Construction of an l-Tyrosine Chassis in Pichia pastoris Enhances Aromatic Secondary Metabolite Production from Glycerol

Bioactive plant-based secondary metabolites such as stilbenoids, flavonoids, and benzylisoquinoline alkaloids (BIAs) are produced from ʟ-tyrosine (ʟ-Tyr) and have a wide variety of commercial applications. Therefore, building a microorganism with high ʟ-Tyr productivity (ʟ-Tyr chassis) is of immense value for large-scale production of various aromatic compounds. The aim of this study was to develop an ʟ-Tyr chassis in the nonconventional yeast Pichia pastoris (Komagataella phaffii) to produce various aromatic secondary metabolites (resveratrol, naringenin, norcoclaurine, and reticuline). Overexpression of feedback-inhibition insensitive variants of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (ARO4ᴷ²²⁹ᴸ) and chorismate mutase (ARO7ᴳ¹⁴¹ˢ) enhanced ʟ-Tyr titer from glycerol in P. pastoris. These engineered P. pastoris strains increased the titer of resveratrol, naringenin, and norcoclaurine by 258, 244, and 3400%, respectively, after expressing the corresponding heterologous pathways. The titer of resveratrol and naringenin further increased by 305 and 249%, resulting in yields of 1825 and 1067 mg/L, respectively, in fed-batch fermentation, which is the highest titer from glycerol reported to date. Furthermore, the resveratrol-producing strain accumulated intermediates in the shikimate pathway. ʟ-Tyr-derived aromatic compounds were produced using crude glycerol byproducts from biodiesel fuel (BDF) production. Constructing an ʟ-Tyr chassis is a promising strategy to increase the titer of various aromatic secondary metabolites and P. pastoris is an attractive host for high-yield production of ʟ-Tyr-derived aromatic compounds from glycerol.