Toruloides Strains Research Articles

Ultraviolet-diethyl sulfate composite mutagenesis of Rhodosporidium toruloides and culture optimization to improve carotenoid production

Rhodosporidium toruloides is a carotenoid-producing oleaginous yeast, and its carotenoids are of considerable value to food, chemical, and medical industries. In this study, to improve carotenoid production capacity, the parent R. toruloides strain (CK) was compound mutagenized using ultraviolet-diethyl sulfate (UV-DES), and cultivation conditions were simultaneously optimized to enhance the carotenoid synthesis ability of the mutant strain (M5). The carotenoid content was significantly increased following UV-DES compound mutagenesis (CK = 249.34 μg/g, M5 = 535.28 μg/g). The carotenoid content of M5 was increased to 763.85 μg/g under optimal culture conditions. In addition, mutagenesis altered the carotenoid components, and torulene and torularhodin levels were also increased following mutation, leading to changes in antioxidant capacity. Real-time quantitative PCR results showed that expression of six genes (CAR1, CAR2, CrtE, CrtI, ME1, HMG1) related to carotenoid accumulation were significantly upregulated by UV-DES composite mutagenesis. In conclusion, combined UV-DES mutagenesis and optimal culture conditions promoted the biosynthesis of carotenoids in R. toruloides.

Cell envelope and stress-responsive pathways underlie an evolved oleaginous Rhodotorula toruloides strain multi-stress tolerance

BackgroundThe red oleaginous yeast Rhodotorula toruloides is a promising cell factory to produce microbial oils and carotenoids from lignocellulosic hydrolysates (LCH). A multi-stress tolerant strain towards four major inhibitory compounds present in LCH and methanol, was derived in our laboratory from strain IST536 (PYCC 5615) through adaptive laboratory evolution (ALE) under methanol and high glycerol selective pressure.ResultsComparative genomic analysis suggested the reduction of the original strain ploidy from triploid to diploid, the occurrence of 21,489 mutations, and 242 genes displaying copy number variants in the evolved strain. Transcriptomic analysis identified 634 genes with altered transcript levels (465 up, 178 down) in the multi-stress tolerant strain. Genes associated with cell surface biogenesis, integrity, and remodelling and involved in stress-responsive pathways exhibit the most substantial alterations at the genome and transcriptome levels. Guided by the suggested stress responses, the multi-stress tolerance phenotype was extended to osmotic, salt, ethanol, oxidative, genotoxic, and medium-chain fatty acid-induced stresses.ConclusionsThe comprehensive analysis of this evolved strain provided the opportunity to get mechanistic insights into the acquisition of multi-stress tolerance and a list of promising genes, pathways, and regulatory networks, as targets for synthetic biology approaches applied to promising cell factories, toward more robust and superior industrial strains. This study lays the foundations for understanding the mechanisms underlying tolerance to multiple stresses in R. toruloides, underscoring the potential of ALE for enhancing the robustness of industrial yeast strains.

Engineering non-conventional yeast Rhodotorula toruloides for ergothioneine production

