The ability of Yarrowia lipolytica W29 immobilized by calcium alginate to degrade oil and chemical oxygen demand （COD） was examined. The degradation rules of oil and COD by immobilized cells with the cell density of...The ability of Yarrowia lipolytica W29 immobilized by calcium alginate to degrade oil and chemical oxygen demand （COD） was examined. The degradation rules of oil and COD by immobilized cells with the cell density of 6.65 × 10^6 CFU/mL degraded 2000 mg/L oil and 2000 mg/L COD within 50 h at 30℃ （pH 7.0, 150 r/min）, similarly to those of free cells, and the degradation efficiencies of oil and COD by immobilized cells were above 80%, respectively. The factors affecting oil and COD degradation by immobilized cells were investigated, the results showed that immobilized cells had high thermostability compared to that of free cells, and substrate concentration significantly affected degrading ability of immobilized cells. Storage stability and reusability tests revealed that the oil degradation ability of immobilized cells was stable after storing at 4~C for 30 d and reuse for 12 times, respectively, the COD degradation rate of immobilized cells was also maintained 82% at the sixth cycle. These results suggested that immobilized Y lipolytica might be applicable to a wastewater treatment system for the removal of oil and COD.展开更多
Astaxanthin is a red-colored carotenoid,used as food and feed additive.Astaxanthin is mainly produced by chemical synthesis,however,the process is expensive and synthetic astaxanthin is not approved for human consumpt...Astaxanthin is a red-colored carotenoid,used as food and feed additive.Astaxanthin is mainly produced by chemical synthesis,however,the process is expensive and synthetic astaxanthin is not approved for human consumption.In this study,we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation.First,we screened 12 different Y.lipolytica isolates for β-carotene production by introducing two genes for β-carotene biosynthesis:bi-functional phytoene synthase/lycopene cyclase(crtYB)and phytoene desaturase(crtI)from the red yeast Xanthophyllomyces dendrorhous.The best strain produced 31.1±0.5 mg/L β-carotene.Next,we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMG1)and geranylgeranyl diphosphate synthase(GGS1/crtE)in the best producing strain and obtained 453.9±20.2 mg/L β-carotene.Additional downregulation of the competing squalene synthase SQS1 increased the β-carotene titer to 797.1±57.2 mg/L.Then we introduced β-carotene ketolase(crtW)from Paracoccus sp.N81106 and hydroxylase(crtZ)from Pantoea ananatis to convert β-carotene into astaxanthin.The constructed strain accumulated 10.4±0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates,5.7±0.5 mg/L canthaxanthin,and 35.3±1.8 mg/L echinenone.Finally,we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW(54.6 mg/L)of astaxanthin in a microtiter plate cultivation.Our study for the first time reports engineering of Y.lipolytica for the production of astaxanthin.The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y.lipolytica-based fermentation process for astaxanthin production.展开更多
Taxadiene is an important precursor for the biosynthesis of highly effective anticancer drug paclitaxel,but its microbial biosynthesis yield is very low.In this study,we employed Yarrowia lipolytica as a microbial hos...Taxadiene is an important precursor for the biosynthesis of highly effective anticancer drug paclitaxel,but its microbial biosynthesis yield is very low.In this study,we employed Yarrowia lipolytica as a microbial host to produce taxadiene.First,a“push–pull”strategy was adopted to increase taxadiene production by 234%.Then taxadiene synthase was fused with five solubilizing tags respectively,leading a maximum increase of 62.3%in taxadiene production when fused with SUMO.Subsequently,a multi-copy iterative integration method was used to further increase taxadiene titer,achieving the maximum titer of 23.7 mg/L in shake flask culture after three rounds of integration.Finally,the taxadiene titer was increased to 101.4 mg/L by optimization of the fed-batch fermentation conditions.This is the first report of taxadiene biosynthesis accomplished in Y.lipolytica,serving as a good example for the sustainable production of taxadiene and other terpenoids in this oleaginous yeast.展开更多
