Recombinant Cyanobacteria for the Asymmetric Reduction of C=C Bonds Fueled by the Biocatalytic Oxidation of Water

Abstract

A recombinant enoate reductase was expressed in cyanobacteria and used for the light-catalyzed, enantioselective reduction of C=C bonds. The coupling of oxidoreductases to natural photosynthesis allows asymmetric syntheses fueled by the oxidation of water. Bypassing the addition of sacrificial cosubstrates as electron donors significantly improves the atom efficiency and avoids the formation of undesired side products. Crucial factors for product formation are the availability of NADPH and the amount of active enzyme in the cells. The efficiency of the reaction is comparable to typical whole-cell biotransformations in E. coli. Under optimized conditions, a solution of 100 mg prochiral 2-methylmaleimide was reduced to optically pure 2-methylsuccinimide (99 % ee, 80 % yield of isolated product). High product yields and excellent optical purities demonstrate the synthetic usefulness of light-catalyzed whole-cell biotransformations using recombinant cyanobacteria.

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

“…Cyclopentanone was converted with a specific activity of 2.3 U/g CDW . Expressing the ene-reductase YqjM in Synechocystis cells, we have previously obtained specific activities of up to 100 U/g CDW , which clearly shows that the NADPH concentration provided by the cells can not be a limiting factor for the BVMO reaction [13]. While the cyanobacterial Baeyer-Villiger oxidation is substantially slower, we would like to emphasise that it is in the same order of magnitude as the same enzyme in E. coli, where 3.5 U/g CDW were achieved [2].…”

“…Indeed, we also found that wildtype Synechocystis cells without the chmo gene catalysed the reduction in 1a and 1c-f to the corresponding alcohols. Previously, we used several cyclohexene derivatives similar to 1b as model compounds for the characterisation of the enereductase YqjM in Synechocystis [13] without observing ketoreduction. This is not surprising because wildtype Synechocytis cells did not reduce 1b.…”

“…PCC6803. The reaction proceeded fast, and was not hampered by side-reactions [13]. We were interested whether the concept can be extended towards the utilization of NADPH for selective oxyfunctionalisation reactions, and if side-reactions and physiological side-effects might be a problem for a catalytic application.…”

Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal

“…Notably, the regulation of cellular processes by green and red light enable the modulation of photosynthesis-dependent biotechnological processes, which require reducing equivalents. In regards to growth in closed photobioreactors under artificial light, electrons originating from photosynthetic water-splitting may partly be re-routed for the reduction of valuable carbon-compounds26 or for the direct production of biohydrogen, especially in engineered cyanobacterial cells27 upon red or green light induction. As this can happen in a time-dependent manner, such light-triggered processes add a new dimension not only to the engineering of cyanobacterial cells for technical applications but also for non-photosynthetic microorganisms based processes.…”

Biocatalytic hydrogenation of the C=C bond in the enone unit of hydroxylated chalcones—process arising from cyanobacterial adaptations

“…Ene-reductases have also been identified in prokaryotic microalgae, i.e. cyanobacteria (Fu et al 2013), and used for light-induced, photocatalytic reduction of C=C bonds (Koninger et al 2016) that conduces to the formation of dihydrochalcones. Because of their high sweet taste, these compounds have potential applications in the food industry for the production of low-calorie, multicomponent, nontoxic and safety sweeteners of natural origin.…”