Effect of light on carotenoid and lipid production in the oleaginous yeast Rhodosporidium toruloides

ABSTRACT

The oleaginous yeast Rhodosporodium toruloides is receiving widespread attention as an alternative energy source for biofuels due to its unicellular nature, high growth rate and because it can be fermented on a large-scale. In this study, R. toruloides was cultured under both light and dark conditions in order to understand the light response involved in lipid and carotenoid biosynthesis. Our results from phenotype and gene expression analysis showed that R. toruloides responded to light by producing darker pigmentation with an associated increase in carotenoid production. Whilst there was no observable difference in lipid production, slight changes in the fatty acid composition were recorded. Furthermore, a two-step response was found in three genes (GGPSI, CAR1, and CAR2) under light conditions and the expression of the gene encoding the photoreceptor CRY1 was similarly affected.     

Compositional profiles of Rhodosporidium toruloides cells under nutrient limitation

Lipid production by the red yeast Rhodosporidium toruloides was explored under nutrient limitation. To determine the compositional profiles of R. toruloides cells, samples were prepared using a continuous cultivation process under nutrient limitation and analyzed via several methods, including Fourier transform infrared spectroscopy and elemental analysis. Under nitrogen limitation, as the dilution rate increased, the cellular lipid content decreased but the carbohydrate and protein contents increased. Under carbon limitation, the cellular lipid, protein, and carbohydrate contents remained relatively constant at the different dilution rates. Moreover, the cellular elemental composition was essentially identical under nitrogen and carbon limitation at a high dilution rate of 0.20 h⁻¹. We also analyzed the consumed carbon to nitrogen (C/N) under different nutrition conditions. The results indicated that the consumed C/N had a major influence on cell metabolism and product formation, which contributed to our understanding of the physiological characteristics of R. toruloides.

     

圆红冬孢酵母基因编辑及天然产物合成的研究进展

圆红冬孢酵母(Rhodotorula toruloides)是一种能够天然合成多种类胡萝卜素和油脂的非模式酵母。该菌能够利用各种廉价原料,耐受甚至同化利用多种有毒木质纤维素水解副产物。目前,该酵母被广泛用于微生物油脂、萜烯类化合物、各种高价值酶、糖醇和聚酮化合物的生产研究。鉴于其广阔的工业应用前景,研究人员对其开展了多维度的理论和技术的探索,包括基因组、转录组、蛋白组、遗传操作平台等。本文着重阐述近年来圆红冬孢酵母的代谢工程和天然产物合成的研究进展,并展望其细胞工厂构建中面临的挑战和可能的应对决策。     

High-Quality RNA Preparation from <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> and cDNA Library Construction Therewith

Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of β-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 μg total RNA per gram of cells was obtained with an average purity measured as A₂₆₀/A₂₈₀ of 2.09. A titer of 10⁵ cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation.     

Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides

The oleaginous yeast Rhodosporidium toruloides is considered a promising candidate for production of chemicals and biofuels thanks to its ability to grow on lignocellulosic biomass, and its high production of lipids and carotenoids. However, efforts to engineer this organism are hindered by a lack of suitable genetic tools. Here we report the development of a CRISPR/Cas9 system for genome editing in R. toruloides based on a fusion 5S rRNA–tRNA promoter for guide RNA (gRNA) expression, capable of greater than 95% gene knockout for various genetic targets. Additionally, multiplexed double-gene knockout mutants were obtained using this method with an efficiency of 78%. This tool can be used to accelerate future metabolic engineering work in this yeast.     

Lipid production from Jerusalem artichoke by <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> Y4

Jerusalem artichoke (JA) is a perennial herbaceous plant widely available as non-grain raw material. Microbial lipid has been suggested as a potential feedstock for large scale biodiesel production. This paper describes lipid production using JA tuber processed by oleaginous yeast Rhodosporidium toruloides Y4. Batch and fed-batch modes were tested with feeding of concentrated JA extracts or JA hydrolysates. Cultivation of R. toruloides Y4 with JA extracts gave a moderate cellular lipid content of 40% (w/w), whereas lipid titer and cellular lipid content reached 39.6 g l⁻¹ and 56.5% (w/w), respectively, when JA hydrolysates were fed. Our results suggested that JA tubers may be further explored as raw material for large scale microbial lipid production.     

Combined mutagenesis of <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> for improved production of carotenoids and lipids

Objectives

To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains.

Results

Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23 g lipids/g cell dry wt and 0.75 mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41 g lipids/g CDW and 0.69 mg carotenoids/g CDW.

Conclusions

A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.

