| A chemostat culture as a tool for the improvement of a recombinant E. coli strain over-producing penicillin G acylase. | |
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MedLine Citation:
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PMID: 11536126 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The recombinant strain RE3(pKA18) of Escherichia coli constitutively overproduces penicillin G acylase (PGA) from plasmid-borne gene pga. The host strain RE3 bears the same pga gene on its chromosome, the expression of which is controlled by the natural mechanism of induction with phenylacetic acid (PA). To evaluate the maximum biosynthetic capacity for PGA, induction of the chromosomal pga by PA was studied in a culture of the recombinant strain. PGA production by batch cultures of RE3(pKA18) and RE3 showed a different response to the addition of PA to the medium: while an addition of PA induces PGA in a culture of strain RE3 as expected, in recombinant cells it lowers the specific activity of PGA and a large amount of PGA is released into the culture medium. To improve the PGA production, the strain RE3(pKA18) was cultured in a carbon-limited chemostat and subjected to selection pressure in a medium supplemented with phenylacetic acid amide (PAA). Phenylacetic acid amide served as a source of nitrogen, an inducer of PGA and a factor exerting positive selection pressure on the maintenance of the recombinant plasmid. After 130 generations of growth in the presence of the inducer, no recombinant strain with constitutive expression of the chromosomal gene pga was detected in the prevailing P(+) subpopulation in the chemostat. Shake-flask experiments with the parent recombinant strain RE3(pKA18), host strain RE3, chemostat evolvant ERE3(epKA18), the cured host ERE3 alone, and its derivative after retransformation with ancestral plasmid ERE3(pKA18) showed that inactivation of the plasmid-borne pga by a frame-shift mutation (plasmid epKA18) occurred in the plasmid-bearing subpopulation accumulated in the chemostat. Marked adaptive changes evolved in the host ERE3 during a 130 generation culture: (1) the specific growth rate of the host increased by 30% in a medium without PA, (2) the copy number of plasmids pKA18 and epKA18 in the host cultured in PA-free medium dropped by about 40%, and (3) the leakage of PGA from the cell in the presence of PA found in strain RE3(pKA18) was not observed in strain ERE3(pKA18). This new recombinant strain with modified traits was constructed by means of retransformation of the evolved host ERE3 with ancestral plasmid pKA18. |
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Authors:
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H Maresová; V Stepánek; P Kyslík |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Biotechnology and bioengineering Volume: 75 ISSN: 0006-3592 ISO Abbreviation: Biotechnol. Bioeng. Publication Date: 2001 Oct |
Date Detail:
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Created Date: 2001-09-05 Completed Date: 2001-12-04 Revised Date: 2006-05-01 |
Medline Journal Info:
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Nlm Unique ID: 7502021 Medline TA: Biotechnol Bioeng Country: United States |
Other Details:
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Languages: eng Pagination: 46-52 Citation Subset: IM |
Copyright Information:
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Copyright 2001 John Wiley & Sons, Inc. |
Affiliation:
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Institute of Microbiology CAS, Videnská 1083, Prague 4, 142 20, Czech Republic. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Culture Media
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pharmacology DNA, Recombinant Escherichia coli / genetics*, metabolism Gene Expression Regulation, Bacterial / drug effects Nitrogen / metabolism Penicillin Amidase / genetics* Phenylacetates / pharmacology Plasmids |
| Chemical | |
Reg. No./Substance:
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0/Culture Media; 0/DNA, Recombinant; 0/Phenylacetates; 103-82-2/phenylacetic acid; 7727-37-9/Nitrogen; EC 3.5.1.11/Penicillin Amidase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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