Metabolic shift induced by synthetic co-cultivation promotes high yield of chain elongated acids from syngas

Bio-catalytic processes for sustainable production of chemicals and fuels receive increased attention within the concept of circular economy. Strategies to improve these production processes include genetic engineering of bio-catalysts or process technological optimization. Alternatively, synthetic microbial co-cultures can be used to enhance production of chemicals of interest. It remains often unclear however how microbe to microbe interactions affect the overall production process and how this can be further exploited for application. In the present study we explored the microbial interaction in a synthetic co-culture of Clostridium autoethanogenum and Clostridium kluyveri, producing chain elongated products from carbon monoxide. Monocultures of C. autoethanogenum converted CO to acetate and traces of ethanol, while during co-cultivation with C. kluyveri, it shifted its metabolism significantly towards solventogenesis. In C. autoethanogenum, expression of the genes involved in the central carbon- and energy-metabolism remained unchanged during co-cultivation compared to monoculture condition. Therefore the shift in the metabolic flux of C. autoethanogenum appears to be regulated by thermodynamics, and results from the continuous removal of ethanol by C. kluyveri. This trait could be further exploited, driving the metabolism of C. autoethanogenum to solely ethanol formation during co-cultivation, resulting in a high yield of chain elongated products from CO-derived electrons. This research highlights the important role of thermodynamic interactions in (synthetic) mixed microbial communities and shows that this can be exploited to promote desired conversions.The research leading to these results has received funding from the Netherlands Ministry of Education, Culture and Science and from the Netherlands Science Foundation (NWO) under the Gravitation Grant nr. 024.002.002 and Programme ‘Closed Cycles’ with Project nr. ALWGK.2016.029.info:eu-repo/semantics/publishedVersio

Time-resolved fluorescence microscopy for measuring specific coenzymes in methanogenic bacteria.

Measurements of time-resolved photobleaching and nanosecond fluorescence decay from microscopic samples of methanogenic bacteria are reported. From cultures of Methanobacterium thermoautotrophicum and Methanosarcina barkeri, decay times of 1 ns and 3 ns were obtained for the specific coenzymes F420 and 7-methylpterin, respectively. In contrast to methylpterin, the fluorescence of F420 was bleached selectively, with a time constant of about 160 s, at an irradiation power density of 5 mW mm-2. Similar time constants were found for samples of sewage sludge containing methanogenic bacteria. Active and inactive bacterial cells could be differentiated by following the course of photobleaching

Time-resolved microfluorescence for measuring coenzyme F₄₂₀ in Methanobacterium thermoautotrophicum.

The time course of photobleaching and the nanosecond fluorescence decay have been measured from microscopic samples of methanogenic bacteria, to our knowledge the first application of these methods in this field. Decay times of about 1 ns and 3 ns were obtained for the specific coenzymes F420 and 7-methylpterin, respectively. In contrast to methylpterin and other fluorescent compounds the intensity of F420 fluorescence was reduced selectively due to photobleaching. This effect, as well as the different decay time constants could be used to discriminate F420 from other fluorescent components. In addition, active and inactive bacterial cells could be differentiated following the course of photobleaching

Fluorescence techniques in biotechnology.

The high specificity and sensitivity of fluorescence techniques have made them important analytical tools in medicine and biotechnology. Besides monitoring and quantitative detection of biomolecules these methods can be used for controlling bacterial activities or for measuring physiological states of cells or tissues. Three topics of importance in biotechnology - immunoassays, photosynthesis and fermentation - are treated in detail

Coenzyme specificity of dehydrogenases and fermentation of pyruvate by clostridia.

Four clostridial species (C. pasteurianum, C. butylicum, C. butyricum and C. tetanomorphum) grow on pyruvate. Two other species (C. roseum and C. rubrum) only ferment this compound; this is probably due to their inability to synthesize hexose phosphates from pyruvate (fructose-1,6-diphosphatase and pyruvate carboxylase are absent). The fermentation of pyruvate by the above clostridia yields acetate, carbon dioxide, hydrogen and small amounts of compounds more reduced than acetate. Hydrogen pressure increases the amount of ethanol, butanol and butyrate formed during the fermentation of pyruvate. Since C. roseum and C. rubrum contain a ferredoxin: NADP reductase it seems likely that NADPH2 is the coenzyme involved in ethanol formation. In accordance with this acetaldehyde and alcohol dehydrogenases exhibit activity with NADPH2. The glyceraldehyde-3-phosphate dehydrogenase of the clostridia under investigation is NAD specific and so is the β-hydroxy-butyryl-CoA dehydrogenase with the exception of C. kluyveri. The specific activity of hydrogenase and the coenzyme specificity of NAD(P) reductase vary among the clostridial species

Polyphosphate/ATP-dependent NAD kinase of Corynebacterium glutamicum: biochemical properties and impact of ppnK overexpression on lysine production

Lindner S, Niederholtmeyer H, Schmitz K, Schoberth SM, Wendisch VF. Polyphosphate/ATP-dependent NAD kinase of Corynebacterium glutamicum: biochemical properties and impact of ppnK overexpression on lysine production. Applied Microbiology and Biotechnology. 2010;87(2):583-593.Nicotinamide adenine dinucleotide phosphate (NADP) is synthesized by phosphorylation of either oxidized or reduced nicotinamide adenine dinucleotide (NAD/NADH). Here, the cg1601/ppnK gene product from Corynebacterium glutamicum genome was purified from recombinant Escherichia coli and enzymatic characterization revealed its activity as a polyphosphate (PolyP)/ATP-dependent NAD kinase (PPNK). PPNK from C. glutamicum was shown to be active as homotetramer accepting PolyP, ATP, and even ADP for phosphorylation of NAD. The catalytic efficiency with ATP as phosphate donor for phosphorylation of NAD was higher than with PolyP. With respect to the chain length of PolyP, PPNK was active with short-chain PolyPs. PPNK activity was independent of bivalent cations when using ATP, but was enhanced by manganese and in particular by magnesium ions. When using PolyP, PPNK required bivalent cations, preferably manganese ions, for activity. PPNK was inhibited by NADP and NADH at concentrations below millimolar. Overexpression of ppnK in C. glutamicum wild type slightly reduced growth and ppnK overexpression in the lysine producing strain DM1729 resulted in a lysine product yield on glucose of 0.136 +/- 0.006 mol lysine (mol glucose)(-1), which was 12% higher than that of the empty vector control strain