Dipeptidyl aminopeptidase processing and biosynthesis of alkaline extracellular protease from Yarrowia lipolytica

Matoba, S; Morano, Kevin A.; Klionsky, Daniel J.; Kim, Keunsung; Ogrydziak, David M.

doi:10.1099/00221287-143-10-3263

Cited by 24 publications

(30 citation statements)

References 25 publications

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“…Purification, characterization, and regulation of proteinases have been reported for seven species (37), and since Saccharomyces cerevisiae does not secrete any proteinase, most studies have focused on two species, Candida albicans, which secretes at least seven acidic aspartyl proteinases (Sap) possibly involved in pathogenicity (10,32), and Yarrowia lipolytica, which secretes both an acidic aspartyl proteinase (AXP) and an alkaline seryl proteinase (AEP), depending on the pH of the growth medium (1). AEP secretion and processing have been extensively studied (14,15,30,31), and AEP was used as a model reporter molecule to investigate protein secretion in this yeast (6,20,52). The regulation of C. albicans and Y. lipolytica extracellular proteinases appears to be quite complex, but the mechanisms involved have some elements in common.…”

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confidence: 99%

Genetic Analysis of Regulatory Mutants Affecting Synthesis of Extracellular Proteinases in the Yeast Yarrowia lipolytica: Identification of a RIM101/pacC Homolog

Lambert

Blanchin‐Roland

Louedec

et al. 1997

Molecular and Cellular Biology

107

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Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes both an acidic proteinase and an alkaline proteinase, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Recessive mutations at four unlinked loci, named PAL1 to PAL4, were isolated which prevent alkaline proteinase derepression under conditions of carbon and nitrogen limitation at pH 6.8. These mutations markedly affect mating and sporulation. A dominant suppressor of all four PAL mutations was isolated from a wild-type genomic library, which turned out to be a C-terminally truncated form of a 585-residue transcriptional factor of the His 2 Cys 2 zinc finger family, which we propose to call YlRim101p. Another C-terminally truncated version of YlRim101p (419 residues) is encoded by the dominant RPH2 mutation previously isolated as expressing alkaline protease independently of the pH. YlRim101p is homologous to the transcriptional activators Rim101p of Saccharomyces cerevisiae, required for entry into meiosis, and PacC of Aspergillus nidulans and Penicillium chrysogenum, which were recently shown to mediate regulation by ambient pH. YlRim101p appears essential for mating and sporulation and for alkaline proteinase derepression. YlRIM101 expression is autoregulated, maximal at alkaline pH, and strongly impaired by PAL mutations.

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confidence: 99%

Genetic Analysis of Regulatory Mutants Affecting Synthesis of Extracellular Proteinases in the Yeast Yarrowia lipolytica: Identification of a RIM101/pacC Homolog

Lambert

Blanchin‐Roland

Louedec

et al. 1997

Molecular and Cellular Biology

107

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“…The AEP precursor undergoes complex processing (27). A diaminopeptidase processes the stretch of nine dipeptides (X-Ala or X-Pro) (28,29), and the endoprotease encoded by the XPR6 gene is required to cleave the pro region, releasing the mature form (14). The pro region is involved in both the inhibition of protease activity and the folding of the propeptide into a conformation compatible with secretion, and secretion at 28°C depends on the glycosylation of the pro region (15,28).…”

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confidence: 99%

Characterization of an Extracellular Lipase Encoded by LIP2 in Yarrowia lipolytica

