Integrated Continuous-Flow Production of Wax Esters Combining Whole-Cell and <i>In Vitro</i> Biocatalysis

A first-of-its-kind, fully continuous synthesis of wax esters from biobased precursors (glucose, fatty acids) was developed using metabolically engineered cells and in vitro enzyme catalysis. The cells, overexpressing fatty acyl-CoA reductase and xylose reductase, could be immobilized onto polyesters and packed in a continuous reactor. The immobilized cells were employed in the bioconversion, incorporating in situ extraction using dodecane as the solvent. Such extractive bioconversion was capable of producing fatty alcohols continuously at a productivity of 8.2 mg/(L·h). The immiscible aqueous-dodecane flow stream from the extractive bioconversion was then separated using an in-line membrane-based separator. The dodecane-rich phase was directed into an enzymatic reactor containing Novozyme 435 for the esterification of fatty alchols and fatty acids into the wax esters. A continuous production of wax esters (6.38–23.35 mg/(L·h)) was achieved as a result of the successful streamlining of the cascade biocatalytic process