Abstract: Hyaluronic acid (HA) is a high-value glycosaminoglycan that is extensively utilized in the food, pharmaceutical, and cosmetic industries. In this study, to establish greener and more cost-effective methods for HA production, Pichia pastoris was employed as a cell factory to evaluate the feasibility of synthesizing HA from methanol, a C1 substrate. Initially, an HA biosynthetic pathway was designed, which was subsequently incorporated into P. pastoriscells. The production of HA was quantified and confirmed using liquid chromatography and mass spectrometry, thereby verifying the successful establishment of an engineered cell factory. Using this system as a basis, with a view toward enhancing HA yield, hyaluronic acid synthases from different sources were screened. The results revealed that the hyaluronic acid synthase seHAS, derived from Streptococcus equi subsp. zooepidemicus, was characterized by the highest efficiency. Using this synthase, an HA titer of 941 mg/L with a molecular weight of approximately 1100 kDa was obtained under shake flask conditions. In addition, the efficacy of selected high-activity HA depolymerases was assessed, and both exogenous addition and co-expression approaches were employed to analyze potential bottlenecks in HA production. It was found that the depolymerase FhChAC was highly expressed in P. pastoris, and regardless of whether it was exogenously supplied or co-expressed, HA polysaccharides accumulated on the cell surface were efficiently released and degraded to oligosaccharides, thereby contributing to a significant increase in overall HA yield. Based on these optimization steps, a final shake flask fermentation yield of 5.6 g/L of unsaturated disaccharide-form HA oligosaccharides was obtained. The findings from this study are expected to provide important theoretical insights and technical guidance for the engineering of P. pastoristo produce HA from methanol.