et al. 2000

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We isolated the LIP2 gene from the lipolytic yeast Yarrowia lipolytica. It was found to encode a 334-amino-acid precursor protein. The secreted lipase is a 301-amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC 3.1.1.3). The Lip2p precursor protein is processed by the KEX2-like endoprotease encoded by XPR6. Deletion of the XPR6 gene resulted in the secretion of an active but less stable proenzyme. Thus, the pro region does not inhibit lipase secretion and activity. However, it does play an essential role in the production of a stable enzyme. Processing was found to be correct in LIP2A (multiple LIP2 copy integrant)-overexpressing strains, which secreted 100 times more activity than the wild type, demonstrating that XPR6 maturation was not limiting. No extracellular lipase activity was detected with the lip2 knockout (KO) strain, strongly suggesting that extracellular lipase activity results from expression of the LIP2 gene. Nevertheless, the lip2 KO strain is still able to grow on triglycerides, suggesting an alternative pathway for triglyceride utilization in Y. lipolytica. This yeast naturally secretes several proteins, depending on the growth conditions (21). For example, if the pH is higher than 6, it secretes an alkaline extracellular protease (AEP) (33,38). Under optimal conditions, up to 1 g of AEP is secreted per liter (37). AEP is encoded by XPR2 (11,27,33). This gene codes for a 454-amino-acid (aa) prepro enzyme precursor containing a 15-aa signal sequence and a stretch of nine X-Ala or X-Pro dipeptides, followed by a 124-aa pro region that includes a glycosylation site (Asn 123 ) and a Lys 156 -Arg 157 processing site, and finally the mature form itself. The AEP precursor undergoes complex processing (27). A diaminopeptidase processes the stretch of nine dipeptides (X-Ala or X-Pro) (28,29), and the endoprotease encoded by the XPR6 gene is required to cleave the pro region, releasing the mature form (14). The pro region is involved in both the inhibition of protease activity and the folding of the propeptide into a conformation compatible with secretion, and secretion at 28°C depends on the glycosylation of the pro region (15, 28).Several enzymes are secreted by Y. lipolytica, and lipase and esterase activities have been detected and analyzed in various studies. Lipase secretion was first reported in 1948 by Peters and Nelson (41, 42), who described a single type of glucoserepressible activity. An extracellular and two cell-bound types of activity corresponding to lipase I (39 kDa) and lipase II (44 kDa) were described by Ota and coworkers (39, 47). The extracellular lipase required oleic acid as a stabilizer-activator, whereas the cell-bound lipases did not and differed in several properties from the extracellular enzyme (40). The rates of production of the extracellular and cell-bound enzymes were reported to depend on the carbon and nitrogen composition of the medium. Extracellular lipase was only detected in cultures grown with an organic nitrogen so...

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“…Thus, the crude alkaline extracellular enzyme has a relative molecular weight about 35 kDa. According to Matoba and Ogrydziak (1989); Barth and Gaillardin (1997), Yarrowia lipolytica can produce an Alkaline extracellular protease with a molecular weight of 32 kDa which processed from a 55 kDa glycosylated precursor. It was also mentioned that the molecular weight of alkaline protease secreted by microorganism ranged between 15 to 45 kDa (Kumar and Takagi, 1999).…”

Section: Gel Electrophoresismentioning

confidence: 99%

Characterization of the Crude Alkaline Extracellular Protease of Yarrowia lipolytica YlTun15

Bessadok¹,

Masri²,

Breuck³

et al. 2017

IPFS

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Abstract:Yarrowia lipolytica YlTun15 (GeneBank Acc. N° MF327143), isolated from farmed Dicentrarchus labrax's gills, secretes an alkaline extracellular protease, which exhibited the highest activity at pH 9 and temperature 45°C. The enzyme activity extracted was tested in the presence of different ion metal and protein inhibitors. .48% respectively at the 5 mM level. The enzyme was almost (activity=1.47%) inhibited by phenylmethylsulfonyl fluoride at the concentration of 5 mM. However, the protease activity was relatively constant in the presence of EDTA and SDS that may conclude that this enzyme was not a metalloprotease and belong to the serine protease category. After 18 months-storage at -20°C, the enzyme activity has decreased to 23.17%. This protease may have a potential application in food and detergent activity.

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Dipeptidyl aminopeptidase processing and biosynthesis of alkaline extracellular protease from Yarrowia lipolytica

Cited by 24 publications

References 25 publications

Genetic Analysis of Regulatory Mutants Affecting Synthesis of Extracellular Proteinases in the Yeast Yarrowia lipolytica: Identification of a RIM101/pacC Homolog

Genetic Analysis of Regulatory Mutants Affecting Synthesis of Extracellular Proteinases in the Yeast Yarrowia lipolytica: Identification of a RIM101/pacC Homolog

Characterization of an Extracellular Lipase Encoded by LIP2 in Yarrowia lipolytica

Characterization of the Crude Alkaline Extracellular Protease of Yarrowia lipolytica YlTun15

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