Dual-Regulation in Peroxisome and Cytoplasm toward Efficient Limonene Biosynthesis with Rhodotorula toruloides.

Rhodotorula toruloides is a potential workhorse for production of various value-added chemicals including terpenoids, oleo-chemicals, and enzymes from low-cost feedstocks. However, the limited genetic toolbox is hindering its metabolic engineering. In the present study, four type I and one novel type II peroxisomal targeting signal (PTS1/PTS2) were characterized and employed for limonene production for the first time in R. toruloides. The implant of the biosynthesis pathway into the peroxisome led to 111.5 mg/L limonene in a shake flask culture. The limonene titer was further boosted to 1.05 g/L upon dual-metabolic regulation in the cytoplasm and peroxisome, which included employing the acetoacetyl-CoA synthase NphT7, adding an additional copy of native ATP-dependent citrate lyase, etc. The final yield was 0.053 g/g glucose, which was the highest ever reported. The newly characterized PTSs should contribute to the expansion of genetic toolboxes forR. toruloides. The results demonstrated that R. toruloides could be explored for efficient production of terpenoids.

Engineering a non-model yeast Rhodotorula mucilaginosa for terpenoids synthesis.

Terpenoids have tremendous biological activities and are widely employed in food, healthcare and pharmaceutical industries. Using synthetic biology to product terpenoids from microbial cell factories presents a promising alternative route compared to conventional methods such as chemical synthesis or phytoextraction. The red yeast Rhodotorula mucilaginosa has been widely studied due to its natural production capacity of carotenoid and lipids, indicating a strong endogenous isoprene pathway with readily available metabolic intermediates. This study constructed several engineered strains of R. mucilaginosa with the aim of producing different terpenoids. Monoterpene α-terpineol was produced by expressing the α-terpineol synthase from Vitis vinifera. The titer of α-terpineol was further enhanced to 0.39 mg/L by overexpressing the endogenous rate-limiting gene of the MVA pathway. Overexpression of α-farnesene synthase from Malus domestica, in combination with MVA pathway rate-limiting gene resulted in significant increase in α-farnesene production, reaching a titer of 822 mg/L. The carotenoid degradation product ß-ionone was produced at a titer of 0.87 mg/L by expressing the ß-ionone synthase from Petunia hybrida. This study demonstrates the potential of R. mucilaginosa as a platform host for the direct biosynthesis of various terpenoids and provides insights for further development of such platforms.

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.

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.

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.

The complete mitochondrial genome of the lipid-producing yeast Rhodotorula toruloides.

Mitochondria are semi-autonomous organelles with their own genome and crucial to cellular material and energy metabolism. Here, we report the complete mitochondrial genome of a lipid-producing basidiomycetous yeast Rhodotorula toruloides NP11. The mitochondrial genome of R. toruloides NP11 was assembled into a circular DNA molecule of 125937bp, encoding 15 proteins, 28 transfer RNAs, 2 ribosomal RNA subunits and 10 open reading frames with unknown function. The G + C content (41%) of the mitochondrial genome is substantially lower than that of the nuclear genome (62%) of R. toruloides NP11. Further reanalysis of the transcriptome data confirmed the transcription of four mitochondrial genes. The comparison of the mitochondrial genomes of R. toruloides NP11 and NBRC0880 revealed a significant genetic divergence. These data can complement our understanding of the genetic background of R. toruloides and provide fundamental information for further genetic engineering of this strain.

Utilization of Amino Acid-Rich Wastes for Microbial Lipid Production.

To produce microbial lipids for biofuel production, carbohydrates and related compounds from biomass have been routinely utilized, yet amino acids (AA) from protein-rich wastes have been overlooked so far. We use the oleaginous yeast Cryptococcus curvatus ATCC 20509 as a lipid producer and evaluate the capacity for lipid production on proteinogenic AA individually or in designated blends under two-staged culture conditions. It was found that cellular lipid contents reached 48.8%, 44.5% and 29.0% when yeast cells were cultivated in media-contained AA blends with compositional profiles similar to those of sheep viscera, meat industry by-products and fish muscle, respectively, and that lipid coefficients were more than 0.10 g g-1. Furthermore, cellular lipid contents were higher than 20% when most AA were used individually. High lipid coefficients of over 0.23 g g-1 were observed when Pro, Trp or Leu were used as a substrate. Results also indicated that higher initial media pH or reduced phosphate concentration was beneficial for lipid production on AA. This work demonstrated the potential to use AA and related wastes as substrates for microbial lipid production by the yeast C. curvatus, which fit well with the protein-based biorefinery concept. Further efforts should be devoted to recognizing the metabolic features, identifying more robust lipid producer and optimizing lipid production processes.

Enhanced acetic acid stress tolerance and ethanol production in Saccharomyces cerevisiae by modulating expression of the de novo purine biosynthesis genes.

