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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. [ABSTRACT FROM AUTHOR]

Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium‐mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T‐DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 105 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and β‐carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1‐13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ‐carotene and β‐carotene, and the proportion of γ‐carotene and β‐carotene were all over 92%. TAIL‐PCR was carried out to found T‐DNA insertion in these mutants, and insertion hotspot was found. RT‐qPCR results showed that CTA1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future. [ABSTRACT FROM AUTHOR]

The 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. [ABSTRACT FROM AUTHOR]

The red yeast Rhodosporidium toruloides is a known lipid producer capable of accumulating large amounts of triacylglycerols and carotenoids. However, it remains challenging to study its carotenoid production profiles owing to limited biochemical information and inefficient genetic tools. Here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) to change its carotenoid production and profiles. We constructed R. toruloides NP11 mutant libraries with ATMT, selected three mutants with different colours, characterized their carotenoid products by high-pressure liquid chromatography-mass spectrometry (HPLC-MS) analysis and assured differences among those strains in terms of carotenoid production and its composition profiles. We then located T-DNA insertion sites using the genome walking technology and provided discussions in terms of the new phenotypes. This study is the first of its kind to change the carotenoid production profiles in R. toruloides . Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The oleaginous yeast Rhodosporidium toruloides is an unconventional yeast species that can accumulate a high content of lipids. Because it belongs to the basidiomycetous group of fungus, limited tools and functional elements are available for genetic engineering of R. toruloides and related red yeasts. Here we report the functional evaluation of five constitutive promoters from this yeast. We assembled a reporter gene expression cassette, consisting of a promoter, the hygromycin gene (HYG) and the nos terminator, and inserted it into the binary vector pZPK. Hygromycin-resistant transformants were obtained when R. toruloides cells were co-cultured with Agrobacterium tumefaciens AGL1 cells harbouring the engineered vector. Genomic integration of the reporter cassette was verified by successful amplification of target DNA fragments. Quantitative PCR analysis suggested that the transformant had only one copy of the reporter cassette. The strength of these promoters was demonstrated at the phenotypic level on the hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. It was found that the strengths of these promoters varied no more than five-fold and followed a decreasing sequence of PPGI, PPGK, PFBA, PTPI, and PGPD. This study established new genetic elements for the construction of superior R. toruloides strains to produce advanced biofuels and related chemicals. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The basidiomycetous yeast Rhodosporidium toruloides represents an excellent producer for microbial lipids and carotenoids. However, further rational engineering of this unconventional yeast remains challenging partially because of the absence of efficient and reliable transformation method. In this study, we developed an Agrobacterium-mediated transformation ( ATMT) protocol for effective gene integration into the R. toruloides genome. Both haploid and diploid strains were successfully modified, and the integration was confirmed by colony PCR, Western blot analysis and genome walking. We further demonstrated that multiple genes could be integrated by consecutive ATMT, leading to engineered strains simultaneously resistant to multiple antibiotics. Our results provided a practical method for functional integration and expression of exogenous genes in R. toruloides, which should facilitate the development of genetic tools and the construction of superior strains to produce biofuel molecules and biochemicals. [ABSTRACT FROM AUTHOR]

The oleaginous yeast Rhodosporidium toruloides has emerged as a robust host for production of microbial lipids as alternative biofuel feedstocks. Oxygen supply is a limiting factor for microbial lipid production, as lipid biosynthesis is highly oxygen-demanding. Vitreoscilla hemoglobin (VHb) is a protein capable of promoting oxygen delivery for anabolism. In this study, we developed R. toruloides with VHb expression for improved lipid production. The VHb expression cassette was integrated into the R. toruloides chromosome via the Agrobacterium-mediated transformation. In shake flask cultures, the engineered strain 4#-13 produced 34% more lipids than the parental strain did. Results obtained under reduced aeration conditions in 3 L bioreactor showed that lipid titer and lipid yield of the engineered strain 4#-13 were 116% and 71%, respectively, higher than those of the parental strain. Under high cell density culture conditions, the engineered strain 4#-13 grew faster and produced 72% more lipids. Our results demonstrated that the VHb gene is functional in R. toruloides for promoting lipid production. The strains described here may be further engineered by integrating extra genetic parts to attain robust producers for more valuable products. This should improve the economics of microbial lipids to facilitate a sustainable production of biodiesel and other lipid-based biofuels. [ABSTRACT FROM AUTHOR]

