The fast-growing marine bacterium Vibrio natriegens represents an emerging strain for molecular biology and biotechnology. Genome sequencing and quantitative PCR analysis revealed that the first chromosome of V. natriegens ATCC 14048 contains two prophage regions (VNP1 and VNP2) that are both inducible by the DNA-damaging agent mitomycin C and exhibit spontaneous activation under standard cultivation conditions. Their activation was also confirmed by live cell imaging of an mCherry fusion to the major capsid proteins of VNP1 and VNP2. Transmission electron microscopy visualized the release of phage particles belonging to the Siphoviridae family into the culture supernatant. Freeing V. natriegens from its proviral load, followed by phenotypic characterization, revealed an improved robustness of the prophage-free variant toward DNA-damaging conditions, reduced cell lysis under hypo-osmotic conditions, and an increased pyruvate production compared to wild-type levels. Remarkably, the prophage-free strain outcompeted the wild type in a competitive growth experiment, emphasizing that this strain is a promising platform for future metabolic engineering approaches. IMPORTANCE The fast-growing marine bacterium Vibrio natriegens represents an emerging model host for molecular biology and biotechnology, featuring a reported doubling time of less than 10 minutes. In many bacterial species, viral DNA (prophage elements) may constitute a considerable fraction of the whole genome and may have detrimental effects on the growth and fitness of industrial strains. Genome analysis revealed the presence of two prophage regions in the V. natriegens genome that were shown to undergo spontaneous induction under standard cultivation conditions. In this study, we generated a prophage-free variant of V. natriegens. Remarkably, the prophage-free strain exhibited a higher tolerance toward DNA damage and hypo-osmotic stress. Moreover, it was shown to outcompete the wild-type strain in a competitive growth experiment. In conclusion, our study presents the prophage-free variant of V. natriegens as a promising platform strain for future biotechnological applications.

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Backgroundl-Histidine biosynthesis is embedded in an intertwined metabolic network which renders microbial overproduction of this amino acid challenging. This is reflected in the few available examples of histidine producers in literature. Since knowledge about the metabolic interplay is limited, we systematically perturbed the metabolism of Corynebacterium glutamicum to gain a holistic understanding in the metabolic limitations for l-histidine production. We, therefore, constructed C. glutamicum strains in a modularized metabolic engineering approach and analyzed them with LC/MS-QToF-based systems metabolic profiling (SMP) supported by flux balance analysis (FBA).ResultsThe engineered strains produced l-histidine, equimolar amounts of glycine, and possessed heavily decreased intracellular adenylate concentrations, despite a stable adenylate energy charge. FBA identified regeneration of ATP from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as crucial step for l-histidine production and SMP identified strong intracellular accumulation of inosine monophosphate (IMP) in the engineered strains. Energy engineering readjusted the intracellular IMP and ATP levels to wild-type niveau and reinforced the intrinsic low ATP regeneration capacity to maintain a balanced energy state of the cell. SMP further indicated limitations in the C1 supply which was overcome by expression of the glycine cleavage system from C. jeikeium. Finally, we rerouted the carbon flux towards the oxidative pentose phosphate pathway thereby further increasing product yield to 0.093 ± 0.003 mol l-histidine per mol glucose.ConclusionBy applying the modularized metabolic engineering approach combined with SMP and FBA, we identified an intrinsically low ATP regeneration capacity, which prevents to maintain a balanced energy state of the cell in an l-histidine overproduction scenario and an insufficient supply of C1 units. To overcome these limitations, we provide a metabolic engineering strategy which constitutes a general approach to improve the production of ATP and/or C1 intensive products.

Zero-growth processes are a promising strategy for the production of reduced molecules and depict a steady transition from aerobic to anaerobic conditions. To investigate the adaptation of Corynebacterium glutamicum to altering oxygen availabilities, we conceived a triple-phase fermentation process that describes a gradual reduction of dissolved oxygen with a shift from aerobiosis via microaerobiosis to anaerobiosis. The distinct process phases were clearly bordered by the bacteria’s physiologic response such as reduced growth rate, biomass substrate yield and altered yield of fermentation products. During the process, sequential samples were drawn at six points and analyzed via RNA-sequencing, for metabolite concentrations and for enzyme activities. We found transcriptional alterations of almost 50% (1421 genes) of the entire protein coding genes and observed an upregulation of fermentative pathways, a rearrangement of respiration, and mitigation of the basic cellular mechanisms such as transcription, translation and replication as a transient response related to the installed oxygen dependent process phases. To investigate the regulatory regime, 18 transcriptionally altered (putative) transcriptional regulators were deleted, but none of the deletion strains showed noticeable growth kinetics under an oxygen restricted environment. However, the described transcriptional adaptation of C. glutamicum resolved to varying oxygen availabilities provides a useful basis for future process and strain engineering.

Es wurde der Einfluß des Aminosäuremusters von Maiskleberdiäten auf die Aktivität der Leucin‐β‐naphthylaminopeptidase (digestiv wirksam), der Tripeptidase (teilweise digestiv wirksam), der Leucinaminopeptidase (stoffwechselwirksam), der Iminopeptidase (Parameter für Darmerkrankungen) und auf die Sauerstoffaufnahme (Qo2) der Rattendünndarmmucosa untersucht. Im Vergleich zu einer Caseinstandarddiät ergab sich für die Aktivität aller 4 Peptidasen und für die Sauerstoffaufnahme der Mucosa bei Verfütterung der Maiskleberdiät eine Erhöhung. Es wird auf Grund dieser Befunde die Möglichkeit eines Adaptationsprozesses der Peptidaseaktivitäten an das qualitativ schlechtere Diätprotein diskutiert. Die gemessene Sauerstoffaufnahme der Darmschleimhaut weist daraufhin, daß sehr wahrscheinlich die für Transport‐ und Resorptionsprozesse bereitzustellenden Energiemengen auch bei minderwertigem Nahrungsprotein nicht absinken, sondern eher ansteigen. In Verallgemeinerung dieser und früherer Ergebnisse unseres Arbeitskreises kann geschlossen werden, daß in therapeutischer oder prophylaktischer Hinsicht ein Einsatz von Enzympräparaten zur Unterstützung der Verdauungstätigkeit monogastrischer Tiere bei Verfütterung von Diätprotein minderer Qualität nicht notwendig erscheint.