Identification of the enzyme involved in the reduction of hydrpxycinnamic acids as an alternative metabolism to decarboxylation in Lactobacillus plantarum

Abstract

The industrial use of hydroxycinnamates has attracted growing interest since these compounds are bioactive molecules possessing potential antioxidant activities and health benefits. Among phenolic acids, hydroxycinnamic acids are the most abundant and they mainly include p-coumaric, caffeic, and ferulic acids. As early as 1997 a decarboxylase activity was described on several hydroxycinnamic acids, e.g. p-coumaric, caffeic, and ferulic acids. The L. plantarum phenolic acid decarboxylase enzyme responsible of that decarboxylation was cloned in E. coli and characterized at molecular level. The decarboxylation of these hydroxycinnamic acids originates the formation of 4-vinyl derivatives. Subsequently, L. plantarum also displayed an uncharacterized vinyl reductase activity, able to reduce the vinyl derivatives into ethyl derivatives which are considered the most important flavour components of fermented soy sauce or, on the other hand, are considered as off flavour and responsible of sensorial wine alterations. In addition, L. plantarum strains also displayed an uncharacterized phenolic acid reductase activity, able to reduce some hydroxycinnamic acids, and to metabolize p-coumaric acid into phloretic acid, caffeic acid into hydrocaffeic acid, ferulic acid into hydroferulic acid, and m-coumaric acid into 3(3-hydroxyphenyl) propionic acid. In this work, the reductase involved in hydroxycinnamic acid reduction in L. plantarum WCFS1 was identified. A transcriptomic analysis made in L. plantarum WCFS1 on the presence of a hydroxycinnamic acid (p-coumaric acid) revealed the induction of several genes annotated as putative reductases. The disruption of these genes allowed the identification of two contiguous reductase-encoding genes which inactivation leads to the absence of hydroxycinnamate reductase activity. Similarly to Lactobacillus johnsonii, both oxidorreductases were purified together when they were recombinantly produced in E. coli. This result suggests the formation in vivo of an heteromeric protein complex. This complex seems to favour the stabilization of the catalytic subunit of the hydroxycinnamate reductase. Extracts from E. coli overproducing the catalytic subunit showed hydroxycinnamate reductase activity. This work constitutes the first description of a new enzymatic activity in bacteria. In relation to hydroxycinnamic compounds, the combined action of the decarboxylase, and the reductase identified in this work, could allow L. plantarum to metabolize compounds abundant in fermented plant-derived food products.