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Systems metabolic engineering of Corynebacterium glutamicum for production of the chemical chaperone ectoine.

Becker J, Schäfer R, Kohlstedt M, Harder BJ, Borchert NS, Stöveken N, Bremer E, Wittmann C - Microb. Cell Fact. (2013)

Bottom Line: Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported.Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L(-1) day(-1) under growth conditions that did not rely on the use of high-salinity media.The present study describes the construction of a stable microbial cell factory for recombinant production of ectoine.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biochemical Engineering, Technische Universität Braunschweig, Braunschweig, Germany. c.wittmann@tu-braunschweig.de.

ABSTRACT

Background: The stabilizing and function-preserving effects of ectoines have attracted considerable biotechnological interest up to industrial scale processes for their production. These rely on the release of ectoines from high-salinity-cultivated microbial producer cells upon an osmotic down-shock in rather complex processor configurations. There is growing interest in uncoupling the production of ectoines from the typical conditions required for their synthesis, and instead design strains that naturally release ectoines into the medium without the need for osmotic changes, since the use of high-salinity media in the fermentation process imposes notable constraints on the costs, design, and durability of fermenter systems.

Results: Here, we used a Corynebacterium glutamicum strain as a cellular chassis to establish a microbial cell factory for the biotechnological production of ectoines. The implementation of a mutant aspartokinase enzyme ensured efficient supply of L-aspartate-beta-semialdehyde, the precursor for ectoine biosynthesis. We further engineered the genome of the basic C. glutamicum strain by integrating a codon-optimized synthetic ectABCD gene cluster under expressional control of the strong and constitutive C. glutamicum tuf promoter. The resulting recombinant strain produced ectoine and excreted it into the medium; however, lysine was still found as a by-product. Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported. Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L(-1) day(-1) under growth conditions that did not rely on the use of high-salinity media.

Conclusions: The present study describes the construction of a stable microbial cell factory for recombinant production of ectoine. We successfully applied metabolic engineering strategies to optimize its synthetic production in the industrial workhorse C. glutamicum and thereby paved the way for further improvements in ectoine yield and biotechnological process optimization.

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Influence of cultivation temperature on the growth and ectoine production performance of C. glutamicum ECT-1. Strain ECT-1 was grown in chemically defined medium with glucose on a miniaturized scale at the indicated growth temperatures. The specific growth rate μ, ectoine secretion (Ectex), and intracellular accumulation of ectoine (Ectint) and hydroxyectoine (EctOHint) were determined. Ectoines were quantified after 10h (27°C, 30°C, 35°C) and 20h (42°C) of cultivation. The data shown represent mean values and corresponding standard deviations from three biological replicates.
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Figure 3: Influence of cultivation temperature on the growth and ectoine production performance of C. glutamicum ECT-1. Strain ECT-1 was grown in chemically defined medium with glucose on a miniaturized scale at the indicated growth temperatures. The specific growth rate μ, ectoine secretion (Ectex), and intracellular accumulation of ectoine (Ectint) and hydroxyectoine (EctOHint) were determined. Ectoines were quantified after 10h (27°C, 30°C, 35°C) and 20h (42°C) of cultivation. The data shown represent mean values and corresponding standard deviations from three biological replicates.

Mentions: The optimal cultivation temperature for the recombinant production of ectoine and hydroxyectoine by strain ECT-1 was assessed by miniaturized cultivations in a temperature range between 27°C and 42°C. Interestingly, this revealed that ectoine production was improved by higher temperature (Figure 3). As compared to the reference cultivation conditions for strain ECT-1 at 30°C, secretion was more than doubled when the temperature was increased to 35°C. The enhanced production performance was also reflected by a slight increase of the intracellular ectoine level. The higher cultivation temperature also positively influenced the intracellular amounts of hydroxyectoine, and the growth performance of strain ECT-1 as reflected by a 28% increase of the specific growth rate (Figure 3). The higher ectoine concentration in the supernatant was taken as positive indication for a better production performance at 35°C.