The management and recycling of plastic waste is a challenging global issue.Polyethylene terephthalate(PET),one of the most widely used synthetic plastics,can be hydrolyzed by a series of enzymes.However,upcycling the...The management and recycling of plastic waste is a challenging global issue.Polyethylene terephthalate(PET),one of the most widely used synthetic plastics,can be hydrolyzed by a series of enzymes.However,upcycling the resulting monomers is also a problem.In this study,we designed a co-cultivation system,in which PET degradation was coupled with polyhydroxybutyrate(PHB)production.First,PETase from Ideonalla sakaiensis was expressed in Yarrowia lipolytica Po1f with a signal peptide from lipase.The engineered PETase-producing Y.lipolytica was confirmed to hydrolyze bis(2-hydroxyethyl)terephthalate(BHET)and PET powder into the monomers terephthalate(TPA)and ethylene glycol(EG).Simultaneously,a TPA-degrading Pseudomonas stutzeri strain isolated from PET waste was transformed with a recombinant plasmid containing the phb CAB operon from Ralstonia eutropha,which encodes enzymes for the biosynthesis of PHB.The two co-cultivated engineered microbes could directly hydrolyze BHET to produce the bioplastic PHB in one fermentation step.During this process,5.16 g/L BHET was hydrolyzed in 12 h,and 3.66 wt%PHB(3.54 g/L cell dry weight)accumulated in 54 h.A total of 0.31g/L TPA was produced from the hydrolyzation of PET in 228 h.Although PHB could not be synthesized directly from PET because of the low hydrolyzing efficiency of PETase,this study provides a new strategy for the biodegradation and upcycling of PET waste by artificial microflora.展开更多
Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different ch...Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different chromosomal sites,shows prominent capability in genomic libraries construction.In this study,we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process,employment of defective Ura marker and optimization of iterative transformations,which enhanced genes integration efficiency by 4.67,22.74 and 1.87 times,respectively.We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE,CrtB and CrtI,with 23.8 times higher production than rDNA integration through homologous recombination(HR).The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites,with the copy number up to 65 and 53,respectively,creating potential for further application of recombinase mediated genome rearrangement in Y.lipolytica.This work provides a high-efficient NHEJ-mediated genomic library technology,which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h.This novel technology also lays an excellent foundation for the development of other genetic technologies in Y.lipolytica.展开更多
The production of extra cellular lipase in Solid State Fermentation (SSF) using Yarrowia lipolytica NCIM 3589 with Palm Kernal cake (Elaeis guineensis) has been studied. Different parameters such as incubation time, i...The production of extra cellular lipase in Solid State Fermentation (SSF) using Yarrowia lipolytica NCIM 3589 with Palm Kernal cake (Elaeis guineensis) has been studied. Different parameters such as incubation time, inoculum level, initial moisture content, carbon level and nitrogen level of the medium were optimized. Screening of various process variables has been accomplished with the help of Plackett-Burman design. The maximum lipase activity of 18.58 units per gram of dry fermented substrate (U/gds) was observed with the substrate of Palm Kernal cake in four days of fermentation.展开更多
Microbial production of chemicals and proteins from biomass-derived andwaste sugar streams is a rapidly growing area of research and development.While the model yeast Saccharomyces cerevisiae is an excellent host for ...Microbial production of chemicals and proteins from biomass-derived andwaste sugar streams is a rapidly growing area of research and development.While the model yeast Saccharomyces cerevisiae is an excellent host for the conversion of glucose to ethanol,production of other chemicals from alternative substrates often requires extensive strain engineering.To avoid complex and intensive engineering of S.cerevisiae,other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product:for example,the efficient production and secretion of proteins,lipids,and primary metabolites that have value as commodity chemicals.Even when using yeasts with beneficial native phenotypes,metabolic engineering to increase yield,titer,and production rate is essential.The non-conventional yeasts Kluyveromyces lactis,K.marxianus,Scheffersomyces stipitis,Yarrowia lipolytica,Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes,including thermotolerance,assimilation of diverse carbon sources,and high protein secretion.However,advanced metabolic engineering in these yeasts has been limited.This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering,and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.展开更多
