The Experts below are selected from a list of 102 Experts worldwide ranked by ideXlab platform
Shuobo Shi - One of the best experts on this subject based on the ideXlab platform.
-
engineering oleaginous yeast Rhodotorula toruloides for overproduction of fatty acid ethyl esters
Biotechnology for Biofuels, 2021Co-Authors: Yang Zhang, Jie Peng, Huimin Zhao, Shuobo ShiAbstract:Background Production of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70% cell mass as lipids, Rhodotorula toruloides represents an attractive host for FAEEs production. Results We first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase/acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs through shake-flask cultivation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, to elevate the performance of Δku70-AbWS* in a bioreactor, both batch and fed-batch cultivation strategies were performed. The FAEEs titer, productivity and yield were 4.03 g/L, 69.5 mg/L/h and 57.9 mg/g (mg FAEEs/g glucose) under batch cultivation, and 9.97 g/L, 90.6 mg/L/h, and 86.1 mg/g under fed-batch cultivation. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing. Conclusion We achieved the highest FAEEs production in yeast with a final titer of 9.97 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid-derived molecules.
-
engineering oleaginous yeast Rhodotorula toruloides for overproduction of fatty acid ethyl esters
Biotechnology for Biofuels, 2021Co-Authors: Yang Zhang, Jie Peng, Huimin Zhao, Shuobo ShiAbstract:Production of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70% cell mass as lipids, Rhodotorula toruloides represents an attractive host for FAEEs production. We first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase/acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs through shake-flask cultivation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, to elevate the performance of Δku70-AbWS* in a bioreactor, both batch and fed-batch cultivation strategies were performed. The FAEEs titer, productivity and yield were 4.03 g/L, 69.5 mg/L/h and 57.9 mg/g (mg FAEEs/g glucose) under batch cultivation, and 9.97 g/L, 90.6 mg/L/h, and 86.1 mg/g under fed-batch cultivation. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing. We achieved the highest FAEEs production in yeast with a final titer of 9.97 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid-derived molecules.
Yanbin Liu - One of the best experts on this subject based on the ideXlab platform.
-
understanding and exploiting the fatty acid desaturation system in Rhodotorula toruloides
Biotechnology for Biofuels, 2021Co-Authors: Yanbin Liu, Chong Mei John Koh, Sihui Amy Yap, Lin CaiAbstract:Rhodotorula toruloides is a robust producer of triacylglycerol owing to its fast growth rate and strong metabolic flux under conditions of high cell density fermentation. However, the molecular basis of fatty acid biosynthesis, desaturation and regulation remains elusive. We present the molecular characterization of four fatty acid desaturase (FAD) genes in R. toruloides. Biosynthesis of oleic acid (OA) and palmitoleic acid (POA) was conferred by a single-copy ∆9 Fad (Ole1) as targeted deletion of which abolished the biosynthesis of all unsaturated fatty acids. Conversion of OA to linoleic acid (LA) and α-linolenic acid (ALA) was predominantly catalyzed by the bifunctional ∆12/∆15 Fad2. FAD4 was found to encode a trifunctional ∆9/∆12/∆15 FAD, playing important roles in lipid and biomass production as well as stress resistance. Furthermore, an abundantly transcribed OLE1-related gene, OLE2 encoding a 149-aa protein, was shown to regulate Ole1 regioselectivity. Like other fungi, the transcription of FAD genes was controlled by nitrogen levels and fatty acids in the medium. A conserved DNA motif, (T/C)(G/A)TTGCAGA(T/C)CCCAG, was demonstrated to mediate the transcription of OLE1 by POA/OA. The applications of these FAD genes were illustrated by engineering high-level production of OA and γ-linolenic acid (GLA). Our work has gained novel insights on the transcriptional regulation of FAD genes, evolution of FAD enzymes and their roles in UFA biosynthesis, membrane stress resistance and, cell mass and total fatty acid production. Our findings should illuminate fatty acid metabolic engineering in R. toruloides and beyond.
-
Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis.
BMC microbiology, 2018Co-Authors: Yanbin Liu, Chong Mei John Koh, Sihui Amy Yap, Mya Myintzu HlaingAbstract:Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence. T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering.
