Heterologous expression of leader-less pga gene in Pichia pastoris: intracellular production of prokaryotic enzyme.

BMC Biotechnology
Helena MaresováPavel Kyslík

Abstract

Penicillin G acylase of Escherichia coli (PGAEc) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of beta-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGAEc in this host and studied yeast production capacity and enzyme authenticity. Leader-less pga gene encoding PGAEcunder the control of AOX1 promoter was cloned in Pichia pastoris X-33. The intracellular overproduction of heterologous PGAEc(hPGAEc) was evaluated in a stirred 10 litre bioreactor in high-cell density, fed batch cultures using different profiles of transient phases. Under optimal conditions, the average volumetric activity of 25900 U l-1 was reached. The hPGAEc was purified, c...Continue Reading

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Citations

Jan 22, 2014·Applied Microbiology and Biotechnology·Helena MarešováPavel Kyslík
Jul 19, 2013·Biotechnology Advances·Michal GrulichPavel Kyslík
Jul 25, 2014·Journal of Analytical Methods in Chemistry·Anirban NandiIndira P Sarethy
Mar 11, 2017·Folia Microbiologica·Helena MarešováPavel Kyslík
May 20, 2020·Applied Microbiology and Biotechnology·Martina BorčinováKarin Kovar

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Methods Mentioned

BETA
PCR
ion exchange chromatography
electrophoresis
Protein assay

Software Mentioned

Intelligent Quantifier
Enzfitter
Image analysis

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