Fatty alcohol biosynthesis by oleaginous microbes was a promising alternative to the petroleum or other non-renewable resources-based process. However, low titer and yield hamper the further industrial and commercial ...Fatty alcohol biosynthesis by oleaginous microbes was a promising alternative to the petroleum or other non-renewable resources-based process. However, low titer and yield hamper the further industrial and commercial applications. Here, we developed an efficient strategy to coordinate fatty alcohol with glycolysis which achieved a ‘pull-and-push’ effect to improve fatty alcohol production. Transcript profiling indicated that genes in carbohydrate metabolism were up-regulated significantly in response to high fatty alcohol production. Based on it, 11 glycolysis promoters were screening to express fatty acyl-CoA reductase(FAR) to relate the fatty alcohol production with the up-regulated carbohydrate metabolism, and the fatty alcohol production reached 557 mg/L when FAR was expressed by the promoter of PFBAin. RNA-seq and qRT-PCR analysis demonstrated that a ‘pull-and-push’ effect caused by the coordination system dynamically enhanced the product synthesis flux from top to bottom, which was also testified and intensified by doubled glucose concentration. After manipulating structural and regulatory genes of lipid metabolism, the final strain achieved up to 5.75 g/L fatty alcohol production from modified YPD medium(containing 91 g/L glucose) in shake flasks, which represented the highest titer reported to date. This work offered a feasible and effective reference for dynamic manipulation of fatty acid-derived chemicals synthesis.展开更多
γ-Linolenic acid-enriched galactosyldiacylglycerols(GDGs-GLA),as the natural form ofγ-linolenic acid in microalgae,have a range of functional activities,including anti-inflammatory,antioxidant,and anti-allergic prop...γ-Linolenic acid-enriched galactosyldiacylglycerols(GDGs-GLA),as the natural form ofγ-linolenic acid in microalgae,have a range of functional activities,including anti-inflammatory,antioxidant,and anti-allergic properties.The low abundance of microalgae and the structural stereoselectivity complexity impede microalgae extraction or chemical synthesis,resulting in a lack of supply of GDGs-GLA with a growing demand.At present,there is a growing interest in engineering oleaginous yeasts for mass production of GDGs-GLA based on their ability to utilize a variety of hydrophobic substrates and a high metabolic flux toward fatty acid and lipid(triacylglycerol,TAG)production.Here,we first introduce the GDGs-GLA biosynthetic pathway in microalgae and challenges in the engineering of the native host.Subsequently,we describe in detail the applications of oleaginous yeasts with Yarrowia lipolytica as the representative for GDGs-GLA biosynthesis,including the development of synthetic biology parts,gene editing tools,and metabolic engineering of lipid biosynthesis.Finally,we discuss the development trend of GDGs-GLA biosynthesis in Y.lipolytica.展开更多
2′-Fucosyllactose(2′-FL)has great application value as a nutritional component and the whole cell biosynthesis of 2′-FL has become the focus of current research.Yarrowia lipolytica has great potential in oligosacch...2′-Fucosyllactose(2′-FL)has great application value as a nutritional component and the whole cell biosynthesis of 2′-FL has become the focus of current research.Yarrowia lipolytica has great potential in oligosaccharide synthesis and large-scale fermentation.In this study,systematic engineering of Y.lipolytica for efficient 2′-FL production was performed.By fusing different protein tags,the synthesis of 2′-FL was optimized and the ubiquitin tag was demonstrated to be the best choice to increase the 2′-FL production.By iterative integration of the related genes,increasing the precursor supply,and promoting NADPH regeneration,the 2′-FL synthesis was further improved.The final 2′-FL titer,41.10 g/L,was obtained in the strain F5-1.Our work reports the highest 2′-FL production in Y.lipolytica,and demonstrates that Y.lipolytica is an efficient microbial chassis for the synthesis of oligosaccharides.展开更多
