Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032 for use as a platform strain for basic research and industrial biotechnology

Abstract

The activity of bacteriophages and phage-related mobile elements is a major source for genome rearrangements and genetic instability of their bacterial hosts. The genome of the industrial amino acid producer Corynebacterium glutamicum ATCC 13032 contains three prophages (CGP1, CGP2, and CGP3) of so far unknown functionality. Several phage genes are regularly expressed, and the large prophage CGP3 (∼190 kbp) has recently been shown to be induced under certain stress conditions. Here, we present the construction of MB001, a prophage-free variant of C. glutamicum ATCC 13032 with a 6% reduced genome. This strain does not show any unfavorable properties during extensive phenotypic characterization under various standard and stress conditions. As expected, we observed improved growth and fitness of MB001 under SOS-response-inducing conditions that trigger CGP3 induction in the wild-type strain. Further studies revealed that MB001 has a significantly increased transformation efficiency and produced about 30% more of the heterologous model protein enhanced yellow fluorescent protein (eYFP), presumably as a consequence of an increased plasmid copy number. These effects were attributed to the loss of the restriction-modification system (cg1996-cg1998) located within CGP3. The deletion of the prophages without any negative effect results in a novel platform strain for metabolic engineering and represents a useful step toward the construction of a C. glutamicum chassis genome of strain ATCC 13032 for biotechnological applications and synthetic biology.

Fig 1

Locations of the three prophages (CGP1, CGP2, and CGP3) in the genome of C. glutamicum (A) and expression patterns of CGP3 genes (B). (A) Circular representation of the C. glutamicum ATCC 13032 chromosome. The concentric circles denote (from outward to inward) coding sequences (CDS) transcribed clockwise and counterclockwise, locations of the three prophages, relative G/C content, and GC skew. A positive deviation in G/C content from the average is shown by bars pointing outward and a negative deviation by bars pointing inward. The same holds for the GC skew plot, where positive skew values are shown in red and negative values in blue. (B) Presentation of the fluorescent output of CGP3 (cg1890-cg2071) and adjacent genes in the genome of C. glutamicum out of more than 100 different microarray experiments stored in our in-house database. Presented are all experiments performed with the current array series. Several prophage genes appear to be regularly expressed (red color). In some of the experiments, the whole prophage region is upregulated, e.g., 1, overexpression of a transcriptional regulator; or 2, induction of the SOS response by addition of mitomycin C. Labeled are the first and the last genes of CGP3 (cg1890 and cg2071) and some examples of frequently expressed prophage genes.

Fig 2

Growth of C. glutamicum ATCC 13032 and the prophage-free strain MB001 on minimal medium with glucose in the presence of mitomycin C (A) or in direct competition (B). (A) The strains were precultivated in BHI medium without mitomycin and inoculated to an OD₆₀₀ of 1 in CGXII minimal medium with or without 1 μM mitomycin C. Each graph shows the average values and standard deviations resulting from four independent cultures. (B) The strains carrying chromosomal fluorescence markers were precultivated in BHI medium, mixed in a 1:1 ratio, and used to inoculate the main cultures in CGXII minimal medium with 2% (wt vol⁻¹) glucose. Production of fluorescence marker proteins was induced with 100 μM IPTG after 2 h and monitored online. On the following day, 100 μl of these cultures was used to inoculate 750 μl fresh CGXII minimal medium. This procedure was repeated three times. Presented is the ratio of fluorescence signals in the stationary phase of each culture (n = 3).

Fig 3

Transformation efficiency of C. glutamicum ATCC 13032, MB001, and the ΔcglMRR strain. A 150-μl cell suspension containing 10⁹ cells was mixed on ice with 500 ng plasmid DNA. After regeneration, 10-μl and 300-μl aliquots were streaked on solid BHIS medium containing 25 mg liter⁻¹ kanamycin. CFU were counted after 2 days incubation at 30°C, and the number of transformants was calculated as CFU per μg DNA and 10⁹ recipient cells.

Fig 4

Production of the heterologous model protein eYFP. Shown is the quantification of eYFP production and plasmid copy number in the C. glutamicum wild type, MB001, and the ΔcglMRR strain, each carrying the plasmid pEKEx2-eyfp. (A) Growth and eYFP fluorescence of the strains cultivated in CGXII minimal medium with 1% (wt vol⁻¹) glucose and 1 mM IPTG in the Biolector at 30°C and 1,200 rpm. Cultivations were performed in seven replicates and sampled during the exponential and stationary growth phases for further analysis of plasmid copy number. Please note that the relatively large error bars of backscatter values below 10 result from technical limitations and the logarithmic presentation. (B) Biomass-specific eYFP fluorescence during the stationary growth phase of the experiment presented in panel A. (C) Plasmid copy number determined by qPCR. The cells were cultivated as described for panel A, and samples were harvested in late exponential and stationary growth phases. (D) Determination of the relative amount of eYFP. The blot was probed with primary antibodies against eYFP and citrate synthase (CS) and an anti-rabbit Cy5-coupled construct as a secondary antibody.