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1.
Electron. j. biotechnol ; 44: 19-24, Mar. 2020. ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1087631

RESUMO

BACKGROUND: Pyruvic acid (PA), a vital α-oxocarboxylic acid, plays an important role in energy and carbon metabolism. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) has considerable potential for the production of PA. An increased NaCl concentration reportedly increases the biomass and PA yield of Y. lipolytica. RESULTS: To increase the yield of PA, the NaCl-tolerant Y. lipolytica A4 mutant was produced using the atmospheric and room temperature plasma method of mutation. The A4 mutant showed growth on medium containing 160 g/L NaCl. The PA yield of the A4 mutant reached 97.2 g/L at 120 h (0.795 g/g glycerol) in a 20-L fermenter with glycerol as the sole carbon source, which was 28.9% higher than that of the parental strain. CONCLUSION: The PA yield from Y. lipolytica can be improved by increasing its NaCl tolerance.


Assuntos
Ácido Pirúvico/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Pressão Osmótica , Leveduras , Carbono/metabolismo , Cloreto de Sódio , Reatores Biológicos , Tolerância ao Sal/genética , Fermentação , Glicerol/metabolismo , Mutação
2.
J Agric Food Chem ; 68(5): 1364-1372, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31903751

RESUMO

Arbutin, a glycoside, is derived from the leaves of several plants, including wheat, pear, and bearberry plants, and has a significant role in the treatment of melanoma, cystitis, and cough. Here, we aimed to modify Yarrowia lipolytica to produce arbutin. To construct the arbutin synthetic pathway in Y. lipolytica, three genes (chorismate pyruvate-lyase (UbiC), 4-hydroxybenzoate 1-hydroxylase (MNX1), and hydroquinone glucosyltransferase (AS)) were codon-optimized and heterologously expressed. To maximize arbutin production, seven arbutin-biosynthesis molecular targets were overexpressed, and we found that the individual strengthening of DHS1 and DHS2 led to an 8.9- and 7.8-fold improvement in arbutin yield, respectively. Through optimization, a maximum arbutin titer of 8.6 ± 0.7 g/L was achieved using the finally engineered strain, po1f-At09. Overall, this is the first report of heterologous arbutin synthesis in Y. lipolytica at a high titer. Furthermore, this work opens a possibility for the overproduction of shikimate pathway derivatives in Y. lipolytica.


Assuntos
Arbutina/biossíntese , Yarrowia/genética , Yarrowia/metabolismo , Arbutina/química , Engenharia Metabólica , Ácido Chiquímico/química , Ácido Chiquímico/metabolismo , Yarrowia/química
3.
Microb Cell Fact ; 18(1): 167, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601223

RESUMO

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the production of recombinant proteins. Recently, regulated promoters from genes EYK1 and EYD1, encoding an erythrulose kinase and an erythritol dehydrogenase, respectively, have been identified and characterized in this yeast. Hybrid promoters up-regulated by polyols such as erythritol and erythrulose have been developed based on tandem copies of upstream activating sequences from EYK1 (UAS1EYK1) and XPR2 (encoding extracellular protease, UAS1XPR2) promoters. RESULTS: The strength of native (pEYD1) and engineered promoters (pEYK1-3AB and pHU8EYK) was compared using the extracellular lipase CalB from Candida antarctica as a model protein and a novel dedicated host strain. This latter is engineered in polyol metabolism and allows targeted chromosomal integration. In process conditions, engineered promoters pEYK1-3AB and pHU8EYK yielded 2.8 and 2.5-fold higher protein productivity, respectively, as compared to the reference pTEF promoter. We also demonstrated the possibility of multicopy integration in the newly developed host strain. In batch bioreactor, the CalB multi-copy strain RIY406 led to a 1.6 fold increased lipase productivity (45,125 U mL-1) within 24 h as compared to the mono-copy strain. CONCLUSIONS: The expression system described herein appears promising for recombinant extracellular protein production in Y. lipolytica.


