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2 edition of Study of the acid extracellular protease promoter of Yarrowia lipolytica found in the catalog.

Study of the acid extracellular protease promoter of Yarrowia lipolytica

Steven Andrew Hipkiss

Study of the acid extracellular protease promoter of Yarrowia lipolytica

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Published by University of Birmingham in Birmingham .
Written in English


Edition Notes

Thesis (M.Phil) - University of Birmingham, School of Biochemistry, Faculty of Science.

Statementby Steven Andrew Hipkiss.
The Physical Object
Pagination171 p. :
Number of Pages171
ID Numbers
Open LibraryOL20162086M

pullulans HN can produce alkaline protease9. In this study, some properties of the alkaline protease immobilized on cell surface of the yeast Yarrowia lipolytica have been investigated and compared with its free form purified from A. pullulans HN Materials and Methods Strains and media The Y. lipolytica yeast strain Po1h (genotype. The invention is related to an inducible promoter for improved and regulated gene expression, useful in synthetic biology and metabolic engineering. In particular, the present invention relates to a nucleotide sequence comprising the regulatory regions of an erythritol- and erythrulose-inducible promoter in yeast and uses thereof in an expression system thus allowing an improved and regulated.   The alkaline protease structural gene (ALP1 gene) was isolated from both the genomic DNA and cDNA of Aureobasidium pullulans 10 by inverse PCR and RT-PCR. An open reading frame of bp encoding a amino-acid protein with calculated molecular weight of kDa was characterized. The gene contained two introns, which had 54 bp and 50 bp, respectively. The promoter . The lipase2 from Yarrowia lipolytica (YLLip2) is a yeast lipase exhibiting high homologous to filamentous fungal lipase family. Though its crystal structure has been resolved, its structure-function relationship has rarely been reported. By contrast, there are two amino acid residues (V94 and I) with significant difference in the substrate binding pocket of YLLip2; they were subjected to.


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Study of the acid extracellular protease promoter of Yarrowia lipolytica by Steven Andrew Hipkiss Download PDF EPUB FB2

The gene encoding an acid extracellular protease (AXP) from Yarrowia lipolytica (Candida olea) was cloned and the complete nucleotide sequence was determined.

The amino acid sequence deduced from the nucleotide sequence reveals that the mature AXP consists of amino acids with an M, of Cited by: We have initiated a study of the promoter region of the alkaline extracellular protease gene (XPR2) from Yarrowia lipolytica to identify upstream sequences possibly involved in carbon, nitrogen.

McEwen RK, Young TW () Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease. Yeast – PubMed CrossRef Google Scholar Nelson G, Young TW () Extracellular acid Cited by: 2.

We isolated the LIP2 gene from the lipolytic yeast Yarrowia was found to encode a amino-acid precursor protein. The secreted lipase is a amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC ).The Lip2p precursor protein is processed by the KEX2-like endoprotease encoded by by:   The promoter from the Y.

lipolytica alkaline extracellular protease XPR2 gene, was one of the first promoters identified and is still widely used (Blanchin-Roland, Cordero Otero and Gaillardin ). While p XPR2 is capable of strong expression of heterologous proteins, it is repressed at pH Cited by: 1.

4. Discussions. Acid whey utilization by Y. lipolytica requires lactose hydrolysis. Similar to many recent studies that expressed extracellular enzymes to achieve the consumption of non-native substrates in Y.

lipolytica (Ledesma-Amaro and Nicaud, b), we chose to introduce secreted β-gal enzymes derived from A. addition, as galactose accounts for 50% of the hydrolyzed products.

Background: The lipase enzyme of the Yarrowia lipolytica yeast is an important enzyme in pharmaceutical, food and environmental biotechnology. Objectives: The aims of the study were cloning and molecular analysis of extracellular lipase genes from DSM native and U6 mutant strains of Y.

lipolytica in Escherichia coli. Materials and Methods: LIP2 gene of Y. lipolytica DSM and its. We isolated the LIP2 gene from the lipolytic yeast Yarrowia lipolytica. It was found to encode a amino-acid precursor protein.

The secreted lipase is a amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC [][1]). The Lip2p precursor protein is processed by the KEX2 -like endoprotease encoded by XPR6. Blanchin-Roland et al. () reported that Y. lipolytica extracellular protease (XPR2) promoter was regulated by a multitude of factors including pH, carbon, nitrogen and peptones.