Growth rate of Yarrowia lipolytica NCIM 3589 was observed in a fermentation medium consisting of peptone, yeast extract, sodium chloride. Logistic equation was fitted to the growth data (time vs. biomass concentration...Growth rate of Yarrowia lipolytica NCIM 3589 was observed in a fermentation medium consisting of peptone, yeast extract, sodium chloride. Logistic equation was fitted to the growth data (time vs. biomass concentration) and compared with the prediction given by Artificial Neural Networks (ANN). ANN was found to be superior in describing growth characteristics. A single MATLAB programme is developed to fit the growth data by logistic equation and ANN.展开更多
Although Yarrowia lipolytica is an important host strain, there have so far been few studies on the production of glutathione by the strain. We therefore performed a study to obtain an improved strain of Y. lipolytica...Although Yarrowia lipolytica is an important host strain, there have so far been few studies on the production of glutathione by the strain. We therefore performed a study to obtain an improved strain of Y. lipolytica ATCC20688, which could produce a high yield of glutathione. First, the capability of glutathione production in the ATCC20688 strain was estimated. In comparison with other yeasts, the yield of this strain was higher than those in Pichia strains. Furthermore, this strain could produce glutathione by assimilating sodium oleate. We next performed mutation and gene cloning to improve the yield. After the yield of glutathione was improved in the isolated methylglyoxal-resistant mutant (MGR3), the glutathione synthetase gene was cloned into the MGR3 strain. By using this recombinant strain, we could reach the maximum yield and intracellular content of glutathione of 54 mg/L-medium and 30 mg/g-dry cell weight, respectively.展开更多
Candida lipolytica with efficient decolorization of Acid Red3 G was screened. An experimental design based on the response surface method was applied to finding the optimal conditions. The results showed that the opti...Candida lipolytica with efficient decolorization of Acid Red3 G was screened. An experimental design based on the response surface method was applied to finding the optimal conditions. The results showed that the optimum dye decolorizing conditions were160 r/min with shake cultivation,initial dye concentration( 400 mg/L),incubation temperature 30 ℃ with inoculum amount 5%,and the medium compositions were glucose 21 g/L,yeast extract 10 g/L,Mg^(2+)20 mmol/L,and Cu^(2+)9. 5 mg/L. The Candida lipolytica culture exhibited decolorization 97. 5% within 48 h. These results demonstrated that the Candida lipolytica culture had high potential to decolorize azo dyes textile-dyeing wastewater.展开更多
基金supported by the National Key Technologies R & D Program of China (No. 2007BAC23B01)the Program of Department of Education, Jiangxi Province, China (No. 2007-41).
文摘The ability of Yarrowia lipolytica W29 immobilized by calcium alginate to degrade oil and chemical oxygen demand （COD） was examined. The degradation rules of oil and COD by immobilized cells with the cell density of 6.65 × 10^6 CFU/mL degraded 2000 mg/L oil and 2000 mg/L COD within 50 h at 30℃ （pH 7.0, 150 r/min）, similarly to those of free cells, and the degradation efficiencies of oil and COD by immobilized cells were above 80%, respectively. The factors affecting oil and COD degradation by immobilized cells were investigated, the results showed that immobilized cells had high thermostability compared to that of free cells, and substrate concentration significantly affected degrading ability of immobilized cells. Storage stability and reusability tests revealed that the oil degradation ability of immobilized cells was stable after storing at 4~C for 30 d and reuse for 12 times, respectively, the COD degradation rate of immobilized cells was also maintained 82% at the sixth cycle. These results suggested that immobilized Y lipolytica might be applicable to a wastewater treatment system for the removal of oil and COD.
基金This research was financially supported by the Novo Nordisk Foundation(grant number NNF15OC0016592)BAD was supported by the ERASMUS Traineeship program.We acknowledge Mette Kristensen for technical assistance on HPLC analysis.