-
Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis
BMC, 2018Co-Authors: Yanbin Liu, Chong Mei John Koh, Sihui Amy Yap, Mya Myintzu HlaingAbstract:Abstract Background Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. Results We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence. Conclusions T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering
-
Additional file 1: of Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis
2018Co-Authors: Yanbin Liu, Chong Koh, Sihui Yap, Mya HlaingAbstract:Figure S1. Optimization of transformation conditions. Unless indicated otherwise, the same volume (100 μL) of R. toruloides strain ATCC 10657 and A. tumefaciens strain AGL1 harboring plasmid pRH201 were co-cultured on IM agar (pH 5.5 and Nylon N+ membrane) for two days, and subsequently selected on YPD agar medium (150 μg/mL hygromycin and 300 μg/mL cefotaxime) for 4 days. (A) The presence (+) and absence (−) of acetosyringone (100 μg/mL). (B) Co-culture time. (C) Volumetric ratio of fungi to Agrobacteria. 100 μL of fungal cells were co-cultured with 10 to 100 μL AGL1 (pRH201) on induction medium before selection. (D) Effect of various promoters for the expression of the synthetic hpt-3 gene. Um gpd1, Rt GPD1 and An gpdA represents the glyceraldehyde-3-phospohate dehydrogenase promoter of U. maydis (0.6 kb), R. toruloides (1.4 kb), and Aspergillus nidulans (0.8 kb), respectively. Ag tef represents the promoter of Ashbya gossypii translation elongation factor (245 bp). Transformation efficiency (TFE) was represented as the relative percentage value against the highest colony forming unit (CFU) observed in the trial. Biological triplicates were used and error bars represent the standard derivations. (PDF 85 kb
-
developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula rhodosporidium yeast species
Microbial Cell Factories, 2016Co-Authors: Yanbin Liu, Sihui Amy Yap, Chong Mei John KohAbstract:Red yeast species in the Rhodotorula/Rhodosporidium genus are outstanding producers of triacylglyceride and cell biomass. Metabolic engineering is expected to further enhance the productivity and versatility of these hosts for the production of biobased chemicals and fuels. Promoters with strong activity during oil-accumulation stage are critical tools for metabolic engineering of these oleaginous yeasts. The upstream DNA sequences of 6 genes involved in lipid biosynthesis or accumulation in Rhodotorula toruloides were studied by luciferase reporter assay. The promoter of perilipin/lipid droplet protein 1 gene (LDP1) displayed much stronger activity (4–11 folds) than that of glyceraldehyde-3-phosphate dehydrogenase gene (GPD1), one of the strongest promoters known in yeasts. Depending on the stage of cultivation, promoter of acetyl-CoA carboxylase gene (ACC1) and fatty acid synthase β subunit gene (FAS1) exhibited intermediate strength, displaying 50–160 and 20–90% levels of GPD1 promoter, respectively. Interestingly, introns significantly modulated promoter strength at high frequency. The incorporation of intron 1 and 2 of LDP1 (LDP1in promoter) enhanced its promoter activity by 1.6–3.0 folds. Similarly, the strength of ACC1 promoter was enhanced by 1.5–3.2 folds if containing intron 1. The intron 1 sequences of ACL1 and FAS1 also played significant regulatory roles. When driven by the intronic promoters of ACC1 and LDP1 (ACC1in and LDP1in promoter, respectively), the reporter gene expression were up-regulated by nitrogen starvation, independent of de novo oil biosynthesis and accumulation. As a proof of principle, overexpression of the endogenous acyl-CoA-dependent diacylglycerol acyltransferase 1 gene (DGA1) by LDP1in promoter was significantly more efficient than GPD1 promoter in enhancing lipid accumulation. Intronic sequences play an important role in regulating gene expression in R. toruloides. Three intronic promoters, LDP1in, ACC1in and FAS1in, are excellent promoters for metabolic engineering in the oleaginous and carotenogenic yeast, R. toruloides.
Chong Mei John Koh - One of the best experts on this subject based on the ideXlab platform.
