BACKGROUND: 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. RESULTS: 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, characterized and compared with the wild-type PGAEc. The alpha-subunit of the hPGAEc formed in the cytosol was processed aberrantly resulting in two forms with C- terminuses extended to the spacer peptide. The enzyme exhibited modified traits: the activity of the purified enzyme was reduced to 49%, the ratios of hydrolytic activities with cephalexin, phenylacetamide or 6-nitro-3-phenylacetylamidobenzoic acid (NIPAB) to penicillin G increased and the enzyme showed a better synthesis/hydrolysis ratio for the synthesis of cephalexin. CONCLUSIONS: Presented results provide useful data regarding fermentation strategy, intracellular biosynthetic potential, and consequences of the heterologous expression of PGAEc in P. pastoris X-33. Aberrant processing of the precursor of PGAEc in the cytosol yielded the mature enzyme with modified traits.

Laboratory of Enzyme Technology Institute of Microbiology vvi Academy of Sciences of the Czech Republic Vídenská 1083 142 20 Prague 4 Czech Republic

Zobrazit více v PubMed

Rajendhran J, Gunasekaran P. Recent biotechnological interventions for developing improved penicillin G acylases. J Biosci Bioeng. 2004;97:1–13. PubMed

Sobotková L, Plháčková K, Kyslík P, Vojtíšek V. Czech Patent No. 278516. 1993.

Kutzbach C, Rauenbusch E. Preparation and general properties of crystalline penicillin acylase from Escherichia coli ATCC 11105. Hoppe-Seylers Z Physiol Chem. 1974;354:45–53. PubMed

Konstantinovic M, Marjanovic N, Ljubijankić G, Glisin V. The penicillin amidase of Arthrobacter viscosus(ATCC 15294) Gene. 1994;143:79–83. doi: 10.1016/0378-1119(94)90608-4. PubMed DOI

Robak M, Szewczuk A. Penicillin amidase from Proteus rettgeri. Acta Biochim Pol. 1981;28:275–284. PubMed

Barbero JL, Buesa JM, González de Buitrago G, Méndez E, Péz-Aranda A, García JL. Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila. Gene. 1986;49(1):69–80. doi: 10.1016/0378-1119(86)90386-0. PubMed DOI

Senthivel SG, Pai JS. Purification of penicillin acylase of Bacillus megaterium. Biotechnol Tech. 1996;10:611–614. doi: 10.1007/BF00157371. DOI

Verhaert RM, Riemens AM, Laan JM van der, van Duin J, Quax WJ. Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis. Appl Environ Microbiol. 1997;63:3412–3418. PubMed PMC

Cheng T, Chen M, Zheng H, Wang J, Yang S, Jiang W. Expression and purification of penicillin G acylase enzymes from four different micro-organisms and a comparative evaluation of their synthesis/hydrolysis ratios for cephalexin. Protein Expression Purif. 2006;46:107–113. doi: 10.1016/j.pep.2005.07.016. PubMed DOI

Bruggink A, Roos EC, de Vroom E. Penicillin acylase in the industrial production of β-lactam antibiotics. Org Process Res Dev. 1998;2:128–133. doi: 10.1021/op9700643. DOI

Gabor EM, Janssen DB. Increasing the synthetic performance of penicillin acylase PAS2 by structure-inspired semi-random mutagenesis. Protein Eng Des Sel. 2004;17(7):571–579. doi: 10.1093/protein/gzh070. PubMed DOI

Ignatova Z, Wischnewski F, Notbohm H, Kasche V. Pro-sequence and Ca2+-binding: Implications for folding and maturation of Ntn-hydrolase penicillin amidase from E. coli. J Mol Biol. 2005;348:999–1014. doi: 10.1016/j.jmb.2005.03.005. PubMed DOI

Hewitt L, Kasche V, Lummer K, Lewis RJ, Murshudov GN, Verma CS, Dodson GG, Wilson KS. Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft. J Mol Biol. 2000;302(4):887–898. doi: 10.1006/jmbi.2000.4105. PubMed DOI

Kasche V, Lummer K, Nurk A, Piotraschke E, Rieks A, Stoeva S, Voelter W. Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli. Biochim Biophys Acta. 1999;1433:76–86. PubMed

Yang Y, Biedendieck R, Wang W, Gamer M, Malten M, Jahn D, Deckwer WD. High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium. Microb Cell Fact. 2006;5:36. doi: 10.1186/1475-2859-5-36. PubMed DOI PMC

Jahic M, Veide A, Charoenrat T, Teeri T, Enfors SO. Process technology for production and recovery of heterologous proteins with Pichia pastoris. Biotechnol Prog. 2006;22:1465–1473. PubMed

Daly R, Hearn MT. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit. 2005;18(2):119–138. doi: 10.1002/jmr.687. PubMed DOI

Gurkan C, Ellar DJ. Recombinant production of bacterial toxins and derivatives in the methylotrophic yeast Pichia pastoris. Microb Cell Fact. 2005;4:33. doi: 10.1186/1475-2859-4-33. PubMed DOI PMC

