Biomimetic enzymatic cascade for fatty alkyl p-hydroxycinnamates synthesis

Abstract

p-Hydroxycinnamic acids (HCAs) are plant and fungal metabolites which display interesting properties. Nevertheless, their limited lipid solubility makes their use in lipid matrices challenging. Alkyl hydroxycinnamates (AHCs), their aliphatic ester derivatives, offer improved solubility and enhanced bioactivity and conventional routes to access them are typically either efficient or sustainable. This study describes the development of a two-step biomimetic enzymatic cascade which aims to provide a sustainable and efficient approach for AHCs synthesis through the activation of HCAs with Coenzyme A (CoASH) in the presence of ATP, catalyzed by a 4-coumarate:CoA ligase from Nicotiana tabacum (Nt4CL2), followed by the acylation of a hydroxy aliphatic molecule (hydroxy fatty acid of fatty alcohol) by a BAHD acyltransferase from Populus trichocarpa (PtFHT1). Optimization of the first step resulted in a 6.6- and 8-fold reduction in ATP and CoASH requirements, compared to the literature. Molecular modeling of PtFHT1 showed its preference for fatty hydroxy acids over hydroxy alcohols as acyl acceptors due to specific electrostatic and hydrogen- bonding interactions. Implementation of the simultaneous cascade in vitro, using 16OHC16 as acyl acceptor, yielded 16-feruloyloxypalmitic acid at a titer of 336 mg/L while achieving in situ regeneration of CoASH, with a total turnover number of 37.5.