文摘Astaxanthin is a red-colored carotenoid,used as food and feed additive.Astaxanthin is mainly produced by chemical synthesis,however,the process is expensive and synthetic astaxanthin is not approved for human consumption.In this study,we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation.First,we screened 12 different Y.lipolytica isolates for β-carotene production by introducing two genes for β-carotene biosynthesis:bi-functional phytoene synthase/lycopene cyclase(crtYB)and phytoene desaturase(crtI)from the red yeast Xanthophyllomyces dendrorhous.The best strain produced 31.1±0.5 mg/L β-carotene.Next,we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMG1)and geranylgeranyl diphosphate synthase(GGS1/crtE)in the best producing strain and obtained 453.9±20.2 mg/L β-carotene.Additional downregulation of the competing squalene synthase SQS1 increased the β-carotene titer to 797.1±57.2 mg/L.Then we introduced β-carotene ketolase(crtW)from Paracoccus sp.N81106 and hydroxylase(crtZ)from Pantoea ananatis to convert β-carotene into astaxanthin.The constructed strain accumulated 10.4±0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates,5.7±0.5 mg/L canthaxanthin,and 35.3±1.8 mg/L echinenone.Finally,we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW(54.6 mg/L)of astaxanthin in a microtiter plate cultivation.Our study for the first time reports engineering of Y.lipolytica for the production of astaxanthin.The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y.lipolytica-based fermentation process for astaxanthin production.
基金supported by the National Key Research and Development Program of China(2019YFA0905000)the National Natural Science Foundation of China(21871085 and 31971380)the Fundamental Research Funds for the Central Universities(222201714026).
文摘Taxadiene is an important precursor for the biosynthesis of highly effective anticancer drug paclitaxel,but its microbial biosynthesis yield is very low.In this study,we employed Yarrowia lipolytica as a microbial host to produce taxadiene.First,a“push–pull”strategy was adopted to increase taxadiene production by 234%.Then taxadiene synthase was fused with five solubilizing tags respectively,leading a maximum increase of 62.3%in taxadiene production when fused with SUMO.Subsequently,a multi-copy iterative integration method was used to further increase taxadiene titer,achieving the maximum titer of 23.7 mg/L in shake flask culture after three rounds of integration.Finally,the taxadiene titer was increased to 101.4 mg/L by optimization of the fed-batch fermentation conditions.This is the first report of taxadiene biosynthesis accomplished in Y.lipolytica,serving as a good example for the sustainable production of taxadiene and other terpenoids in this oleaginous yeast.
基金funding from the National Natural Science Foundation of China(grant numbers:Institute of Microbiology,Chi-nese Academy of Sciences:31961133016Beijing Institute of Technol-ogy:31961133015Shandong University:31961133014)and the Na-tional Key Research and Development Program of China(grant num-ber:2019YFA0706900)and was supported by European Union’s Hori-zon 2020 research and innovation programme under grant agreement No.870292(BIOICEP).
文摘The management and recycling of plastic waste is a challenging global issue.Polyethylene terephthalate(PET),one of the most widely used synthetic plastics,can be hydrolyzed by a series of enzymes.However,upcycling the resulting monomers is also a problem.In this study,we designed a co-cultivation system,in which PET degradation was coupled with polyhydroxybutyrate(PHB)production.First,PETase from Ideonalla sakaiensis was expressed in Yarrowia lipolytica Po1f with a signal peptide from lipase.The engineered PETase-producing Y.lipolytica was confirmed to hydrolyze bis(2-hydroxyethyl)terephthalate(BHET)and PET powder into the monomers terephthalate(TPA)and ethylene glycol(EG).Simultaneously,a TPA-degrading Pseudomonas stutzeri strain isolated from PET waste was transformed with a recombinant plasmid containing the phb CAB operon from Ralstonia eutropha,which encodes enzymes for the biosynthesis of PHB.The two co-cultivated engineered microbes could directly hydrolyze BHET to produce the bioplastic PHB in one fermentation step.During this process,5.16 g/L BHET was hydrolyzed in 12 h,and 3.66 wt%PHB(3.54 g/L cell dry weight)accumulated in 54 h.A total of 0.31g/L TPA was produced from the hydrolyzation of PET in 228 h.Although PHB could not be synthesized directly from PET because of the low hydrolyzing efficiency of PETase,this study provides a new strategy for the biodegradation and upcycling of PET waste by artificial microflora.