BackgroundErgothioneine (EGT) is a distinctive sulfur-containing histidine derivative, which has been recognized as a high-value antioxidant and cytoprotectant, and has a wide range of applications in food, medical, and cosmetic fields. Currently, microbial fermentation is a promising method to produce EGT as its advantages of green environmental protection, mild fermentation condition, and low production cost. However, due to the low-efficiency biosynthetic process in numerous cell factories, it is still a challenge to realize the industrial biopreparation of EGT. The non-conventional yeast Rhodotorula toruloides is considered as a potential candidate for EGT production, thanks to its safety for animals and natural ability to synthesize EGT. Nevertheless, its synthesis efficiency of EGT deserves further improvement.ResultsIn this study, out of five target wild-type R. toruloides strains, R. toruloides 2.1389 (RT1389) was found to accumulate the highest EGT production, which could reach 79.0 mg/L at the shake flask level on the 7th day. To achieve iterative genome editing in strain RT1389, CRISPR-assisted Cre recombination (CACR) method was established. Based on it, an EGT-overproducing strain RT1389-2 was constructed by integrating an additional copy of EGT biosynthetic core genes RtEGT1 and RtEGT2 into the genome, the EGT titer of which was 1.5-fold increase over RT1389. As the supply of S-adenosylmethionine was identified as a key factor determining EGT production in strain RT1389, subsequently, a series of gene modifications including S-adenosylmethionine rebalancing were integrated into the strain RT1389-2, and the resulting mutants were rapidly screened according to their EGT production titers with a high-throughput screening method based on ergothionase. As a result, an engineered strain named as RT1389-3 was selected with a production titer of 267.4 mg/L EGT after 168 h in a 50 mL modified fermentation medium.ConclusionsThis study characterized the EGT production capacity of these engineered strains, and demonstrated that CACR and high-throughput screening method allowed rapid engineering of R. toruloides mutants with improved EGT production. Furthermore, this study provided an engineered RT1389-3 strain with remarkable EGT production performance, which had potential industrial application prospects.

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 100mM, whereas proline exerts effect at 2mM. 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.

Scale-up of microbial lipid and bioethanol production from oilcane

Microbial oils are a sustainable biomass-derived substitute for liquid fuels and vegetable oils. Oilcane, an engineered sugarcane with superior feedstock characteristics for biodiesel production, is a promising candidate for bioconversion. This study describes the processing of oilcane stems into juice and hydrothermally pretreated lignocellulosic hydrolysate and their valorization to ethanol and microbial oil using Saccharomyces cerevisiae and engineered Rhodosporidium toruloides strains, respectively. A bioethanol titer of 106 g/L was obtained from S. cerevisiae grown on oilcane juice in a 3 L fermenter, and a lipid titer of 8.8 g/L was obtained from R. toruloides grown on oilcane hydrolysate in a 75 L fermenter. Oil was extracted from the R. toruloides cells using supercritical CO2, and the observed fatty acid profile was consistent with previous studies on this strain. These results demonstrate the feasibility of pilot-scale lipid production from oilcane hydrolysate as part of an integrated bioconversion strategy.

Tolerance of engineered Rhodosporidium toruloides to sorghum hydrolysates during batch and fed-batch lipid production

BackgroundOleaginous yeasts are a promising candidate for the sustainable conversion of lignocellulosic feedstocks into fuels and chemicals, but their growth on these substrates can be inhibited as a result of upstream pretreatment and enzymatic hydrolysis conditions. Previous studies indicate a high citrate buffer concentration during hydrolysis inhibits downstream cell growth and ethanol fermentation in Saccharomyces cerevisiae. In this study, an engineered Rhodosporidium toruloides strain with enhanced lipid accumulation was grown on sorghum hydrolysate with high and low citrate buffer concentrations.ResultsBoth hydrolysis conditions resulted in similar sugar recovery rates and concentrations. No significant differences in cell growth, sugar utilization rates, or lipid production rates were observed between the two citrate buffer conditions during batch fermentation of R. toruloides. Under fed-batch growth on low-citrate hydrolysate a lipid titer of 16.7 g/L was obtained.ConclusionsCitrate buffer was not found to inhibit growth or lipid production in this engineered R. toruloides strain, nor did reducing the citrate buffer concentration negatively affect sugar yields in the hydrolysate. As this process is scaled-up, $131 per ton of hydrothermally pretreated biomass can be saved by use of the lower citrate buffer concentration during enzymatic hydrolysis.Graphical

An Evolved Strain of the Oleaginous Yeast Rhodotorula toruloides, Multi-Tolerant to the Major Inhibitors Present in Lignocellulosic Hydrolysates, Exhibits an Altered Cell Envelope.

