Modularly engineering Rhodotorula toruloides for α-terpineol production.

α-Terpineol is a monoterpenoid alcohol that has been widely used in the flavor, fragrance, and pharmaceutical industries because of its sensory and biological properties. However, few studies have focused on the microbial production of α-terpineol. The oleaginous yeast Rhodotorula toruloides is endowed with a natural mevalonate pathway and is a promising host in synthetic biology and biorefinery. The primary objective of this work was to engineer R. toruloides for the direct biosynthesis of α-terpineol. The improvement in monoterpenoid production was achieved through the implementation of modular engineering strategies, which included the enhancement of precursor supply, blocking of downstream pathways, and disruption of competing pathways. The results of these three methods showed varying degrees of favorable outcomes in enhancing α-terpineol production. The engineered strain 5L6HE5, with competitive pathway disruption and increased substrate supply, reached the highest product titer of 1.5 mg/L, indicating that reducing lipid accumulation is an efficient method in R. toruloides engineering for terpenoid synthesis. This study reveals the potential of R. toruloides as a host platform for the synthesis of α-terpineol as well as other monoterpenoid compounds.

Biodiesel production from Rhodosporidium toruloides by acidic ionic liquids catalyzed hydrothermal liquefaction.

Hydrothermal liquefaction (HTL) has been proved to be an efficient method to disrupt cell walls and extract lipids from oleaginous yeast. However, many steps are needed for converting bio-oil into fatty acid methyl esters after HTL. Herein, acidic ionic liquid 1-butyl-3-methylimidazolium hydrogen sulfate ([Bmim][HSO4]) was introduced as the catalyst in HTL to convert oleaginous yeast Rhodosporidium toruloides to biodiesel in one step. [Bmim][HSO4] has dual effects on cell wall disruption and transesterification in reactions. As a result, the biodiesel yield achieved as high as 15.1 ± 3.2 % in optimal condition. The biodiesel was composed of long chain fatty acid methyl esters, and the higher heating value was 40.62 ± 0.05 MJ·kg-1. After the catalyst recycled 4 times, the catalytic efficiency still kept at 62.8 ± 2.1 %. The results indicated catalytic HTL was a direct and efficient method for biodiesel production from Rhodosporidium toruloides.

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.

Reduction of lipid-accumulation of oleaginous yeast Rhodosporidium toruloides through CRISPR/Cas9-mediated inactivation of lipid droplet structural proteins.

The basidiomycetous yeast Rhodosporidium toruloides is an important chassis organism for producing microbial lipids and terpenoids. However, excess carbon flux flows towards lipid synthesis than terpenoid synthesis. Thus, it is essential to limit lipid accumulation so that R. toruloides can be explored as an advanced cell factory for producing non-lipid derivatives. In this study, we knocked out two lipid droplet (LD) structural proteins (Ldp1 and Cals) of R. toruloides NP11 through the CRISPR/Cas9 system to reduce lipid production. The results showed that lipid content of LD protein-disrupted strains dropped by over 40%. LDP1-disrupted mutants harbored small-sized LDs. This study provided valuable information to study about microbial lipid metabolism and platform strains for constructing advanced cell factories.

A seven-gene prognostic signature predicts overall survival of patients with lung adenocarcinoma (LUAD).

BACKGROUND: Lung adenocarcinoma (LUAD) is one of the most common types in the world with a high mortality rate. Despite advances in treatment strategies, the overall survival (OS) remains short. Our study aims to establish a reliable prognostic signature closely related to the survival of LUAD patients that can better predict prognosis and possibly help with individual monitoring of LUAD patients. METHODS: Raw RNA-sequencing data were obtained from Fudan University and used as a training group. Differentially expressed genes (DEGs) for the training group were screened. The univariate, least absolute shrinkage and selection operator (LASSO), and multivariate cox regression analysis were conducted to identify the candidate prognostic genes and construct the risk score model. Kaplan-Meier analysis, time-dependent receiver operating characteristic (ROC) curve were used to evaluate the prognostic power and performance of the signature. Moreover, The Cancer Genome Atlas (TCGA-LUAD) dataset was further used to validate the predictive ability of prognostic signature. RESULTS: A prognostic signature consisting of seven prognostic-related genes was constructed using the training group. The 7-gene prognostic signature significantly grouped patients in high and low-risk groups in terms of overall survival in the training cohort [hazard ratio, HR = 8.94, 95% confidence interval (95% CI)] [2.041-39.2]; P = 0.0004), and in the validation cohort (HR = 2.41, 95% CI [1.779-3.276]; P < 0.0001). Cox regression analysis (univariate and multivariate) demonstrated that the seven-gene signature is an independent prognostic biomarker for predicting the survival of LUAD patients. ROC curves revealed that the 7-gene prognostic signature achieved a good performance in training and validation groups (AUC = 0.91, AUC = 0.7 respectively) in predicting OS for LUAD patients. Furthermore, the stratified analysis of the signature showed another classification to predict the prognosis. CONCLUSION: Our study suggested a new and reliable prognostic signature that has a significant implication in predicting overall survival for LUAD patients and may help with early diagnosis and making effective clinical decisions regarding potential individual treatment.

