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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.
World J Microbiol Biotechnol ; 36(1): 11, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31879822

RESUMO

Because of their protein cross-linking properties, transglutaminases are widely used in several industrial processes, including the food and pharmaceutical industries. Transglutaminases obtained from animal tissues and organs, the first sources of this enzyme, are being replaced by microbial sources, which are cheaper and easier to produce and purify. Since the discovery of microbial transglutaminase (mTGase), the enzyme has been produced for industrial applications by traditional fermentation process using the bacterium Streptomyces mobaraensis. Several studies have been carried out in this field to increase the enzyme industrial productivity. Researches on gene expression encoding transglutaminase biosynthesis were performed in Streptomyces lividans, Escherichia coli, Corynebacterium glutamicum, Yarrowia lipolytica, and Pichia pastoris. In the first part of this review, we presented an overview of the literature on the origins, types, mediated reactions, and general characterizations of these important enzymes, as well as the studies on recombinant microbial transglutaminases. In this second part, we focus on the application versatility of mTGase in three broad areas: food, pharmacological, and biotechnological industries. The use of mTGase is presented for several food groups, showing possibilities of applications and challenges to further improve the quality of the end-products. Some applications in the textile and leather industries are also reviewed, as well as special applications in the PEGylation reaction, in the production of antibody drug conjugates, and in regenerative medicine.


Assuntos
Biotecnologia , Indústria Alimentícia , Têxteis , Transglutaminases , Animais , Corynebacterium glutamicum/genética , Bases de Dados Factuais , Escherichia coli/genética , Fermentação , Alimentos , Tecnologia de Alimentos , Pichia/genética , Proteínas Recombinantes , Streptomyces/enzimologia , Transglutaminases/biossíntese , Transglutaminases/genética , Yarrowia/genética
4.
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
5.
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
6.
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
7.
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
8.
Genes (Basel) ; 10(7)2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295964

RESUMO

Although the pan-genome concept originated in prokaryote genomics, an increasing number of eukaryote species pan-genomes have also been analysed. However, there is a relative lack of software intended for eukaryote pan-genome analysis compared to that available for prokaryotes. In a previous study, we analysed the pan-genomes of four model fungi with a computational pipeline that constructed pan-genomes using the synteny-dependent Pan-genome Ortholog Clustering Tool (PanOCT) approach. Here, we present a modified and improved version of that pipeline which we have called Pangloss. Pangloss can perform gene prediction for a set of genomes from a given species that the user provides, constructs and optionally refines a species pan-genome from that set using PanOCT, and can perform various functional characterisation and visualisation analyses of species pan-genome data. To demonstrate Pangloss's capabilities, we constructed and analysed a species pan-genome for the oleaginous yeast Yarrowia lipolytica and also reconstructed a previously-published species pan-genome for the opportunistic respiratory pathogen Aspergillus fumigatus. Pangloss is implemented in Python, Perl and R and is freely available under an open source GPLv3 licence via GitHub.


Assuntos
Aspergillus fumigatus/genética , Genoma Fúngico , Software , Yarrowia/genética , Genômica
9.
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
10.
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
11.
Arch Microbiol ; 201(7): 991-998, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31025056

RESUMO

Caffeine is a compound that can exert physiological-beneficial effects in the organism. Nevertheless, there are controversies about its protective-antioxidant and/or its negative genotoxic effect. To abound on the analysis of the possible genotoxic/antioxidant effect of caffeine, we used as research model the yeast Yarrowia lipolytica parental strain, and mutant strains (∆rad52 and ∆ku80), which are deficient in the DNA repair mechanisms. Caffeine (5 mM) showed a cytostatic effect on all strains, but after 72 h of incubation the parental and ∆ku80 strains were able to recover of this inhibitory effect on growth, whereas ∆rad52 was unable to recover. When cells were pre-incubated with caffeine and H2O2 or incubated with a mixture of both agents, a higher inhibitory effect on growth of mutant strains was observed and this effect was noticeably greater for the Δrad52 strain. The toxic effect of caffeine appears to be through a mechanism of DNA damage (genotoxic effect) that involves DSB generation since, in all tested conditions, the growth of Δrad52 strain (cells deficient in HR DNA repair mechanism) was more severely affected.