The presence of toxic compounds in lignocellulosic hydrolysates (LCH) is among the main barriers affecting the efficiency of lignocellulose-based fermentation processes, in particular, to produce biofuels, hindering the production of intracellular lipids by oleaginous yeasts. These microbial oils are promising sustainable alternatives to vegetable oils for biodiesel production. In this study, we explored adaptive laboratory evolution (ALE), under methanol- and high glycerol concentration-induced selective pressures, to improve the robustness of a Rhodotorula toruloides strain, previously selected to produce lipids from sugar beet hydrolysates by completely using the major C (carbon) sources present. An evolved strain, multi-tolerant not only to methanol but to four major inhibitors present in LCH (acetic acid, formic acid, hydroxymethylfurfural, and furfural) was isolated and the mechanisms underlying such multi-tolerance were examined, at the cellular envelope level. Results indicate that the evolved multi-tolerant strain has a cell wall that is less susceptible to zymolyase and a decreased permeability, based on the propidium iodide fluorescent probe, in the absence or presence of those inhibitors. The improved performance of this multi-tolerant strain for lipid production from a synthetic lignocellulosic hydrolysate medium, supplemented with those inhibitors, was confirmed.

Production of Enriched Biomass by Carotenogenic Yeasts Cultivated on by-Products of Poultry Processing—A Screening Study

Carotenogenic yeasts are a group of microorganisms producing valuable metabolites such as carotenoids, ergosterol, ubiquinone or fatty acids. Their exceptional adaptability allows them to grow in diverse conditions. Owing to their extracellular lipase activity, they are capable of processing many lipid-type waste substrates. This study discusses the processing of poultry waste, specifically fat and feathers by using carotenogenic yeasts. Poultry fat does not require any pre-treatment to be utilized by yeast, but hydrolytic pre-treatment is required for the utilization of the nitrogen contained in feathers. Glycerol was used as a supplementary substrate to support the culture in the early stages of growth. Seven yeast strains were used for the experiments, of which the strain Rhodotorula mucilaginosa CCY19-4-25 achieved exceptional results of biomass production: 29.5 g/L on poultry fat + 10% glycerol at C/N ratio 25 and 28.3 g/L on media containing poultry fat + 25% glycerol at C/N 50. The bioreactor cultivation of the Rhodosporidium toruloides strain in media containing glycerol and feather hydrolysate as a nitrogen substrate achieved a biomass yield of 34.92 g/L after 144 h of cultivation. The produced enriched yeast biomass can be used as a component for poultry feeding; thus, the study is performed under the biorefinery concept.

Diversity investigation of cultivable yeasts associated with honeycombs and identification of a novel Rhodotorula toruloides strain with the robust concomitant production of lipid and carotenoid

Oleaginous yeasts-derived microbial lipids provide a promising alternative feedstock for the biodiesel industry. However, hyperosmotic stress caused by high sugar concentration during fermentation significantly prevents high cell density and productivity. Isolation of new robust osmophilic oleaginous species from specific environment possibly resolves this issue to some extent. In this study, the cultivable yeast composition of honeycombs was investigated. Totally, 11 species of honeycomb-associated cultivable yeast were identified and characterized. Among them, an osmophilic yeast strain, designated as Rhodotorula toruloides C23 was featured with excellent lipogenic and carotenogenic capacity and remarkable cell growth using glucose, xylose or glycerol as feedstock, with simultaneous production of 24.41 g/L of lipids and 15.50 mg/L of carotenoids from 120 g/L glucose in 6.7-L fermentation. Comparative transcriptomic analysis showed that C23 had evolved a dedicated molecular regulation mechanism to maintain their high simultaneous accumulation of intracellular lipids and carotenoids and cell growth under high sugar concentration.

Evaluation of engineered low-lignin poplar for conversion into advanced bioproducts

