Dual-Promoter-Driven Secretion of Recombinant Candida antarctica Lipase B from Magnetically Immobilized Komagataella phaffii for Biodiesel Production

Abstract Conventional biodiesel synthesis is both chemical- and energy-intensive, whereas lipase-catalyzed transesterification provides a more sustainable alternative. Nevertheless, the industrial application of native Candida antarctica lipase B (CalB) is limited by its low catalytic activity and high production costs. To overcome these challenges, we developed a recombinant Komagataella phaffii expression platform to efficiently secrete recombinant CalB (rCalB). To the best of our knowledge, this study demonstrates a proof-of-concept platform that integrates CalB secretion through a dual-promoter expression system regulated by AOX1/GAP in PDI-assisted and magnetically immobilized K. phaffii cells for biodiesel production. This integrated platform enabled extracellular production of rCalB and magnetic recovery of the engineered yeast cells. The maximum biodiesel yield was reached after 96 h using a methanol-to-oil ratio of 4:1 and a catalyst amount of 8 mL (2.4 EU) at 30 °C. The Gas Chromatography Flame Ionization Detector analysis results indicated that the produced biodiesel had higher amounts of C16 and C18 fatty acid methyl esters. These results highlight the potential to produce biodiesel via transesterification catalyzed by rCalB secreted from magnetically immobilized yeast cells.