In addition, upstream activating sequence (UAS) regions have been suggested as the major player in mediating the response to those factors (Wagner and Alper, ). 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. terminal fusion of the secretion signal of the extracellular alkaline protease (XPR2) to heterologous proteins.

For high-level expression, a strong hybrid promoter consisting of four copies of an upstream activator sequence (UAS1) from the XPR2 locus [27], fused to the LEU2 minimal promoter, can be used. In a comparative study, Y. lipolytica. This study found that pLIP2 is a promoter of choice as compared to pPOX2 to drive gene expression for recombinant protein production by Y.

lipolytica used as cell factory. In recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing recombinant proteins.

of the GPMFBA1 chimeric promoter confirmed that the 5′-region within the FBA1 gene could en-hance GPM1 promoter activity. Using the strong FBA1 IN promoter, we expressed S. cerevisiae SUC2 gene in Y. lipolytica. SUC+ phenotype of Y.

lipolytica was conferred by expression of either the Suc2, with its native signal sequence, or the. Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins.

Previous results have indicated that the alkaline protease response to pH was dependent on YlRimp, YlRim8p/YlPalF, and. Alkaline extracellular protease 1 Publication Yarrowia lipolytica (strain CLIB / E ) (Yeast) (Candida lipolytica) (UASs) are essential for promoter activity under conditions of repression or full induction.

The distal UAS (UAS1) is located at position to Young TW, Wadeson A, Glover DJ, Quincey RV, Butlin MJ, Kamei EA.

The extracellular acid protease gene of Yarrowia lipolytica: sequence and pH-regulated transcription. Microbiology. Oct; (Pt 10)– Porta A, Ramon AM, Fonzi WA. These vectors were used to express the Y.

lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached U ml −1 in shake flasks, while a strain containing multiple integrations reached U ml −1 in.

PCR amplification of CPO gene from Yarrowia lipolytica Po1f transformants. The ethidium stained (%) agarose gel showing, Yarrowia lipolytica Po1f strains transformed with expression cassette pINAPOX2-LIP2-CPO (lane 1, 2, 3 and 4), pINAPOX2-Lacc-CPO (lane 5 and 6), and Yarrowia lipolytica Po1f strain as negative control (lane 7).

The Yarrowia lipolytica PMR1 gene (YlPMR1) is a Saccharomyces cerevisiae PMR1 homolog which encodes a putative secretory pathway Ca2+-ATPase. In this study, we investigated the effects of a YlPMR1 disruption on the processing and secretion of native and foreign proteins in Y.

lipolytica and found variable responses by the YlPMR1 -disrupted mutant depending on the protein. Yarrowia lipolytica: recent achievements in heterologous protein ): Po1f, Po1g, and Po1h were further deleted for the acid extracellular protease (axpallele). Po1g was also fitted with an integrated pBR docking platform (ura3- composed of a promoter (P), the open reading frame (ORF).

The XPR2 gene encoding an alkaline extracellular protease (AEP) from Yarrowia lipolytica was cloned, and its complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AEP consists of amino acids with a relative molecular weight of 30, The industrial yeast Yarrowia lipolytica secretes high amounts of an alkaline extracellular protease encoded by the XPR2 gene.

The industrial use of the XPR2 promoter was however hindered by its complex regulation. We designed hybrid promoters, based on tandem copies of the XPR2 promoter UAS1 region. In contrast to native XPR2 promoter, these. Yarrowia lipolytica possesses 16 genes encoding for lipases and one study suggests that 15 of the 16 lipases are capable of acting intracellularly (Syal and Gupta, ).

Regardless of location, the first step involves the cleavage of an ester bond to form a free fatty acid and a diacylglycerol (DAG). The development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism.

Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeast Yarrowia lipolytica. These synthetic promoters are comprised of two modular. Yarrowia lipolytica has shown promise for converting glycerol to SCO and citric acid (André et al., ; Makri et al., ).

Recently, lipid accumulation has been found in the Y. lipolytica strains grown on glycerol in the range of –% of cell dry weight (Sriwongchai et al., ). promoter of Y.

lipolytica extracellular protease (XPR2) can be Figure 1. Promoter architecture. (A) Eukaryotic promoters contain a core promoter sequence that may have a TATA box, a proximal promoter sequence, and enhancer sequences located farther upstream.

(B) Hybrid promoters were created by placing eight UAS1B sequences (UAS1B8). Yarrowia lipolytica strain PO1f (ATCC MYA), a leucine and uracil auxotroph devoid of any secreted protease activity (28), was used for all studies.

lipolytica PO1f containing plasmids was routinely cultivated at 30°C with constant agitation in yeast synthetic complete (YSC)-LEU medium, con. The Y. lipolytica strains used in this study are derived from trainsareroutinelygrown on YPD medium (1% (w/v) yeast extract, 1%.