BackgroundLignocellulosic resources are promising feedstocks for the manufacture of bio-based products and bioenergy. However, the inherent recalcitrance of biomass to conversion into simple sugars currently hinders the deployment of advanced bioproducts at large scale. Lignin is a primary contributor to biomass recalcitrance as it protects cell wall polysaccharides from degradation and can inhibit hydrolytic enzymes via non-productive adsorption. Several engineering strategies have been designed to reduce lignin or modify its monomeric composition. For example, expression of bacterial 3-dehydroshikimate dehydratase (QsuB) in poplar trees resulted in a reduction in lignin due to redirection of metabolic flux toward 3,4-dihydroxybenzoate at the expense of lignin. This reduction was accompanied with remarkable changes in the pools of aromatic compounds that accumulate in the biomass.ResultsThe impact of these modifications on downstream biomass deconstruction and conversion into advanced bioproducts was evaluated in the current study. Using ionic liquid pretreatment followed by enzymatic saccharification, biomass from engineered trees released more glucose and xylose compared to wild-type control trees under optimum conditions. Fermentation of the resulting hydrolysates using Rhodosporidium toruloides strains engineered to produce α-bisabolene, epi-isozizaene, and fatty alcohols showed no negative impact on cell growth and yielded higher titers of bioproducts (as much as + 58%) in the case of QsuB transgenics trees.ConclusionOur data show that low-recalcitrant poplar biomass obtained with the QsuB technology has the potential to improve the production of advanced bioproducts.

Growth Potential of Selected Yeast Strains Cultivated on Xylose-Based Media Mimicking Lignocellulosic Wastewater Streams: High Production of Microbial Lipids by Rhodosporidium toruloides

The potential of Rhodosporidium toruloides, Candida oleophila, Metschnikowia pulcherima, and Cryptococcus curvatus species to produce single-cell-oil (SCO) and other valuable metabolites on low-cost media, based on commercial-type xylose, was investigated. Rhodosporidium strains were further evaluated in shake-flasks using different lignosulphonate (LS) concentrations, in media mimicking waste streams derived from the paper and pulp industry. Increasing the LS concentration up to 40 g/L resulted in enhanced dry cell weight (DCW) while SCO production increased up to ~5.0 g/L when R. toruloides NRRL Y-27012 and DSM 4444 were employed. The intra-cellular polysaccharide production ranged from 0.9 to 2.3 g/L in all fermentations. Subsequent fed-batch bioreactor experiments with R. toruloides NRRL Y-27012 using 20 g/L of LS and xylose, led to SCO production of 17.0 g/L with maximum lipids in DCW (YL/X) = 57.0% w/w. The fatty acid (FA) profile in cellular lipids showed that oleic (50.3–63.4% w/w) and palmitic acid (23.9–31.0%) were the major FAs. Only SCO from batch trials of R. toruloides strains contained α-linolenic acid. Media that was supplemented with various LS concentrations enhanced the unsaturation profile of SCO from R. toruloides NRRL Y-27012. SCO from R. toruloides strains could replace plant-based commodity oils in oleochemical-operations and/or it could be micro- and nano-encapsulated into novel food-based formulas offering healthier food-products.

Genetic manipulation of the interconversion between diacylglycerols and triacylglycerols in Rhodosporidium toruloides

The basidiomycetous yeast Rhodosporidium toruloides (R. toruloides) is an excellent producer for neutral lipids, including triacylglycerols (TAG). Partially because genetic tools for this yeast were less developed, limited efforts were shown to explore its capacity for the production of higher-value lipids such as diacylglycerols (DAG). Here, four genes linked to the interconversion between DAG and TAG were manipulated to promote the production of DAG and free fatty acids (FFA). Among them, three TAG synthesis-related genes, DGA1, LRO1, and ARE1, were down-regulated successively via the RNA interference technology, and an endogenous TAG lipase encoded by TGL5 was fused with LDP1 and over-expressed to convert TAG into DAG and FFA. Results showed that those engineered R. toruloides strains grew normally under nutrient-rich conditions but notably slower than the parental strain NP11 in the lipid production stage. When cultivated in nitrogen-limited media, engineered strains were able to produce total lipids with improved contents of DAG and FFA by up to two-fold and three-fold, respectively. Further correlation analysis between lipid composition and cell density indicated that the formation of TAG correlated positively with cell growth; however, other lipids including DAG did negatively. This study offered valuable information and strains to engineer R. toruloides for advanced production of fatty acid derivatives.

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.

Engineering the Oleaginous Yeast Rhodosporidium toruloides for Improved Resistance Against Inhibitors in Biomass Hydrolysates.

