Delactosed permeate (DLP), a lactose- and protein-rich waste derived from whey processing, represents an abundant, low-cost substrate for microbial fermentation. Leveraging DLP as a fermentation feedstock not only reduces industrial waste but also adds economic value to the dairy supply chain. However, wild-type Issatchenkia orientalis lacks the intrinsic ability to metabolize lactose. To address this limitation, we first engineered I. orientalis to express a heterologous lactose catabolic pathway, comprising lactose permease and β-galactosidase, enabling the intracellular hydrolysis of lactose into glucose and galactose. While this modification allowed growth on lactose, galactose accumulation in the medium indicated a bottleneck in galactose utilization. To overcome this, we further integrated the Leloir pathway genes, enabling assimilation of galactose. The resulting strain demonstrated growth on both pure lactose and DLP. Building upon this platform, we subsequently introduced both the lactose degradation and galactose utilization pathways into previously engineered I. orientalis strains capable of producing organic acids such as lactic acid, succinic acid, and 3-hydroxypropionic acid. This work expands the metabolic repertoire of I. orientalis and highlights its potential as a robust microbial cell factory for sustainable bioproduction from dairy waste streams.