Assuntos
Proteínas Fúngicas , Lipase , Microrganismos Geneticamente Modificados , Proteínas Recombinantes , Yarrowia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica/genética , Lipase/genética , Lipase/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Yarrowia/genética , Yarrowia/metabolismo
4.
Microb Cell Fact ; 18(1): 176, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615519

RESUMO

BACKGROUND: Yarrowia lipolytica is an unconventional yeast with a huge industrial potential. Despite many advantages for biotechnological applications, it possesses enormous demand for oxygen, which is a bottleneck in large scale production. In this study a codon optimized bacterial hemoglobin from Vitreoscilla stercoraria (VHb) was overexpressed in Y. lipolytica for efficient growth and erythritol synthesis from glycerol in low-oxygen conditions. Erythritol is a natural sweetener produced by Y. lipolytica under high osmotic pressure and at low pH, and this process requires high oxygen demand. RESULTS: Under these conditions the VHb overexpressing strain showed mostly yeast-type cells resulting in 83% higher erythritol titer in shake-flask experiments. During a bioreactor study the engineered strain showed higher erythritol productivity (QERY = 0.38 g/l h) and yield (YERY = 0.37 g/g) in comparison to the control strain (QERY = 0.30 g/l h, YERY = 0.29 g/g). Moreover, low stirring during the fermentation process resulted in modest foam formation. CONCLUSIONS: This study showed that overexpression of VHb in Y. lipolytica allows for dynamic growth and efficient production of a value-added product from a low-value substrate.


Assuntos
Eritritol/biossíntese , Hemoglobinas , Microrganismos Geneticamente Modificados/metabolismo , Yarrowia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Clonagem Molecular , Fermentação , Glicerol/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Engenharia Metabólica , Oxigênio/metabolismo , Vitreoscilla/metabolismo , Yarrowia/genética , Yarrowia/metabolismo
5.
Nat Commun ; 10(1): 4055, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492836

RESUMO

Long-chain polyunsaturated fatty acids (LC-PUFAs), particularly the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects. Consequently, sustainable sources have to be developed to meet the increasing demand for these PUFAs. Here, we demonstrate the design and construction of artificial PUFA biosynthetic gene clusters (BGCs) encoding polyketide synthase-like PUFA synthases from myxobacteria adapted for the oleaginous yeast Yarrowia lipolytica. Genomic integration and heterologous expression of unmodified or hybrid PUFA BGCs yielded different yeast strains with specific LC-PUFA production profiles at promising yield and thus valuable for the biotechnological production of distinct PUFAs. Nutrient screening revealed a strong enhancement of PUFA production, when cells were phosphate limited. This represents, to the best of our knowledge, highest concentration of DHA (16.8 %) in total fatty acids among all published PUFA-producing Y. lipolytica strains.


Assuntos
Proteínas de Bactérias/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos Insaturados/biossíntese , Myxococcales/enzimologia , Yarrowia/metabolismo , Proteínas de Bactérias/genética , Biotecnologia/métodos , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Graxo Sintases/genética , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Insaturados/metabolismo , Engenharia Metabólica/métodos , Myxococcales/genética , Reprodutibilidade dos Testes
6.
Appl Microbiol Biotechnol ; 103(20): 8585-8596, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31511932

RESUMO

Oleaginous microorganisms are of high biotechnological interest being considered as alternative sources of oil (single cell oil-SCO). Current research for increasing productivity of oleaginous microorganisms is focused on the overexpression of genes implicated in lipid synthesis, the inactivation of genes implicated in storage lipid turnover, and on the suppression of competitive to lipid biosynthesis pathways. An alternative strategy, described here, relies on evolution of Yarrowia lipolytica under alternating environments that promote growth, encourage storage lipid synthesis, and reward high energy-containing cells. Derived populations were characterized biochemically, especially on their ability to accumulate lipids, and compared with the starting strain. Interestingly, lipid-accumulating ability early in the evolution was decreased compared with the starting strain. Subsequently, oleaginous lineages dominated, leading to populations able to accumulate lipids in high amounts. A population obtained after 77 generations was able to accumulate 44% w/w of lipid, which was 30% higher than that of the starting strain. We conclude that evolution-based strategies can be utilized as a robust tool for improving lipid accumulation capacity in oleaginous microorganisms.


