Transcriptional and physiological analysis of the model cyanobacterium Synechocystis PCC 6803 under ethanologenic and external ethanol conditions

Bis zum heutigen Zeitpunkt ist wenig über die physiologischen Effekte von Ethanol auf Cyanobakterien bekannt. Dies ist nicht überraschend, da es unwahrscheinlich ist, dass Cyanobakterien in ihrer natürlichen Umwelt auf Wachstums inhibierende Konzentrationen stoßen, und deswegen war die Stressantwort auf Ethanol nur von geringerem Interesse für die Forschungsgemeinschaft. Nichts desto weniger sind durch neue Entwicklungen im Biofuel- Sektor, insbesondere im Kontext der Produktion von Ethanol mit Hilfe von genetisch manipulierten Cyanobakterien, Kenntnisse über die zelluläre Toleranz und Zellantwort gegenüber dem gewünschten Produkt von grundlegender Bedeutung. Microarray-Experimente, die einen Einblick in die zelluläre Antwort durch Änderung der Genexpression auf Ethanolproduktion bringen sollten, zeigten, dass Gene des Phycocyanin-Operons als die am signifikantesten und stärksten betroffenen funktionalen genetischen Elemente. Weitere Microarray-Experimente mit verschiedenen Konzentrationen von extern zugefügtem Ethanol zeigten eine zeitverzögerte (24h) Hochregulation von PS II-Genen und dem Transkript cpcG2. Diese Arbeit beschreibt weiterhin die Ergebnisse eines Experiments zur "Evolution im Labor", das die intrinsische Kapazität von Synechocystis sp. PCC 6803 zur Erweiterung der Toleranz gegenüber Ethanol aufzeigen sollte. Die erhöhte Ethanoltoleranz führte zu einer Optimierung der endogenen Ethanolproduktion. Derartige Versuche zur Stammoptimierung durch "Evolution im Labor" sollten daher geeignete Mittel sein, um bestimmte Eigenschaften von Organismen für biotechnologische Ziele zu verbessern. In der Gesamtheit geben die Ergebnisse dieser Arbeit Einblicke in die Antwort der Synechocystis-Zellen auf Ethanol auf den Ebenen des Stoffwechsels und der Genexpression und stellen eine wertvolle Datensammlung für zukünftige Versuche mit dem Ziel dar, die Ethanolproduktionsrate in Cyanobakterien durch genetic engineering zu erhöhen.Until recently, little has been known about the effects of ethanol on the physiology of cyanobacteria. This is not surprising as it is unlikely that cyanobacteria encounter growth inhibiting concentrations of ethanol in their natural environment, and thus the ethanol stress response used to be of limited interest to the scientific community. Nevertheless, for recent biotechnological approaches in the field of biofuel production, and in particular for the attempts to produce ethanol with the help of genetically modified microalgae and cyanobacteria, knowledge of cellular tolerance and response to the desired product is pivotal. Microarray analysis demonstrating that a specific part of the phycocyanin operon is the most significantly and strongly affected functional genetic subsystem under ethanol producing conditions. Additional microarray experiments with different concentrations of external ethanol showed a time-delayed (24h) characterized by a prominent up-regulation of PS II genes with phycocyanin linker proteins playing a major role in the transcriptional response. Another aspect of this work was an artificial evolution experiment, which was performed to delineate the intrinsic capacity of Synechocystis sp. PCC6803 to tolerate ethanol. In addition, the evolved strain proved to be a superior background for endogenous ethanol production showing that artificial evolution experiments are a suitable method to improve certain features of organisms for biotechnological purposes. Overall, the results of this work give new insight into physiological and gene regulatory responses of Synechocystis sp. PCC6803 exposed to ethanol and will be a very valuable dataset for future attempts to improve cyanobacterial ethanol production by the means of genetic engineering

Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803

BACKGROUND: The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. RESULTS: The transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d(-1) and 0.0303% (v/v) ethanol d(-1) were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. CONCLUSIONS: Changes in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected