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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

Sugars and CA concentrations during growth of Y. lipolytica SUC+ strains in a bioreactor with minimum medium and sucrose as the carbon source. a Sucrose hydrolysis kinetics. b Glucose concentration. c Fructose concentration. d CA concentration. Graph presented are average of three replicates
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Fig5: Sugars and CA concentrations during growth of Y. lipolytica SUC+ strains in a bioreactor with minimum medium and sucrose as the carbon source. a Sucrose hydrolysis kinetics. b Glucose concentration. c Fructose concentration. d CA concentration. Graph presented are average of three replicates

Mentions: Invertase secretion allows sucrose hydrolysis and leads to glucose and fructose appearance in the medium, which are subsequently uptaken by the yeast. In the bioreactor study, the rate of sucrose hydrolysis and the subsequent glucose and fructose disappearance from the medium were also analyzed. Sucrose is hydrolyzed at different rates by the four invertase-expressing strains. Sucrose degradation rate (Rs in g/l/h) calculated for JMY2529 is the slowest one (Rs = 2.50) with sucrose being fully hydrolyzed after 52 h (Fig. 5a). It is faster for B56-5 (Rs = 6.15), where sucrose is fully hydrolyzed after 24 h (Fig. 5a), and even faster for JMY2531 (Rs = 7.63) and for JMY2593 (Rs = 7.07), which hydrolyses sucrose within 14 h (Fig. 5a). These last two strains have a very similar profile for sucrose hydrolysis. The profile of strain B56-5 is slightly different. It has a slow hydrolysis rate at the beginning of the growth until around 12 h when the hydrolysis rate increases. However, it does not affect growth compared to JMY2531 and JMY2593 (Fig. 3). These sucrose hydrolysis data are in line with the extracellular invertase activity level of these different strains. At mid-exponential phase, both strains JMY2531 and JMY2593 have a high and similar extracellular invertase activity and present a rapid sucrose cleavage, JMY2529 has very low activity and a low sucrose cleavage activity, with B56-5 being in between (Fig. 4a). JMY2531 and JMY2593 have the same sucrose hydrolysis rate, which confirms that expression of SUC2-1462 cassette alone is sufficient for maximum hydrolysis rate in that condition.Fig. 5


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)

Sugars and CA concentrations during growth of Y. lipolytica SUC+ strains in a bioreactor with minimum medium and sucrose as the carbon source. a Sucrose hydrolysis kinetics. b Glucose concentration. c Fructose concentration. d CA concentration. Graph presented are average of three replicates
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Sugars and CA concentrations during growth of Y. lipolytica SUC+ strains in a bioreactor with minimum medium and sucrose as the carbon source. a Sucrose hydrolysis kinetics. b Glucose concentration. c Fructose concentration. d CA concentration. Graph presented are average of three replicates
Mentions: Invertase secretion allows sucrose hydrolysis and leads to glucose and fructose appearance in the medium, which are subsequently uptaken by the yeast. In the bioreactor study, the rate of sucrose hydrolysis and the subsequent glucose and fructose disappearance from the medium were also analyzed. Sucrose is hydrolyzed at different rates by the four invertase-expressing strains. Sucrose degradation rate (Rs in g/l/h) calculated for JMY2529 is the slowest one (Rs = 2.50) with sucrose being fully hydrolyzed after 52 h (Fig. 5a). It is faster for B56-5 (Rs = 6.15), where sucrose is fully hydrolyzed after 24 h (Fig. 5a), and even faster for JMY2531 (Rs = 7.63) and for JMY2593 (Rs = 7.07), which hydrolyses sucrose within 14 h (Fig. 5a). These last two strains have a very similar profile for sucrose hydrolysis. The profile of strain B56-5 is slightly different. It has a slow hydrolysis rate at the beginning of the growth until around 12 h when the hydrolysis rate increases. However, it does not affect growth compared to JMY2531 and JMY2593 (Fig. 3). These sucrose hydrolysis data are in line with the extracellular invertase activity level of these different strains. At mid-exponential phase, both strains JMY2531 and JMY2593 have a high and similar extracellular invertase activity and present a rapid sucrose cleavage, JMY2529 has very low activity and a low sucrose cleavage activity, with B56-5 being in between (Fig. 4a). JMY2531 and JMY2593 have the same sucrose hydrolysis rate, which confirms that expression of SUC2-1462 cassette alone is sufficient for maximum hydrolysis rate in that condition.Fig. 5

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