Rhodotorula glutinis X-20 through combining process optimization and selenium transport.

Selenium-enriched yeast can transform toxic inorganic selenium into absorbable organic selenium, which is of great significance for human health and pharmaceutical industry. A yeast Rhodotorula glutinis X-20 we obtained before has good selenium-enriched ability, but its selenium content is still low for industrial application. In this study, strategies of process optimization and transport regulation of selenium were thus employed to further improve the cell growth and selenium enrichment. Through engineering phosphate transporters from Saccharomyces cerevisiae into R. glutinis X-20, the selenium content was increased by 21.1%. Through using mixed carbon culture (20 g L-1, glycerol: glucose 3:7), both biomass and selenium content were finally increased to 5.3 g L-1 and 5349.6 µg g-1 (cell dry weight, DWC), which were 1.14 folds and 6.77 folds compared to their original values, respectively. Our results indicate that high selenium-enrichment ability and biomass production can be achieved through combining process optimization and regulation of selenium transport.

Cloning and functional characterization of a novel metallothionein gene in Antarctic sea-ice yeast (Rhodotorula mucilaginosa).

Metallothionein (MT) is a low-molecular-weight protein with a high metal binding capacity and plays a key role in organism adaptation to heavy metals. In this study, a metallothionein gene was successfully cloned and sequenced from Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5. Nucleotide sequencing and analysis revealed that the gene had four exons interrupted by three introns. MTs complementary DNA (named as RmMT) had an open reading frame of 321 bp encoding a 106 amino acid protein with a predicted molecular weight of 10.3 kDa and pI of 8.49. The number of amino acids and distribution of cysteine residues indicated that RmMT was a novel family of fungal MTs. Quantitative real-time polymerase chain reaction analysis showed that RmMT expression was elevated under copper-induced stress. The RmMT gene was transferred into E. coli and the RmMT expressing bacteria showed improved tolerance to copper ion and increased accumulation of heavy metals, such as Cu2+ , Pb2+ , Zn2+ , Cd2+ , and Ag+ . Moreover, in vitro studies, purified recombinant RmMT demonstrated that it could be used as a good scavenger of superoxide anion, hydroxyl, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals. In summary, these results demonstrate that RmMT plays a key role in the tolerance and bioaccumulation of heavy metals.

Carotenoid production in sugarcane juice and synthetic media supplemented with nutrients by Rhodotorula rubra l02

ABSTRACT In order for the use of biological carotenoids to become feasible, it is necessary to have adequate low cost sources and improved methods of cultivation. The aim of this study was to evaluate the effect of supplementation with nitrogen, phosphorus, zinc, and magnesium, on the biomass and carotenoid volumetric production by yeast Rhodotorula rubra L02 using a complex medium (sugarcane juice) and synthetic media (sucrose and maltose) as substrates. The experimental design used for each substrate was randomized in blocks with 16 treatments and 3 repetitions. The treatments were compound for 15 different combinations of nutrients (N; Mg; Zn; P, N + Mg; N + Zn; N + P; Mg + Zn; Mg + P; Zn + P; N + P + Zn; N + P + Mg; N + Zn + Mg; P + Zn + Mg; N + Zn + Mg + P) alone and combined, and a control. The results were submitted to analysis of variance and Tukey test at 5% significance level. Among the treatments evaluated, the highest production of dry biomass, with both maltose and sucrose, was observed for Mg (1.60 g/L and 1.94 g/L, respectively). Additionally, another treatment that stood out in terms of biomass production was the control treatment with maltose (1.54 g/L). After the incubation time, killer activity was not observed since there was no formation of inhibition halo around the L02 yeast.

Ergothioneine production using Methylobacterium species, yeast, and fungi.

Ergothioneine (EGT) is a sulfur-containing, anti-oxidative amino acid derived from histidine. EGT is synthesized in bacteria and fungi but not in animals and plants, and is now recognized as important for human health. Its cost-effective fermentative production has not been elucidated due to the lack of information for productive microorganisms. In this study, we doubled the gene copy for EGT synthesis and deleted the histidine ammonia-lyase gene in a potent EGT-producing methylotrophic bacterium Methylobacterium aquaticum strain 22A, and optimized its culture conditions, resulting in increased EGT production of 7.0 mg EGT/g dry cell weight and 100 µg EGT/5 mL/7 days. In addition, through screening we found EGT-producing eukaryotic strains of Aureobasidium pullulans and Rhodotorula mucilaginosa, which can produce 1.0 and 3.2 mg EGT/g dry cell weight, 70 and 120 µg EGT/5 mL/7 days, respectively. This study proposes practical uses of potent EGT-producing recombinant Methylobacterium species and non-recombinant yeast and fungal strains.