Choi YJ, Miguez CB, Lee BH. Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96. Appl Environ Microbiol. 2004;70(6):3213–3221. doi: 10.1128/AEM.70.6.3213-3221.2004. PubMed DOI PMC

Ljubijankić G, Storici F, Glišin V, Bruschi CV. Synthesis and secretion of Providencia rettgeri and Escherichia coli heterodimeric penicillin amidases in Saccharomyces cerevisiae. Gene. 1999;228:225–232. doi: 10.1016/S0378-1119(98)00584-8. PubMed DOI

Ljubijankić G, Gvozdenović J, Ševo M, Degrassi G. High-level secretory expression of penicillin amidase from Providencia rettgeri in Saccharomyces cerevisiae: purification and characterization. Biotechnol Prog. 2002;18:330–336. doi: 10.1021/bp010182g. PubMed DOI

Ševo M, Degrassi G, Skoko N, Venturi V, Ljubijankić G. Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris. FEMS Yeast Res. 2002;1:271–277. doi: 10.1016/S1567-1356(01)00040-X. PubMed DOI

Senerovic L, Stankovic N, Pizzo P, Basso A, Gardossi L, Vasiljevic B, Ljubijankić G, Tisminetzky S, Degrassi G. High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability. Biotechnol Bioeng. 2006;93:344–354. doi: 10.1002/bit.20728. PubMed DOI

Gabor EM, de Vries EJ, Janssen DB. A novel penicillin acylase from the environmental gene pool with improved synthetic properties. Enzyme Microb Technol. 2005;36:182–190. doi: 10.1016/j.enzmictec.2004.04.021. DOI

Valešová R, Hollerová-Sobotková L, Štěpánek V, Kyslík P. Optimisation of the host-plasmid interaction in the recombinant Escherichia coli strains overproducing penicillin G acylase. Enzyme Microb Technol. 2004;35:74–80. doi: 10.1016/j.enzmictec.2004.03.015. DOI

Plhačková K, Štěpánek V, Kyslík P, Sobotková L. Czech Patent No. 282712. 1997.

Böck A, Wirth R, Schmid G, Schumacher G, Lang G, Buckel P. The penicillin acylase from Escherichia coli ATCC 11105 consists of two dissimilar subunits. FEMS Microbiol Lett. 1983;20:135–139. doi: 10.1111/j.1574-6968.1983.tb00103.x. DOI

Ignatova Z, Stoeva S, Galunsky B, Hörnle C, Nurk A, Piotraschke E, Voelter W, Kasche V. Proteolytic processing of penicillin amidase from Alcaligenes faecaliscloned in Escherichia coli yields several active forms. Biotechnol Lett. 1998;20:977–982. doi: 10.1023/A:1005446719483. DOI

Zhou Z, Zhang AH, Wang JR, Chen ML, Li RB, Yang S, Yuan ZY. Improving the specific synthetic activity of a penicillin G acylase using DNA family shuffling. Acta Biochim Biophys Sin. 2003;35:573–579. PubMed

Erarslan A, Terzi I, Guray A, Bermek E. Purification and kinetics of penicillin G acylase from a mutant strain of Escherichia coli ATCC 11105. J Chem Technol Biotechnol. 1991;51:27–40. PubMed

Cai G, Zhu S, Yang S, Zhao G, Jiang W. Cloning, overexpression, and characterization of a novel thermostable penicillin G acylase from Achromobacter xylosoxidans: probing the molecular basis for its high thermostability. Appl Environ Microbiol. 2004;70:2764–2770. doi: 10.1128/AEM.70.5.2764-2770.2004. PubMed DOI PMC

Sobotková L, Štěpánek V, Plháčková K, Kyslík P. Development of a high-expression system for penicillin G acylase based on the recombinant Escherichia coli strain RE3(pKA18) Enzyme Microb Technol. 1996;19:389–397. doi: 10.1016/S0141-0229(96)00052-X. DOI

Sambrook J, Fitsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Press; 1989.

Balasingham K, Warburton D, Dunnill P, Lilly MD. The isolation and kinetics of penicillin amidase from Escherichia coli. Biochim Biophys Acta. 1972;276:250–256. PubMed

Weatherburn MW. Linear regression analysis of the released ammonia, determined by conversion to indophenol. Anal Chem. 1967;39:971–974. doi: 10.1021/ac60252a045. DOI

Volf P, Skarupova S, Man P. Characterization of the lectin from females of Phlebotomus duboscqi sand flies. Eur J Biochem. 2002;269:6294–6301. doi: 10.1046/j.1432-1033.2002.03349.x. PubMed DOI

Zhang X, Rogowska-Wrzesinska A, Roepstorff P. On-target sample preparation of 4-sulfophenyl isothiocyanate-derivatized peptides using AnchorChip Targets. J Mass Spectrom. 2008;43(3):346–359. doi: 10.1002/jms.1327. PubMed DOI

Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris

Potential of Pichia pastoris for the production of industrial penicillin G acylase