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.

Cell-free protein synthesis is a versatile protein production system. Performance of the protein synthesis depends on highly active cytoplasmic extracts. Extracts from E. coli are believed to work best; they are routinely obtained from exponential growing cells, aiming to capture the most active translation system. Here, we report an active cell-free protein synthesis system derived from cells harvested at non-growth, stressed conditions. We found a downshift of ribosomes and proteins. However, a characterization revealed that the stoichiometry of ribosomes and key translation factors was conserved, pointing to a fully intact translation system. This was emphasized by synthesis rates, which were comparable to those of systems obtained from fast-growing cells. Our approach is less laborious than traditional extract preparation methods and multiplies the yield of extract per cultivation. This simplified growth protocol has the potential to attract new entrants to cell-free protein synthesis and to broaden the pool of applications. In this respect, a translation system originating from heat stressed, non-growing E. coli enabled an extension of endogenous transcription units. This was demonstrated by the sigma factor depending activation of parallel transcription. Our cell-free expression platform adds to the existing versatility of cell-free translation systems and presents a tool for cell-free biology.

Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems.

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedbackresistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y Iso/S ) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical Y Iso/S . Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non-growing conditions. K E Y W O R D Sisobutanol, ketoacid decarboxylase, metabolic engineering, microaerobic, Pseudomonas putida Abbreviations: 2-KIV, 2-ketoisovalerate; AlsS, acetolactate synthase; BHI, brain-heart infusion; KDC, ketoacid decarboxylase; LB, Lysogeny broth.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Pseudomonas putida KT2440 is emerging as a promising microbial host for biotechnological industry due to its broad range of substrate affinity and resilience to physicochemical stresses. Its natural tolerance towards aromatics and solvents qualifies this versatile microbe as promising candidate to produce next generation biofuels such as isobutanol. In this study, we scaled‐up the production of isobutanol with P. putida from shake flask to fed‐batch cultivation in a 30 L bioreactor. The design of a two‐stage bioprocess with separated growth and production resulted in 3.35 gisobutanol L–1. Flux analysis revealed that the NADPH expensive formation of isobutanol exceeded the cellular catabolic supply of NADPH finally causing growth retardation. Concomitantly, the cell counteracted to the redox imbalance by increased formation of 2‐ketogluconic thereby providing electrons for the respiratory ATP generation. Thus, P. putida partially uncoupled ATP formation from the availability of NADH. The quantitative analysis of intracellular pyridine nucleotides NAD(P)+ and NAD(P)H revealed elevated catabolic and anabolic reducing power during aerobic production of isobutanol. Additionally, the installation of micro‐aerobic conditions during production doubled the integral glucose‐to‐isobutanol conversion yield to 60 mgisobutanol gglucose–1 while preventing undesired carbon loss as 2‐ketogluconic acid.

Background and Aims Performance and hemocompatibility are the two main functions of a hemodialyzer. Synthetic dialysis membranes made from polysulfone (PSU) or polyethersulfone (PES) are mainly used for dialysis treatments and are blended with the hydrophilic agent polyvinylpyrrolidone (PVP), to improve hemocompatibility during dialysis treatments. A novel PSU-based dialyzer (FX CorAL) with an increased and stabilized PVP content has been developed and has shown strong performance and a favourable hemocompatibility profile within previous short term clinical studies. In the present clinical study, we now investigated the performance and hemocompatibility of the FX CorAL vs. two comparators over a longer follow-up time and applied an extended panel of hemocompatibility biomarkers. This allowed us to analyse treatment-specific performance as well as an extensive intra- and interdialytic hemocompatibility profile. Method eMPORA III was a prospective, open, controlled, multicentre crossover trial with randomized treatment sequences conducted in DE, CZ and HU. It randomized stable patients receiving regular post-dilution online HDF to FX CorAL 600, FX CorDiax 600 (both Fresenius Medical Care) and xevonta Hi 15 (B. Braun), each for 4 weeks. The primary outcome was β2-m removal rate (RR) during 4 hrs HDF. Non-inferiority (margin: 5%) and superiority of FX CorAL 600 versus comparators were tested at α = 2.5% (one-sided), with adjustment for multiple tests. Secondary endpoints were RR and/or clearance of β2-m and other molecules, as well as intra- and interdialytic changes of markers of complement activation (C3a, sC5b-9), cell activation / inflammation (white blood cells (WBC), PMN elastase, IL-6, IL-8, LTB-4, sICAM-1, hsCRP), platelet activation (platelet count, β-TG, TxB-2), and oxidative stress (MDA, GSH-Px). Intradialytic hemocompatibility markers were analysed descriptively as differences vs. the values determined at the start of the HDF session (LS means). Results Eighty-two patients were included and analysed in the safety population, with n = 76 presenting data for the primary outcome (ITT population). FX CorAL 600 showed the highest β2-m RR (LS mean: 76.31%), followed by FX CorDiax 600 (75.71%) and xevonta Hi 15 (74.49%). Non-inferiority to its comparators was statistically significant (p<0.0001 each). Superiority was shown vs. xevonta Hi 15 (p<0.0001), but not vs. FX CorDiax 600 (p = 0.0606). The secondary endpoints β2-m clearance as well as myoglobin clearance and RR affirmed these results; small molecule clearances and/or RR were similar between dialyzers. Analyses of hemocompatibility markers within one HDF session, found the following significant differences: Complement activation • C3a 15 min: FX CorAL: 31%; FX CorDiax: 59% (p = 0.003 vs. FX CorAL); xevonta: 41% (p = 0.029 vs. FX CorAL) • sC5b-9 60 min: FX CorAL: 24%; FX CorDiax: 25% (p = 0.337); xevonta: 41% (p<0.0001) Cell activation/ Inflammation • WBC 15 min: FX CorAL: -7.7%; FX CorDiax: -11.1% (p = 0.015); xevonta: -10.1% (p = 0.246) • Monocytes 15 min: FX CorAL: -30.9%; FX CorDiax: -30.3% (p = 0.533); xevonta: -38.2% (p = 0.012) • Neutrophils 15 min: FX CorAL: -3.0%; FX CorDiax: -7.5% (p = 0.019); xevonta: -6.1% (p = 0.258) • PMN elastase 60 min: FX CorAL: 18%; FX CorDiax: 42% (p = 0.003); xevonta: 52% (p = 0.0003) • LTB-4 15 min: FX CorAL: 170%; FX CorDiax: 217% (p = 0.047); xevonta: 186% (p = 0.539) Platelet activation • β-TG 60 min: FX CorAL: -15%; FX CorDiax: 4% (p<0.0001); xevonta: 18% (p<0.0001) Hemocompatibility markers showed no interdialytic changes, i.e., no significant changes within treatment periods. Nine serious adverse events occurred in this study, none of which was dialyser related. Conclusion FX CorAL 600 efficiently removed middle and small molecules and was non-inferior to both comparators and significantly superior to xevonta Hi 15 in β2-m RR. The typical drop in WBC, monocyte, and neutrophil count during dialysis as well as the rise of complement (C3a, sC5b-9) and cell / platelet activation markers (PMN elastase, LTB-4 and β-TG) were lower or comparable during treatment with FX CorAL vs. both comparators, indicating superior hemocompatibility properties.

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