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

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.

journal_name

Appl Microbiol Biotechnol

journal_title

Applied microbiology and biotechnology

authors

Kroll J,Klinter S,Steinbüchel A

doi

10.1007/s00253-010-2899-2

subject

Has Abstract

pub_date

2011-02-01 00:00:00

pages

593-604

issue

eissn

0175-7598

issn

1432-0614

journal_volume

pub_type

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