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.

• MeSH
• Achromobacter genetika   metabolismus   MeSH
• bioreaktory mikrobiologie   MeSH
• exprese genu MeSH
• genetické vektory MeSH
• genová dávka MeSH
• klonování DNA MeSH
• kodon genetika   MeSH
• penicilinamidasa genetika   metabolismus   MeSH
• Pichia genetika   metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• průmyslová mikrobiologie metody   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• transformace genetická MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kodon MeSH
• penicilinamidasa MeSH
• rekombinantní proteiny MeSH

This paper summarizes research results and their industrial applications obtained by continuous culture in the former Czechoslovakia. Past achievements as well as recent trends and developments worldwide are presented. The term "Prague School of continuous culture" is put forward and its international activity is outlined. The impact of this school was pervasive across the entire field of applied microbiology and biotechnology in Czechoslovakia and, perhaps, even beyond the country's borders. Continuous culture is a very mature field, and since its establishment it has become a powerful research tool. The present activity in this field amounts to a renaissance of continuous culture, emphasizing new dimensions in bioinformatics and systems biology.

• MeSH
• akademie a ústavy MeSH
• biotechnologie dějiny   trendy   MeSH
• dějiny 20. století MeSH
• fermentace * MeSH
• mezinárodní spolupráce MeSH
• průmyslová mikrobiologie dějiny   trendy   MeSH
• systémová biologie MeSH
• výpočetní biologie MeSH
• výzkum MeSH
• Check Tag
• dějiny 20. století MeSH
• Publikační typ
• časopisecké články MeSH
• historické články MeSH
• Geografické názvy
• Československo MeSH

The potential for production of penicillin G-acylase (PGA), encoded by the chromosomal gene pgai, of four strains belonging to a genealogical line derived from the strain Escherichia coli W, was evaluated in a medium with and without the inducer phenylacetic acid (PA). These strains were used as hosts of the recombinant plasmid pKA18, in which the structural gene pgac isolated from the strain RE3, the best host strain of a line giving the highest production, was cloned. The presence of the inducer reduced the copy number of the plasmid in all recombinant strains. Only in recombinant strain RE3 (pKA18) the reduction of the gene dosage of pgac resulted also in the reduction of the amount of PGA synthesized by the cells. The reduced activity of the cells did not result from a segregation of plasmid-free clones. Also the growth rate was decreased by 20 and 40% in the host and recombinant strains, respectively. The host strain RE3 showing the highest production of PGA was also the best host of the recombinant plasmid in terms of the segregational stability and copy number (198 copies per chromosome). The recombinant strain RE3 (pKA18) also provided the highest production of PGA.

• MeSH
• Escherichia coli klasifikace   genetika   metabolismus   MeSH
• klonování DNA MeSH
• penicilinamidasa biosyntéza   genetika   MeSH
• plazmidy genetika   MeSH
• regulace genové exprese u bakterií MeSH
• rekombinantní DNA MeSH
• rekombinantní proteiny biosyntéza   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• Názvy látek
• penicilinamidasa MeSH
• rekombinantní DNA MeSH
• rekombinantní proteiny MeSH