Comparative Analysis of Catabolic and Anabolic Dehydroshikimate Dehydratases for 3,4-DHBA Production in <i>Escherichia 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)...
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MDPI AG
2022-07-01
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| Online Access: | https://www.mdpi.com/2076-2607/10/7/1357 |
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| author | Ekaterina A. Shmonova Ekaterina A. Savrasova Elizaveta N. Fedorova Vera G. Doroshenko |
| author_facet | Ekaterina A. Shmonova Ekaterina A. Savrasova Elizaveta N. Fedorova Vera G. Doroshenko |
| author_sort | Ekaterina A. Shmonova |
| collection | DOAJ |
| description | 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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| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-09T13:20:47Z |
| publishDate | 2022-07-01 |
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| spelling | doaj.art-c96bcf1556824722b3ed926e8a512aa72023-11-30T21:30:26ZengMDPI AGMicroorganisms2076-26072022-07-01107135710.3390/microorganisms10071357Comparative Analysis of Catabolic and Anabolic Dehydroshikimate Dehydratases for 3,4-DHBA Production in <i>Escherichia coli</i>Ekaterina A. Shmonova0Ekaterina A. Savrasova1Elizaveta N. Fedorova2Vera G. Doroshenko3Ajinomoto-Genetika Research Institute, 117545 Moscow, RussiaAjinomoto-Genetika Research Institute, 117545 Moscow, RussiaAjinomoto-Genetika Research Institute, 117545 Moscow, RussiaAjinomoto-Genetika Research Institute, 117545 Moscow, RussiaThe 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.https://www.mdpi.com/2076-2607/10/7/1357microbial production3,4-DHBAprotocatechuic aciddehydroshikimate dehydratase |
| spellingShingle | Ekaterina A. Shmonova Ekaterina A. Savrasova Elizaveta N. Fedorova Vera G. Doroshenko Comparative Analysis of Catabolic and Anabolic Dehydroshikimate Dehydratases for 3,4-DHBA Production in <i>Escherichia coli</i> Microorganisms microbial production 3,4-DHBA protocatechuic acid dehydroshikimate dehydratase |
| title | Comparative Analysis of Catabolic and Anabolic Dehydroshikimate Dehydratases for 3,4-DHBA Production in <i>Escherichia coli</i> |
| title_full | Comparative Analysis of Catabolic and Anabolic Dehydroshikimate Dehydratases for 3,4-DHBA Production in <i>Escherichia coli</i> |
| title_fullStr | Comparative Analysis of Catabolic and Anabolic Dehydroshikimate Dehydratases for 3,4-DHBA Production in <i>Escherichia coli</i> |
| title_full_unstemmed | Comparative Analysis of Catabolic and Anabolic Dehydroshikimate Dehydratases for 3,4-DHBA Production in <i>Escherichia coli</i> |
| title_short | Comparative Analysis of Catabolic and Anabolic Dehydroshikimate Dehydratases for 3,4-DHBA Production in <i>Escherichia coli</i> |
| title_sort | comparative analysis of catabolic and anabolic dehydroshikimate dehydratases for 3 4 dhba production in i escherichia coli i |
| topic | microbial production 3,4-DHBA protocatechuic acid dehydroshikimate dehydratase |
| url | https://www.mdpi.com/2076-2607/10/7/1357 |
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