Assuntos
Cafeína/toxicidade , Reparo do DNA/genética , Yarrowia/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Mutação/genética , Yarrowia/genética , Yarrowia/crescimento & desenvolvimento
12.
Appl Microbiol Biotechnol ; 103(8): 3511-3520, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30863877

RESUMO

Sandalwood oil is a valuable resource derived from Santalum album. It has antibacterial, cosmetic, and sedative effects. α-Santalene is the precursor of α-santalol, the main component of sandalwood oil. Yarrowia lipolytica is an oleaginous yeast, which has been metabolically engineered to produce valuable compounds such as terpenoids and biofuel. This study presents a method for the heterologous synthesis of α-santalene by Y. lipolytica. Using Y. lipolytica ATCC 201249, a codon-optimized plant-origin α-santalene synthase (STS) was integrated into the genome, and a yield of 5.19 mg/L α-santalene was obtained after fermentation. Upstream genes in the MVA pathway (ERG8, ERG10, ERG12, ERG13, ERG19, ERG20, HMG1, and tHMG1) were overexpressed, and we found that the key genes ERG8, HMG1, and tHMG1 can increase the supply of FPP and the yield of α-santalene. ERG8 and HMG1 were overexpressed in multiple-copy formats, and the plasmid pERG8HMG1 and ERG8-HMG1 expression modules were optimized as single-copy and multiple-copy formats, respectively. The overexpression of single-copy plasmid pERG8HMG1 led to α-santalene yield of 13.31 mg/L. The optimal feeding strategy was determined by initial carbon source concentration optimizations and five feeding methods. Using 50 g/L glucose as the initial carbon source, maintaining the carbon source concentration at 5-20 g/L during the feeding process is most conducive to increased production. These results were verified in a 5-L fermenter by batch and fed-batch fermentation. The OD of fed-batch fermentation broth reached 79.09, and the production of α-santalene reached 27.92 mg/L; 5.38 times of the initial yield, without by-products farnesol and trans-α-bergamotene.


Assuntos
Fermentação , Engenharia Metabólica , Sesquiterpenos/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Reatores Biológicos/microbiologia , Vias Biossintéticas/genética , Carbono/química , Carbono/metabolismo , Meios de Cultura/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Glucose/química , Glucose/metabolismo
13.
Curr Opin Biotechnol ; 57: 73-81, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30875587

RESUMO

Current transportation fuels derived from petroleum can also be made from microbial systems. In particular, oleaginous yeast have naturally evolved high flux pathways for fatty acids in the form of neutral lipids, which can be converted into a variety of drop-in fuels. Here, we describe the recent advances in the use of the four most popular oleaginous yeasts for making lipids and other potential fuels - Yarrowia lipolytica, Lipomyces starkeyi, Rhodosporidium toruloides, and Cutaneotrichosporon oleaginosus. The paper is divided into three major sections focusing on (1) the important natural complex phenotypes of each yeast; (2) the development of metabolic engineering tools for each yeast; and (3) demonstrations of metabolic engineering in each yeast. At the end of each section, we provide our assessment, of which yeast is most promising in the near and long term for bioenergy production.


Assuntos
Biocombustíveis/microbiologia , Lipídeos/biossíntese , Yarrowia/metabolismo , Ácidos Graxos/metabolismo , Engenharia Genética , Engenharia Metabólica , Yarrowia/genética
14.
J Agric Food Chem ; 67(9): 2581-2588, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30757901

RESUMO

Compound K (CK) is a rare, tetracyclic, triterpenoid compound with important medical properties, such as antitumor and anti-inflammatory activities. Herein, an efficient biosynthetic pathway of CK was constructed in metabolically engineered Yarrowia lipolytica for the first time, and the engineered strain, YL-CK0, produced 5.1 mg/L CK. The production of CK was further increased by 5.96-fold to 30.4 mg/L with overexpression of key genes in the MVA pathway and fusion of cytochrome P450 monooxygenase (PPDS) and NADPH-P450 reductase. Finally, 161.8 mg/L CK production was achieved by fed-batch fermentation in a 5 L fermenter using the strain YL-MVA-CK. To the best of our knowledge, this is the first report on heterologous CK synthesis with the highest titer in Y. lipolytica. This study demonstrates the feasibility of producing high-value triterpenoid compounds using Y. lipolytica as a platform.


Assuntos
Ginsenosídeos/biossíntese , Engenharia Metabólica , Yarrowia/genética , Anti-Inflamatórios , Antineoplásicos , Reatores Biológicos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Expressão Gênica , Fusão Gênica , Engenharia Metabólica/métodos , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Yarrowia/metabolismo
15.
Methods Mol Biol ; 1923: 153-168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30737739

RESUMO

Yarrowia lipolytica has emerged as an alternative expression system for heterologous protein production and enzyme evolution. Several different expression systems dedicated for this species have been developed, ranging from the simple cloning of expression vectors to recently developed high-throughput methodologies using efficient cloning and assembly such as Gateway and Golden Gate strategies. The latter strategies, due to their modular character, enable multiple vector construction and the construction of expression cassettes containing different genes or a gene under different promoters of various strengths.Here, we present the Golden Gate cloning strategy for the construction of multiple expression cassettes, the transformation into Y. lipolytica, and the selection of efficient enzyme-producing strains using an insect alpha-amylase as a reporter detected via a thermal cycler-based microassay.


