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Expression of native and mutant extracellular lipases fromYarrowia lipolytica in Saccharomyces cerevisiae.

Darvishi F - Microb Biotechnol (2012)

Bottom Line: These strains can utilize olive oil and lipids as low-cost substrates to produce bioethanol, single cell protein and other biotechnologically valuable products.The LIP2 gene of Y. lipolytica was expressed in S. cerevisiae as a heterologous protein without any modifications.Strong components of the Y. lipolytica expression/secretion system could be used for high-level production of recombinant proteins in S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science, University of Maragheh, Maragheh, 55181-83111, Iran. f.darvishi@ymail.com

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Schematic representation of extracellular lipase gene (LIP2), p426GPD, pFDP100 and pFDP101 vectors.A. Schematic representation of extracellular lipase gene (LIP2) and situation of forward primer (Ylip2START) and reverse primer (Ylip2STOP). Shown are the putative 13‐aa signal sequence (SS), followed by a stretch of four dipeptides (DP); a short 12‐aa pro region (PRO), including the Lys‐Arg (KR) cleavage site of the KEX2‐like XPR6 endoprotease; and the mature 301‐aa lipase (MATURE). The diamonds indicate the positions of the potential signals for asparagine‐linked glycosylation (Asn‐X‐Thr/Ser). The promoter (P) and terminator (T) regions, consisting of 1.06 and 0.97 kb fragments situated upstream and downstream from the ylLIP2 ORF respectively.B. Schematic representation structure of the p426GPD Saccharomyces cerevisiae expression vector. GPD promoter, glycerol phosphate dehydrogenase promoter; Lac promoter, lac promoter; T3, T3 phage promoter; T7 promoter, T7 phage promoter; AmpR, ampicillin resistance gene; URA3, URA3 marker; 2‐micron ORI, yeast 2 μ expression replication origin; pMB1 ORI, origin replication of E. coli; F1 ORI, origin of replication.C. The structure of pFDP100 vector containing extracellular lipase ORF of native Y. lipolytica strain DSM3286 (Y.DSM3286 Lip2).D. The structure of pFDP101 vector containing extracellular lipase ORF of the mutant Y. lipolytica strain U6 (Y.U6 Lip2).
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f1: Schematic representation of extracellular lipase gene (LIP2), p426GPD, pFDP100 and pFDP101 vectors.A. Schematic representation of extracellular lipase gene (LIP2) and situation of forward primer (Ylip2START) and reverse primer (Ylip2STOP). Shown are the putative 13‐aa signal sequence (SS), followed by a stretch of four dipeptides (DP); a short 12‐aa pro region (PRO), including the Lys‐Arg (KR) cleavage site of the KEX2‐like XPR6 endoprotease; and the mature 301‐aa lipase (MATURE). The diamonds indicate the positions of the potential signals for asparagine‐linked glycosylation (Asn‐X‐Thr/Ser). The promoter (P) and terminator (T) regions, consisting of 1.06 and 0.97 kb fragments situated upstream and downstream from the ylLIP2 ORF respectively.B. Schematic representation structure of the p426GPD Saccharomyces cerevisiae expression vector. GPD promoter, glycerol phosphate dehydrogenase promoter; Lac promoter, lac promoter; T3, T3 phage promoter; T7 promoter, T7 phage promoter; AmpR, ampicillin resistance gene; URA3, URA3 marker; 2‐micron ORI, yeast 2 μ expression replication origin; pMB1 ORI, origin replication of E. coli; F1 ORI, origin of replication.C. The structure of pFDP100 vector containing extracellular lipase ORF of native Y. lipolytica strain DSM3286 (Y.DSM3286 Lip2).D. The structure of pFDP101 vector containing extracellular lipase ORF of the mutant Y. lipolytica strain U6 (Y.U6 Lip2).

Mentions: In this study, the S. cerevisiae expression vector p426GPD with strong constitutive glycerol phosphate dehydrogenase (GPD) promoter was used for LIP2 gene expression (Fig. 1B). The native LIP2 gene from Y. lipolytica DSM3286 and the mutant LIP2 gene from the mutant Y. lipolytica U6 were cloned into the vector without any modifications. The resulting constructs designated pFDP100 and pFDP101 contained the native and mutant LIP2 gene respectively (Fig. 1C and D). The vectors were transformed to the S. cerevisiae strain CEN.PK 113‐5D, and then, the new recombinant S. cerevisiae containing native and mutant extracellular lipase named S. cerevisiae FDS100 and FDS101 respectively.