-
understanding and exploiting the fatty acid desaturation system in Rhodotorula toruloides
Biotechnology for Biofuels, 2021Co-Authors: Yanbin Liu, Chong Mei John Koh, Sihui Amy Yap, Lin CaiAbstract:Rhodotorula toruloides is a robust producer of triacylglycerol owing to its fast growth rate and strong metabolic flux under conditions of high cell density fermentation. However, the molecular basis of fatty acid biosynthesis, desaturation and regulation remains elusive. We present the molecular characterization of four fatty acid desaturase (FAD) genes in R. toruloides. Biosynthesis of oleic acid (OA) and palmitoleic acid (POA) was conferred by a single-copy ∆9 Fad (Ole1) as targeted deletion of which abolished the biosynthesis of all unsaturated fatty acids. Conversion of OA to linoleic acid (LA) and α-linolenic acid (ALA) was predominantly catalyzed by the bifunctional ∆12/∆15 Fad2. FAD4 was found to encode a trifunctional ∆9/∆12/∆15 FAD, playing important roles in lipid and biomass production as well as stress resistance. Furthermore, an abundantly transcribed OLE1-related gene, OLE2 encoding a 149-aa protein, was shown to regulate Ole1 regioselectivity. Like other fungi, the transcription of FAD genes was controlled by nitrogen levels and fatty acids in the medium. A conserved DNA motif, (T/C)(G/A)TTGCAGA(T/C)CCCAG, was demonstrated to mediate the transcription of OLE1 by POA/OA. The applications of these FAD genes were illustrated by engineering high-level production of OA and γ-linolenic acid (GLA). Our work has gained novel insights on the transcriptional regulation of FAD genes, evolution of FAD enzymes and their roles in UFA biosynthesis, membrane stress resistance and, cell mass and total fatty acid production. Our findings should illuminate fatty acid metabolic engineering in R. toruloides and beyond.
-
Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis.
BMC microbiology, 2018Co-Authors: Yanbin Liu, Chong Mei John Koh, Sihui Amy Yap, Mya Myintzu HlaingAbstract:Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence. T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering.
-
Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis
BMC, 2018Co-Authors: Yanbin Liu, Chong Mei John Koh, Sihui Amy Yap, Mya Myintzu HlaingAbstract:Abstract Background Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. Results We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence. Conclusions T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering
-
developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula rhodosporidium yeast species
Microbial Cell Factories, 2016Co-Authors: Yanbin Liu, Sihui Amy Yap, Chong Mei John KohAbstract:Red yeast species in the Rhodotorula/Rhodosporidium genus are outstanding producers of triacylglyceride and cell biomass. Metabolic engineering is expected to further enhance the productivity and versatility of these hosts for the production of biobased chemicals and fuels. Promoters with strong activity during oil-accumulation stage are critical tools for metabolic engineering of these oleaginous yeasts. The upstream DNA sequences of 6 genes involved in lipid biosynthesis or accumulation in Rhodotorula toruloides were studied by luciferase reporter assay. The promoter of perilipin/lipid droplet protein 1 gene (LDP1) displayed much stronger activity (4–11 folds) than that of glyceraldehyde-3-phosphate dehydrogenase gene (GPD1), one of the strongest promoters known in yeasts. Depending on the stage of cultivation, promoter of acetyl-CoA carboxylase gene (ACC1) and fatty acid synthase β subunit gene (FAS1) exhibited intermediate strength, displaying 50–160 and 20–90% levels of GPD1 promoter, respectively. Interestingly, introns significantly modulated promoter strength at high frequency. The incorporation of intron 1 and 2 of LDP1 (LDP1in promoter) enhanced its promoter activity by 1.6–3.0 folds. Similarly, the strength of ACC1 promoter was enhanced by 1.5–3.2 folds if containing intron 1. The intron 1 sequences of ACL1 and FAS1 also played significant regulatory roles. When driven by the intronic promoters of ACC1 and LDP1 (ACC1in and LDP1in promoter, respectively), the reporter gene expression were up-regulated by nitrogen starvation, independent of de novo oil biosynthesis and accumulation. As a proof of principle, overexpression of the endogenous acyl-CoA-dependent diacylglycerol acyltransferase 1 gene (DGA1) by LDP1in promoter was significantly more efficient than GPD1 promoter in enhancing lipid accumulation. Intronic sequences play an important role in regulating gene expression in R. toruloides. Three intronic promoters, LDP1in, ACC1in and FAS1in, are excellent promoters for metabolic engineering in the oleaginous and carotenogenic yeast, R. toruloides.