Assuntos
Regiões Promotoras Genéticas/genética , Yarrowia/genética , Proteínas Fúngicas/genética , Vetores Genéticos/genética
16.
BMC Biotechnol ; 19(1): 11, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30744615

RESUMO

BACKGROUND: Citric acid is considered as the most economically feasible product of microbiological production, therefore studies on cheap and renewable raw materials for its production are highly desirable. In this study citric acid was synthesized by genetically engineered strains of Yarrowia lipolytica from widely available, renewable polysaccharide - inulin. Hydrolysis of inulin by the Y. lipolytica strains was established by expressing the inulinase gene (INU1 gene; GenBank: X57202.1) with its native secretion signal sequence was amplified from genomic DNA from Kluyveromyces marxianus CBS6432. To ensure the maximum citric acid titer, the optimal cultivation strategy-repeated-batch culture was applied. RESULTS: The strain Y. lipolytica AWG7 INU 8 secreted more than 200 g dm- 3 of citric acid during repeated-batch culture on inulin, with a productivity of 0.51 g dm- 3 h- 1 and a yield of 0.85 g g- 1. CONCLUSIONS: The citric acid titer obtained in the proposed process is the highest value reported in the literature for Yarrowia yeast. The obtained results suggest that citric acid production from inulin by engineered Y. lipolytica may be a very promising technology for industrial citric acid production.


Assuntos
Ácido Cítrico/síntese química , Engenharia Genética , Glicosídeo Hidrolases/genética , Inulina/química , Yarrowia/genética , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Glicosídeo Hidrolases/metabolismo , Hidrólise , Microbiologia Industrial , Kluyveromyces/genética
17.
Appl Microbiol Biotechnol ; 103(7): 3167-3179, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30734122

RESUMO

Lipogenesis is a complicated process involving global transcriptional reprogramming of lipogenic pathways. It is commonly believed that nitrogen starvation triggers a metabolic shift that reroutes carbon flux from Krebs cycles to lipogenesis. In this study, we systematically surveyed and dynamically profiled the transcriptional activity of 22 lipogenic promoters aiming to delineate a picture how nitrogen starvation regulates lipogenesis in Y. lipolytica. These lipogenic promoters drive the expression of critical pathways that are responsible for the generation of reducing equivalents (NADPH), carbon backbones (acetyl-CoA, malonyl-CoA, DHAP, etc.), synthesis and degradation of fatty acids. Specifically, our investigated promoters span across an array of metabolic pathways, including glycolysis, Krebs cycle, pentose phosphate pathway, mannitol cycle, glutamine-GABA cycle, fatty acid and lipid synthesis, glyoxylate, ß-oxidation, and POM (pyruvate-oxaloacetate-malate) cycle. Our work provides evidences that mannitol cycle, glutamine-GABA cycle and amino acid degradation, pyruvate oxidation, and acetate assimilation pathways are lipogenesis-related steps involved in generating cytosolic NADPH and acetyl-CoA precursors. This systematic investigation and dynamic profiling of lipogenic promoters may help us better understand lipogenesis, facilitate the formulation of structure-based kinetic models, as well as develop efficient cell factories for fuels and chemical production in oleaginous species.


Assuntos
Lipogênese , Regiões Promotoras Genéticas , Transcrição Genética , Yarrowia/genética , Yarrowia/metabolismo , Ciclo do Ácido Cítrico/genética , Ácidos Graxos/metabolismo , Regulação Fúngica da Expressão Gênica , Glicólise/genética , Metabolismo dos Lipídeos , Lipídeos/biossíntese , Redes e Vias Metabólicas , Oxirredução , Via de Pentose Fosfato/genética
18.
Methods Mol Biol ; 1927: 155-177, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788791