Exogenous l-proline improved Rhodosporidium toruloides lipid production on crude glycerol.

BACKGROUND: Crude glycerol as a promising feedstock for microbial lipid production contains several impurities that make it toxic stress inducer at high amount. Under stress conditions, microorganisms can accumulate l-proline as a safeguard. Herein, l-proline was assessed as an anti-stress agent in crude glycerol media. RESULTS: Crude glycerol was converted to microbial lipids by the oleaginous yeast Rhodosporidium toruloides CGMCC 2.1389 in a two-staged culture mode. The media was supplied with exogenous l-proline to improve lipid production efficiency in high crude glycerol stress. An optimal amount of 0.5 g/L l-proline increased lipid titer and lipid yield by 34% and 28%, respectively. The lipid titer of 12.2 g/L and lipid content of 64.5% with a highest lipid yield of 0.26 g/g were achieved with l-proline addition, which were far higher than those of the control, i.e., lipid titer of 9.1 g/L, lipid content of 58% and lipid yield of 0.21 g/g. Similarly, l-proline also improved cell growth and glycerol consumption. Moreover, fatty acid compositional profiles of the lipid products was found suitable as a potential feedstock for biodiesel production. CONCLUSION: Our study suggested that exogenous l-proline improved cell growth and lipid production on crude glycerol by R. toruloides. The fact that higher lipid yield as well as glycerol consumption indicated that l-proline might act as a potential anti-stress agent for the oleaginous yeast strain.

Automated Pressurized Liquid Extraction of Microbial Lipids from Oleaginous Yeasts.

The lipids produced by oleaginous microbes are considered sustainable resources for biofuels. To facilitate controlled lipid production and lipid analysis, more efficient lipid extraction methods are required. This study describes the automated pressurized liquid extraction (APLE) method for lipid extraction from dried cells of the oleaginous yeast species Rhodosporidium toruloides and Cryptococcus curvatus. Cells were mixed with diatomite in a mortar, added to the sample chamber, and treated with a mixture of chloroform and methanol at 105 °C. More than 95% lipids were extracted. Analysis by using high-performance thin-layer chromatography showed that the neutral lipid contents in the obtained samples by APLE method were similar to those by the ball milling-assisted extraction method. The lipids had an essentially identical fatty acid composition compared with lipids extracted with the acid-heating extraction (AHE) method. This demonstrated that lipids can be efficiently extracted from oleaginous yeasts in less time and without harsh pretreatment procedures.

Effects of selected ionic liquids on lipid production by the oleaginous yeast Rhodosporidium toruloides.

Lignocellulosic biomass pretreatment with ionic liquids (ILs) has been emerged as a new technology, but the effects of residual ILs on the downstream biotransformation remain largely unknown. Here, three typical ILs were tested for their effects on lipid production by the oleaginous yeast Rhodosporidium toruloides AS 2.1389. When cultures were maintained at pH 6.0 in the presence of 30mM ILs, [Emim]Cl, [Emim][DEP], or [Emim][OAc], minor inhibition effects were observed. When cultures were performed in the presence of 60mM ILs or without pH control, inhibition was largely dependent on ILs. Detailed analysis indicated that the anion of [Emim][OAc] was assimilated, leading to a rapid alkaline-pH shift and enhanced inhibition on cell growth and lipid production. Our results demonstrated that R. toruloides is a robust lipid producer tolerating ILs at low concentrations, and that care should be taken in bioprocess control and data analysis when ILs are involved.