Comparative Analysis of Catabolic and Anabolic Dehydroshikimate Dehydratases for 3,4-DHBA Production in IEscherichia coli/I

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 Bacillus 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 Corynebacterium glutamicum (QsuB) and Neurospora crassa (Qa-4) had higher K[sub.m] (1 and 0.6 mM, respectively) and k[sub.cat] (61 and 220 s[sup.−1], respectively) than biosynthetic AsbF from Bacillus thuringiensis (K[sub.m]~0.04 mM, k[sub.cat]~1 s[sup.−1]). 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]~0.08 mM), and Escherichia coli MG1655 ΔaroE PlacUV5-asbF[sub.attφ80] strain provided only 0.2 g/L 3,4-DHBA in test-tube fermentation. Isogenic strains MG1655 ΔaroE PlacUV5-qsuB[sub.attφ80] and MG1655 ΔaroE PlacUV5-qa-4[sub.attφ80] expressing QsuB and Qa-4 with IC[sub.50] ~0.35 mM and ~0.64 mM, respectively, accumulated 2.7 g/L 3,4-DHBA under the same conditions.