Transmembrane Shuttling of Photosynthetically Produced Electrons to Propel Extracellular Biocatalytic Redox Reactions in a Modular Fashion

Abstract

Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and timeconsuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme-or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with > 99 % optical purity.

Photobiocatalytic Strategies for Organic Synthesis

“…Winkler and Kroutil recognized that heterologous expression and substrate transport across the cyanobacteria wall presented a challenge in more broadly implementing photosynthetic organisms in biocatalytic reactions. To overcome this issue, they developed a shuttling strategy where acetone would shuttle reducing equivalents from a cyanobacterium to biocatalysts in solution (Figure A) . The cyanobacterium ( Synechococcus elongatus PCC 7942) heterologously expressing the alcohol dehydrogenase from Lactobacillus kefir (Lk-ADH) reduces acetone to i-PrOH using photosynthetically produced NADPH (Module A).…”

Harnessing photosynthetic microorganisms for enhanced bioremediation of microplastics: A comprehensive review

“…The methods available for their engineering allow the expression of different heterologous enzymes: the gene expressions can be tuned by either exploiting self-replicative plasmids or integrative vectors under different synthetic and native promoters of varying strength [ [184] , [185] , [186] , [187] , [188] ]. The ability of cyanobacteria to recycle NADPH, using light-driven water oxidation to supply electrons, makes them attractive as photobiocatalysts for different uses [ [189] , [190] , [191] , [192] , [193] , [194] , [195] , [196] , [197] ]. Particularly, their capacity to utilise light for the biotransformation of specific substrates into the desired products is a unique advantage compared to heterotrophic bacteria.…”

Exciting Enzymes: Current State and Future Perspective of Photobiocatalysis

“…To circumvent the challenging genetic manipulation of cyanobacteria, efforts to shuttle the electrons for cofactor recycling to the extracellular space headed the development of the coupling of formate dehydrogenase to formate‐exporting cells of the microalgae Chlamydomonas reinhardtii , [75] and, quite recently, employing acetone/isopropanol as mediators for the export of reducing equivalents from the cyanobacterium Synechococcus elongatus PCC 7942 to extracellularly allocated oxidoreductases [76] …”

“…[41] To circumvent the challenging genetic manipulation of cyanobacteria, efforts to shuttle the electrons for cofactor recycling to the extracellular space headed the development of the coupling of formate dehydrogenase to formate-exporting cells of the microalgae Chlamydomonas reinhardtii, [75] and, quite recently, employing acetone/isopropanol as mediators for the export of reducing equivalents from the cyanobacterium Synechococcus elongatus PCC 7942 to extracellularly allocated oxidoreductases. [76] To investigate all these opportunities of photobiocatalysis, one crucial challenge remains. Being a comparably young discipline, well-characterized illumination equipment or photobioreactors are not broadly available [77] and the community still needs to define a good practice regarding the reporting of all experimental data that is required for the reproduction of photo(bio)catalytic reactions (see next sections).…”