Metabolic and enzyme rewiring enables high-production of vanillin in unconventional yeast

Vanillin is an aromatic flavor compound widely used in the food, pharmaceutical, and cosmetic industries. Microbial biosynthesis offers a sustainable alternative to traditional plant extraction and chemical synthesis; however, the susceptibility of vanillin to redox reactions and the weak enzyme activity in cells severely limit the vanillin production capacity by microbial biosynthesis. This study presents the first successful attempt at de novo synthesis of vanillin in the unconventional yeast Komagataella phaffii. The initial titer was quite low (0.5 mg/L), but removal of 14 endogenous oxidoreductases to block vanillin conversion resulted in an 11.1-fold improvement in vanillin production. The combination of pathway rewiring and cofactor (nicotinamide adenine dinucleotide phosphate [NADPH] and S-adenosylmethionine) regeneration redirected the metabolic flux toward vanillin synthesis and achieved a further 19.9-fold improvement in vanillin production. Rational rewiring of the rate-limiting enzyme, caffeic acid O-methyltransferase (NtCOMT), generated a dominant mutant NtCOMT^N312A/H315N from 70 variants, which promoted activity by 49.7 % and prevented intermediate accumulation. These strategies eventually enabled the co-coupling of de novo biosynthesis and caffeic acid conversion, achieving the highest reported production of vanillin (1055.9 mg/L) by K. phaffii fermentation in a bioreactor. These findings highlight the potential of unconventional yeast as a chassis host for aromatic aldehyde synthesis and the construction of a versatile microbial platform for the production of carbonyl compounds.