The red yeast Rhodosporidium toruloides is an excellent microbial host for production of carotenoids, neutral lipids and valuable enzymes. In recent years, genetic tools for gene expression and gene disruption have been developed for this red yeast. However, methods remain limited in terms of fine-tuning gene expression. In this study, we first demonstrated successful implementation of RNA interference (RNAi) in R. toruloides NP11, which was applied to down-regulate the expression of autophagy related gene 8 (ATG8), and fatty acid synthase genes (FAS1 and FAS2), respectively. Compared with the control strain, RNAi-engineered strains showed a silencing efficiency ranging from 11% to 92%. The RNAi approach described here ensures selective inhibition of the target gene expression, and should expand our capacity in the genetic manipulation of R. toruloides for both fundamental research and advanced cell factory development. [ABSTRACT FROM AUTHOR]

The red yeast Rhodosporidium toruloide is a versatile host for production of lipids and carotenoids. Genetic tools are underdeveloped for red yeasts due to their unique genetics and physiology. Currently expression of a heterogonous gene in red yeasts is largely based on integration of the designed cassette by Agrobacterium mediated transformation, yet this method is somewhat restricted when multiple genes are required to be expressed due to the lack of functional genetic elements. Here we demonstrate that virus 2A sequence is effective to mediate co-expression of multiple enzymes in R. toruloides. Two different 2A sequences, Porcine teschovirus -1 2A (P2A) and foot-and-mouth disease virus 2A (F2A), were evaluated. It was found that P2A sequence was more effective for co-expression of two antibiotic selection markers. Co-expression of three antibiotic resistance proteins was successful from a single promoter mediated by P2A sequence. When three heterogeneous enzymes responsible for β-carotene biosynthesis were co-expressed, recombinant R. toruloides strains produced up to 4.5-fold more β-carotene than that of the parental one. The use of 2A sequence can facilitate cassette construction to engineer advanced cell factories for production of lipids and related oleochemicals. [ABSTRACT FROM AUTHOR]

Background: Lipid accumulation by oleaginous microorganisms is of great scientific interest and biotechnological potential. While nitrogen limitation has been routinely employed, low-cost raw materials usually contain rich nitrogenous components, thus preventing from efficient lipid production. Inorganic phosphate (Pi) limitation has been found sufficient to promote conversion of sugars into lipids, yet the molecular basis of cellular response to Pi limitation and concurrent lipid accumulation remains elusive. Results: Here, we performed multi-omic analyses of the oleaginous yeast Rhodosporidium toruloides to shield lights on Pi-limitation-induced lipid accumulation. Samples were prepared under Pi-limited as well as Pi-repleted chemostat conditions, and subjected to analysis at the transcriptomic, proteomic, and metabolomic levels. In total, 7970 genes, 4212 proteins, and 123 metabolites were identified. Results showed that Pi limitation facilitates up-regulation of Pi-associated metabolism, RNA degradation, and triacylglycerol biosynthesis while down-regulation of ribosome biosynthesis and tricarboxylic acid cycle. Pi limitation leads to dephosphorylation of adenosine monophosphate and the allosteric activator of isocitrate dehydrogenase key to lipid biosynthesis. It was found that NADPH, the key cofactor for fatty acid biosynthesis, is limited due to reduced flux through the pentose phosphate pathway and transhydrogenation cycle and that this can be overcome by over-expression of an endogenous malic enzyme. These phenomena are found distinctive from those under nitrogen limitation. Conclusions: Our data suggest that Pi limitation activates Pi-related metabolism, RNA degradation, and TAG biosynthesis while inhibits ribosome biosynthesis and TCA cycle, leading to enhanced carbon fluxes into lipids. The information greatly enriches our understanding on microbial oleaginicity and Pi-related metabolism. Importantly, systems data may facilitate designing advanced cell factories for production of lipids and related oleochemicals. [ABSTRACT FROM AUTHOR]