Production of Protocatechuic Acid from p-Hydroxyphenyl (H) Units and Related Aromatic Compounds Using an Aspergillus niger Cell Factory

Protocatechuic acid (3,4-dihydroxybenzoic acid) is a chemical building block for polymers and plastics. In addition, protocatechuic acid has many properties of great pharmaceutical interest. Much research has been performed in creating bacterial protocatechuic acid production strains, but no protocatechuic acid-producing fungal cell factories have been described. The filamentous fungus Aspergillus niger can produce protocatechuic acid as an intermediate of the benzoic acid metabolic pathway. Recently, the p-hydroxybenzoate-m-hydroxylase (phhA) and protocatechuate 3,4-dioxygenase (prcA) of A. niger have been identified. It has been shown that the prcA deletion mutant is still able to grow on protocatechuic acid. This led to the identification of an alternative pathway that converts protocatechuic acid to hydroxyquinol (1,3,4-trihydroxybenzene). However, the gene involved in the hydroxylation of protocatechuic acid to hydroxyquinol remained unidentified. Here, we describe the identification of protocatechuate hydroxylase (decarboxylating) (PhyA) by using whole-genome transcriptome data. The identification of phyA enabled the creation of a fungal cell factory that is able to accumulate protocatechuic acid from benzyl alcohol, benzaldehyde, benzoic acid, caffeic acid, cinnamic acid, cinnamyl alcohol, m-hydroxybenzoic acid, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-anisyl alcohol, p-anisaldehyde, p-anisic acid, p-coumaric acid, and protocatechuic aldehyde. IMPORTANCE Aromatic compounds have broad applications and are used in many industries, such as the cosmetic, food, fragrance, paint, plastic, pharmaceutical, and polymer industries. The majority of aromatic compounds are synthesized from fossil sources, which are becoming limited. Plant biomass is the most abundant renewable resource on Earth and can be utilized to produce chemical building blocks, fuels, and bioplastics through fermentations with genetically modified microorganisms. Therefore, knowledge about the metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable aromatic compounds such as protocatechuic acid. Protocatechuic acid has many pharmaceutical properties but also can be used as a chemical building block to produce polymers and plastics. Here, we show that the fungus Aspergillus niger can be engineered to produce protocatechuic acid from plant-derived aromatic compounds and contributes to creating al