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A novel plasmid add...
A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium
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- Kroll, Jens (författare)
- Westfälische Wilhelms-Universität Münster, Germany,Institut für Molekulare Mikrobiologie und Biotechnologie
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- Klinter, Stefan (författare)
- Westfälische Wilhelms-Universität Münster, Germany,Institut für Molekulare Mikrobiologie und Biotechnologie
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- Steinbüchel, Alexander (författare)
- Westfälische Wilhelms-Universität Münster, Germany,Institut für Molekulare Mikrobiologie und Biotechnologie
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(creator_code:org_t)
- 2010-10-05
- 2011
- Engelska.
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 89:3, s. 593-604
- Relaterad länk:
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https://urn.kb.se/re...
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visa fler...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- Introduction: Hitherto the production of the biopolymer cyanophycin (CGP) using recombinant Escherichia coli strains and cheap mineral salts medium yielded only trace amounts of CGP (<0.5%, w/w) of the cell dry matter (CDM). This was probably due to the instability of the plasmids encoding the cyanophycin synthetase. Material and methods: In this study, we developed an anabolism-based media-dependent plasmid addiction system (PAS) to enhance plasmid stability, and we established a process based on a modified mineral salts medium yielding a CGP content of 42% (w/w) at the maximum without the addition of amino acids to the medium for the first time. This PAS is based on different lysine biosynthesis pathways and consists of two components: (1) a knockout of the chromosomal dapE disrupts the native succinylase pathway in E. coli and (2) the complementation by the plasmid-encoded artificial aminotransferase pathway mediated by the dapL gene from Synechocystis sp. PCC 6308, which allows the synthesis of the essential lysine precursor L,L-2,6-diaminopimelate. In addition, this plasmid also harbors cphAC595S, an engineered cyanophycin synthetase gene responsible for CGP production. Results: Cultivation experiments in Erlenmeyer flask and also in bioreactors in mineral salts medium without antibiotics revealed an at least 4.5-fold enhanced production of CGP in comparison to control cultivations without PAS. Discussion: Fermentation experiments with culture volume of up to 400 l yielded a maximum of 18% CGP (w/w) and a final cell density of 15.2 g CDM/l. Lactose was used constantly as an effective inducer and carbon source. Thus, we present a convenient option to produce CGP with E. coli at a technical scale without the need to add antibiotics or amino acids using the mineral salts medium designed in this study.
Ämnesord
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology (hsv//eng)
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik -- Bioprocessteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology -- Bioprocess Technology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Mikrobiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Microbiology (hsv//eng)
Nyckelord
- Cyanophycin
- DapE
- DapL
- Mineral salts medium
- Plasmid addiction system
- Succinylase pathway
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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