Conversion of lignocellulosic biomass into lipids and related chemicals has attracted much attention in the past two decades, and the oleaginous yeast Rhodosporidium toruloides has been widely used in this area. While R. toruloides species naturally have physiological advantages in terms of substrate utilization, lipid accumulation, and inhibitor resistance, reduced lipid production and cell growth are noticed when biomass hydrolysates are used as feedstocks. To improve the robustness of R. toruloides, here, we devised engineered strains by overexpressing genes responsible for phenolic compound degradation. Specifically, gene expression cassettes of the manganese peroxidase gene (MNP) and versatile peroxidase gene (VP) were constructed and integrated into the genome of R. toruloides NP11. A series of engineered strains were evaluated for lipid production in the presence of typical phenolic inhibitors. The results showed that R. toruloides strains with proper expression of MNP or VP indeed grew faster in the presence of vanillin and 5-hydroxymethylfurfural than the parental strain. When cultivated in concentrated mode biomass hydrolysates, the strain VP18 had improved performance as the cell mass and lipid content increased by 30% and 25%, respectively. This study provides more robust oleaginous yeast strains for microbial lipid production from lignocellulosic biomass, and similar efforts may be used to devise more advanced lipid producers.

Lipid and carotenoid production by Rhodosporodium toruloides ATCC 204091 using C5 and C6 sugars obtained from lignocellulosic hydrolysate

Aim: This study investigated the production of lipids and carotenoids and associated growth properties by the oleaginous red yeast Rhodosporodium toruloides strain ATCC 204091, using C5 and C6 sugar streams of lignocellulosic waste hydrolysate separately and in combination (C5+C6). Methodology: Cell density, wet and dry biomass weight, concentration of total sugars and reducing sugars were determined at various time intervals during cell growth in media containing C5, C6 and mixed sugars. Lipid and carotenoids were extracted and the media were compared with respect to production. Results: Production of lipid (22.25%) and carotenoids (19 mg l-1) was highest in C6 sugar, as compared to mixed sugars and C5 sugar. Interpretation: Due to the versatility of Rhodosporodium toruloides strain ATCC 204091 for utilizing C6 and C5 sugars present in waste hydrolysates, it has been projected as a good choice for cultivation in “waste” hydrolysates.

Microbial lipid production from crude glycerol and hemicellulosic hydrolysate with oleaginous yeasts

BackgroundCrude glycerol (CG) and hemicellulose hydrolysate (HH) are low—value side-products of biodiesel transesterification and pulp—and paper industry or lignocellulosic ethanol production, respectively, which can be converted to microbial lipids by oleaginous yeasts. This study aimed to test the ability of oleaginous yeasts to utilise CG and HH and mixtures of them as carbon source.ResultsEleven out of 27 tested strains of oleaginous yeast species were able to grow in plate tests on CG as sole carbon source. Among them, only one ascomycetous strain, belonging to Lipomyces starkeyi, was identified, the other 10 strains were Rhodotorula spec. When yeasts were cultivated in mixed CG/ HH medium, we observed an activation of glycerol conversion in the Rhodotorula strains, but not in L. starkeyi. Two strains—Rhodotorula toruloides CBS 14 and Rhodotorula glutinis CBS 3044 were further tested in controlled fermentations in bioreactors in different mixtures of CG and HH. The highest measured average biomass and lipid concentration were achieved with R. toruloides in 10% HH medium mixed with 55 g/L CG—19.4 g/L and 10.6 g/L, respectively, with a lipid yield of 0.25 g lipids per consumed g of carbon source. Fatty acid composition was similar to other R. toruloides strains and comparable to that of vegetable oils.ConclusionsThere were big strain differences in the ability to convert CG to lipids, as only few of the tested strains were able to grow. Lipid production rates and yields showed that mixing GC and HH have a stimulating effect on lipid accumulation in R. toruloides and R. glutinis resulting in shortened fermentation time to reach maximum lipid concentration, which provides a new perspective on converting these low-value compounds to microbial lipids.

