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.

BACKGROUND AND PURPOSEAtorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). EXPERIMENTAL APPROACHLigand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation. KEY RESULTSAll atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors. CONCLUSIONS AND IMPLICATIONSAtorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound.

The stereochemistry of 2,3-butanediol (2,3-BD) synthesis in microbial fermentations is important for many applications. In this work, we showed that Corynebacterium glutamicum endowed with the Lactococcus lactis genes encoding α-acetolactate synthase and decarboxylase activities produced meso-2,3-BD as the major end product, meaning that (R)-acetoin is a substrate for endogenous 2,3-butanediol dehydrogenase (BDH) activity. This is curious in view of the reported absolute stereospecificity of C. glutamicum BDH for (S)-acetoin (Takusagawa et al. Biosc Biotechnol Biochem 65:1876-1878, 2001). To resolve this discrepancy, the enzyme encoded by butA was produced in Escherichia coli and purified, and the stereospecific properties of the pure protein were examined. Activity assays monitored online byH-NMR using racemic acetoin and an excess of NADH showed an initial, fast production of (2S,3S)-2,3-BD, followed by a slow (∼20-fold lower apparent rate) formation of meso-2,3-BD. Kinetic parameters for (S)-acetoin, (R)-acetoin, meso-2,3-BD and (2S,3S)-BD were determined by spectrophotometric assays. V values for (S)-acetoin and (R)-acetoin were 119 ± 15 and 5.23 ± 0.06 μmol min mg protein, and K values were 0.23 ± 0.02 and 1.49 ± 0.07 mM, respectively. We conclude that C. glutamicum BDH is not absolutely specific for (S)-acetoin, though this is the preferred substrate. Importantly, the low activity of BDH with (R)-acetoin was sufficient to support high yields of meso-2,3-BD in the engineered strain C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,butA ). Additionally, we found that the BDH activity was nearly abolished upon inactivation of butA (from 0.30 ± 0.03 to 0.004 ± 0.001 μmol min mg protein), indicating that C. glutamicum expresses a single BDH under the experimental conditions examined.

The engineering of synthetic metabolic routes can provide valuable lessons on the roles of different biochemical constraints in shaping pathway activity. In this study, we designed and engineered a novel glycerol assimilation pathway in Escherichia coli. While the synthetic pathway was based only on well‐characterized endogenous reactions, we were not able to establish robust growth using standard concentrations of glycerol. Long‐term evolution failed to improve growth via the pathway, indicating that this limitation was not regulatory but rather relates to fundamental aspects of cellular metabolism. We show that the activity of the synthetic pathway is fully controlled by three key physicochemical constraints: thermodynamics, kinetics and metabolite toxicity. Overcoming a thermodynamic barrier at the beginning of the pathway requires high glycerol concentrations. A kinetic barrier leads to a Monod‐like growth dependency on substrate concentration, but with a very high substrate saturation constant. Finally, the flat thermodynamic profile of the pathway enforces a pseudoequilibrium between glycerol and the reactive intermediate dihydroxyacetone, which inhibits growth when the feedstock concentration surpasses 1000 mm. Overall, this study serves to demonstrate the use of synthetic biology to elucidate key design principles of cellular metabolism.