Effect of weakening 1,3-β-glucan synthesis on sophorolipids biosynthesis in Komagataella phaffii

Komagataella phaffii (formerly 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, PAS_chr2-1_0263 gene responsible for β-glucan synthase was inactivated to investigate the impact of β-glucan deficiency on sophorolipid (SL) biosynthesis. Furthermore, we systematically evaluated the combined effects of this mutation and faa1 inactivation on SL production and host performance. Firstly, the SL biosynthesis-related genes (comprising cyp52M1, ugtA1, ugtB1, sble, at, and mdr) were overexpressed for the first time, demonstrating that P_GAP-driven K. phaffii GS115 can synthesize seven structural types of SLs with a total titer of 4.09 g/L. Subsequently, to facilitate subsequent gene editing, the DNA repair-related 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 increases intracellular UDP-glucose levels, both of which contribute to enhanced SL biosynthesis. This study demonstrates for the first time that attenuating 1,3-β-glucan synthesis in K. phaffii 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.