A dimorphic yeast in the phylum Ascomycota. This species is strictly aerobic, degrades hydrophobic substrates, assimilates glucose, fructose and mannose, and utilizes ethanol and glycerol as carbon sources.

lipolytica is a biocatalyst and used commercially in biotransformation to catalyze hydrolysis, esterification, interesterification and transesterification reactions. In this study, a novel rDNA based plasmid was developed for display of heterologous proteins on the cell surface of Yarrowia lipolytica using the C-terminal end of the glycosylphosphatidylinositol (GPI) anchored Y.

lipolytica cell wall protein 1 (YlCWP1). mCherry was used as a model protein to assess the efficiency of the constructed plasmid. lipolytica transformants harbouring the. Biotechnological Applications of the Yeast Yarrowia lipolytica Appl Environ Microbiol 80(5)– doi/ aem McEwen RK, Young TW () Secretion and pH.

To produce hIFN a2b in Y. lipolytica and to target its production into the culture medium, we had previously expressed this gene under the control of POX2 promo-ter inducible by oleic acid.

Different sequences of the signal secretion signal of Y. lipolytica extracellular lipase encoded by the LIP2 gene were tested [13]. Best results. Microbial strains and growth conditions. Yarrowia lipolytica Po1h (MATA ura xpr axp) provided by C.

Madzak (INRA, Thiverval-Grignon, France) was used as the parental strain for the construction of Y. lipolytica Δmhb1 (MATA ΔmhbURA3 ura xpr axp). lipolytica strains E (MATA ura leu lys) and E (MATA ura leu lys xpr2.

Saccharomyces cerevisiae cannot produce extracellular lipase and utilize low‐cost lipid substrates. This study aimed to express extracellular lipase from Yarrowia lipolytica in S. cerevisiae, construct recombinant oily substrate consumer strains, and compare the roles of native and mutant Y.

lipolytica extracellular lipases in S. LIP2 gene of Y. lipolytica DSM and its. The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases.

To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light.

The acid protease structural gene was amplified from the genomic DNA of Saccharomycopsis fibuligera A When the gene was cloned into the multiple cloning site of the surface display vector pINAYlCWP and expressed in the cells of Yarrowia lipolytica, the cells displaying the acid protease could form clear zone on the plate-containing milk indicating that they had extracellular acid.

Recent advances in bioengineering of the oleaginous yeast Yarrowia lipolytica Murtaza Shabbir Hussain, Gabriel M Rodriguez, Difeng Gao, Michael Spagnuolo, Lauren promoter level, genetic tools can be engineered by constructing strong hybrid responsive and alkaline extracellular protease (XPR2).

A list of the native promoters commonly. The gene (RIM) encodes a protein of amino acid residues, which shows highest similarity to PacC proteins from Fusarium oxysporum and Aspergillus niger. The gene had the capacity to restore protease activity to rim mutants from Yarrowia lipolytica, confirming its homologous function, and was expressed at both acid and neutral pH.

Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non‐reverting auxotrophic markers, deletions of protease‐encoding genes, and carrying a docking platform.

To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. In this study, Streptomyces hygroscopicus pro-TGase was efficiently expressed and secreted by a food-grade host, Yarrowia lipolytica, without antibiotic markers.

The pro-TGase gene was cloned into integrative vectors pINA (monocopy) and pINA (multicopy), and was used to transform the Y. lipolytica Po1g or Po1h strain, respectively. Regulation, purification, characterization, and processing of yeast extracellular proteases are reviewed. Results obtained from the sequences of cloned genes, especially the Saccharomyces cerevisae Bar protease, the Candida albicans acid protease, and the Yarrowia lipolytica alkaline protease, have been emphasized.

Biotechnological applications.Aims: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica‐overproducing mutant in order to optimize its production in large‐scale bioreactors.

Methods and Results: The level of lipase production and LIP2 induction, measured using an LIP2–LacZ reporter gene, were compared for different carbon and nitrogen sources and for.YLEX Expression Kit based on INRA INAPG licensed patent* provides an easy approach for cloning and expressing a gene of interest in the yeast, Yarrowia this kit, high level of heterologous protein may be expressed intracellularly or be secreted from the cell into the medium by selecting the supplied expression vector pYLEX1 or pYLSC1.