Mixed Infection of Respiratory Tract in a Dog Caused by Rhodotorula mucilaginosa and Trichosporon jirovecii: A Case Report.

This report describes the isolation of two environmental fungi: Rhodotorula mucilaginosa and Trichosporon jirovecii accompanied by Pseudomonas aeruginosa and Escherichia coli from a dog with bronchotracheitis. All microorganisms were isolated routinely from a mucopurulent discharge, obtained during bronchoscopy from laryngotracheal area. The initial identification of yeasts was confirmed by API Candida and by molecular analysis of internal transcribed spacer region. Administered antimicrobial treatment with Marbofloxacin and Canizol has brought the improvement in the dogs' health status. The final results of control mycological culture were negative. Most probably underlying hypothyroidism and the use of steroids were the factors predisposing this patient to opportunistic infection of mixed aetiology. As far as we are concerned, this is the first case of dogs' respiratory tract infection caused by R. mucilaginosa and T. jirovecii.

Functional identification of glutamate cysteine ligase and glutathione synthetase in the marine yeast Rhodosporidium diobovatum.

Glutathione (GSH) fulfills a variety of metabolic functions, participates in oxidative stress response, and defends against toxic actions of heavy metals and xenobiotics. In this study, GSH was detected in Rhodosporidium diobovatum by high-performance liquid chromatography (HPLC). Then, two novel enzymes from R. diobovatum were characterized that convert glutamate, cysteine, and glycine into GSH. Based on reverse transcription PCR, we obtained the glutathione synthetase gene (GSH2), 1866 bp, coding for a 56.6-kDa protein, and the glutamate cysteine ligase gene (GSH1), 2469 bp, coding for a 90.5-kDa protein. The role of GSH1 and GSH2 for the biosynthesis of GSH in the marine yeast R. diobovatum was determined by deletions using the CRISPR-Cas9 nuclease system and enzymatic activity. These results also showed that GSH1 and GSH2 were involved in the production of GSH and are thus being potentially useful to engineer GSH pathways. Alternatively, pET-GSH constructed using vitro recombination could be used to detect the function of genes related to GSH biosynthesis. Finally, the fermentation parameters determined in the present study provide a reference for industrial GSH production in R. diobovatum.

Cloning and characterization of iron-superoxide dismutase in Antarctic yeast strain Rhodotorula mucilaginosa AN5.

A novel superoxide dismutase gene from Antarctic yeast Rhodotorula mucilaginosa AN5 was cloned, sequenced, and then expressed in Escherichia coli. The R. mucilaginosa AN5 SOD (RmFeSOD) gene was 639 bp open reading frame in length, which encoded a protein of 212 amino acids with a deduced molecular mass of 23.5 kDa and a pI of 7.89. RmFeSOD was identified as iron SOD type with a natural status of homodimer. The recombinant RmFeSOD showed good pH stability in the pH 1.0-9.0 after 1 h incubation. Meanwhile, it was found to behave relatively high thermostability, and maintained more than 80% activity at 50 °C for 1 h. By addition of 1 mM metal ions, the enzyme activity increased by Zn2+ , Cu2+ , Mn2+ , and Fe3+ , and inhibited only by Mg2+ . RmFeSOD showed relatively low tolerance to some compounds, such as PMSF, SDS, Tween-80, Triton X-100, DMSO, ß-ME, and urea. However, DTT showed no inhibition to enzyme activity. Using copper stress experiment, the RmFeSOD recombinant E. coli exhibited better growth than non-recombinant bacteria, which revealed that RmFeSOD might play an important role in the adaptability of heavy metals.

Two acetyl-CoA synthetase isoenzymes are encoded by distinct genes in marine yeast Rhodosporidium diobovatum.

OBJECTIVES: Two genes encoding two acetyl-CoA synthetase (ACS) isoenzymes have been identified in the marine yeast Rhodosporidium diobovatum MCCC 2A00023. RESULTS: ACS1 encoded a polypeptide with a sequence of 578 amino acid residues, a predicted molecular weight of 63.73 kDa, and pI of 8.14, while the ACS2 encoded a polypeptide containing 676 amino acid residues with a deduced molecular mass of 75.61 kDa and a pI of 5.95. Biological activity of Acs1p and Acs2p was confirmed by heterologous expression in Escherichia coli. A 1.5-kb DNA fragment of the ACS1 gene and a 2.7-kb DNA fragment of the ACS2 gene were deleted using the RNA guide CRISPR-Cas9 system. The strain lacking ACS1 was unable to grow on acetate and ethanol media, while the ACS2 deletant was unable to grow on glucose medium. ACS1-ACS2 double mutants of R. diobovatum were non-viable. CONCLUSIONS: ACS isoenzymes are essential to the yeast metabolism, and other sources of ACSs cannot compensate for the lack of ACSs encoded by the two genes.