Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803

AbstractThe cytochrome b6f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b6f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase

Expression und Regulation von Membranproteinen am Beispiel der Cytochrom bd\it {bd}-Oxidase aus Synechocystis sp.\textit {Synechocystis sp.} PCC 6803

Durch Etablierung einer schnellen reproduzierbaren RNA Präparation aus $\textit {Synechocystis sp.}$ PCC 6803 wurde die standardmäßige Nutzung der qRT-PCR ermöglicht, die zur Charakterisierung von Membranproteinen, sowie der Untersuchung von Expressionsproblemen im initialen Schritt genutzt werden kann. Die heterologe Expression eines Membranproteins wurde am Bsp. von Bacteriorhodopsin gezeigt. Zum ersten Mal konnte ein direkter Beweis für die Präsenz der Cytochrom $\it {bd}$-Oxidase in Synechocystis erbracht und gezeigt werden, dass die Regulation wahrscheinlich auf Translationsebene stattfindet. CydA konnte durch einen Peptid-Antikörper (AK) detektiert werden, dessen Monospezifität Experimente zur Lokalisation des Komplexes in $\it {Synechocystis}$ erlaubt. Die Aufreinigung getaggten Proteins aus $\it {E.coli}$ lieferte nahezu reines CydB zu AK-Gewinnung. Funktionelle Untersuchungen einer Komplementationsmutante bestätigen die Beteiligung des slr1379/slr1380 kodierten Komplex am photosynthetischen Elektronentransport

Photosynthetic production of enantioselective biocatalysts

$\bf Background:$ Global resource depletion poses a dramatic threat to our society and creates a strong demand for alternative resources that do not compete with the production of food. Meeting this challenge requires a thorough rethinking of all steps of the value chain regarding their sustainability resource demand and the possibility to substitute current, petrol-based supply-chains with renewable resources. This regards also the production of catalysts for chemical synthesis. Phototrophic microorganisms have attracted considerable attention as a biomanufacturing platform for the sustainable production of chemicals and biofuels. They allow the direct utilization of carbon dioxide and do not compete with food production. Photosynthetic enzyme production of catalysts would be a sustainable supply of these important components of the biotechnological and chemical industries. This paper focuses on the usefulness of recombinant cyanobacteria for the photosynthetic expression of enantioselective catalysts. As a proof of concept, we used the cyanobacterium $\it Synechocystis$ sp. PCC 6803 for the heterologous expression of two highly enantioselective enzymes. $\bf Results:$ We investigated the expression yield and the usefulness of cyanobacterial cell extracts for conducting stereoselective reactions. The cyanobacterial enzyme expression achieved protein yields of 3% of total soluble protein (%TSP) while the expression in $\textit {E. coli}$ yielded 6-8% TSP. Cell-free extracts from a recombinant strain expressing the recombinant esterase ST0071 from the thermophilic organism $\textit {Sulfolobus tokodai}$ ST0071 and arylmalonate decarboxylase from $\textit {Bordetella bronchiseptica}$ showed excellent enantioselectivity (>99% ee) and yield (>91%) in the desymmetrisation of prochiral malonates. $\bf Conclusions:$ We were able to present the proof-of-concept of photoautotrophic enzyme expression as a viable alternative to heterotrophic expression hosts. Our results show that the introduction of foreign genes is straightforward. Cell components from $\it Synechocystis$ did not interfere with the stereoselective transformations, underlining the usability of photoautotrophic organisms for the production of enzymes. Given the considerable commercial value of recombinant biocatalysts, cyanobacterial enzyme expression has thus the potential to complement existing approaches to use phototrophic organisms for the production of chemicals and biofuels