Protein expression and secretion in the yeast<i>Yarrowia lipolytica</i>

Nicaud, Jean‐Marc; Madzak, Catherine; Broek, P. van den; Gisler, Christophe; Duboc, Philippe; Niederberger, Peter; Gaillardin, Claude

doi:10.1111/j.1567-1364.2002.tb00106.x

Cited by 94 publications

(129 citation statements)

References 23 publications

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“…The sequences of the primers were as follows: POX2-ATG, 5Ј-TCCGCCTAGGCACAATGAAC CCCAACAACACTGGCACCATTG-3Ј; POX2-STOP, 5Ј-CAGGCCCGCGGG GCCCTATTCCTCATCAAGCTCGCAAATGTC-3Ј; POX3-ATG, 5Ј-GATCC GCCTAGGCACAATGATCTCCCCCAACCTCACAGCT-3Ј; and POX3-STOP, 5Ј-GAGGCCCGCGGGGCCCTATTCCTCGTCCAGCACGCAAATG-3Ј. The PCR fragments were cleaved with BlnI and SfiI and inserted into the plasmid pYEG1 (22), giving rise to the plasmids pYEG1-POX2 and pYEG1-POX3, respectively. These plasmids allow the expression of the POX2 and POX3 genes under the control of the POX2 promoter.…”

Section: Methodsmentioning

confidence: 99%

Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast Yarrowia lipolytica

Mlı́čková

Roux

Athenstaedt

et al. 2004

Appl Environ Microbiol

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Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal ␤-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (⌬pox2 ⌬pox3 ⌬pox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.The yeast Yarrowia lipolytica can grow on alkanes or fatty acids as the sole carbon source (for a review, see reference 5). Alkanes must be converted to fatty acids before they can serve as substrates for different metabolic pathways. In mammalian cells, the degradation of fatty acids occurs through ␤-oxidation in mitochondria and peroxisomes. In yeasts, the enzymes in this pathway are present only in peroxisomes. Fatty acid degradation is a multiple-step process requiring four different enzymatic activities. The first step is catalyzed by an acyl-coenzyme A (CoA) oxidase (Aoxp). Saccharomyces cerevisiae contains only one Aox (28), but Y. lipolytica contains five Aox isoenzymes with different substrate specificities and different activity levels ( Fig. 1) (27,28). Aox3p is specific for short-chain acyl-CoAs (17), Aox2p preferentially oxidizes long-chain acylCoAs (18), and the Aox4p and Aox5p activities do not appear to be sensitive to the length of the aliphatic chain of the acyl-CoA (29). Previous studies revealed that the mutants MTLY36 (⌬pox2 ⌬pox3 ⌬pox5) and MTLY37 (⌬pox2 ⌬pox3 ⌬pox4 ⌬pox5) either had a growth defect or did not grow at all, respectively, when cultivated on oleic acid minimal medium (28).Fatty acids can also be utilized as storage molecules when they are incorporated into triacylglycerols. All eukaryotic organisms and some gram-positive bacteria store triacylglycerols in intracellular compartments that are variously termed lipid particles, lipid droplets (LDs), lipid bodies (LBs), oil bodies, oleosomes, or spherosomes (in plants). The structure of these lipid-rich compartments is similar in all cell types and is rather simple: LBs consist of a hydrophobic core formed from neutral lipids, mainly triacylglycerols and/or steryl esters...

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Section: Methodsmentioning

confidence: 99%

Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast Yarrowia lipolytica

Mlı́čková

Roux

Athenstaedt

et al. 2004

Appl Environ Microbiol

Self Cite

197

135

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“…The yeast Y. lipolytica can produce large amounts of proteolytic enzymes; among them, alkaline protease (AEP) and acid protease (AXP) are the most common. Alkaline proteases, however, are the major protein secreted, reaching several grams per litre under optimized conditions (Nicaud et al, 2002). The secretion of this enzyme can occur in parallel to lipase and it is induced at neutral/basic pH.…”

Section: Effect Of Nitrogen Source On Lipase Productionmentioning

confidence: 99%

NITROGEN SOURCES ON TPOMW VALORIZATION THROUGH SOLID STATE FERMENTATION PERFORMED BY Yarrowia lipolytica

Lopes

Farias

Belo

et al. 2016

Braz. J. Chem. Eng.

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-This manuscript reports the valorization of two-phase olive mill waste (TPOMW) as raw material and carbon source for solid state fermentation using Yarrowia lipolytica as biocatalyst. Due to its chemical characteristics, a combination of different raw materials (TPOMW and wheat bran, WB) was evaluated and two distinct nitrogen sources were applied as supplementation for lipase production. A TPOMW/WB ratio of 1:1 and supplementation with ammonium sulfate was chosen as the best condition. The productivity in 24 h reached 7.8 U/g*h and, after four days of process, only decreased about 35%. Process pH ranged from 5.5 -5.9, remaining in an acid range. Thus, the successful use of TPOMW, a watery solid by-product with high content of lipids, as raw material for Yarrowia lipolytica growth and lipase production provided an environmental friendly alternative to valorize such waste.

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“…Plasmid pYEG1 [17] was digested by BlnI and SfiI and ligated with the PCR fragments digested with the same enzymes, giving rise to plasmid pYEG1-POX2 and pYEG1-POX3. Both expression cassettes were introduced into MTLY36 and MTLY40 by the lithium acetate method [18].…”

Section: Strain Constructionsmentioning

confidence: 99%

Genetic engineering of the β-oxidation pathway in the yeast Yarrowia lipolytica to increase the production of aroma compounds

Groguenin¹

2004

Journal of Molecular Catalysis B: Enzymatic

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The yeast Yarrowia lipolytica possesses five acyl-CoA oxidases (Aox1p to 5), the enzyme catalysing the first reaction of ␤-oxidation. The understanding of the specific role of each acyl-CoA oxidase is important to construct a yeast strain growing at a good rate and able to produce without degrading the aroma compound ␥-decalactone. In this study we observed that Aox4p exhibits a slight activity on a broad spectrum of substrates and that it is involved in lactone degradation. We constructed a strain lacking this activity. Its growth was only slightly altered and it produced 10 times more lactone than the wild type in 48 h.

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Protein expression and secretion in the yeastYarrowia lipolytica

Cited by 94 publications

References 23 publications

Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast Yarrowia lipolytica

Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast Yarrowia lipolytica

NITROGEN SOURCES ON TPOMW VALORIZATION THROUGH SOLID STATE FERMENTATION PERFORMED BY Yarrowia lipolytica

Genetic engineering of the β-oxidation pathway in the yeast Yarrowia lipolytica to increase the production of aroma compounds

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