     

A new combined approach to improved lipid production using a strictly aerobic and oleaginous yeast

Microbial lipids have potential applications in energy, and food industry, because most of those lipids are triacylglycerol with long‐chain fatty‐acids that are comparable to conventional vegetable oils and can be obtained without arable land requirement. Rhodosporidium toruloides is a strictly aerobic strain, where oxygen plays a crucial role in growth, maintenance, and metabolite production, such as lipids and carotenoids. Dissolved oxygen concentration is one of the major factors affecting yeast physiological and biochemical characteristics. In this context, different approaches have been developed to increase available oxygen by the increasing the aeration and the addition of an oxygen‐vector. The growth of R. toruloides in 2‐L mechanical stirred tank reactor equipped with 1 or 2 porous spargers and a 70 C/N ratio, revealed a lipid content of 0.47 and 0.52 g/g and a lipidic productivity of 0.16 and 0.17 g/L day, respectively. The oxygen‐vector addition, increased the lipidic productivity for 0.20 g/L day and a lipid contend of 0.51 g of lipids/g of biomass. The combined approach, combining high aeration (AA), and 1% of n‐dodecane addition (DA), produced a significant improvement in the lipid accumulation (62%, w/w), when compared with the DA (51%, w/w) and the AA (52%, w/w) approaches. The increasing of lipids accumulation and smaller culture time are key factors for the success of scale‐up and profitability of a bioprocess.     

Renewable microbial lipid production from <Emphasis Type="Italic">Oleaginous Yeast</Emphasis>: some surfactants greatly improved lipid production of <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis>

Microbial oil is drawing increasing interest worldwide as an alternative non-food oil feedstock for biodiesel industry. Nowadays researchers have been increasingly focused on the improvement of microbial oil production process. Oleaginous yeast Rhodosporidium toruloides (R. toruloides) is considered an important candidate due to its excellent capabilities of lipid accumulation, broad adaptabilities to various carbon substrates, and the potential of co-production of some pigments. In present work, the individual effects of non-ionic, cationic, and anionic surfactant on cell growth and lipid accumulation of R. toruloides were investigated for the first time. Interesting results were noticed when some anionic surfactants were supplemented. The most significant effect was observed with addition of 0.2 % (w/v) sodium lignosulfonate, that biomass concentration, lipid concentration, and lipid yield was increased by 25.1, 44.9, and 15.7 %, respectively. The fatty acid compositions of R. toruloides lipids remained unchanged, which is similar to that of vegetable oils, and is considered potential feedstock for biodiesel preparation.     

Efficient concomitant production of lipids and carotenoids by oleaginous red yeast <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> cultured in palm oil mill effluent and application of lipids for biodiesel production

Rhodotorula glutinis TISTR 5159 is oleaginous red yeast that accumulates both lipids and carotenoids. It was cultured in palm oil mill effluent (POME) with only the addition of ammonium sulfate and Tween 20 as a suitable nitrogen source and surfactant, respectively. Response surface methodology (RSM) was applied to optimize initial chemical oxygen demand (COD) in POME, C/N ratio, and Tween 20 concentration for concomitant production of lipids and carotenoids. Among three investigated factors, C/N ratio contributed a significant effect upon lipid and carotenoids production. Analysis of response surface plots revealed that the optimum C/N ratio for the biomass was 140, while that for lipid content and carotenoids were higher at 180 and 170, respectively. The high level of the nitrogen source (with a low C/N ratio) enhanced the biomass, making the accumulation of lipids and carotenoids less preferable. Hence, the two-stage process was attempted as an optimal way for cell growth in the first stage and product accumulation in the second stage. The lipid yield and carotenoid production obtained in the two-stage process were higher than those in the one-stage process. In the semi-continuous fermentation, R. glutinis TISTR 5159 accumulated high lipid content and produced a considerably high concentration of carotenoids during long-term cultivation. Additionally, efficient COD removal by R. glutinis TISTR 5159 was observed. The biodiesel produced from yeast lipids was composed mainly of oleic and palmitic acids, similar to those from plant oil.     

一株降解N-酰基高丝氨酸内酯酵母菌菌株的分离鉴定及其降解特性

利用N酰基高丝氨酸内酯(N-acyl-homoserine lactone,简称AHL)为唯一碳源和能源,筛选得到一株能够降解AHL的菌株R1。常规鉴定和18S rDNA序列分析表明,菌株R1属于红冬孢酵母菌(Rhodosporidium toruloides),定名为R.toruloidesR1。结果显示R.toruloidesR1能利用所测试的3种AHL作为唯一碳源和能源生长,具有降解AHL的能力,其对AHL依赖型胡萝卜欧文氏软腐病菌(Erwinia carotovora subsp.carotovora)的致病有一定的抑制作用。     

Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid

This paper explores the use of the hydrolysate from the dilute sulfuric acid pretreatment of wheat straw for microbial oil production. The resulting hydrolysate was composed of pentoses (24.3 g/L) and hexoses (4.9 g/L), along with some other degradation products, such as acetic acid, furfural, and hydroxymethylfurfural (HMF). Five oleaginous yeast strains, Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium toruloides, Lipomyces starkeyi, and Yarrowia lipolytica, were evaluated by using this hydrolysate as substrates. The results showed that all of these strains could use the detoxified hydrolysate to produce lipids while except R. toruloides non-detoxified hydrolysate could also be used for the growth of all of the selective yeast strains. C. curvatus showed the highest lipid concentrations in medium on both the detoxified (4.2 g/L) and non-detoxified (5.8 g/L) hydrolysates. And the inhibitory effect studies on C. curvatus indicated HMF had insignificant impacts at a concentration of up to 3 g/L while furfural inhibited cell growth and lipid content by 72.0% and 62.0% at 1 g/L, respectively. Our work demonstrates that lipid production is a promising alternative to utilize hemicellulosic sugars obtained during pretreatment of lignocellulosic materials.     

