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Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones.

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Title: Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones.
Authors: Thomson, Nicholas M.1, Saika, Azusa2, Ushimaru, Kazunori2, Sangiambut, Smith1, Tsuge, Takeharu2, Summers, David K.3, Sivaniah, Easan1 es10009@cam.ac.uk
Source: Applied & Environmental Microbiology. Mar2013, Vol. 79 Issue 6, p1948-1955. 8p.
Subjects: Polyhydroxyalkanoates, Necator, Escherichia coli, Molecular chaperones, Enzyme activation
Abstract: The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter-1), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter-1) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer. [ABSTRACT FROM AUTHOR]
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Database: Engineering Source
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Abstract:The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter-1), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter-1) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer. [ABSTRACT FROM AUTHOR]
ISSN:00992240
DOI:10.1128/AEM.02881-12