Publication: Effective enhancement of Pseudomonas stutzeri D-phenylglycine aminotransferase functional expression in Pichia pastoris by co-expressing Escherichia coli GroEL-GroES
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2012
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eng
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Mahidol University
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BioMed Central
Bibliographic Citation
Microbial Cell Factories. Vol. 11, (2012), 47
Suggested Citation
Kanidtha Jariyachawalid, Poramaet Laowanapiban, Vithaya Meevootisom, Suthep Wiyakrutta Effective enhancement of Pseudomonas stutzeri D-phenylglycine aminotransferase functional expression in Pichia pastoris by co-expressing Escherichia coli GroEL-GroES. Microbial Cell Factories. Vol. 11, (2012), 47. doi:10.1186/1475-2859-11-47 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/2727
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Title
Effective enhancement of Pseudomonas stutzeri D-phenylglycine aminotransferase functional expression in Pichia pastoris by co-expressing Escherichia coli GroEL-GroES
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Abstract
Background: D-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible
stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and
D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is
a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form.
Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble
inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for
high level production of the D-PhgAT.
Results: Intracellular expression of the codon-optimized synthetic dpgA gene under the PAOX1 promoter in
P. pastoris resulted in inactive D-PhgAT associated with insoluble cellular fraction and very low level of D-PhgAT
activity in the soluble fraction. Manipulation of culture conditions such as addition of sorbitol to induce intracellular
accumulation of osmolytes, addition of benzyl alcohol to induce chaperone expression, or lowering incubation
temperature to slow down protein expression and folding rates all failed to increase the active D-PhgAT yield.
Co-expression of E. coli chaperonins GroEL-GroES with the D-PhgAT dramatically improved the soluble active
enzyme production. Increasing gene dosage of both the dpgA and those of the chaperones further increased
functional D-PhgAT yield up to 14400-fold higher than when the dpgA was expressed alone. Optimization of
cultivation condition further increased D-PhgAT activity yield from the best co-expressing strain by 1.2-fold.
Conclusions: This is the first report on the use of bacterial chaperones co-expressions to enhance functional
intracellular expression of bacterial enzyme in P. pastoris. Only two bacterial chaperone genes groEL and groES were
sufficient for dramatic enhancement of functionally active D-PhgAT expression in this yeast. With the optimized
gene dosage and chaperone combinations, P. pastoris can be attractive for intracellular expression of bacterial
proteins since it can grow to a very high cell density which is translated into the higher volumetric product yield
than the E. coli or other bacterial systems.
