Metabolic engineering of Acinetobacter baylyi ADP1 for naringenin production

Abstract Naringenin, a flavanone and a precursor for a variety of flavonoids, has potential applications in the health and pharmaceutical sectors. The biological production of naringenin using genetically engineered microbes is considered as a promising strategy. The naringenin synthesis pathway involving chalcone synthase (CHS) and chalcone isomerase (CHI) relies on the efficient supply of key substrates, malonyl-CoA and coumaroyl-CoA. In this research, we utilized a soil bacterium, Acinetobacter baylyi ADP1, which exhibits several characteristics that make it a suitable candidate for naringenin biosynthesis; the strain naturally tolerates and can uptake and metabolize coumarate, a primary compound in alkaline-pretreated lignin and a precursor for naringenin production. A. baylyi ADP1 also produces intracellular lipids, such as wax esters, thereby being able to provide an excess of malonyl-CoA for naringenin biosynthesis. Moreover, the genomic engineering of this strain is notably straightforward. In the course of the construction of a naringenin-producing strain, the coumarate catabolism was eliminated by a single gene knockout (ΔhcaA) and various combinations of plant-derived CHS and CHI were evaluated. The best performance was obtained by a novel combination of genes encoding for a CHS from Hypericum androsaemum and a CHI from Medicago sativa, that enabled the production of 18 mg/L naringenin in batch cultivations from coumarate. Furthermore, the implementation of a fed-batch system led to a significant 3.7-fold increase (66 mg/L) in naringenin production. These findings underscore the potential of A. baylyi ADP1 as a host for naringenin biosynthesis as well as advancement of lignin-based bioproduction.