RESUMO

Yarrowia lipolytica is an industrial oleaginous yeast that has many attractive physiological and metabolic characteristics for various biotechnological applications. Although it has a long history of industrial applications, the number of genetic tools available to effectively and efficiently engineer Y. lipolytica still falls behind the vast number of tools available for common organisms such as Escherichia coli and Saccharomyces cerevisiae. In this protocol, we have developed a complete and versatile genetic toolkit tailored for facile genetic manipulation in Y. lipolytica. We created a versatile DNA assembly platform YaliBrick, which can streamline the cloning of large multigene pathways with reused genetic parts. We established a sensitive luciferase reporter assay to characterize a set of 12 native promoters. In addition, we used YaliBrick to generate different gene configurations in multigene constructs. The five-gene biosynthetic pathway of the anticancer, antimicrobial pigment violacein was rapidly assembled in 1 week to demonstrate the simplicity and effectiveness of integrating pathway-balancing strategies with our YaliBrick vectors. In the end, we incorporated CRISPR-Cas9 into our YaliBrick vectors and achieved indel mutation and frameshift gene deletion at the CAN1 (arginine permease) genomic loci of Yarrowia lipolytica. The reported protocol provides a standard procedure to streamline and accelerate metabolic pathway engineering in Yarrowia lipolytica.


Assuntos
Edição de Genes , Engenharia Genética , Engenharia Metabólica , Redes e Vias Metabólicas , Yarrowia/genética , Yarrowia/metabolismo , Sistemas CRISPR-Cas , Clonagem Molecular , DNA Fúngico , Edição de Genes/métodos , Expressão Gênica , Biblioteca Gênica , Genes Reporter , Engenharia Metabólica/métodos , Plasmídeos/genética , Regiões Promotoras Genéticas , RNA Catalítico , RNA Guia , Transformação Genética
19.
Bioresour Technol ; 281: 84-89, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30802819

RESUMO

Alginate oligosaccharides (AOS) showed various biological activities. Traditional protocol for producing AOS was a multiple-step and high-pollution procedure. In this study, a rapid and efficient AOS producing method was developed directly from Laminaria japonica. Natural sun-dried L. japonica with a feed ratio of 1:7 (w/v) was pretreated using cellulase with a dry weight of 3%, for releasing the fermentable sugars (8.5 g/L glucose and 15.2 g/L mannitol). Then, the engineered yeast Yarrowia lipolytica strain with alginate lyase activity was grown using an algae-based medium. After fermentation for 72 h, glucose and mannitol were completely consumed, and 71.8 mM AOS was extracted from the fermentation supernatant. The degree of polymerization (DP) was ranging from 2 to 3. The recovery yield of AOS was about 91.7%. The purity of the extracted AOS was 92.6%. Overall, our work provided new insights for the development of green biotechnologies for oligosaccharide production from seaweed.


Assuntos
Fermentação , Laminaria/metabolismo , Oligossacarídeos/metabolismo , Alginatos/metabolismo , Hidrólise , Alga Marinha/metabolismo , Yarrowia/genética , Yarrowia/metabolismo
20.
BMC Microbiol ; 19(1): 3, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30616522

RESUMO

BACKGROUND: To combat salinity, plants need easily accessible, safe and sustainable mechanisms for optimum growth. Recently, endophytes proved to be the promising candidates that helped the host plant to thrive under stress conditions. Therefore, the aim was to discover endophytic strain(s) and their mechanism of action to alleviate salt stress in maize. RESULTS: Keeping the diverse role of endophytes in view, 9 endophytic fungi from the spines of Euphorbia milli L. were isolated. Among the isolated fungal isolates, isolate FH1 was selected for further study on the basis of high antioxidant activity and capability to produce high indole-3-acetic acid (IAA), indole-3-acetamide (IAM), phenol and flavonoid contents. The 18S rDNA sequence homology and phylogenetic analysis of the fungal isolate FH1 revealed to be Yarrowia lipolytica. Furthermore, the inoculation of Y. lipolytica FH1 had significantly promoted plant growth attributes in treated maize as compared to positive (salt stress) and negative (salt stress free) controls. Likewise, differences in chlorophyll, carotenes, electrolyte leakage, leaf relative water, peroxidase, catalase, ABA, IAA and proline contents were observed between treated maize and controls. Interestingly, Y. lipolytica FH1 inoculated plants showed lower endogenous ABA and higher endogenous IAA contents. CONCLUSION: From the results, we have concluded that Y. lipolytica inoculation has promoted the growth of maize plants through controlled metabolism and hormonal secretions (ABA and IAA) under salinity stress. Because of the fact, Y. lipolytica can be tried as an eco-friendly bio-fertilizer to achieve optimum crop productivity under saline conditions.


Assuntos
Endófitos/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Estresse Salino/fisiologia , Yarrowia/metabolismo , Zea mays/microbiologia , Ácido Abscísico/metabolismo , Ácidos Indolacéticos/metabolismo , Filogenia , RNA Ribossômico 18S/genética , Yarrowia/classificação , Yarrowia/genética
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