Biosynthesis of the fragrance compound cinnamyl isobutyrate in Escherichia coli.

Biosynthesis of the fragrance compound cinnamyl isobutyrate was achieved in Escherichia coli through acyltransferase selection and artificial pathway construction.The acyltransferase CATec3 Y20F responsible for cinnamyl isobutyrate formation was identified. Genes involved in cinnamyl alcohol biosynthesis, including Arabidopsis thaliana phenylalanine ammonia-lyase (AtPAL), Hypericum calycinum cinnamate:CoA ligase (HcCNL), Lolium perenne cinnamyl-CoA reductase (LpCCR1), and the branched-chain α-keto acid dehydrogenase complex (BKD) for isobutyryl-CoA biosynthesis, were introduced along with CATec3 Y20F into E. coli BL21 (DE3). The resulting recombinant strain, BD02, produced 2.6 mg/L cinnamyl isobutyrate and 428.9 mg/L cinnamyl acetate. Subsequently, alsS (encoding acetolactate synthase) from Bacillus subtilis and ilvC (encoding ketol-acid reductoisomerase)/ilvD (encoding dihydroxyacid dehydratase) from E. coli were overexpressed to increase the isobutyryl-CoA level, generating the recombinant strain BD03, which achieved a cinnamyl isobutyrate titer of 7.5 mg/L and 112.4 mg/L of cinnamyl acetate. Thereafter, the panB gene (encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase), the pfkA gene (encoding phosphofructokinase), and the mdh gene (encoding malate dehydrogenase) were knocked out to enhance the supply of isobutyryl-CoA, resulting in the recombinant strain BD04. The strain BD04 produced 17.7 mg/L of cinnamyl isobutyrate and 67.2 mg/L of cinnamyl acetate. De novo biosynthesis of cinnamyl isobutyrate was achieved for the first time in engineered E. coli, thereby establishing a proof-of-concept for its microbial biosynthesis. This process was accompanied by the generation of cinnamyl acetate, which provides a reference for the microbial synthesis of diverse cinnamyl esters.