Assuntos
Metabolismo dos Lipídeos , Lipídeos/análise , Inoculações Seriadas , Yarrowia/crescimento & desenvolvimento , Yarrowia/metabolismo , Técnicas Microbiológicas
7.
Appl Microbiol Biotechnol ; 103(18): 7729-7740, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31367856

RESUMO

Plastic production and waste generation will continue to rise as nations worldwide grow economically. In this work, we detail a pyrolysis-based bioconversion process for polypropylene (PP) to produce value-added fatty acids (FAs). PP pellets were depolymerized by pyrolysis, generating oil that consisted of mainly branched chain fatty alcohols and alkenes. The oil was mixed with biodegradable surfactants and trace nutrients and mechanically homogenized. The resulting medium, OP4, was used for fermentation by Yarrowia lipolytica strain 78-003. Y. lipolytica assimilated > 80% of the substrate over 312 h, including 86% of the fatty alcohols. Y. lipolytica produced up to 492 mg L-1 lipids, compared with 216 mg L-1 during growth in surfactant-based control medium. C 18 compounds, including oleic acid, linoleic acid, and stearic acid, were the predominant products, followed by C 16 compounds palmitic and palmitoleic acids. Two percent of the products was C 20 compounds. The majority of the products were unsaturated FAs. Growth on hydrophobic substrates (OP4 medium, hexadecane) was compared with growth on hydrophilic substrates (glucose, starch). The resulting FA profiles revealed an absence of short-chain fatty acids during growth on hydrophobic media, findings consistent with ex novo FA biosynthesis. Overall, FA profiles by Y. lipolytica during growth in OP4 medium were similar to FA profiles while growing on natural substrates. The process described here offers an alternative approach to managing postconsumer plastic waste.


Assuntos
Ácidos Graxos/biossíntese , Temperatura Alta , Polipropilenos/metabolismo , Yarrowia/metabolismo , Alcanos/metabolismo , Meios de Cultura/química , Fermentação , Glucose/metabolismo , Polimerização , Pirólise , Yarrowia/crescimento & desenvolvimento
8.
J Agric Food Chem ; 67(35): 9851-9857, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31418561

RESUMO

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.


Assuntos
Ácido Araquidônico/biossíntese , Engenharia Metabólica , Yarrowia/genética , Yarrowia/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Glucose/metabolismo , Haptófitas/enzimologia
9.
J Ind Microbiol Biotechnol ; 46(12): 1715-1724, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31428944

RESUMO

Ionic liquids show promise for deconstruction of lignocellulosic biomass prior to fermentation. Yet, imidazolium ionic liquids (IILs) can be toxic to microbes even at concentrations present after recovery. Here, we show that dominant overexpression of an Ilt1p homolog (encoded by YlILT1/YALI0C04884) from the IIL-tolerant yeast Yarrowia lipolytica confers an improvement in 1-ethyl-3-methylimidazolium acetate tolerance in Saccharomyces cerevisiae compared to the endogenous Ilt1p (ScILT1/YDR090C). We subsequently enhance tolerance in S. cerevisiae through directed evolution of YlILT1 using growth-based selection, leading to identification of mutants that grow in up to 3.5% v/v ionic liquid. Lastly, we demonstrate that strains expressing YlILT1 variants demonstrate improved growth rate and ethanol production in the presence of residual IIL. This shows that dominant overexpression of a heterologous protein (wild type or evolved) from an IIL-tolerant yeast can increase tolerance in S. cerevisiae at concentrations relevant to bioethanol production from IIL-treated biomass.


Assuntos
Imidazóis/farmacologia , Líquidos Iônicos/farmacologia , Saccharomyces cerevisiae/metabolismo , Yarrowia/metabolismo , Biomassa , Etanol/metabolismo , Evolução Molecular , Fermentação , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Yarrowia/genética
10.
Environ Sci Pollut Res Int ; 26(30): 31234-31242, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31463748

