Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys⁻), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg).

One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet specific requirements from various applications in food, nutrition, and cosmetic industries. Oleic acid (C18:1) and stearic acid (C18:0) are two common long fatty acids in the side chain of triglycerides in plant fats and oils that have similar chemical composition and structures, except for an unsaturated bond between C9 and C10 in oleic acid. Two lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL), show activity in reactions involving oleate and stearate, and share high sequence and structural identity. In this research, the preference for one of these two similar fatty acid side chains was investigated for the two lipases and was related to the respective enzyme structure. From transesterification reactions with 1:1 (molar ratio) mixed ethyl stearate (ES) and ethyl oleate (EO), both RML and ROL showed a higher activity towards EO than ES, but RML showed around 10% higher preference for ES compared with ROL. In silico results showed that stearate has a less stable interaction with the substrate binding crevice in both RML and ROL and higher tendency to freely move out of the substrate binding region, compared with oleate whose structure is more rigid due to the existence of the double bond. However, Trp88 from RML which is an Ala at the identical position in ROL shows a significant stabilization effect in the substrate interaction in RML, especially with stearate as a ligand.

Lipases are vital in modifying lipid substrates across industries such as food, cosmetics, and pharmaceuticals. Among their features, fatty acid selectivity is particularly important for industrial applications. Rhizopus oryzae lipase (ROL) stands out for its high selectivity and broad applicability. In this study, we engineered single‐residue variants of ROL by targeting Ala89 and Phe95 in its lid region. Additionally, a lid‐swap chimera was created by replacing ROL's 15‐residue lid with that of the homologous lipase from Rhizomucor miehei (RML). These variants were expressed and characterized to assess changes in substrate selectivity. Our results highlight the lid's key role in determining fatty acid preference. Notably, mutating Phe95 to smaller residues (Ile or Ala) significantly increased selectivity toward medium‐chain fatty acid (MCFA) esters. In contrast, substituting Ala89 with bulkier residues (Phe or Trp) reduced activity—except in the lid‐swap variant. Interestingly, although the lid‐swap variant contains Trp89, the surrounding smaller, non‐conserved residues may alleviate steric hindrance. This chimera retained high activity but shifted its preference from MCFAs to long‐chain fatty acids (LCFAs), a novel observation. Overall, the engineered variants exhibited distinct substrate preferences without compromising thermostability, suggesting their potential for tailored applications in food, nutrition, and cosmetic industries.