A light-driven NAD(P)H regeneration system for microbial
bio-production
Yuki HONDA
Department of Chemistry, Faculty of Science, Nara Women’s University
Report: A recombinant Escherichia coli strain genetically engineered to enhance its
porphyrin synthesis accumulated significant amounts of porphyrin dye (mainlyZn-uroporphyrin III) in both culture media and cells. A reaction mixture consisting ofZn-uroporphyrin III purified from culture with a sacrificial reagent converted NAD+ tobiologically inactive NAD dimer under light irradiation. At present, I have not achieved anew photocatalytic NADH regeneration system. On the other hand, this biologicallysynthesized porphyrin succeeded in photocatalytically reducing methyl viologen, which hasoften been used as an artificial coenzyme. Methyl viologen photoreduction was successfully
combined with a hydrogenase reaction, demonstrating that a light-driven hydrogenproduction system could be constructed by combining hydrogenase with the photoreduction
reaction catalyzed by the porphyrin dye biologically synthesized by E. coli.
Research aims
Coenzyme regeneration is a major hurdle to realize industrial bioprocesses using
biocatalysts that require a steady supply of reducing power from NADH, NADPH, or anelectron mediator. The objectiveof this research is to construct a
new coenzyme regenerationsystem; specifically, this researchaims tocoenzyme

tackle light-driven
regeneration using

biologicallysynthesizedmolecular photocatalysts.
Methods
To createalight-driven

coenzyme regeneration system,
this research focused on the useof porphyrin dyes, which aremolecular
photosensitizers

Fig. 1 Porphyrin dye production bygenetically engineered E. coli. A, culture;B, purification of dye; C, HPLC analysis ofwidely used in artificial photosynthesis research. The following three projects were
undertaken: 1) production of dyes by enhancing porphyrin biosynthesis pathways, 2)
photo-regeneration of NADH using biologically synthesized dyes, and 3) photoreduction of
methylviologen by dyes and application of this reaction to photocatalytic hydrogenproduction.
Results
1. Production of dyes by enhancing the porphyrin biosynthesis pathway
Porphyrin biosynthesis was enhanced in E. coli BL21(DE3) cells by introducing the genes
encoding aminolevulinic acid synthase from Rhodobacter capsulatus along with HemB,HemC, and HemD genes from E. coli. Red porphyrin dyes were successfully produced by
the engineered E. coli (Fig. 1A). The biosynthesized dyes were purified from culture
supernatants using an ion exchange resin, and a purified dye solution was obtained (Fig. 1B).
HPLC analysis revealed that the main product was Zn-uroporphyrin III (Fig. 1C).
2. Photo-regeneration of NADH by biologically synthesized dyes
Photocatalytic reduction of NAD+ using the purified dye was investigated. In buffersolution containing the dye molecule, NAD+, and the electron donor, triethanolamine(TEOA), an increase in absorbance at 340 nm, which is an absorption peak for monitoring
NADH formation, wasobserved under lightirradiation using an LED
light at 405 and 420 nm.
Subsequent investigations
revealed that the increasein absorbance at 340 nm
was caused by formationof enzymatically inactive
NAD dimer due to therapid reaction of two
molecules of NAD・, thesingle electron reduced
form of NAD+. We
continue to investigate
developmentofanNADH by combining thissystemwithotherFig. 2 Light-driven hydrogen formation using the purifiedporphyrin dye. A, absorbance change in the reaction by lightirradiation; B, amount of reduced MV formed; C, reaction
system of hydrogen formation; D, amount of hydrogen formed
under light.

enzymatic reactions. Construction of a light-driven NADH regeneration system has not yet
been successful.
3. Photoreduction of methylviologen by dyes, and application of this reaction tophotocatalytic hydrogen production
Using purified biologically produced porphyrin, photocatalytic reduction of
methylviologen (MV2 +), which is frequently used as an electron donor to metalloenzymes,
was investigated. As shown in Fig. 2A and B, MV2+ was reduced under light irradiation. To
demonstrate that MV photoreduction can be coupled with enzymatic reactions, we applied
MV reduction by porphyrin to hydrogen production by E. coli expressing[FeFe]-hydrogenase genes (Fig. 2C). Hydrogen was successfully formed by a reaction
system consisting of purified dye, MV, and E. coli expressing hydrogenase genes,
demonstrating a light-driven enzyme reaction using biologically synthesized porphyrin dyes.

Conclusion
A significant yield of dye molecules was produced by enhancing the porphyrinbiosynthesis pathway in genetically engineered E. coli. Light-driven hydrogen production
was achieved by coupling photocatalytic reduction of the artificial coenzyme MV withenzymatic hydrogen production by hydrogenases, using biosynthesized porphyrin. Althoughan NADH regeneration system has not yet been devised, this project demonstrates thefeasibility of light-driven production of useful chemicals using biosynthesized porphyrins as
molecular photocatalysts.
In addition, enhancement of the porphyrin synthesis pathway indicates efficientheterologous expression of heme enzyme genes in E. coli 1); this is a related achievement ofour project.
References
1) Honda, Y., Nanasawa, K., and Fujii, H. (2018) Coexpression of 5‐aminolevulinic acidsynthase gene facilitates heterologous production of thermostable cytochrome P450,
CYP119, in holo form in Escherichia coli. ChemBioChem 19: 2156-2159.