BACKGROUND: Yeast strains that are tolerant to multiple environmental stresses are highly desired for various industrial applications. Despite great efforts in identifying key genes involved in stress tolerance of budding yeast Saccharomyces cerevisiae, the effects of de novo purine biosynthesis genes on yeast stress tolerance are still not well explored. Our previous studies showed that zinc sulfate addition improved yeast acetic acid tolerance, and key genes involved in yeast stress tolerance were further investigated in this study. RESULTS: Three genes involved in de novo purine biosynthesis, namely, ADE1, ADE13, and ADE17, showed significantly increased transcription levels by zinc sulfate supplementation under acetic acid stress, and overexpression of these genes in S. cerevisiae BY4741 enhanced cell growth under various stress conditions. Meanwhile, ethanol productivity was also improved by overexpression of the three ADE genes under stress conditions, among which the highest improvement attained 158.39% by ADE17 overexpression in the presence of inhibitor mixtures derived from lignocellulosic biomass. Elevated levels of adenine-nucleotide pool "AXP" ([ATP] + [ADP] + [AMP]) and ATP content were observed by overexpression of ADE17, both under control condition and under acetic acid stress, and is consistent with the better growth of the recombinant yeast strain. The global intracellular amino acid profiles were also changed by overexpression of the ADE genes. Among the changed amino acids, significant increase of the stress protectant γ-aminobutyric acid (GABA) was revealed by overexpression of the ADE genes under acetic acid stress, suggesting that overexpression of the ADE genes exerts control on both purine biosynthesis and amino acid biosynthesis to protect yeast cells against the stress. CONCLUSION: We proved that the de novo purine biosynthesis genes are useful targets for metabolic engineering of yeast stress tolerance. The engineered strains developed in this study with improved tolerance against multiple inhibitors can be employed for efficient lignocellulosic biorefinery to produce biofuels and biochemicals.

RNA interference in the oleaginous yeast Rhodosporidium toruloides.

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.

Capturing CO2 to reversible ionic liquids for dissolution pretreatment of cellulose towards enhanced enzymatic hydrolysis.

To overcome the natural recalcitrance of cellulose for glucose production in aqueous media catalyzed by enzyme, in this study, a dissolution pretreatment strategy was developed by using in situ formed CO2-based reversible ionic compounds (RICs)/DMSO mixed organic electrolytes under mild conditions. The influences of the constitution of RICs, CO2 pressure, dissolution pretreatment time on the physic-chemical structure of cellulose were investigated systematically by FTIR, XRD, SEM, AFM towards in-depth understanding of the correlations between the pretreatment conditions, micro-scale structure and enzymatic saccharification of cellulose. The results showed that the tetramethyl guanidine (TMG) based RICs solvent system [TMGH]2+[O2COCH2CH2OCO2]2-/DMSO (XRICs = 0.1, XRICs: the mole fraction of the formed RICs in the mixture) presented the best performance, which was evidenced by 100% glucose yield after the dissolution-regeneration pretreatment strategy under mild conditions (T = 60 °C, Pco2 = 2.0 MPa, t = 2 h). Furthermore, the solvent system have good recyclability and usability.

Fast dissolution pretreatment of the corn stover in gamma-valerolactone promoted by ionic liquids: Selective delignification and enhanced enzymatic saccharification.

The dissolution of corn stover was investigated in gamma-valerolactone (GVL) assisted by ionic liquids. An enhanced subsequent enzymatic saccharification was reached with a total reducing sugar yield of 0.69 g.g-1 and a glucose of 0.38 g.g-1 within 24 h. The treatment effects on the physical-chemical features of corn stover in terms of the natural recalcitrance to the subsequent biological digest were systematically investigated using composition analysis, X-ray diffraction (XRD), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The structures of the associated enzymatic hydrolysis lignin (EHL) and ionic liquid extracted lignin (IEL) were characterized by gel permeation chromatography (GPC), fourier transform infra-red spectroscopy (FTIR), phosphorous nuclear magnet resonance spectrometry (31P NMR), and heteronuclear single quantum coherence spectroscopy (HSQC) for an in-depth understanding of the delignification process and the basic structural information for further lignin valorization.

Efficient co-expression of multiple enzymes from a single promoter mediated by virus 2A sequence in the oleaginous yeast Rhodosporidium toruloides.

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 ß-carotene biosynthesis were co-expressed, recombinant R. toruloides strains produced up to 4.5-fold more ß-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.

Systems analysis of phosphate-limitation-induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides.

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.

Selective Production of Renewable para-Xylene by Tungsten Carbide Catalyzed Atom-Economic Cascade Reactions.

Tungsten carbide was employed as the catalyst in an atom-economic and renewable synthesis of para-xylene with excellent selectivity and yield from 4-methyl-3-cyclohexene-1-carbonylaldehyde (4-MCHCA). This intermediate is the product of the Diels-Alder reaction between the two readily available bio-based building blocks acrolein and isoprene. Our results suggest that 4-MCHCA undergoes a novel dehydroaromatization-hydrodeoxygenation cascade process by intramolecular hydrogen transfer that does not involve an external hydrogen source, and that the hydrodeoxygenation occurs through the direct dissociation of the C=O bond on the W2 C surface. Notably, this process is readily applicable to the synthesis of various (multi)methylated arenes from bio-based building blocks, thus potentially providing a petroleum-independent solution to valuable aromatic compounds.

