Multidimensional Engineering of <i>Cyberlindnera jadinii</i> for Efficient Invertase Production

The yeast <i>Cyberlindnera jadinii</i> has long been used in food and feed industries. While it shows strong intracellular protein synthesis, its limited secretion capacity hampers its use as a protein production host. To overcome this, we used the endogenous invertase CjINV1 as a model and applied multidimensional engineering to the tetraploid wild-type strain NBRC0988. This involved multicopy integration of gene expression cassettes into the chromosome via a landing pad strategy, transcriptional enhancement through combinatorial optimization of promoter and terminator elements, and chitin-based cell wall remodeling to facilitate protein release. The engineered strain showed markedly enhanced protein secretion in a 5-L bioreactor, yielding 3456 ± 81 mg/L extracellular protein after 168 h cultivation, with invertase specific activity of 789.3 ± 52.3 U/mg. To our knowledge, this represents the highest reported yield for yeast-based invertase production to date, highlighting <i>C. jadinii</i> as a promising platform for recombinant protein production.