基金supported by Major Program of the National Natural Science Foundation of China(21621004)the Natural Science Foundation of Tianjin City(19JCQNJC09200)the Young Elite Scientists Sponsorship Program by Tianjin(TJSQNTJ-2018-16).
文摘Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different chromosomal sites,shows prominent capability in genomic libraries construction.In this study,we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process,employment of defective Ura marker and optimization of iterative transformations,which enhanced genes integration efficiency by 4.67,22.74 and 1.87 times,respectively.We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE,CrtB and CrtI,with 23.8 times higher production than rDNA integration through homologous recombination(HR).The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites,with the copy number up to 65 and 53,respectively,creating potential for further application of recombinase mediated genome rearrangement in Y.lipolytica.This work provides a high-efficient NHEJ-mediated genomic library technology,which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h.This novel technology also lays an excellent foundation for the development of other genetic technologies in Y.lipolytica.
文摘The production of extra cellular lipase in Solid State Fermentation (SSF) using Yarrowia lipolytica NCIM 3589 with Palm Kernal cake (Elaeis guineensis) has been studied. Different parameters such as incubation time, inoculum level, initial moisture content, carbon level and nitrogen level of the medium were optimized. Screening of various process variables has been accomplished with the help of Plackett-Burman design. The maximum lipase activity of 18.58 units per gram of dry fermented substrate (U/gds) was observed with the substrate of Palm Kernal cake in four days of fermentation.
基金This work was supported by NSF CBET-1510697 and -1403264.
文摘Microbial production of chemicals and proteins from biomass-derived andwaste sugar streams is a rapidly growing area of research and development.While the model yeast Saccharomyces cerevisiae is an excellent host for the conversion of glucose to ethanol,production of other chemicals from alternative substrates often requires extensive strain engineering.To avoid complex and intensive engineering of S.cerevisiae,other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product:for example,the efficient production and secretion of proteins,lipids,and primary metabolites that have value as commodity chemicals.Even when using yeasts with beneficial native phenotypes,metabolic engineering to increase yield,titer,and production rate is essential.The non-conventional yeasts Kluyveromyces lactis,K.marxianus,Scheffersomyces stipitis,Yarrowia lipolytica,Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes,including thermotolerance,assimilation of diverse carbon sources,and high protein secretion.However,advanced metabolic engineering in these yeasts has been limited.This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering,and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.
基金supported by the National Natural Science Foundation of China (21621004)Young Elite Scientists Sponsorship Program by Tianjin (TJSQNTJ-2018-16)
文摘Fatty alcohol biosynthesis by oleaginous microbes was a promising alternative to the petroleum or other non-renewable resources-based process. However, low titer and yield hamper the further industrial and commercial applications. Here, we developed an efficient strategy to coordinate fatty alcohol with glycolysis which achieved a ‘pull-and-push’ effect to improve fatty alcohol production. Transcript profiling indicated that genes in carbohydrate metabolism were up-regulated significantly in response to high fatty alcohol production. Based on it, 11 glycolysis promoters were screening to express fatty acyl-CoA reductase(FAR) to relate the fatty alcohol production with the up-regulated carbohydrate metabolism, and the fatty alcohol production reached 557 mg/L when FAR was expressed by the promoter of PFBAin. RNA-seq and qRT-PCR analysis demonstrated that a ‘pull-and-push’ effect caused by the coordination system dynamically enhanced the product synthesis flux from top to bottom, which was also testified and intensified by doubled glucose concentration. After manipulating structural and regulatory genes of lipid metabolism, the final strain achieved up to 5.75 g/L fatty alcohol production from modified YPD medium(containing 91 g/L glucose) in shake flasks, which represented the highest titer reported to date. This work offered a feasible and effective reference for dynamic manipulation of fatty acid-derived chemicals synthesis.
基金the National Key Research and Development Program of China(2021YFC2103200)the Natural Science Foundation of Zhejiang Province(LR20B060003)the Natural Science Foundation of China(22278361).