RESUMO

Yarrowia lipolytica (Y. lipolytica) is an oleaginous yeast that can utilize hydrophobic substrates as carbon source to produce single-cell lipids for biodiesel production. This study attempts to increase the lipid accumulation ability of Y. lipolytica by first gradually elevating pure oil substrate concentration during the cultivation and then adding short-chain carbon compounds, such as glucose and sodium acetate, to a culture substance according to the optimal oil concentration. Results showed that Y. lipolytica cultured under 40.0 g L-1 oil concentration showed higher lipids (2.97 g L-1) and lipid content (37.35%, DW) compared with that cultured under 20.0 g L-1, where the corresponding values were 1.91 g L-1 and 24.46%. By contrast, the lipid content of Y. lipolytica increased from 37.35 to 41.50% when the substrate was changed from 40.0 g L-1 pure oil to 5% sodium acetate combined with 95% oil under the same total carbon concentration. However, lipid accumulation did not increase even though 15% sodium acetate or 5% glucose, or 15% glucose was added to the combined substrate. Moreover, the lipid biomodification of Y. lipolytica was evident when it was cultured under the oil concentration of 20.0 g L-1. Therefore, the lipid accumulation of Y. lipolytica can be elevated through the gradient increase of oil concentration and by adding a suitable amount of easily degradable carbon source. Furthermore, the lipid biomodification of Y. lipolytica improves biodiesel quality.


Assuntos
Biocombustíveis , Carbono/metabolismo , Metabolismo dos Lipídeos , Yarrowia/metabolismo , Biomassa , Glucose/metabolismo , Acetato de Sódio/metabolismo
11.
J Microbiol Biotechnol ; 29(7): 1071-1077, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31337188

RESUMO

Natural gamma-decalactone (GDL) produced by biotransformation is an essential food additive with a peach-like aroma. However, the difficulty of effectively controlling the concentration of the substrate ricinoleic acid (RA) in water limits the biotransformation productivity, which is a bottleneck for industrialization. In this study, expanded vermiculite (E-V) was utilized as a carrier of RA to increase its distribution in the medium. E-V and three commonly used organic compounds were compared with respect to their effects on the biotransformation process, and the mechanism was revealed. Scanning electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis indicated that RA was physically adsorbed onto the surface of and inside E-V instead of undergoing a chemical reaction, which increased the opportunity for interactions between microorganisms and the substrate. The highest concentration of GDL obtained in the medium with E-V was 6.2 g/l, which was 50% higher than that in the reference sample. In addition, the presence of E-V had no negative effect on the viability of the microorganisms. This study provides a new method for producing natural GDL through biotransformation on an industrial scale.


Assuntos
Silicatos de Alumínio/química , Biotransformação , Lactonas/metabolismo , Ácidos Ricinoleicos/química , Ácidos Ricinoleicos/metabolismo , Adsorção , Microbiologia Industrial , Viabilidade Microbiana , Yarrowia/metabolismo , Yarrowia/fisiologia
12.
Appl Biochem Biotechnol ; 189(3): 933-959, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31152353

RESUMO

Yarrowia lipolytica lipase obtained by solid-state fermentation was characterized and applied in the synthesis of esters with commercial value in the food industry. The effect of different conditions on the hydrolysis activity of this biocatalyst was evaluated in the presence of metal ions, solvents, detergents, several pH and temperature parameters, and different substrates. Storage stability was also studied. The solid biocatalyst produced in soybean meal was used in synthesis reactions aiming to produce short-, medium-, and long-chain esters. Results showed that the best fermentation condition to produce the biocatalyst was using soybean oil (3% w/w), moisture content (55% w/v), and inoculum of 2.1 mgdry biomass/gsoybean meal at 28 °C for 14 h. High substrate conversion for ethyl octanoate, cetyl stearate, and stearyl palmitate synthesis was achieved in the presence of non-polar solvents in less than 6 h using a substrate molar ratio of 1:1 at 38 °C with 10-15% (w/v) of biocatalyst. This work showed the high potential of Y. lipolytica lipase to be used in the synthesis of different esters. Also, that it can be considered an attractive and economical process alternative to obtain high-added value products.


