Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is a natural bioactive phenolic acid potentially valuable as a pharmaceutical raw material owing to its diverse pharmacological activities. Corynebacterium glutamicum forms PCA as a key intermediate in a native pathway to assimilate shikimate/quinate through direct conversion of the shikimate pathway intermediate 3-dehydroshikimate (DHS), which is catalyzed by qsuB-encoded DHS dehydratase (the DHS pathway). PCA can also be formed via an alternate pathway extending from chorismate by introducing heterologous chorismate pyruvate lyase that converts chorismate into 4-hydroxybenzoate (4-HBA), which is then converted into PCA catalyzed by endogenous 4-HBA 3-hydroxylase (the 4-HBA pathway). In this study, we generated three plasmid-free C. glutamicum strains overproducing PCA based on the markerless chromosomal recombination by engineering each or both of the above mentioned two PCA-biosynthetic pathways combined with engineering of the host metabolism to enhance the shikimate pathway flux and to block PCA consumption. Aerobic growth-arrested cell reactions were performed using the resulting engineered strains, which revealed that strains dependent on either the DHS or 4-HBA pathway as the sole PCA-biosynthetic route produced 43.8 and 26.2 g/L of PCA from glucose with a yield of 35.3% and 10.0% (mol/mol), respectively, indicating that PCA production through the DHS pathway is significantly efficient compared to that produced through the 4-HBA pathway. Remarkably, a strain simultaneously using both DHS and 4-HBA pathways achieved the highest reported PCA productivity of 82.7 g/L with a yield of 32.8% (mol/mol) from glucose in growth-arrested cell reaction. These results indicated that simultaneous engineering of both DHS and 4-HBA pathways is an efficient method for PCA production. The generated PCA-overproducing strain is plasmid-free and does not require supplementation of aromatic amino acids and vitamins due to the intact shikimate pathway, thereby representing a promising platform for the industrial bioproduction of PCA and derived chemicals from renewable sugars.

原儿茶酸（3, 4-二羟基苯甲酸，PCA）是一种天然的生物活性酚酸，由于其多种药理活性而具有潜在的医药原料价值。谷氨酸棒杆菌形成 PCA 作为天然途径中的关键中间体，通过直接转化莽草酸途径中间体 3-脱氢莽草酸 (DHS) 来同化莽草酸/奎宁，该途径由qsuB编码的 DHS 脱水酶（DHS 途径）催化。PCA 也可以通过从分支酸延伸的替代途径形成，通过引入异源分支酸丙酮酸裂解酶，将分支酸转化为 4-羟基苯甲酸 (4-HBA)，然后由内源性 4-HBA 3-羟化酶 (4-HBA) 催化转化为 PCA。 HBA 途径）。在这项研究中，我们产生了三个无质粒谷氨酸棒杆菌通过改造上述两种 PCA 生物合成途径中的每一种或两种，结合宿主代谢的改造，以增强莽草酸途径通量并阻止 PCA 消耗，基于无标记染色体重组过度生产 PCA 的菌株。使用产生的工程菌株进行有氧生长受阻细胞反应，结果表明依赖 DHS 或 4-HBA 途径作为唯一 PCA 生物合成途径的菌株从葡萄糖中产生 43.8 和 26.2 g/L 的 PCA，产量为分别为 35.3% 和 10.0% (mol/mol)，表明与通过 4-HBA 途径生产的 PCA 相比，通过 DHS 途径生产的 PCA 显着有效。值得注意的是，同时使用 DHS 和 4-HBA 途径的菌株达到了最高报告的 PCA 生产力 82。在生长停滞的细胞反应中，7 g/L 的葡萄糖产率为 32.8% (mol/mol)。这些结果表明，同时设计 DHS 和 4-HBA 途径是生产 PCA 的有效方法。生成的 PCA 过量生产菌株不含质粒，并且由于完整的莽草酸途径不需要补充芳香族氨基酸和维生素，从而为 PCA 和可再生糖衍生化学品的工业生物生产提供了一个有前景的平台。