High lipids accumulation in <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> by applying single and multiple nutrients limitation in a simple chemically defined medium

Microbial oil produced by the oleaginous yeast Rhodosporidium toruloides ATCC 204091 (formerly referred to as Rhodotorula glutinis) has a similar fatty acid composition to the vegetable oils and represents a potential alternative for biodiesel production. Finding strategies to improve the oil production by this yeast is desirable, as it is one of this nutrient’s limitations during the accumulation phase, as well as one of the main factors influencing the process. Therefore, the effect of single or combined nutrient limitation on lipid accumulation by R. toruloides was investigated. Biomass production and lipid accumulation by R. toruloides was improved using experimental designs in a two-step batch culture on a chemically-defined culture medium with high initial glucose concentration. For the first culture step, a Box–Behnken design was applied to optimize the main medium components’ concentrations, while maintaining a high biomass production. A biomass concentration of 44.3 g/L was reached with a medium composed of (g/L): glucose, 100; KH₂PO₄, 4.6; NaNO₃, 13.4; MgSO₄ ^.7H₂O, 0.2; and CaCl₂ ^.2H₂O, 0.11. For the second culture step, the biomass was transferred to lipid accumulation media. A 2³ factorial experimental design was conducted to investigate the effect of N, P and S limitations (individually or jointly) on lipid production from glucose (100 g/L). Lipid accumulation on dry cell mass was 77.04, 65.42, 70.13 and 69.84% for N, P, S and simultaneous nutrients’ limitations, respectively.     

Expression of a cephalosporin C esterase gene in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> for the direct production of deacetylcephalosporin C

A recombinant fungal microorganism capable of producing deacetylcephalosporin C was constructed by transforming a cephalosporin C esterase gene from Rhodosporidium toruloides into Acremonium chrysogenum. The cephalosporin C esterase gene can be expressed from its endogenous R. toruloides promoter or from the Aspergillus nidulans trpC promoter under standard Acremonium chrysogenum fermentation conditions. The expression of an active cephalosporin C esterase enzyme in A. chrysogenum results in the conversion of cephalosporin C to deacetylcephalosporin C in vivo, a novel fermentation process for the production of deacetylcephalosporin C. The stability of deacetylcephalosporin C in the fermentation broth results in a 40% increase in the cephalosporin nucleus.     

Homologous gene targeting of a carotenoids biosynthetic gene in <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Objectives

To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method.

Results

The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11.

Conclusions

Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.

     

New at-line flow cytometric protocols for determining carotenoid content and cell viability during Rhodosporidium toruloides NCYC 921 batch growth

Rhodosporidium toruloides NCYC 921 batch growth was monitored as a means to evaluate the yeast biomass potential as a source for the production of carotenoids and other lipids.Carotenoid content, cell viability and size were assessed by multiparameter flow cytometry. The saponifiable lipid fraction was assayed by gas–liquid chromatography.The carotenoid production increased during the stationary phase, reaching 78 μg/g while the total fatty acid content attained 32% (w/w) at the end of the fermentation. The fatty acid profile was suitable for biodiesel purposes.As the yeast cells entered the stationary phase, the proportion of cells with depolarised mitochondrial membrane and cells with permeabilised cytoplasmic membrane increased, attaining 65% and 14%, respectively. Nevertheless, a high proportion of cells (82%) showed esterase activity.These results demonstrated that flow cytometry can be a powerful at-line technique to monitor the total carotenoids and cell viability during the yeast growth, being useful for the yeast process optimisation at lab and pilot scales.     

Model‐assisted identification of metabolic engineering strategies for Jatropha curcas lipid pathways

Efficient approaches to increase plant lipid production are necessary to meet current industrial demands for this important resource. While Jatropha curcas cell culture can be used for in vitro lipid production, scaling up the system for industrial applications requires an understanding of how growth conditions affect lipid metabolism and yield. Here we present a bottom‐up metabolic reconstruction of J. curcas supported with labeling experiments and biomass characterization under three growth conditions. We show that the metabolic model can accurately predict growth and distribution of fluxes in cell cultures and use these findings to pinpoint energy expenditures that affect lipid biosynthesis and metabolism. In addition, by using constraint‐based modeling approaches we identify network reactions whose joint manipulation optimizes lipid production. The proposed model and computational analyses provide a stepping stone for future rational optimization of other agronomically relevant traits in J. curcas.