This study deals with the potential of Pichia pastoris X-33 for the production of penicillin G acylase (PGAA) from Achromobacter sp. CCM 4824. Synthetic gene matching the codon usage of P. pastoris was designed for intracellular and secretion-based production strategies and cloned into vectors pPICZ and pPICZα under the control of AOX1 promoter. The simple method was developed to screen Pichia transformants with the intracellularly produced enzyme. The positive correlation between acylase production and pga gene dosage for both expression systems was demonstrated in small scale experiments. In fed-batch bioreactor cultures of X-33/PENS2, an extracellular expression system, total PGAA expressed from five copies reached 14,880 U/L of an active enzyme after 142 h; however, 60% of this amount retained in the cytosol. The maximum PGAA production of 31,000 U/L was achieved intracellularly from nine integrated gene copies of X-33/PINS2 after 90 h under methanol induction. The results indicate that in both expression systems the production level of PGAA is similar but there is a limitation in secretion efficiency.

Department of Genetics and Microbiology Faculty of Science Charles University Prague Viničná 5 12840 Prague 2 Czech Republic

Fermenta Biotech Ltd Thane India

Institute of Microbiology of the CAS v v i Vídeňská 1083 14220 Prague 4 Czech Republic

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PLoS One. 2014 May 07;9(5):e96974 PubMed

Biotechnol J. 2010 Apr;5(4):413-20 PubMed

Yeast. 2005 Mar;22(4):249-70 PubMed

Biotechnol Bioeng. 2004 Feb 20;85(4):367-75 PubMed

Biotechnol Adv. 2013 Dec;31(8):1319-32 PubMed

Biochim Biophys Acta. 1972 Jul 13;276(1):250-6 PubMed

Microb Cell Fact. 2006 Apr 06;5:17 PubMed

Protein Expr Purif. 2001 Feb;21(1):71-80 PubMed

Microb Cell Fact. 2005 Nov 10;4:31 PubMed

FEMS Yeast Res. 2002 Jan;1(4):271-7 PubMed

Biotechnol Rep (Amst). 2015 Mar 26;6:112-118 PubMed

Appl Microbiol Biotechnol. 2011 Feb;89(4):1127-35 PubMed

Microb Cell Fact. 2005 Dec 07;4:33 PubMed

Biotechnol Adv. 2015 Nov 1;33(6 Pt 2):1177-93 PubMed

Nucleic Acids Res. 1986 Jul 25;14(14):5713-27 PubMed

Microb Cell Fact. 2010 Apr 26;9:24 PubMed

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003 May;35(5):416-22 PubMed

Microb Cell Fact. 2010 Sep 13;9:68 PubMed

BMC Biotechnol. 2010 Feb 03;10:7 PubMed

BMC Biotechnol. 2011 May 10;11:47 PubMed

Appl Microbiol Biotechnol. 2015 Apr;99(7):2925-38 PubMed

Microb Cell Fact. 2009 Feb 10;8:13 PubMed

Appl Environ Microbiol. 2004 May;70(5):2764-70 PubMed

Appl Microbiol Biotechnol. 2014 Apr;98(7):2867-79 PubMed

Front Microbiol. 2015 Oct 19;6:1140 PubMed

Protein Expr Purif. 2001 Feb;21(1):60-4 PubMed

Methods Mol Biol. 2007;389:65-76 PubMed

Biotechnol Bioeng. 2001 Oct 5;75(1):46-52 PubMed

J Appl Microbiol. 2000 Jul;89(1):152-7 PubMed

Eur J Biochem. 1990 Aug 28;192(1):143-51 PubMed

J Biotechnol. 2009 Jul 15;142(3-4):250-8 PubMed

PLoS One. 2013;8(3):e58393 PubMed

Nat Biotechnol. 2004 Nov;22(11):1399-408 PubMed

Appl Microbiol Biotechnol. 2014 Jun;98(12):5301-17 PubMed

Biotechnol Bioeng. 2006 Feb 5;93(2):344-54 PubMed

Appl Microbiol Biotechnol. 2014 Feb;98(3):1195-203 PubMed

Microb Cell Fact. 2006 Nov 28;5:36 PubMed

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