Efficient sustainable production of protocatechuic acid from glucose by engineered Bacillus licheniformis

Protocatechuic acid (PCA) is a natural phenolic acid with various biological activities, which is widely used in the fields of pharmaceuticals, functional foods and biobased plastics. Microbial fermentation provides a green and sustainable process for PCA production. However, the current yield of PCA by microorganisms remain limited. Here, a Bacillus licheniformis cell factory was developed for green and sustainable efficient synthesis of PCA. Firstly, a heterologous PCA biosynthetic pathway was established in B. licheniformis DW2 through the screening and expression optimization of 3-dehydroshikimate (3-DHS) dehydratase, and obtained a starting strain PCA6 with a PCA titer of 0.58 g/L. Subsequently, the conversion of intracellular PCA into catechol was unexpectedly discovered and blocked, correspondingly identified an endogenous PCA decarboxylase complex BsdBCD, and excavated a novel transcription factor BsdA that responds to PCA activation. The biosynthesis level of PCA was significantly enhanced by engineering B. licheniformis, such as weakening competitive pathway, enhancing precursor supply, strengthening 3-DHS synthesis flux, identifying PCA transporter MDR and promoting PCA efflux, and obtained an optimal strain PCA22 with a PCA titer of 17.83 g/L. Finally, PCA22 produced 21.68 g/L PCA, and a yield of 0.54 g/g from glucose via optimizing fermentation process, which represented the highest yield of PCA from glucose to date. This study provided a green and sustainable biotechnology for the efficient synthesis of PCA, and promoting the further development of green chemical industry.