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A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium

  • Biotechnological Products and Process Engineering
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Abstract

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.

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Acknowledgements

The provision of plasmid pET30a::dapL Ss by Prof. Dr. Thomas Leustek is gratefully acknowledged.

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Authors and Affiliations

  1. Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, 48149, Münster, Germany

    Jens Kroll, Stefan Klinter & Alexander Steinbüchel

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  1. Jens Kroll
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  2. Stefan Klinter
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  3. Alexander Steinbüchel
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Correspondence to Alexander Steinbüchel.

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Kroll, J., Klinter, S. & Steinbüchel, A. A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium. Appl Microbiol Biotechnol 89, 593–604 (2011). https://doi.org/10.1007/s00253-010-2899-2

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