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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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Growth kinetics of SUC+Y. lipolytica strains in medium with sucrose as the carbon source in a 5-l bioreactor. Biomass accumulation during cultures is presented on log10 scale
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Fig3: Growth kinetics of SUC+Y. lipolytica strains in medium with sucrose as the carbon source in a 5-l bioreactor. Biomass accumulation during cultures is presented on log10 scale

Mentions: To begin with, the growth kinetic of the four strains described above was determined by following biomass production in a 5-l stirred-tank bioreactor with sucrose as the sole carbon source and containing YE (0.03 % final concentration) as inducer of pXPR2 promoter for SUC2-302 cassette. In this experimental setup, strains JMY2531, JMY2593, and B56-5 have a similar growth rate and reach stationary phase within 14 h, whereas strain JMY2529, expressing only one copy of SUC2-302, has a slower growth rate and reaches stationary phase within 24 h but with a similar final cell density (Fig. 3). Strains JMY2531 and JMY2593 have maximum growth rates of 0.139 and 0.161, respectively. The strain B56-5 has a maximum growth rate of 0.132, while the strain JMY2529 has a growth rate of only 0.096 (Table 2). This is in line with what has been seen in microtiter plates, except that growth of JMY2529 is less delayed and that of B56-5 is not delayed compared to JMY2531 and JMY2593 in the bioreactor.Fig. 3


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)

Growth kinetics of SUC+Y. lipolytica strains in medium with sucrose as the carbon source in a 5-l bioreactor. Biomass accumulation during cultures is presented on log10 scale
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Growth kinetics of SUC+Y. lipolytica strains in medium with sucrose as the carbon source in a 5-l bioreactor. Biomass accumulation during cultures is presented on log10 scale
Mentions: To begin with, the growth kinetic of the four strains described above was determined by following biomass production in a 5-l stirred-tank bioreactor with sucrose as the sole carbon source and containing YE (0.03 % final concentration) as inducer of pXPR2 promoter for SUC2-302 cassette. In this experimental setup, strains JMY2531, JMY2593, and B56-5 have a similar growth rate and reach stationary phase within 14 h, whereas strain JMY2529, expressing only one copy of SUC2-302, has a slower growth rate and reaches stationary phase within 24 h but with a similar final cell density (Fig. 3). Strains JMY2531 and JMY2593 have maximum growth rates of 0.139 and 0.161, respectively. The strain B56-5 has a maximum growth rate of 0.132, while the strain JMY2529 has a growth rate of only 0.096 (Table 2). This is in line with what has been seen in microtiter plates, except that growth of JMY2529 is less delayed and that of B56-5 is not delayed compared to JMY2531 and JMY2593 in the bioreactor.Fig. 3

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