Assuntos
Ésteres/síntese química , Fermentação , Indústria Alimentícia , Lipase/química , Lipase/metabolismo , Yarrowia/enzimologia , Biocatálise , Técnicas de Química Sintética , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Esterificação , Ésteres/química , Concentração de Íons de Hidrogênio , Hidrólise , Solventes/química , Especificidade por Substrato , Temperatura , Yarrowia/metabolismo
13.
Microb Cell Fact ; 18(1): 99, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151440

RESUMO

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is an organism of choice for the tailored production of various compounds such as biofuels or biopolymers. When properly engineered, it is capable of producing medium-chain-length polyhydroxyalkanoate (mcl-PHA), a biobased and biodegradable polymer that can be used as bioplastics or biopolymers for environmental and biomedical applications. RESULTS: This study describes the bioproduction and the main properties of two different mcl-PHA polymers. We generated by metabolic engineering, strains of Y. lipolytica capable of accumulating more than 25% (g/g) of mcl-PHA polymers. Depending of the strain genetic background and the culture conditions, we produced (i) a mcl-PHA homopolymer of 3-hydroxydodecanoic acids, with a mass-average molar mass (Mw) of 316,000 g/mol, showing soft thermoplastic properties with potential applications in packaging and (ii) a mcl-PHA copolymer made of 3-hydroxyoctanoic (3HO), decanoic (3HD), dodecanoic (3HDD) and tetradecanoic (3TD) acids with a Mw of 128,000 g/mol, behaving like a thermoplastic elastomer with potential applications in biomedical material. CONCLUSION: The ability to engineer Y. lipolytica to produce tailored PHAs together with the range of possible applications regarding their biophysical and mechanical properties opens new perspectives in the field of PHA bioproduction.


Assuntos
Engenharia Metabólica , Poli-Hidroxialcanoatos/biossíntese , Yarrowia/metabolismo , Microrganismos Geneticamente Modificados , Poli-Hidroxialcanoatos/química , Yarrowia/genética
14.
Methods Mol Biol ; 1995: 103-120, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148123

RESUMO

Improvement in biorefining technologies coupled with development of novel fermentation strategies and analysis will be paramount in establishing supplementary and sustainable biofuel pathways. Oleaginous microorganisms that are capable of accumulating triacylglycerides (TAGs) and fatty acid methyl esters (FAMEs), such as Rhodococcus and Yarrowia species, can be used to produce second-generation biofuels from non-food competing carbon sources. These "microbiorefineries" provide a pathway to upgrade agricultural and industrial waste streams to fungible fuels or precursors to chemicals and materials. Here we provide a general overview on cultivating Rhodococcus and Yarrowia on agro-waste/industrial biomass pretreatment waste streams to produce single-cell oils/lipids and preparing samples for FAME detection.


Assuntos
Lignina/metabolismo , Lipídeos/análise , Lipogênese , Rhodococcus/metabolismo , Yarrowia/metabolismo , Agricultura , Biocombustíveis/análise , Biocombustíveis/microbiologia , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Microbiologia Industrial/métodos , Resíduos Industriais , Óleos/análise , Óleos/metabolismo , Rhodococcus/química , Rhodococcus/crescimento & desenvolvimento , Triglicerídeos/análise , Triglicerídeos/metabolismo , Yarrowia/química , Yarrowia/crescimento & desenvolvimento
15.
Molecules ; 24(9)2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31064128

RESUMO

A novel recombinant strain has been constructed for converting glycerol into a specific conjugated linoleic acid isomer (trans-10, cis-12 CLA) using Yarrowia lipolytica as host. The lipid accumulation pathway was modified for increasing lipid content. Overexpression of the diacylglycerol transferase (DGA1) gene improved the intracellular lipid yield by approximately 45% as compared to the original strain. The corresponding intracellular lipid yield of recombinant strain WXYL037 reached 52.2% of the cell dry weight. In combination with integration of Δ12 desaturase from Mortierella alpina (MA12D) and DGA1, the linoleic acid (LA) production content reached 0.88 g/L, which was 2-fold that of the original strain. Furthermore, with overexpressed DGA1, MA12D and Propionibacterium acnes isomerase (PAI), the titer of trans-10, cis-12 CLA in WXYL037 reached 110.6 mg/L after 72 h of shake flask culture, representing a 201.8% improvement when compared with that attained in the WXYL030 strain, which manifested overexpressed PAI. With optimal medium, the maximum CLA content and lipid yield of Y. lipolytica Po1g were 132.6 mg/L and 2.58 g/L, respectively. This is the first report of the production of trans-10, cis-12 CLA by the oleaginous yeast Y. lipolytica using glycerol as the sole carbon source through expression of DGA1 combined with MA12D and PAI.


