Engineering glycolytic pathway for improved Lacto-N-neotetraose production in pichia pastoris

Lacto-N-neotetraose (LNnT) is a primary solid component of human milk oligosaccharides (HMOs) with various promising health effects for infants. LNnT production by GRAS (generally recognized as safe) microorganisms has attracted considerable attention. However, few studies have emphasized Pichia Pastoris as a cell factory for LNnT's production. Here, we have reported the first-ever synthesis of LNnT employing P. pastoris as the host. Initially, LNnT biosynthetic pathway genes β-1,3-N-acetylglucosaminyltransferase (lgtA) and β-1,4-galactostltransferase (lgtB) along with lactose permease (lac12) and galactose epimerase (gal10) were integrated into the genome of P. pastoris, but only 0.139 g/L LNnT was obtained. Second, the titer of LNnT was improved to 0.162 g/L via up-regulating genes to strengthen the supply of precursors, UDP-GlcNAc (Uridine diphosphate N-acetylglucosamine) and UDP-Gal (Uridine diphosphate galactose), for LNnT biosynthesis. Third, by knocking out critical mediator pfk (6-phosphofructokinase) genes in glycolysis, the major glucose metabolic flux was rewired to the LNnT biosynthesis pathway. As a result, the strain accumulated 0.867 g/L LNnT in YPG medium supplemented with glucose and lactose. Finally, LNnT production was increased to 1.24 g/L in a 3 L bioreactor. The work aimed to explore the potential of P. pastoris as a for LNnT production.