Systems metabolic engineering of Corynebacterium glutamicum for production of the chemical chaperone ectoine.

Becker J, Schäfer R, Kohlstedt M, Harder BJ, Borchert NS, Stöveken N, Bremer E, Wittmann C - Microb. Cell Fact. (2013)

Influence of cultivation temperature on the growth and ectoine production performance of C. glutamicum ECT-1. Strain ECT-1 was grown in chemically defined medium with glucose on a miniaturized scale at the indicated growth temperatures. The specific growth rate μ, ectoine secretion (Ectex), and intracellular accumulation of ectoine (Ectint) and hydroxyectoine (EctOHint) were determined. Ectoines were quantified after 10h (27°C, 30°C, 35°C) and 20h (42°C) of cultivation. The data shown represent mean values and corresponding standard deviations from three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225761&req=5

Figure 3: Influence of cultivation temperature on the growth and ectoine production performance of C. glutamicum ECT-1. Strain ECT-1 was grown in chemically defined medium with glucose on a miniaturized scale at the indicated growth temperatures. The specific growth rate μ, ectoine secretion (Ectex), and intracellular accumulation of ectoine (Ectint) and hydroxyectoine (EctOHint) were determined. Ectoines were quantified after 10h (27°C, 30°C, 35°C) and 20h (42°C) of cultivation. The data shown represent mean values and corresponding standard deviations from three biological replicates.
Mentions: The optimal cultivation temperature for the recombinant production of ectoine and hydroxyectoine by strain ECT-1 was assessed by miniaturized cultivations in a temperature range between 27°C and 42°C. Interestingly, this revealed that ectoine production was improved by higher temperature (Figure 3). As compared to the reference cultivation conditions for strain ECT-1 at 30°C, secretion was more than doubled when the temperature was increased to 35°C. The enhanced production performance was also reflected by a slight increase of the intracellular ectoine level. The higher cultivation temperature also positively influenced the intracellular amounts of hydroxyectoine, and the growth performance of strain ECT-1 as reflected by a 28% increase of the specific growth rate (Figure 3). The higher ectoine concentration in the supernatant was taken as positive indication for a better production performance at 35°C.

Bottom Line: Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported.Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L(-1) day(-1) under growth conditions that did not rely on the use of high-salinity media.The present study describes the construction of a stable microbial cell factory for recombinant production of ectoine.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biochemical Engineering, Technische Universität Braunschweig, Braunschweig, Germany. c.wittmann@tu-braunschweig.de.

ABSTRACT

Background: The stabilizing and function-preserving effects of ectoines have attracted considerable biotechnological interest up to industrial scale processes for their production. These rely on the release of ectoines from high-salinity-cultivated microbial producer cells upon an osmotic down-shock in rather complex processor configurations. There is growing interest in uncoupling the production of ectoines from the typical conditions required for their synthesis, and instead design strains that naturally release ectoines into the medium without the need for osmotic changes, since the use of high-salinity media in the fermentation process imposes notable constraints on the costs, design, and durability of fermenter systems.

Results: Here, we used a Corynebacterium glutamicum strain as a cellular chassis to establish a microbial cell factory for the biotechnological production of ectoines. The implementation of a mutant aspartokinase enzyme ensured efficient supply of L-aspartate-beta-semialdehyde, the precursor for ectoine biosynthesis. We further engineered the genome of the basic C. glutamicum strain by integrating a codon-optimized synthetic ectABCD gene cluster under expressional control of the strong and constitutive C. glutamicum tuf promoter. The resulting recombinant strain produced ectoine and excreted it into the medium; however, lysine was still found as a by-product. Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported. Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L(-1) day(-1) under growth conditions that did not rely on the use of high-salinity media.

Conclusions: The present study describes the construction of a stable microbial cell factory for recombinant production of ectoine. We successfully applied metabolic engineering strategies to optimize its synthetic production in the industrial workhorse C. glutamicum and thereby paved the way for further improvements in ectoine yield and biotechnological process optimization.

Show MeSH
Related in: MedlinePlus