Ievgeniia A Tiukova - One of the best experts on this subject based on the ideXlab platform.
-
proteome analysis of xylose metabolism in Rhodotorula toruloides during lipid production
Biotechnology for Biofuels, 2019Co-Authors: Ievgeniia A Tiukova, Jule Brandenburg, Johanna Blomqvist, Sabine Sampels, Nils Egil Mikkelsen, Morten Skaugen, Magnus O Arntzen, Jens NielsenAbstract:Rhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids. Rhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose. The results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.
-
proteome analysis of xylose metabolism in Rhodotorula toruloides during lipid production
bioRxiv, 2019Co-Authors: Ievgeniia A Tiukova, Jule Brandenburg, Johanna Blomqvist, Sabine Sampels, Nils Egil Mikkelsen, Morten Skaugen, Magnus O Arntzen, Jens NielsenAbstract:Abstract Background Rhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids. Results R. toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose-and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose. Conclusions The results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through PPP although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.
-
Genome-scale model of Rhodotorula toruloides metabolism.
Biotechnology and Bioengineering, 2019Co-Authors: Ievgeniia A Tiukova, Sylvain Prigent, Jens Nielsen, Mats Sandgren, Eduard J KerkhovenAbstract:The basidiomycete red yeast Rhodotorula toruloides is a promising platform organism for production of biooils. We present rhto-GEM, the first genome-scale model (GEM) of R. toruloides metabolism, that was largely reconstructed using RAVEN toolbox. The model includes 852 genes, 2,731 reactions, and 2,277 metabolites, while lipid metabolism is described using the SLIMEr formalism allowing direct integration of lipid class and acyl chain experimental distribution data. The simulation results confirmed that the R. toruloides model provides valid growth predictions on glucose, xylose, and glycerol, while prediction of genetic engineering targets to increase production of linolenic acid, triacylglycerols, and carotenoids identified genes-some of which have previously been engineered to successfully increase production. This renders rtho-GEM valuable for future studies to improve the production of other oleochemicals of industrial relevance including value-added fatty acids and carotenoids, in addition to facilitate system-wide omics-data analysis in R. toruloides. Expanding the portfolio of GEMs for lipid-accumulating fungi contributes to both understanding of metabolic mechanisms of the oleaginous phenotype but also uncover particularities of the lipid production machinery in R. toruloides.
Yang Zhang - One of the best experts on this subject based on the ideXlab platform.
-
engineering oleaginous yeast Rhodotorula toruloides for overproduction of fatty acid ethyl esters
Biotechnology for Biofuels, 2021Co-Authors: Yang Zhang, Jie Peng, Huimin Zhao, Shuobo ShiAbstract:Background Production of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70% cell mass as lipids, Rhodotorula toruloides represents an attractive host for FAEEs production. Results We first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase/acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs through shake-flask cultivation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, to elevate the performance of Δku70-AbWS* in a bioreactor, both batch and fed-batch cultivation strategies were performed. The FAEEs titer, productivity and yield were 4.03 g/L, 69.5 mg/L/h and 57.9 mg/g (mg FAEEs/g glucose) under batch cultivation, and 9.97 g/L, 90.6 mg/L/h, and 86.1 mg/g under fed-batch cultivation. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing. Conclusion We achieved the highest FAEEs production in yeast with a final titer of 9.97 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid-derived molecules.
-
engineering oleaginous yeast Rhodotorula toruloides for overproduction of fatty acid ethyl esters
Biotechnology for Biofuels, 2021Co-Authors: Yang Zhang, Jie Peng, Huimin Zhao, Shuobo ShiAbstract:Production of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70% cell mass as lipids, Rhodotorula toruloides represents an attractive host for FAEEs production. We first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase/acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs through shake-flask cultivation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, to elevate the performance of Δku70-AbWS* in a bioreactor, both batch and fed-batch cultivation strategies were performed. The FAEEs titer, productivity and yield were 4.03 g/L, 69.5 mg/L/h and 57.9 mg/g (mg FAEEs/g glucose) under batch cultivation, and 9.97 g/L, 90.6 mg/L/h, and 86.1 mg/g under fed-batch cultivation. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing. We achieved the highest FAEEs production in yeast with a final titer of 9.97 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid-derived molecules.