posted on 2023-12-29, 14:57authored byYi-Jung Sung, Takumi Takahashi, Yi-Hsuan Lin, Tony Z. Jia, Yin-Ru Chiang, Po-Hsiang Wang
Flavin adenine dinucleotide (FAD)
is a universal cellular
cofactor
involved in biological redox and radical metabolism reactions. FAD
biosynthesis from riboflavin typically proceeds through two ATP-dependent
enzymatic reactions, with flavin mononucleotide (FMN) as the intermediate.
Traditional in vivo methods employ microorganisms
for FAD synthesis at an industrial scale; however, these approaches
often suffer from complex purification processes. Considering the
atomic economy and percentage yield, in vitro enzymatic
FAD synthesis using enzymes could be a more efficient and sustainable
alternative. While catalytically efficient, the requirements of expensive
ATP (substrate) limit the industrialization of enzymatic FAD synthesis.
To overcome the ATP requirements, here we develop a two-enzyme cascade
for ATP regeneration from adenosine using wastewater microalgal polyphosphate
as the P-donor. With the ATP regeneration system, the bifunctional
riboflavin kinase/FAD synthetase and pyrophosphatase completely convert
saturated riboflavin into FAD within 2 h with a titer of ∼1.2
g/L (1.5 mmol/L). Notably, orthophosphate, the only byproduct of this
enzymatic process, can be recycled to synthesize polyphosphate by
wastewater microalgae, which can then be fed back into the system
as the P-donor in the ATP regeneration step, resulting in a FAD synthesis
process with almost net-zero waste generation.