Lignin valorization for protocatechuic acid production in engineered Saccharomyces cerevisiae

Abstract

The biological valorization of lignin to aromatic compounds has attracted extensive attention as this route actively contributes to the bioeconomy. However, the diversity of lignin-derived monomers causes difficulties in the biological upgrading of lignin. Protocatechuic acid is an important potential precursor in polymer synthesis and the pharmaceutical industry. This study aims to effectively funnel lignin derived monomers to a compound platform, protocatechuic acid, by designing a cell factory of engineered Saccharomyces cerevisiae. A biological funnel pathway was successfully constructed in S. cerevisiae to produce protocatechuic acid from p-coumaric acid and ferulic acid. A S. cerevisiae chassis was optimized to reduce the further metabolism of intermediates and the generation of byproducts with the deletion of ADH6, ADH7, BDH2, and FDC1 genes. The key genes fcs, ech, and vdh in the CoA-dependent non-β-oxidation pathway were screened to produce the precursor of protocatechuic acid, including p-hydroxybenzoic acid and vanillic acid from p-coumaric acid and ferulic acid, respectively. Following that, the regulatory module of tetrahydrofolic acid metabolism was introduced into the biological funnel pathway with the overexpression of MET6 and metf1 genes and knockout of MHT1 and SAM4 genes, by which 120 mg L⁻¹ protocatechuic acid was produced from ferulic acid. Excitingly, by integrating the multi-copy heterologous pobA gene in the yPCA12 strain, the titer of protocatechuic acid was improved to 720 mg L⁻¹ from p-coumaric acid. Interestingly, the yPCA12 strain also exhibited a good ligninolytic capacity of the actual lignin streams. Together with fermentation optimization, the highest titer of 810 mg L⁻¹ protocatechuic acid was obtained from alkaline pretreatment liquor. Overall, a biological funnel pathway of lignin-derived monomers was constructed in S. cerevisiae to produce a compound platform, protocatechuic acid, for valuable product synthesis. The design of the S. cerevisiae cell factory could be a promising potential route to make lignin valorization sustainable.