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Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications.

Lazar Z, Rossignol T, Verbeke J, Crutz-Le Coq AM, Nicaud JM, Robak M - J. Ind. Microbiol. Biotechnol. (2013)

Bottom Line: This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate.Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context.The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, Chełmońskiego 37/41, 51-630, Wroclaw, Poland.

ABSTRACT
Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.

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Related in: MedlinePlus

a Schematic representation of integration of NotI digested PTURA-LEU2ex-zeta cassette from JMP1226 by double crossing-over at the URA3 locus of Po1d, giving rise to JMY2033, followed by single crossing-over of the SUC2-1462 cassette from JMP1462 at zeta platform giving rise to JMY2593. b Schematic representation of the 2 invertase expression cassette SUC2-302 and SUC2-1462. Signal sequence (ss); S. cerevisiae SUC2p (ScSUC2); truncated form of S. cerevisiae SUC2p deleted of the 11 first amino acids (ScSUC2short)
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Fig1: a Schematic representation of integration of NotI digested PTURA-LEU2ex-zeta cassette from JMP1226 by double crossing-over at the URA3 locus of Po1d, giving rise to JMY2033, followed by single crossing-over of the SUC2-1462 cassette from JMP1462 at zeta platform giving rise to JMY2593. b Schematic representation of the 2 invertase expression cassette SUC2-302 and SUC2-1462. Signal sequence (ss); S. cerevisiae SUC2p (ScSUC2); truncated form of S. cerevisiae SUC2p deleted of the 11 first amino acids (ScSUC2short)

Mentions: The zeta docking cassette for locus specific integration [5] was synthesized by Geneart (Life Technologies, Saint Aubin, France) and cloned at the I-CeuI restriction site of a vector containing a 1-kb promoter and 1-kb terminator region of URA3 surrounding the LEU2ex excisable marker and a I-CeuI restriction site (unpublished data, Fig. 1). This construction gives rise to JMP1226 (PTURA3-LEU2ex-zeta, Table 1). This cassette allows the integration by double crossing-over at the URA3 locus of a LEU2ex-zeta docking platform (Fig. 1) providing a locus-specific integration site as previously described [5].Fig. 1


Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications.

Lazar Z, Rossignol T, Verbeke J, Crutz-Le Coq AM, Nicaud JM, Robak M - J. Ind. Microbiol. Biotechnol. (2013)

a Schematic representation of integration of NotI digested PTURA-LEU2ex-zeta cassette from JMP1226 by double crossing-over at the URA3 locus of Po1d, giving rise to JMY2033, followed by single crossing-over of the SUC2-1462 cassette from JMP1462 at zeta platform giving rise to JMY2593. b Schematic representation of the 2 invertase expression cassette SUC2-302 and SUC2-1462. Signal sequence (ss); S. cerevisiae SUC2p (ScSUC2); truncated form of S. cerevisiae SUC2p deleted of the 11 first amino acids (ScSUC2short)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824566&req=5

Fig1: a Schematic representation of integration of NotI digested PTURA-LEU2ex-zeta cassette from JMP1226 by double crossing-over at the URA3 locus of Po1d, giving rise to JMY2033, followed by single crossing-over of the SUC2-1462 cassette from JMP1462 at zeta platform giving rise to JMY2593. b Schematic representation of the 2 invertase expression cassette SUC2-302 and SUC2-1462. Signal sequence (ss); S. cerevisiae SUC2p (ScSUC2); truncated form of S. cerevisiae SUC2p deleted of the 11 first amino acids (ScSUC2short)
Mentions: The zeta docking cassette for locus specific integration [5] was synthesized by Geneart (Life Technologies, Saint Aubin, France) and cloned at the I-CeuI restriction site of a vector containing a 1-kb promoter and 1-kb terminator region of URA3 surrounding the LEU2ex excisable marker and a I-CeuI restriction site (unpublished data, Fig. 1). This construction gives rise to JMP1226 (PTURA3-LEU2ex-zeta, Table 1). This cassette allows the integration by double crossing-over at the URA3 locus of a LEU2ex-zeta docking platform (Fig. 1) providing a locus-specific integration site as previously described [5].Fig. 1

Bottom Line: This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate.Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context.The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, Chełmońskiego 37/41, 51-630, Wroclaw, Poland.

ABSTRACT
Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.

Show MeSH
Related in: MedlinePlus