文摘γ-Linolenic acid-enriched galactosyldiacylglycerols(GDGs-GLA),as the natural form ofγ-linolenic acid in microalgae,have a range of functional activities,including anti-inflammatory,antioxidant,and anti-allergic properties.The low abundance of microalgae and the structural stereoselectivity complexity impede microalgae extraction or chemical synthesis,resulting in a lack of supply of GDGs-GLA with a growing demand.At present,there is a growing interest in engineering oleaginous yeasts for mass production of GDGs-GLA based on their ability to utilize a variety of hydrophobic substrates and a high metabolic flux toward fatty acid and lipid(triacylglycerol,TAG)production.Here,we first introduce the GDGs-GLA biosynthetic pathway in microalgae and challenges in the engineering of the native host.Subsequently,we describe in detail the applications of oleaginous yeasts with Yarrowia lipolytica as the representative for GDGs-GLA biosynthesis,including the development of synthetic biology parts,gene editing tools,and metabolic engineering of lipid biosynthesis.Finally,we discuss the development trend of GDGs-GLA biosynthesis in Y.lipolytica.
基金supported by the Key R&D Program of Shandong Province(2020CXGC010602).
文摘2′-Fucosyllactose(2′-FL)has great application value as a nutritional component and the whole cell biosynthesis of 2′-FL has become the focus of current research.Yarrowia lipolytica has great potential in oligosaccharide synthesis and large-scale fermentation.In this study,systematic engineering of Y.lipolytica for efficient 2′-FL production was performed.By fusing different protein tags,the synthesis of 2′-FL was optimized and the ubiquitin tag was demonstrated to be the best choice to increase the 2′-FL production.By iterative integration of the related genes,increasing the precursor supply,and promoting NADPH regeneration,the 2′-FL synthesis was further improved.The final 2′-FL titer,41.10 g/L,was obtained in the strain F5-1.Our work reports the highest 2′-FL production in Y.lipolytica,and demonstrates that Y.lipolytica is an efficient microbial chassis for the synthesis of oligosaccharides.
文摘Growth rate of Yarrowia lipolytica NCIM 3589 was observed in a fermentation medium consisting of peptone, yeast extract, sodium chloride. Logistic equation was fitted to the growth data (time vs. biomass concentration) and compared with the prediction given by Artificial Neural Networks (ANN). ANN was found to be superior in describing growth characteristics. A single MATLAB programme is developed to fit the growth data by logistic equation and ANN.
文摘Although Yarrowia lipolytica is an important host strain, there have so far been few studies on the production of glutathione by the strain. We therefore performed a study to obtain an improved strain of Y. lipolytica ATCC20688, which could produce a high yield of glutathione. First, the capability of glutathione production in the ATCC20688 strain was estimated. In comparison with other yeasts, the yield of this strain was higher than those in Pichia strains. Furthermore, this strain could produce glutathione by assimilating sodium oleate. We next performed mutation and gene cloning to improve the yield. After the yield of glutathione was improved in the isolated methylglyoxal-resistant mutant (MGR3), the glutathione synthetase gene was cloned into the MGR3 strain. By using this recombinant strain, we could reach the maximum yield and intracellular content of glutathione of 54 mg/L-medium and 30 mg/g-dry cell weight, respectively.
基金National Science and Technology Major Project of the Ministry of Science and Technology of China(No.2014ZX09J14103-09C-003)
文摘Candida lipolytica with efficient decolorization of Acid Red3 G was screened. An experimental design based on the response surface method was applied to finding the optimal conditions. The results showed that the optimum dye decolorizing conditions were160 r/min with shake cultivation,initial dye concentration( 400 mg/L),incubation temperature 30 ℃ with inoculum amount 5%,and the medium compositions were glucose 21 g/L,yeast extract 10 g/L,Mg^(2+)20 mmol/L,and Cu^(2+)9. 5 mg/L. The Candida lipolytica culture exhibited decolorization 97. 5% within 48 h. These results demonstrated that the Candida lipolytica culture had high potential to decolorize azo dyes textile-dyeing wastewater.
