Engineering chloramphenicol acetyltransferase (CAT) for enhanced retinyl acetate biosynthesis in Escherichia coli

Abstract

Retinyl acetate, a stable and commercially valuable vitamin A derivative, is in high demand across the pharmaceutical and cosmetic industries. However, microbial production of retinyl acetate remains constrained by inefficient terminal conversion of retinol to retinyl acetate and by the use of wild-type chloramphenicol acetyltransferase (CAT), which can confer a chloramphenicol-resistance phenotype in host cells. To overcome these limitations, an Escherichia coli strain was engineered to increase retinyl acetate production by repurposing CAT as a retinol acetyltransferase. Screening of CAT homologs representing major CAT enzyme families identified CAT.ec1 from E. coli as a promising Type A candidate for subsequent structure-guided engineering. Structure-guided site-directed mutagenesis of CAT.ec1 identified candidate substrate-pocket positions associated with improved retinyl acetate formation. The Y133I V170L variant increased retinyl acetate titer by 4.1-fold relative to the wild-type CAT.ec1 control and increased the retinyl acetate fraction from 39.5 ± 1.4% to 87.2 ± 0.5%. Incorporation of Y133I V170L into a simplified two-plasmid production system further increased the retinyl acetate fraction from 62.6 ± 0.3% to 94.4 ± 0.1% relative to the corresponding wild-type CAT.ec1 control. In qualitative plate assays, engineered variants, including Y133I V170L, showed reduced or undetectable growth under the tested chloramphenicol conditions while retaining improved retinyl acetate production. These findings provide a foundation for further biochemical characterization, strain optimization, and evaluation of engineered CAT variants in industrially relevant retinoid production systems.