A new process for obtaining hydroxytyrosol using transformed Escherichia coli whole cells with phenol hydroxylase gene from Geobacillus thermoglucosidasius

摘要

Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence 'Campus Mare Nostrum', University of Murcia, Campus Espinardo, E-30100'Murcia, Spain;Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence 'Campus Mare Nostrum', University of Murcia, Campus Espinardo, E-30100'Murcia, Spain,Murcia Biomedical Research Institute (IMIB), 30120 Murcia, Spain;Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence 'Campus Mare Nostrum', University of Murcia, Campus Espinardo, E-30100'Murcia, Spain,Murcia Biomedical Research Institute (IMIB), 30120 Murcia, Spain;%Phenol hydroxylase gene cloning from the thermophilic bacteria Geobacillus thermoglucosidasius was used to develop an effective method to convert tyrosol into the high-added-value compound hydroxytyrosol by hydroxylation. Phenol hydroxylase is a two-component enzyme encoded by pheAl and pheA2 genes and strictly dependent on NADH and FAD. These two genes were subcloned together as a 2 kb fragment into Escherichia coli Rosetta cells, and the transformants were able to grow and effectively transform up to 5 mM of phenol and tyrosol using IPTG (isopropyl-β-D-thiogalactopyranoside) as inducer. In addition, when a new fragment with a 340 pb upstream pheAl gene was subcloned, a similar biotransformation rate was attained without IPTG, confirming that this fragment encodes for a phenol hydroxylase promoter that can be recognised by E. coli. Both transformants brought about the total bioconversion of mon-ophenols at a high concentration (5 mM), which represents an increase, both in concentration and in yield, compared with that previously described in the bibliography. The use of the transformant with its constitutive promoter was more interesting from a biotechnological point of view, since it is not necessary to use IPTG. It also gave rise to greater operational stability.