| Summary: | The production of 3,4-dihydroxybenzoic acid (3,4-DHBA or protocatechuate) is a relevant task owing to 3,4-DHBA’s pharmaceutical properties and its use as a precursor for subsequent synthesis of high value-added chemicals. The microbial production of 3,4-DHBA using dehydroshikimate dehydratase (DSD) (EC: 4.2.1.118) has been demonstrated previously. DSDs from soil-dwelling organisms (where DSD is involved in quinate/shikimate degradation) and from <i>Bacillus</i> spp. (synthesizing the 3,4-DHBA-containing siderophore) were compared in terms of the kinetic properties and their ability to produce 3,4-DHBA. Catabolic DSDs from <i>Corynebacterium glutamicum</i> (QsuB) and <i>Neurospora crassa</i> (Qa-4) had higher K<sub>m</sub> (1 and 0.6 mM, respectively) and k<sub>cat</sub> (61 and 220 s<sup>−1</sup>, respectively) than biosynthetic AsbF from <i>Bacillus thuringiensis</i> (K<sub>m</sub>~0.04 mM, k<sub>cat</sub>~1 s<sup>−1</sup>). Product inhibition was found to be a crucial factor when choosing DSD for strain development. AsbF was more inhibited by 3,4-DHBA (IC<sub>50</sub>~0.08 mM), and <i>Escherichia coli</i> MG1655 Δ<i>aroE</i> P<i><sub>lacUV5</sub></i>-<i>asbF</i><sub>attφ80</sub> strain provided only 0.2 g/L 3,4-DHBA in test-tube fermentation. Isogenic strains MG1655 Δ<i>aroE</i> P<i><sub>lacUV5</sub></i>-<i>qsuB</i><sub>attφ80</sub> and MG1655 Δ<i>aroE</i> P<i><sub>lacUV5</sub></i>-<i>qa-4</i><sub>attφ80</sub> expressing QsuB and Qa-4 with IC<sub>50</sub> ~0.35 mM and ~0.64 mM, respectively, accumulated 2.7 g/L 3,4-DHBA under the same conditions.
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