Effect of weakening 1,3-β-glucan synthesis on sophorolipids biosynthesis in Pichia pastoris

Pichia pastoris is a widely used host for heterologous protein expression and biotransformation, and simplifying its cell wall polysaccharides is a promising strategy for designing advanced chassis strains. Previously, we constructed two superior chassis hosts by inactivating the β -glucan biosynthesis genes PAS_chr1-3_0225 and PAS_chr1-3_0661 . In this study, we inactivated another β -glucan synthase, encoded by PAS_chr2-1_0263 , to investigate the impact of β -glucan deficiency on the biosynthesis of sophorolipids (SLs), a class of glycolipid biosurfactants of growing interest, and to examine how its deletion, together with that of a β -oxidation gene, affects the supply of SL precursors, since this synthase also redirects UDP-glucose flux toward SL synthesis. Firstly, the SL biosynthesis gene cluster (comprising cyp52M1 , ugtA1 , ugtB1 , sble , acet , and mdr ) was overexpressed for the first time, demonstrating that P _GAP -driven P. pastoris GS115 can synthesize seven structural types of SLs with a total titer of 4.09 g/L. Subsequently, to facilitate subsequent gene editing, the homologous recombination genes ku70 and mph1 were deleted; this deletion did not depress SL production. PAS_chr2-1_0263 was then knocked out to obtain a novel chassis host, and the SL productivity, oil-to-SLs ratio, and precursor UDP-glucose level were systematically investigated. The results showed that knocking out PAS_chr2-1_0263 reduced glucan content by 24.21% while increasing total SLs to 7.69 g/L, a 43.8% increase, and improving the conversion ratios of both oil and glucose to SLs. Moreover, the combined deletion of PAS_chr2-1_0263 and faa1 , which encodes fatty acid acyl-CoA synthase, further elevated the SL titer to 11.53 g/L and achieved even higher glucose-to-SLs and oil-to-SLs conversion rates. These findings indicate that weakening 1,3- β -glucan synthesis not only improves the utilization ratio of glucose but also enhances the supply of SL precursors, both of which contribute to enhanced SL biosynthesis. This study demonstrates for the first time that attenuating 1,3- β -glucan synthesis in P. pastoris is an effective strategy to boost SL biosynthesis and improve host performance, and this novel chassis possesses excellent potential for the biotransformation of other glycolipids.