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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

Kinetics of invertase activity in bioreactor of Y. lipolytica SUC+ strains in minimum medium with sucrose as the carbon source. a Extracellular invertase activity. b Intracellular invertase activity. Graphs presented are the average of three replicates. c Graphic legend and SUC2 signal sequence (SS) present in each strain
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Fig4: Kinetics of invertase activity in bioreactor of Y. lipolytica SUC+ strains in minimum medium with sucrose as the carbon source. a Extracellular invertase activity. b Intracellular invertase activity. Graphs presented are the average of three replicates. c Graphic legend and SUC2 signal sequence (SS) present in each strain

Mentions: Extracellular and intracellular invertase activities were evaluated in the bioreactor process. Extracellular invertase activity increases rapidly for strains JMY2531 and JMY2593 expressing SUC2-1462, whereas it is slower for strain B56-5 expressing two copies of SUC2-302 and stays very low for strain JMY2529 expressing one copy (Fig. 4a). This is in line with the higher secretion capacity of SUC2-1462 construction hypothesized previously. Similarly, the strain JMY2593 reaches the highest level of extracellular activity after the 72-h fermentation with 4,519 U/l, and strain JMY2529 the lowest with 597 U/l (Fig. 4a; Table 2). On the contrary, the increase in intracellular invertase activity is very similar between the strains B56-5, JMY2531, and JMY2593, and stays low for JMY2529 (Fig. 4b). Overall, JMY2593 ended with a slightly lower intracellular activity after the 72-h fermentation compared to B56-5 and JMY2531 (respectively 12,960, 16,130, and 14,890 U/l), while JMY2529 stays much lower with 4,380 U/l (Fig. 4b; Table 2). Considering the sum of intracellular and extracellular invertase activity (Table 2), JMY2531, JMY2593, and B56-5 have similar production levels. However, the proportion of secreted invertase for JMY2593 is much higher (25.8 %). It should be noted that the total activity of both SUC2 constructions is not cumulative; the sum of total invertase activity of strain JMY2529 and strain JMY2593 does not correspond to the total invertase activity of JMY2531 combining both forms. Expressing simultaneously SUC2-302 and SUC2-1462 leads to a surprisingly lower extracellular invertase activity compared to JMY2593 expressing only SUC2-1462 (Table 2). It can be speculated that the presence of the secretion signal of SUC2-302, which has a defect in secretion capacity, may interfere with the secretion of the SUC2-1462 by overloading the secretion pathway. The other alternative is that the genetic environment of the integration site for SUC2-1462 cassette may impact the Suc2p expression/secretion level between these two strains. This also confirms that one integrated copy of SUC2-1462 alone is sufficient for optimal growth on sucrose (Fig. 2b, c). Similarly, strain B56-5, with two copies of SUC2-302, does not specifically produce two times more invertase compared to JMY2529 expressing only one copy (Table 2), a phenomenon that has previously been observed [17]. However, the overall proportion of secreted invertase is similar (11.9 and 12.3 %, respectively). By comparing JMY2529 and JMY2593 (which are expressing each only one form of invertase and which have the same genetic background) the last strain (expressing SUC2-1462) produces and secretes more invertase in terms of units per liter. Intracellular enzyme activity is 3.5 times higher and extracellular 7.5 times higher, which represents a massive improvement of invertase production. It can be concluded from this data that the secretion signal of SUC2-1462 is much more efficient than for SUC2-302. Hong and collaborators [14] very recently determined intracellular and extracellular invertase activity as a consequence of different variants of sequence signal in Y. lipolytica: the xpr2p prepro sequence signal followed by mature Suc2p, and native Suc2p with its own sequence signal, both under the hybrid strong promoter FBA1in. They end up with similar conclusions on secretion efficacy of the native Suc2p secretion signal sequence. However, they detected only the invertase activity extracellularly, which has been around 100 times lower than in our case in terms of U/l. This probably results from sampling time point differences and growth conditions, as they were measuring invertase activity in exponential growth phase at OD around 1.4–1.8, in flask, while data in Table 2 correspond to 72-h growth in bioreactor, corresponding to approximately OD = 30. Presented kinetics of invertase activity (Fig. 4a, b) have reveal that at the beginning of the exponential phase (6–8 h), extracellular invertase activities are very low within values in units per liter, in the same range of what Hong and collaborators [14] observed at a similar growth stage. However, it appears that in the bioreactor process, extracellular invertase rapidly reaches a much higher level (Fig. 4a). On the other hand, intracellular activities are already 3–5 times higher than in Hong et al.’s [14] study, even in the exponential phase. The presence of the second ATG allowing production of the minor cytosolic form in S. cerevisiae lacking the secretion signal sequence may explain the presence of such an intracellular invertase activity. Hong and collaborators [14] had a similar construction and failed to detect such activity. However, growth parameters in the bioreactor process allow reaching a much higher rate of invertase production, which can overload the secretion system. A more probable hypothesis is that a large part of the over-secreted invertase stays locked in to the periplasmic space during external invertase recovering experiments (supernatant), and is therefore attributed to the cell’s extract fraction. Preliminary protoplastization experiments revealed a very high release of invertase activity, thus strengthening this hypothesis (data not shown). However, we cannot exclude that the genetic environment of the cassette integration site might affect the expression of SUC2. Moreover, it cannot be excluded that the different genetic background between the strain used by Hong et al. [14] and strains described in this study may also affect the invertase secretion profile.Fig. 4


