No abstract

Clostridium pasteurianum produces industrially valuable chemicals such as n‐butanol and 1,3‐propanediol from fermentations of glycerol and glucose. Metabolic engineering for increased yields of selective compounds is not well established in this microorganism. In order to study carbon fluxes and to selectively increase butanol yields, we integrated the latest advances in genome editing to obtain an electrocompetent Clostridium pasteurianum strain for further engineering. Deletion of the glycerol dehydratase large subunit (dhaB) using an adapted S. pyogenes Type II CRISPR/Cas9 nickase system resulted in a 1,3‐propanediol‐deficient mutant producing butanol as the main product. Surprisingly, the mutant was able to grow on glycerol as the sole carbon source. In spite of reduced growth, butanol yields were highly increased. Metabolic flux analysis revealed an important role of the newly identified electron bifurcation pathway for crotonyl‐CoA to butyryl‐CoA conversion in the regulation of redox balance. Compared to the parental strain, the electron bifurcation pathway flux of the dhaB mutant increased from 8 to 46% of the overall flux from crotonyl‐CoA to butyryl‐CoA and butanol, indicating a new, 1,3‐propanediol‐independent pattern of glycerol fermentation in Clostridium pasteurianum.

In this contribution, we studied the effect of electro‐fermentation on the butanol production of Clostridium pasteurianum strains by a targeted metabolomics approach. Two strains were examined: an electrocompetent wild type strain (R525) and a mutant strain (dhaB mutant) lacking formation of 1,3‐propanediol (PDO). The dhaB‐negative strain was able to grow on glycerol without formation of PDO, but displayed a high initial intracellular NADH/NAD ratio which was lowered subsequently by upregulation of the butanol production pathway. Both strains showed a 3–5 fold increase of the intracellular NADH/NAD ratio when exposed to cathodic current in a bioelectrochemical system (BES). This drove an activation of the butanol pathway and resulted in a higher molar butanol to PDO ratio for the R525 strain. Nonetheless, macroscopic electron balances suggest that no significant amount of electrons derived from the BES was harvested by the cells. Overall, this work points out that electro‐fermentation can be used to trigger metabolic pathways and improve product formation, even when the used microbe cannot be considered electroactive. Accordingly, further studies are required to unveil the underlying (regulatory) mechanisms.

Autotrophic or mixotrophic use of one‐carbon (C1) compounds is gaining importance for sustainable bioproduction. In an effort to integrate the reductive glycine pathway (rGP) as a highly promising pathway for the assimilation of CO2 and formate, genes coding for glycine synthase system from Gottschalkia acidurici were successfully introduced into Clostridium pasteurianum, a non‐model host microorganism with industrial interests. The mutant harboring glycine synthase exhibited assimilation of exogenous formate and reduced CO2 formation. Further metabolic data clearly showed large impacts of expression of glycine synthase on the product metabolism of C. pasteurianum. In particular, 2‐oxobutyrate (2‐OB) was observed for the first time as a metabolic intermediate of C. pasteurianum and its secretion was solely triggered by the expression of glycine synthase. The perturbation of C1 metabolism is discussed regarding its interactions with pathways of the central metabolism, acidogenesis, solventogenesis, and amino acid metabolism. The secretion of 2‐OB is considered as a consequence of metabolic and redox instabilities due to the activity of glycine synthase and may represent a common metabolic response of Clostridia in enhanced use of C1 compounds.

For efficient production of recombinant proteins by mammalian cells in a bioreactor, optimal growth rates are required and represent the most important process parameter. We present the first successful attempt to monitor the growth behavior and cell cycle state of a mammalian production relevant cell line under bioreactor cultivation conditions up to 1.2 l, utilizing a fluorescent read-out without the need of additional staining or marking. For this purpose, we developed two new production relevant cell line derivatives (CHO-K1 FUCCI CM & CHO-K1 FUCCI CN) and corresponding analytical methods. The approach is easily scalable, applicable to mammalian recombinant protein production cell lines, and it allows for real-time monitoring using appropriate fluorescence probes. It is based on the Ubiquitination-based Cell Cycle Indicator (FUCCI) system developed by Miyawaki et al. CHO-K1 was chosen as a model cell line due to its close relationship to several production cell lines. We defined a new process parameter i , a quantitative and numerically robust representation of the cell cycle distribution, and demonstrate it to be linearly correlated with the cell cycle state and inversely related to the real time growth rate. Detection of growth rate limitations is possible earlier than using cell-count-based approaches. Analytics were compatible with bulk fluorescence methods, using a plate reader as well as a flow cytometer. For future real time applications in industry scale bioreactors we recommend the use of on-line or at-line fluorescence probes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1408-1417, 2017.