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Rationale: Elastin‐like polypeptides (ELPs) are elastic and thermoresponsive biopolymers composed of VPGXG repeats (X can be any amino acid except proline), used in biomedical applications, for example, tissue engineering and drug delivery. As different variants of ELP are mostly produced fermentatively, there is a need for the development of analysis methods that allow for absolute protein quantification in both complex matrices and purified samples and MW determination of the final products. Methods: ELPs were intracellularly expressed in Escherichia coli quantified after cell lysis and enzymatic digestion using a proline‐specific protease ProAlanase (Promega) at acidic conditions. Resulting peptides were separated by liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high‐resolution mass spectrometry using an Orbitrap mass spectrometer. The addition of a stable isotopically labeled internal standard enabled quantification in complex matrices. Prior to intact mass analysis, ELPs were purified from fermentation broth by inverse temperature cycling. Intact protein analysis was performed using reversed‐phase liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high‐resolution mass spectrometry using a time‐of‐flight mass spectrometer. Results: Absolute quantification of ELPs was achieved by utilizing ELP‐specific properties, that is, proline‐rich, soluble at low pH and low temperature. The repetitive nature of ELPs allows for sensitivity increase and use of higher dilution factors to minimize the matrix effects. Despite the lack of amino acids with charged side chains (Arg, His, Lys, Asp, and Glu) in ELP, we demonstrated successful intact protein analysis using reversed‐phase LC coupled to electrospray ionization TOF MS. Moreover, truncated protein forms could be chromatographically separated and characterized as well as N‐terminal modifications. Conclusions: Both methods combined enabled quantitative and qualitative characterization of fermentatively produced ELPs. [ABSTRACT FROM AUTHOR]

Large-scale microbial industrial fermentations have significantly higher absolute pressure and dissolved CO2 concentrations than otherwise comparable laboratory-scale processes. Yet the effect of increased dissolved CO2 (dCO2) levels is rarely addressed in the literature. In the current work, we have investigated the impact of industrial levels of dCO2 (measured as the partial pressure of CO2, pCO2) in an Escherichia coli-based fed-batch process producing the human milk oligosaccharide 2′-fucosyllactose (2′-FL). The study evaluated the effect of high pCO2 levels in both carbon-limited (C-limited) and carbon/nitrogen-limited (C/N-limited) fed-batch processes. High-cell density cultures were sparged with 10%, 15%, 20%, or 30% CO2 in the inlet air to cover and exceed the levels observed in the industrial scale process. While the 10% enrichment was estimated to achieve similar or higher pCO2 levels as the large-scale fermentation it did not impact the performance of the process. The product and biomass yields started being affected above 15% CO2 enrichment, while 30% impaired the cultures completely. Quantitative proteomics analysis of the C-limited process showed that 15% CO2 enrichment affected the culture on the protein level, but to a much smaller degree than expected. A more significant impact was seen in the dual C/N limited process, which likely stemmed from the effect pCO2 had on nitrogen availability. The results demonstrated that microbial cultures can be seriously affected by elevated CO2 levels, albeit at higher levels than expected. [ABSTRACT FROM AUTHOR]

The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating Complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of Complex I, a wild-type O. parapolymorpha strain and a congenic Complex I-deficient mutant were grown on glucose in aerobic batch, chemostat and retentostat cultures in bioreactors. In batch cultures, both strains exhibited a fully respiratory metabolism and showed the same growth rate and biomass yield, indicating that, under these conditions, the contribution of NADH oxidation via Complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the Complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ~0.001 h-1, both O. parapolymorpha strains showed a high viability. Maintenance-energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a 'stringent response'-like behavior. Quantitative transcriptome and proteome analyses indicated condition39 dependent expression patterns of Complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations. [ABSTRACT FROM AUTHOR]