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)

Kinetics of invertase activity in bioreactor of Y. lipolytica SUC+ strains in minimum medium with sucrose as the carbon source. a Extracellular invertase activity. b Intracellular invertase activity. Graphs presented are the average of three replicates. c Graphic legend and SUC2 signal sequence (SS) present in each strain
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
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Fig4: Kinetics of invertase activity in bioreactor of Y. lipolytica SUC+ strains in minimum medium with sucrose as the carbon source. a Extracellular invertase activity. b Intracellular invertase activity. Graphs presented are the average of three replicates. c Graphic legend and SUC2 signal sequence (SS) present in each strain
Mentions: Extracellular and intracellular invertase activities were evaluated in the bioreactor process. Extracellular invertase activity increases rapidly for strains JMY2531 and JMY2593 expressing SUC2-1462, whereas it is slower for strain B56-5 expressing two copies of SUC2-302 and stays very low for strain JMY2529 expressing one copy (Fig. 4a). This is in line with the higher secretion capacity of SUC2-1462 construction hypothesized previously. Similarly, the strain JMY2593 reaches the highest level of extracellular activity after the 72-h fermentation with 4,519 U/l, and strain JMY2529 the lowest with 597 U/l (Fig. 4a; Table 2). On the contrary, the increase in intracellular invertase activity is very similar between the strains B56-5, JMY2531, and JMY2593, and stays low for JMY2529 (Fig. 4b). Overall, JMY2593 ended with a slightly lower intracellular activity after the 72-h fermentation compared to B56-5 and JMY2531 (respectively 12,960, 16,130, and 14,890 U/l), while JMY2529 stays much lower with 4,380 U/l (Fig. 4b; Table 2). Considering the sum of intracellular and extracellular invertase activity (Table 2), JMY2531, JMY2593, and B56-5 have similar production levels. However, the proportion of secreted invertase for JMY2593 is much higher (25.8 %). It should be noted that the total activity of both SUC2 constructions is not cumulative; the sum of total invertase activity of strain JMY2529 and strain JMY2593 does not correspond to the total invertase activity of JMY2531 combining both forms. Expressing simultaneously SUC2-302 and SUC2-1462 leads to a surprisingly lower extracellular invertase activity compared to JMY2593 expressing only SUC2-1462 (Table 2). It can be speculated that the presence of the secretion signal of SUC2-302, which has a defect in secretion capacity, may interfere with the secretion of the SUC2-1462 by overloading the secretion pathway. The other alternative is that the genetic environment of the integration site for SUC2-1462 cassette may impact the Suc2p expression/secretion level between these two strains. This also confirms that one integrated copy of SUC2-1462 alone is sufficient for optimal growth on sucrose (Fig. 2b, c). Similarly, strain B56-5, with two copies of SUC2-302, does not specifically produce two times more invertase compared to JMY2529 expressing only one copy (Table 2), a phenomenon that has previously been observed [17]. However, the overall proportion of secreted invertase is similar (11.9 and 12.3 %, respectively). By comparing JMY2529 and JMY2593 (which are expressing each only one form of invertase and which have the same genetic background) the last strain (expressing SUC2-1462) produces and secretes more invertase in terms of units per liter. Intracellular enzyme activity is 3.5 times higher and extracellular 7.5 times higher, which represents a massive improvement of invertase production. It can be concluded from this data that the secretion signal of SUC2-1462 is much more efficient than for SUC2-302. Hong and collaborators [14] very recently determined intracellular and extracellular invertase activity as a consequence of different variants of sequence signal in Y. lipolytica: the xpr2p prepro sequence signal followed by mature Suc2p, and native Suc2p with its own sequence signal, both under the hybrid strong promoter FBA1in. They end up with similar conclusions on secretion efficacy of the native Suc2p secretion signal sequence. However, they detected only the invertase activity extracellularly, which has been around 100 times lower than in our case in terms of U/l. This probably results from sampling time point differences and growth conditions, as they were measuring invertase activity in exponential growth phase at OD around 1.4–1.8, in flask, while data in Table 2 correspond to 72-h growth in bioreactor, corresponding to approximately OD = 30. Presented kinetics of invertase activity (Fig. 4a, b) have reveal that at the beginning of the exponential phase (6–8 h), extracellular invertase activities are very low within values in units per liter, in the same range of what Hong and collaborators [14] observed at a similar growth stage. However, it appears that in the bioreactor process, extracellular invertase rapidly reaches a much higher level (Fig. 4a). On the other hand, intracellular activities are already 3–5 times higher than in Hong et al.’s [14] study, even in the exponential phase. The presence of the second ATG allowing production of the minor cytosolic form in S. cerevisiae lacking the secretion signal sequence may explain the presence of such an intracellular invertase activity. Hong and collaborators [14] had a similar construction and failed to detect such activity. However, growth parameters in the bioreactor process allow reaching a much higher rate of invertase production, which can overload the secretion system. A more probable hypothesis is that a large part of the over-secreted invertase stays locked in to the periplasmic space during external invertase recovering experiments (supernatant), and is therefore attributed to the cell’s extract fraction. Preliminary protoplastization experiments revealed a very high release of invertase activity, thus strengthening this hypothesis (data not shown). However, we cannot exclude that the genetic environment of the cassette integration site might affect the expression of SUC2. Moreover, it cannot be excluded that the different genetic background between the strain used by Hong et al. [14] and strains described in this study may also affect the invertase secretion profile.Fig. 4

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