A chromosomally encoded T7 RNA polymerase‐dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single‐cell level

Abstract

Corynebacterium 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 T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac 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 T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 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.

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

“…S2 in the supplemental material) using nonexposed cIPTG were found to be moderate in BHI medium (up to 1.5-fold increase in comparison to control strains) and CGXII medium (up to 2.2-fold). As reported in the literature (50), basal expression levels of the system used here were found to be elevated in CGXII minimal medium and increased with ongoing cultivation times. The dynamic range of induction, however, was high and comparable in BHI medium (63-fold for cIPTG and 61-fold for IPTG).…”

“…In BHI medium, a second population with lower fluorescence intensity occurred after 20 h of overexpression, whereas prolonged expression in CGXII medium obviously resulted in the formation of a second population with increased YFP accumulation. In principle, expression heterogeneity has recently been described for a similar expression setup in C. glutamicum (50). Notably, flow cytometric single-cell analysis using propidium iodide-based LIVE/DEAD staining (43) further suggested that light induction did not affect membrane integrity and thus cell viability (see Fig.…”

Identification of the cAMP phosphodiesterase CpdA as novel key player in cAMP‐dependent regulation in Corynebacterium glutamicum

“…PCR fragments covering the respective genes were generated using the oligonucleotides listed in Supporting Information Table S3 and included overlaps between the PCR fragments and the pEKEx2 backbone. Plasmid pMKEx1 was constructed by integration of the expression cassette of pET-TEV (Bussmann et al, 2010) into plasmid pJC1, as described before for pMKEx2 (Kortmann et al, 2015). The resulting products were used to transform E. coli and the plasmids were isolated and checked for correctness by sequencing.…”

Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum

“…Also, extensive bioprocess knowledge with C.   glutamicum and methods for genetic manipulation are available because this microbe is a major producer for amino acids at industrial scale for decades [8]. Recently, the construction of a C.   glutamicum strain harboring the DE3/T7 expression system was reported, allowing to control gene expression levels for intracellular protein production [9]. This system is based on the prophage-cured C.   glutamicum strain MB001, which was shown earlier to be beneficial for intracellular protein production [10].…”