General medium for the autotrophic cultivation of acetogens.

Syngas fermentation, a microbial process in which synthesis gas serves as a substrate for acetogens, has attracted increasing interest in the last few years. For the purposeful selection of acetogens for various applications, it would be useful to characterize and compare the process performances of as many autotrophic strains as possible under identical process conditions. Unfortunately, all the media compositions so far recommended for syngas fermentation differ considerably with respect to each individual strain. Therefore, a general medium for syngas fermentation was designed. The suitability of this new general-acetogen medium (GA-medium) was proven based on the autotrophic batch cultivation of Acetobacterium fimetarium, Acetobacterium wieringae, Blautia hydrogenotrophica, Clostridium magnum, Eubacterium aggregans, Sporomusa acidovorans, Sporomusa ovata and Terrisporobacter mayombei in anaerobic flasks with an initial gas phase of H2:CO2 (66:34) (P = 200 kPa). A comparison of the autotrophic batch processes with this medium revealed T. mayombei as the bacterium with the highest maximum growth rate of 5.77 day(-1) which was more than 10 times higher than the lowest identified maximum growth rate of A. fimetarium. The maximum growth rates of A. wieringae, C. magnum and S. acidovorans were all in the same order of magnitude around 1.7 day(-1). The newly designed GA-medium offers the possibility to compare autotrophic process performances of different acetogens under similar conditions absent the effects of various media compositions.

High-level soluble expression of a bacterial N-acyl-d-glucosamine 2-epimerase in recombinant Escherichia coli.

N-Acyl-d-glucosamine 2-epimerase (AGE) is an important enzyme for the biocatalytic synthesis of N-acetylneuraminic acid (Neu5Ac). Due to the wide range of biological applications of Neu5Ac and its derivatives, there has been great interest in its large-scale synthesis. Thus, suitable strategies for achieving high-level production of soluble AGE are needed. Several AGEs from various organisms have been recombinantly expressed in Escherichia coli. However, the soluble expression level was consistently low with an excessive formation of inclusion bodies. In this study, the effects of different solubility-enhancement tags, expression temperatures, chaperones and host strains on the soluble expression of the AGE from the freshwater cyanobacterium Anabaena variabilis ATCC 29413 (AvaAGE) were examined. The optimum combination of tag, expression temperature, co-expression of chaperones and host strain (His6-tag, 37°C, GroEL/GroES, E. coli BL21(DE3)) led to a 264-fold improvement of the volumetric epimerase activity, a measure of the soluble expression, compared to the starting conditions (His6-maltose-binding protein-tag, 20°C, without chaperones, E. coli BL21(DE3)). A maximum yield of 22.5mg isolated AvaAGE per liter shake flask culture was obtained.

Comparative reaction engineering analysis of different acetogenic bacteria for gas fermentation.

The production of chemicals by syngas fermentation is a promising alternative to heterotrophic fermentation processes. The autotrophic process performances of the so far not well studied acetogens Acetobacterium fimetarium, Acetobacterium wieringae, Blautia hydrogenotrophica, Clostridium magnum, Eubacterium aggregans, Sporomusa acidovorans, Sporomusa ovata and Terrisporobacter mayombei were characterized. Acetobacterium woodii was used as reference strain. Standardized batch experiments with continuous supply of the gaseous substrates CO2 and H2 were performed in fully controlled stirred-tank bioreactors. A. wieringae and S. ovata showed by far the highest growth rates and maximum biomass concentrations among the acetogens under study. Aside from the reference strain A. woodii, highest volumetric (17.96gL(-1)d(-1)) as well as cell specific acetate formation rates (21.03gg(-1)d(-1)) were observed with S. ovata resulting in a final acetate concentration of 32.2gL(-1). Accumulation of formate with up to 4.8gL(-1) was observed with all acetogens. Ethanol was produced autotrophically with up to 0.42gL(-1) by four of the acetogenic bacteria under study (A. wieringae, C. magnum, S. acidovorans and S. ovata) and also by A. woodii. Butyrate was formed with up to 0.14gL(-1) by three of the acetogenic bacteria under study (C. magnum, B. hydrogenotrophica and E. aggregans). Due to its superior process performances S. ovata may be a promising host for redirecting carbon fluxes by applying metabolic engineering and tools of synthetic biology to produce non-natural chemicals from syngas.

New N-acyl-D-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate.

N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-D-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-D-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-D-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117±2 U mg(-1) at 37 °C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the Km for the substrate than on the enzyme activity. At high substrate concentrations (≥200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess.