Enzymatic Oxyfunctionalization Driven by Photosynthetic Water-Splitting in the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

Photosynthetic water-splitting is a powerful force to drive selective redox reactions. The need of highly expensive redox partners such as NADPH and their regeneration is one of the main bottlenecks for the application of biocatalysis at an industrial scale. Recently, the possibility of using the photosystem of cyanobacteria to supply high amounts of reduced nicotinamide to a recombinant enoate reductase opened a new strategy for overcoming this hurdle. This paper presents the expansion of the photosynthetic regeneration system to a Baeyer-Villiger monooxygenase. Despite the potential of this strategy, this work also presents some of the encountered challenges as well as possible solutions, which will require further investigation. The successful enzymatic oxygenation shows that cyanobacterial whole-cell biocatalysis is an applicable approach that allows fuelling selective oxyfunctionalisation reactions at the expense of light and water. Yet, several hurdles such as side-reactions and the cell-density limitation, probably due to self-shading of the cells, will have to be overcome on the way to synthetic applications.

Artifizielle Lichtsammelkomplexe ermöglichen Rieske‐Oxygenase‐ katalysierte Hydroxylierungen in nicht‐photosynthetischen Zellen

“…Um diese Herausforderung zu lösen, werden derzeit viele alternative Ansätze in Betracht gezogen . Neben der Verknüpfung der Photochemie mit Enzymen in vitro zur Cofaktor‐Regeneration, haben autotrophe und chemolithoautotrophe Organismen in jüngster Zeit Aufmerksamkeit erhalten, da sie dazu in der Lage sind, anorganische Verbindungen als Elektronendonoren zu nutzen . Licht‐getriebene Ganzzellreaktionen in Cyanobakterien zeigen die gleichen Reaktionsgeschwindigkeiten wie E. coli .…”

“…In der Tat waren die erhaltenen Produktbildungen mit den Leervektor‐Kontrollen unter dunklen und hellen Bedingungen viel geringer. Wir führen den Umsatz im Dunkeln auf die Produktion von Kohlenhydraten zurück, die unter “Hungerbedingungen” zur Regeneration von NAD(P)H verbraucht werden . Unter Lichtbedingungen wurde jedoch eine photochemische Hintergrundreaktion beobachtet, die je nach angewandter Lichtintensität zwischen 0.15 und 3.5 m m und je nach angewandter Photosensibilisator/Elektronendonor‐Kombination variierte (Abbildung A, Abbildung S19).…”

Biocatalysis Fueled by Light: On the Versatile Combination of Photocatalysis and Enzymes

“…When cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus NCIMB 9874 was introduced for recombinant expression into the cyanobacterial host, drastically increased reaction rates under illumination with a LED light source (coldwhite, 10 W) were observed . Undesired keto reduction through the action of an endogenous ADH, however, was encountered in this whole‐cell approach.…”

Hydrogen‐Driven Cofactor Regeneration for Stereoselective Whole‐Cell C=C Bond Reduction in Cupriavidus necator

“…[5] We noted that am arked improvement in the specific activities could be achieved by increasing the availableh eadspace to allow for better mixing. However,t he formed 1b was clearly converted to its corresponding alcohol, contributing up to 10 %o ft otal product formation.S imilarly,t he ADHs did not convert the smallerc ompounds 2a and 2b.T his preference for cyclohexanone wasa lso observed for the endogenous ADHs of the cyanobacterium Synechocystis sp.…”

“…As ar esult, biocatalysis is becoming an important pillar of catalysis fort he preparation of specialty and platform chemicals. [3] Despite the availability of other autotrophicc hassis, such as light-driven oxygenic cyanobacteria, [4,5] the implementation of chemolithoautotrophs provides an avenue for the utilization of other waste streams such as syngas and H 2 -and CO 2 -rich factory emissions,a lbeit with some caveats such as the requirement for strictly anoxic conditions and often unamenable genetics. Limiting factors for whole-cellb iotransformations are, besides the expression level of recombinante nzymes and the transport of substrates and products, often the intracellular levels of reduced nicotinamide cofactors.…”