Background2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable pseudoaromatic dicarboxylic acid, represents a promising building block for the manufacture of biodegradable polyesters. Microbial production of PDC has been extensively investigated, but low titers and yields have limited industrial applications.ResultsIn this study, a multi-step biosynthesis strategy for the microbial production of PDC was demonstrated using engineeredEscherichia coliwhole-cell biocatalysts. The PDC biosynthetic pathway was first divided into three synthetic modules, namely the 3-dehydroshikimic acid (DHS) module, the protocatechuic acid (PCA) module and the PDC module. Several effective enzymes, including 3-dehydroshikimate dehydratase for the PCA module as well as protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase for the PDC module were isolated and characterized. Then, the highly efficient whole-cell bioconversion systems for producing PCA and PDC were constructed and optimized, respectively. Finally, the efficient multi-step biosynthesis of PDC from glucose was achieved by smoothly integrating the above three biosynthetic modules, resulting in a final titer of 49.18 g/L with an overall 27.2% molar yield, which represented the highest titer for PDC production from glucose reported to date.ConclusionsThis study lays the foundation for the microbial production of PDC, including one-step de novo biosynthesis from glucose as well as the microbial transformation of monoaromatics.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02350-y.