The productivity of industrial fermentation processes is essentially limited by the biomass specific substrate consumption rate (q) of the applied microbial production system. Since q depends on the growth rate (μ), we highlight the potential of the fastest growing non-pathogenic bacterium, , as novel candidate for future biotechnological processes. grows rapidly in BHIN complex medium with a μ of up to 4.43 h (doubling time of 9.4 min) as well as in minimal medium supplemented with various industrially relevant substrates. Bioreactor cultivations in minimal medium with glucose showed that possesses an exceptionally high q under aerobic (3.90 ± 0.08 g g h) and anaerobic (7.81 ± 0.71 g g h) conditions. Fermentations with resting cells of genetically engineered under anaerobic conditions yielded an overall volumetric productivity of 0.56 ± 0.10 g alanine L min (i.e. 34 g L h). These inherent properties render a promising new microbial platform for future industrial fermentation processes operating with high productivity. Low conversion rates are one major challenge to realize microbial fermentation processes for the production of commodities operating competitively to existing petrochemical approaches. For this reason, we screened for a novel platform organism possessing superior characteristics to traditionally employed microbial systems. We identified the fast growing which exhibits a versatile metabolism and shows striking growth and conversion rates, as a solid candidate to reach outstanding productivities. Due to these inherent characteristics can speed up common laboratory routines, is suitable for already existing production procedures, and forms an excellent foundation to engineer next generation bioprocesses.
Dynamic 13C-tracer-based flux analyses of in vivo reaction networks still require a continuous development of advanced quantification methods applying state-of-the-art mass spectrometry platforms. Utilizing alkaline HILIC chromatography, we adapt strategies for a systematic quantification study in non- and 13C-labeled multicomponent endogenous Corynebacterium glutamicum extracts by LC-QTOF high resolution (HRMS) and LC-QQQ tandem mass spectrometry (MS/MS). Without prior derivatization, a representative cross-section of 17 central carbon and anabolic key intermediates were analyzed with high selectivity and sensitivity under optimized ESI-MS settings. In column detection limits for the absolute quantification range were between 6.8–304.7 (QQQ) and 28.7–881.5 fmol (QTOF) with comparable linearities (3–5 orders of magnitude) and enhanced precision using QQQ-MRM detection. Tailor-made preparations of uniformly (U)13C-labeled cultivation extracts for isotope dilution mass spectrometry enabled the accurate quantification in complex sample matrices and extended linearities without effect on method parameters. Furthermore, evaluation of metabolite-specific m+1-to-m+0 ratios (ISR1:0) in non-labeled extracts exhibited sufficient methodical spectral accuracies with mean deviations of 3.89 ± 3.54% (QTOF) and 4.01 ± 3.01% (QQQ). Based on the excellent HILIC performance, conformity analysis of time-resolved isotopic enrichments in 13C-tracer experiments revealed sufficient spectral accuracy for QQQ-SIM detection. However, only QTOF-HRMS ensures determination of the full isotopologue space in complex matrices without mass interferences.
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.
Iron is a vital mineral for almost all living organisms and has a pivotal role in central metabolism. Despite its great abundance on earth, the accessibility for microorganisms is often limited, because poorly soluble ferric iron (Fe3+) is the predominant oxidation state in an aerobic environment. Hence, the reduction of Fe3+ is of essential importance to meet the cellular demand of ferrous iron (Fe2+) but might become detrimental as excessive amounts of intracellular Fe2+ tend to undergo the cytotoxic Fenton reaction in the presence of hydrogen peroxide. We demonstrate that the complex formation rate of Fe3+ and phenolic compounds like protocatechuic acid was increased by 46% in the presence of HCO3− and thus accelerated the subsequent redox reaction, yielding reduced Fe2+. Consequently, elevated CO2/HCO3− levels increased the intracellular Fe2+ availability, which resulted in at least 50% higher biomass-specific fluorescence of a DtxR-based Corynebacterium glutamicum reporter strain, and stimulated growth. Since the increased Fe2+ availability was attributed to the interaction of HCO3− and chemical iron reduction, the abiotic effect postulated in this study is of general relevance in geochemical and biological environments. IMPORTANCE In an oxygenic environment, poorly soluble Fe3+ must be reduced to meet the cellular Fe2+ demand. This study demonstrates that elevated CO2/HCO3− levels accelerate chemical Fe3+ reduction through phenolic compounds, thus increasing intracellular Fe2+ availability. A number of biological environments are characterized by the presence of phenolic compounds and elevated HCO3− levels and include soil habitats and the human body. Fe2+ availability is of particular interest in the latter, as it controls the infectiousness of pathogens. Since the effect postulated here is abiotic, it generally affects the Fe2+ distribution in nature.
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