A process for p-hydroxystyrene production from glycerol based on cell-free biosynthesis system

As an essential chemical, p-hydroxystyrene has attracted much attention and is widely used in the field of semiconductors. However, the current production methods have some disadvantages, such as low molar conversion yield, p-hydroxystyrene toxicity, and high substrate cost. To overcome these problems, an five enzymes cell-free cascade, consisting of alditol oxidase from Streptomyces coelicolor A3 (ScALDO), dehydratase from Paralcaligenes ureilyticus (PuDHT), tyrosine phenol lyase from Citrobacter freundii (CfTPL), tyrosine ammonia lyase from Rhodotorula glutinis (RgTAL) and phenylacrylate decarboxylase from Enterobacter sp. (EsPAD), was constructed for the de novo biosynthesis of p-hydroxystyrene from glycerol. Through process strategy optimization, a molar conversion yield of more than 84.2% was finally obtained from 10 mM crude glycerol. It is worth mentioning that the reaction system performed an excellent titer using 50 mM crude glycerol, and the p-hydroxystyrene concentration reached 30.32 mM, which exceeded the maximum titer of fermentation. This showed that our system is robust to p-hydroxystyrene and crude glycerol. In addition, a ‘two-step purification process’ was designed to produce pure p-hydroxystyrene. This is the first study focused on the cell-free biosynthesis of p-hydroxystyrene, and the highest conversion yield and titer thus far was obtained for p-hydroxystyrene de novo biosynthesis.