Open AccessA chromosomally encoded T 7 RNA polymerase‐dependent gene expression system for C orynebacterium glutamicum : construction and comparative evaluation at the single‐cell level
Author(s) -
Kortmann Maike,
Kuhl Vanessa,
Klaffl Simon,
Bott Michael
Publication year - 2015
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/1751-7915.12236
Subject(s) - corynebacterium glutamicum , biology , t7 rna polymerase , microbiology and biotechnology , lac operon , rna polymerase , polymerase , gene , escherichia coli , stringent response , gene expression , homologous recombination , genetics , bacteriophage
Summary C orynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl‐β‐ d ‐1‐thiogalactopyranosid ( IPTG )‐inducible T 7 expression system in the prophage‐free strain C . glutamicum MB 001. For this purpose, part of the DE 3 region of E scherichia coli BL 21( DE 3) including the T 7 RNA polymerase gene 1 under control of the lac UV 5 promoter was integrated into the chromosome, resulting in strain MB 001( DE 3). Furthermore, the expression vector pMKEx 2 was constructed allowing cloning of target genes under the control of the T 7 lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG , the specific fluorescence increased 450‐fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG ‐induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T 7‐based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp , the functionality of the corynebacterial T 7 expression system was also successfully demonstrated by overexpression of the C . glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg −1 . It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C . glutamicum .