Rhodosporidium toruloides - A potential red yeast chassis for lipids and beyond.

ABSTRACTThe red yeast Rhodosporidium toruloides naturally produces microbial lipids and carotenoids. In the past decade or so, many studies demonstrated R. toruloides as a promising platform for lipid production owing to its diverse substrate appetites, robust stress resistance and other favorable features. Also, significant progresses have been made in genome sequencing, multi-omic analysis and genome-scale modeling, thus illuminating the molecular basis behind its physiology, metabolism and response to environmental stresses. At the same time, genetic parts and tools are continuously being developed to manipulate this distinctive organism. Engineered R. toruloides strains are emerging for enhanced production of conventional lipids, functional lipids as well as other interesting metabolites. This review updates those progresses and highlights future directions for advanced biotechnological applications.

Scale-up of two-step acid-catalysed glycerol pretreatment for production of oleaginous yeast biomass from sugarcane bagasse by Rhodosporidium toruloides

Two-step dilute acid and acid-catalysed glycerol pretreatment was developed to maximise sugar yield from sugarcane bagasse. At the laboratory scale, dilute acid pretreatment at 130 °C followed by acid-catalysed glycerol pretreatment at 170 °C led to a total sugar (C5 + C6) yield of 82%, 31% higher than that from one-step acid-catalysed glycerol pretreatment. At the pilot scale, the two-step dilute acid and acid-catalysed glycerol pretreatment led to a maximum sugar yield of 74%, 13% higher than that from one-step pretreatment with 52% reduction in glycerol usage. The enzymatic hydrolysate containing glucose and residual glycerol were used to produce microbial oils by a Rhodosporidium toruloides strain. A fed-batch cultivation strategy led to the production of 44.8 g/L cell mass, including 26.6 g/L oil, 8.6 g/L protein and 12.7 mg/L carotenoid. The cell mass and oil yields were 19% higher than those from batch cultivation as feedstock inhibition and catabolite repression were alleviated.

Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides.

The basidiomycetous yeast Rhodosporidium toruloides (R. toruloides) has been explored as a promising host for the production of lipids and carotenoids. However, the rational manipulation of this yeast remains difficult due to lack of efficient genetic tools. Here, the development of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas9) system for genome editing in R. toruloides is described. First, R. toruloides strains are generated with sufficient production of the Cas9 protein of Staphylococcus aureus origin by integrating a cassette containing a codon-optimized Cas9 gene into the genome. In parallel, two U6 genes are identified, predicting two U6 promoters and confirming better transcription of single-guide RNA (sgRNA) with the U6b promoter. Next, sgRNA cassettes are designed targeting CRTI, CAR2, and CLYBL gene, respectively, transforming into those Cas9-expressed strains, and finding over 60% transformants with successful insertion and deletion (indel) mutations. Furthermore, when the sgRNA cassette includes donor DNA flanked by two homologous arms of the gene CRTI, gene knockout occurs via homologous recombination. Thus, the CRISPR/Cas9 system is now established as a powerful genome-editing tool in R. toruloides, which should facilitate functional genomic study and advanced cell factory development.

Enhancement of Torularhodin Production in Rhodosporidium toruloides by Agrobacterium tumefaciens-Mediated Transformation and Culture Condition Optimization.

Nine transformants of Rhodosporidium toruloides with significant changes in the carotenoid profile were obtained by Agrobacterium tumefaciens-mediated transformation, including a white, three red, and four yellow mutants. A red mutant A1-15-BRQ that showed a high torularhodin production was selected for culture condition optimization. Results indicated that the torularhodin yield was boosted with glucose as the carbon source, at a carbon/nitrogen ratio of 22, a loading volume of 75 mL, and 28 °C. The torularhodin yield of 21.3 mg/L consisting of 94.4% total carotenoids was obtained by Box-Behnken design experiments. The torularhodin yield was 17.0-fold higher than that of the wild type, with time shortened from 9 to 3 days. This study reports an effective strategy for improving torularhodin production and provides a candidate R. toruloides strain for highly selective production of torularhodin.