Expression of native and mutant extracellular lipases fromYarrowia lipolytica in Saccharomyces cerevisiae.

Darvishi F - Microb Biotechnol (2012)

Schematic representation of extracellular lipase gene (LIP2), p426GPD, pFDP100 and pFDP101 vectors.A. Schematic representation of extracellular lipase gene (LIP2) and situation of forward primer (Ylip2START) and reverse primer (Ylip2STOP). Shown are the putative 13‐aa signal sequence (SS), followed by a stretch of four dipeptides (DP); a short 12‐aa pro region (PRO), including the Lys‐Arg (KR) cleavage site of the KEX2‐like XPR6 endoprotease; and the mature 301‐aa lipase (MATURE). The diamonds indicate the positions of the potential signals for asparagine‐linked glycosylation (Asn‐X‐Thr/Ser). The promoter (P) and terminator (T) regions, consisting of 1.06 and 0.97 kb fragments situated upstream and downstream from the ylLIP2 ORF respectively.B. Schematic representation structure of the p426GPD Saccharomyces cerevisiae expression vector. GPD promoter, glycerol phosphate dehydrogenase promoter; Lac promoter, lac promoter; T3, T3 phage promoter; T7 promoter, T7 phage promoter; AmpR, ampicillin resistance gene; URA3, URA3 marker; 2‐micron ORI, yeast 2 μ expression replication origin; pMB1 ORI, origin replication of E. coli; F1 ORI, origin of replication.C. The structure of pFDP100 vector containing extracellular lipase ORF of native Y. lipolytica strain DSM3286 (Y.DSM3286 Lip2).D. The structure of pFDP101 vector containing extracellular lipase ORF of the mutant Y. lipolytica strain U6 (Y.U6 Lip2).
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Related In: Results  -  Collection

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f1: Schematic representation of extracellular lipase gene (LIP2), p426GPD, pFDP100 and pFDP101 vectors.A. Schematic representation of extracellular lipase gene (LIP2) and situation of forward primer (Ylip2START) and reverse primer (Ylip2STOP). Shown are the putative 13‐aa signal sequence (SS), followed by a stretch of four dipeptides (DP); a short 12‐aa pro region (PRO), including the Lys‐Arg (KR) cleavage site of the KEX2‐like XPR6 endoprotease; and the mature 301‐aa lipase (MATURE). The diamonds indicate the positions of the potential signals for asparagine‐linked glycosylation (Asn‐X‐Thr/Ser). The promoter (P) and terminator (T) regions, consisting of 1.06 and 0.97 kb fragments situated upstream and downstream from the ylLIP2 ORF respectively.B. Schematic representation structure of the p426GPD Saccharomyces cerevisiae expression vector. GPD promoter, glycerol phosphate dehydrogenase promoter; Lac promoter, lac promoter; T3, T3 phage promoter; T7 promoter, T7 phage promoter; AmpR, ampicillin resistance gene; URA3, URA3 marker; 2‐micron ORI, yeast 2 μ expression replication origin; pMB1 ORI, origin replication of E. coli; F1 ORI, origin of replication.C. The structure of pFDP100 vector containing extracellular lipase ORF of native Y. lipolytica strain DSM3286 (Y.DSM3286 Lip2).D. The structure of pFDP101 vector containing extracellular lipase ORF of the mutant Y. lipolytica strain U6 (Y.U6 Lip2).
Mentions: In this study, the S. cerevisiae expression vector p426GPD with strong constitutive glycerol phosphate dehydrogenase (GPD) promoter was used for LIP2 gene expression (Fig. 1B). The native LIP2 gene from Y. lipolytica DSM3286 and the mutant LIP2 gene from the mutant Y. lipolytica U6 were cloned into the vector without any modifications. The resulting constructs designated pFDP100 and pFDP101 contained the native and mutant LIP2 gene respectively (Fig. 1C and D). The vectors were transformed to the S. cerevisiae strain CEN.PK 113‐5D, and then, the new recombinant S. cerevisiae containing native and mutant extracellular lipase named S. cerevisiae FDS100 and FDS101 respectively.

Bottom Line: These strains can utilize olive oil and lipids as low-cost substrates to produce bioethanol, single cell protein and other biotechnologically valuable products.The LIP2 gene of Y. lipolytica was expressed in S. cerevisiae as a heterologous protein without any modifications.Strong components of the Y. lipolytica expression/secretion system could be used for high-level production of recombinant proteins in S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science, University of Maragheh, Maragheh, 55181-83111, Iran. f.darvishi@ymail.com

Show MeSH
Related in: MedlinePlus