Multistep metabolic engineering of Pichia pastoris for biosynthesis of N‑acetylneuraminic acid

Free or protein-conjugated N-Acetylneuraminic acid (Neu5Ac) is widely distributed in organisms and possesses diverse biological functions, and for example lactoferrin with Neu5Ac-mediated sialylation exhibits significantly enhanced functional activity compared with non-sialylated lactoferrin. Pichia pastoris serves as an excellent host for lactoferrin; however, efficient biosynthesis of Neu5Ac is essential for efficiently expressing lactoferrin with Neu5Ac-mediated sialylation. In this study, P. pastoris was engineered into a high-yield Neu5Ac host for expressing highly active lactoferrin with Neu5Ac modification. Heterologous Neu5Ac biosynthesis genes (gfa1, gna1, yqaB, age, and neuB) were first introduced into a P. pastoris strain in which pfk1 and zwf attenuation combined with pfk2 deletion redirected carbon flux toward fructose-6-phosphate, yielding the Neu5Ac-producing strain P. pastoris N1 with Neu5Ac production of 9.19 mg/L. Neu5Ac titers were subsequently boosted 2.8-fold via precursor enhancement through pyk attenuation and gs overexpression. Implementation of glucose-glycerol co-utilization (gut1, gut2, gt2 overexpression) dramatically increased Neu5Ac production to 285.41 mg/L. Optimization via increased copy number of gfa1, age, and neuB, coupled with their overexpression under the strong promoter P_GAP, culminated in approximately 111.5-fold improvement (reaching 1.03 g/L) over the N1 strain. The effective integration of diverse metabolic engineering strategies, including heterologous pathway introduction, precursor enhancement, and co-substrate utilization, established a valuable and transferable framework for reprogramming P. pastoris to synthesize Neu5Ac and other high-value compounds. KEY POINTS: • P. pastoris was engineered to synthesize Neu5Ac. • Neu5Ac production achieved 1.03 g/L via remodeling of key metabolic pathways. • The strain harbored potential for synthesis of free Neu5Ac or sialylated proteins.