Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO ₂ has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an ldh gene from Bacillus subtilis (encoding an l -lactate dehydrogenase) into the genome of Synechocystis sp. strain PCC6803 leads to l -lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the single ldh mutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.

The societal importance of renewable carbon-based commodities and energy carriers has elicited a particular interest for high performance phototrophic microorganisms. Selection of optimal strains is often based on direct comparison under laboratory conditions of maximal growth rate or additional valued features such as lipid content. Instead of reporting growth rate in culture, estimation of photosynthetic efficiency (quantum yield of PSII) by pulse-amplitude modulated (PAM) fluorimetry is an often applied alternative method. Here we compared the quantum yield of PSII and the photonic yield on biomass for the green alga Chlorella sorokiniana 211-8K and the cyanobacterium Synechocystis sp. PCC 6803. Our data demonstrate that the PAM technique inherently underestimates the photosynthetic efficiency of cyanobacteria by rendering a high F₀ and a low F_M, specifically after the commonly practiced dark pre-incubation before a yield measurement. Yet when comparing the calculated biomass yield on light in continuous culture experiments, we obtained nearly equal values for both species. Using mutants of Synechocystis sp. PCC 6803, we analyzed the factors that compromise its PAM-based quantum yield measurements. We will discuss the role of dark respiratory activity, fluorescence emission from the phycobilisomes, and the Mehler-like reaction. Based on the above observations we recommend that PAM measurements in cyanobacteria are interpreted only qualitatively.

ArcBA is a two-component regulatory system of Escherichia coli involved in sensing oxygen availability and the concomitant transcriptional regulation of oxidative and fermentative catabolism. Based on in vitro data, it has been postulated that the redox state of the ubiquinone pool is the determinant for ArcB kinase activity. Here we report on the in vivo regulation of ArcB activation, as determined using a lacZ reporter specifically responsive to phosphorylated ArcA. Our results indicate that upon deletion of a ubiquinone biosynthetic enzyme, regulation of ArcB in the anaerobic-aerobic transition is not affected. In contrast, interference with menaquinone biosynthesis leads to inactivation of ArcB during anaerobic growth; this phenotype is fully rescued by addition of a menaquinone precursor. This clearly demonstrates that the menaquinones play a major role in ArcB activation. ArcB shows a complex pattern of regulation when E. coli is titrated through the entire aerobiosis range; ArcB is activated under anaerobic and subaerobic conditions and is much less active under fully aerobic and microaerobic conditions. Furthermore, there is no correlation between ArcB activation and the redox state of the ubiquinone pool, but there is a restricted correlation between the total cellular ubiquinone content and ArcB activity due to the considerable increase in the size of the ubiquinone pool with increasing degrees of aerobiosis. These results lead to the working hypothesis that the in vivo activity of ArcB in E. coli is modulated by the redox state of the menaquinone pool and that the ubiquinone/ubiquinol ratio in vivo surely is not the only determinant of ArcB activity.

Molecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by “designer microbes.” The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make “designer microbes” includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made.