Assuntos
Ácidos Linoleicos Conjugados/biossíntese , Transferases/genética , Transferases/metabolismo , Yarrowia/metabolismo , Extratos Celulares/química , Escherichia coli/metabolismo , Fermentação , Glicerol/química , Isomerismo , Lipídeos/química , Propionibacterium acnes/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
16.
Microb Cell Fact ; 18(1): 83, 2019 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-31103047

RESUMO

BACKGROUND: As renewable biomass, lignocellulose remains one of the major choices for most countries in tackling global energy shortage and environment pollution. Efficient utilization of xylose, an important monosaccharide in lignocellulose, is essential for the production of high-value compounds, such as ethanol, lipids, and isoprenoids. Protopanaxadiol (PPD), a kind of isoprenoids, has important medical values and great market potential. RESULTS: The engineered protopanaxadiol-producing Yarrowia lipolytica strain, which can use xylose as the sole carbon source, was constructed by introducing xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis, overexpressing endogenous xylulose kinase (ylXKS) and heterologous PPD synthetic modules, and then 18.18 mg/L of PPD was obtained. Metabolic engineering strategies such as regulating cofactor balance, enhancing precursor flux, and improving xylose metabolism rate via XR (K270R/N272D) mutation, the overexpression of tHMG1/ERG9/ERG20 and transaldolase (TAL)/transketolase (TKL)/xylose transporter (TX), were implemented to enhance PPD production. The final Y14 strain exhibited the greatest PPD titer from xylose by fed-batch fermentation in a 5-L fermenter, reaching 300.63 mg/L [yield, 2.505 mg/g (sugar); productivity, 2.505 mg/L/h], which was significantly higher than the titer of glucose fermentation [titer, 167.17 mg/L; yield, 1.194 mg/g (sugar); productivity, 1.548 mg/L/h]. CONCLUSION: The results showed that xylose was more suitable for PPD synthesis than glucose due to the enhanced carbon flux towards acetyl-CoA, the precursor for PPD biosynthetic pathway. This is the first report to produce PPD in Y. lipolytica with xylose as the sole carbon source, which developed a promising strategy for the efficient production of high-value triterpenoid compounds.


Assuntos
Sapogeninas/metabolismo , Xilose/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Biomassa , Vias Biossintéticas , D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Fermentação , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Organismos Geneticamente Modificados
17.
Nucleic Acids Res ; 47(11): 5746-5760, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30968120

RESUMO

Message-specific translational regulation mechanisms shape the biogenesis of multimeric oxidative phosphorylation (OXPHOS) enzyme in mitochondria from the yeast Saccharomyces cerevisiae. These mechanisms, driven mainly by the action of mRNA-specific translational activators, help to coordinate synthesis of OXPHOS catalytic subunits by the mitoribosomes with both the import of their nucleus-encoded partners and their assembly to form the holocomplexes. However, little is known regarding the role that the mitoribosome itself may play in mRNA-specific translational regulation. Here, we show that the mitoribosome small subunit protein Cox24/mS38, known to be necessary for mitoribosome-specific intersubunit bridge formation and 15S rRNA H44 stabilization, is required for efficient mitoribogenesis. Consequently, mS38 is necessary to sustain the overall mitochondrial protein synthesis rate, despite an adaptive ∼2-fold increase in mitoribosome abundance in mS38-deleted cells. Additionally, the absence of mS38 preferentially disturbs translation initiation of COX1, COX2, and COX3 mRNAs, without affecting the levels of mRNA-specific translational activators. We propose that mS38 confers the mitochondrial ribosome an intrinsic capacity of translational regulation, probably acquired during evolution from bacterial ribosomes to facilitate the translation of mitochondrial mRNAs, which lack typical anti-Shine-Dalgarno sequences.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica , Ribossomos Mitocondriais/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Arabidopsis/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Kluyveromyces/metabolismo , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/química , Oryza/metabolismo , Fosforilação Oxidativa , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , RNA Mitocondrial , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Yarrowia/metabolismo
18.
Yeast ; 36(5): 319-327, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30945772