Exchanging the order of carotenogenic genes linked by porcine teschovirus-1 2A peptide enable to optimize carotenoid metabolic pathway in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae serves as a promising host for the production of a wide range of chemical compounds and fuels. Currently, simultaneous expression of several genes could be achieved via the use of 2A viral peptides, yet detailed characterizations to assess the discrepancy of different orders of genes linked by 2A peptides are rarely sufficient. In this study, we investigated the effects of the order of genes linked by porcine teschovirus-1 2A (P2A) peptide on the metabolic pathway in S. cerevisiae. A heterologous carotenoid biosynthetic system involving nine kinds of polycistronic expression of codon-optimized carotenogenic genes GGPPS, CARB and CARRP from Blakeslea trispora was introduced into S. cerevisiae. The order of genes in the polycistronic segment was exchanged; ß-carotene production by engineered yeasts was significantly different. The highest ß-carotene yield was achieved in transformants carrying the plasmid, with CARB as the first gene in the polycistronic construct regardless of the location of GGPPS, CARRP. In addition, we found that ß-carotene production was coupled with the growth in engineered strain with the highest ß-carotene content during the shake flask fermentation and fed-batch fermentation. A novel microbial heterologous carotenoid production system was established by optimizing the order of genes linked by P2A peptide sequences in a polycistronic expression construct. The observation of the importance of the order in a polycistronic construct may be used to increase yields in other P2A peptide-containing expression systems.

Deletion of acetate transporter gene ADY2 improved tolerance of Saccharomyces cerevisiae against multiple stresses and enhanced ethanol production in the presence of acetic acid.

The aim of this work was to study the effects of deleting acetate transporter gene ADY2 on growth and fermentation of Saccharomyces cerevisiae in the presence of inhibitors. Comparative transcriptome analysis revealed that three genes encoding plasma membrane carboxylic acid transporters, especially ADY2, were significantly downregulated under the zinc sulfate addition condition in the presence of acetic acid stress, and the deletion of ADY2 improved growth of S. cerevisiae under acetic acid, ethanol and hydrogen peroxide stresses. Consistently, a concomitant increase in ethanol production by 14.7% in the presence of 3.6g/L acetic acid was observed in the ADY2 deletion mutant of S. cerevisiae BY4741. Decreased intracellular acetic acid, ROS accumulation, and plasma membrane permeability were observed in the ADY2 deletion mutant. These findings would be useful for developing robust yeast strains for efficient ethanol production.

Characterization the carotenoid productions and profiles of three Rhodosporidium toruloides mutants from Agrobacterium tumefaciens-mediated transformation.

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.

Fast and efficient genetic transformation of oleaginous yeast Rhodosporidium toruloides by using electroporation.

Metabolic engineering of Rhodosporidium toruloides, a robust lipid and caroteinoid producer, is of great importance for oleochemicals and carotenoids production. However, the Agrobacterium-mediated gene transformation is tedious and time consuming. Here, we described a fast and efficient genetic transformation of R. toruloides using electroporation with linear DNA fragments, and the process was optimized. The results showed that 2 × 103 transformants can be obtained at 0.7 kV/µg linear DNA by using hygromycin and bleomycin as selection markers after the competent cells pretreated with 25 mM DTT and 100 mM LiAc. Our results would facilitate mutant library construction and metabolic engineering of R. toruloides for production of oleochemicals and carotenoids. We further demonstrated that all transformants arose due to illegitimate integration of transforming DNA fragments by colony PCR.

Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi.

Oleaginous yeast Lipomyces starkeyi, a promising strain of great biotechnical importance, is able to accumulate over 60% of its cell biomass as triacylglycerols (TAGs). It is promising to directly produce the derivatives of TAGs, such as long-chain fatty acid methyl esters and alkanes, in L. starkeyi. However, techniques for genetic modification of this oleaginous yeast are lacking, thus, further research is needed to develop genetic tools and functional elements. Here, we used two exogenous promoters (pGPD and pPGK) from oleaginous yeast Rhodosporidium toruloides to establish a simpler Agrobacterium-mediated transformation (AMT) method for L. starkeyi. Hygromycin-resistant transformants were obtained on antibiotic-contained plate. Mitotic stability test, genotype verification by PCR, and protein expression confirmation all demonstrated the success of this method. Furthermore, the strength of these two promoters was evaluated at the phenotypic level on a hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. The PGK promoter strength was 2.2-fold as that of GPD promoter to initiate the expression of the hygromycin-resistance gene. This study provided an easy and efficient genetic manipulation method and elements of the oleaginous yeast L. starkeyi for constructing superior strains to produce advanced biofuels.