Selective Esterification Design of Lipases for TAG Synthesis Based on the Unique Structure of Curved DHA

The synthesis of long-chain n-3 polyunsaturated fatty acid (n-3 PUFA) triacylglycerols (TAGs) via lipase-catalyzed esterification is crucial for optimal human nutrition. However, existing lipases lack selectivity for long-chain n-3 PUFAs, hindering the efficient synthesis of highly polyunsaturated TAGs. By two-dimensional NMR and quantum chemical calculations, polyunsaturated DHA was found to have a unique curved conformation. We rationally designed the lipase MAS1 from marine Streptomyces sp. Strain W007 was based on the bent conformation of DHA, using virtual saturation mutagenesis and binding free energy calculations. Consequently, D150V with more hydrogen bonds and a lower binding free energy to DHA (−28.3820 kcal/mol) was obtained. The esterification selectivity of D150V to DHA was 2.18-fold that of the wild type, whereas its selectivity to behenic acid exhibited minimal improvement. Collectively, our findings present a significant advancement in lipase design for selective long-chain n-3 PUFA esterification, thereby opening avenues for lipase modification and industrial applications.