RESUMO

Citric acid (CA) productivity by Yarrowia lipolytica dependents on strain type, carbon source, carbon to nitrogen (C/N) molar ratio as well as physicochemical conditions (pH, temperature, oxygen transfer rate, etc.). In the current study, 10 different Y. lipolytica strains were first challenged in shake-flask culture for CA production in a glucose-based media under nitrogen-limited conditions. For the most promising one, strain K57, CA productivity was monitored during culture in batch bioreactor for three initial C/N molar ratio (167, 367, and 567 Cmol/Nmol). The highest CA yield (0.77 g/g glucose), titre (72.3 g/L CA), and productivity (0.04 g/g.h) were found for C/N ratio of 367. However, the highest growth rate was obtained for an initial C/N ratio of 167. From these results, Y. lipolytica strain K57 could be considered to produce CA at higher titre on glucose-based medium in batch bioreactor than others Y. lipolytica strain reported in the literature.


Assuntos
Reatores Biológicos , Ácido Cítrico/metabolismo , Yarrowia/crescimento & desenvolvimento , Yarrowia/metabolismo , Técnicas de Cultura Celular por Lotes , Meios de Cultura/química , Fermentação , Glucose/química , Nitrogênio/metabolismo
19.
Molecules ; 24(7)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925836

RESUMO

: Cane molasses is one of the main by-products of sugar refineries, which is rich in sucrose. In this work, low-cost cane molasses was introduced as an alternative substrate for isomaltulose production. Using the engineered Yarrowia lipolytica, the isomaltulose production reached the highest (102.6 g L-¹) at flask level with pretreated cane molasses of 350 g L-¹ and corn steep liquor of 1.0 g L-¹. During fed-batch fermentation, the maximal isomaltulose concentration (161.2 g L-¹) was achieved with 0.96 g g-¹ yield within 80 h. Simultaneously, monosaccharides were completely depleted, harvesting the high isomaltulose purity (97.4%) and high lipid level (12.2 g L-¹). Additionally, the lipids comprised of 94.29% C16 and C18 fatty acids, were proved suitable for biodiesel production. Therefore, the bioprocess employed using cane molasses in this study was low-cost and eco-friendly for high-purity isomaltulose production, coupling with valuable lipids.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Fermentação , Engenharia Genética/métodos , Isomaltose/análogos & derivados , Lipídeos/química , Melaço , Saccharum/química , Yarrowia/metabolismo , Biocombustíveis , Biotransformação/efeitos dos fármacos , Carbono/farmacologia , Ácidos Graxos/análise , Fermentação/efeitos dos fármacos , Isomaltose/isolamento & purificação , Lipídeos/biossíntese , Yarrowia/efeitos dos fármacos
20.
Appl Microbiol Biotechnol ; 103(8): 3549-3558, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30852660

RESUMO

Biosynthesis of Ds(+)-threo-isocitric acid from ethanol in the Yarrowia lipolytica batch and repeated-batch cultures was studied. Repeated-batch cultivation was found to provide for a good biosynthetic efficiency of the producer for as long as 748 h, probably due to maintenance of high activities of enzymes involved in the biosynthesis of isocitric acid. Under optimal repeated-batch cultivation conditions, the producer accumulated 109.6 g/L Ds(+)-threo-isocitric acid with a production rate of 1.346 g/L h. The monopotassium salt of isocitric acid isolated from the culture liquid and purified to 99.9% was found to remove neurointoxication, to restore memory, and to improve the learning of laboratory rats intoxicated with lead and molybdenum salts. Taking into account the fact that the neurotoxic effect of heavy metals is mainly determined by oxidative stress, the aforementioned favorable action of isocitric acid on the intoxicated rats can be explained by its antioxidant activity among other pharmacological effects.


Assuntos
Antioxidantes/metabolismo , Antioxidantes/farmacologia , Técnicas de Cultura Celular por Lotes/métodos , Isocitratos/metabolismo , Isocitratos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/isolamento & purificação , Reatores Biológicos/microbiologia , Etanol/metabolismo , Isocitratos/isolamento & purificação , Masculino , Metais Pesados/toxicidade , Ratos , Yarrowia/metabolismo
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