Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase ( vdh ) gene

Abstract

The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceeds via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillate and protocatechuate, which is further degraded by the ortho-cleavage pathway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which catalyses the NAD(+)-dependent oxidation of vanillin to vanillate, was inactivated by the insertion of omega elements into the vdh gene, which was characterized recently. Omega elements conferring resistance against kanamycin (omega Km) or gentamycin (omega Gm) were constructed by polymerase chain reaction amplification of the aminoglycoside 3'-O-phosphotransferase gene and the gentamycin-3-acetyltransferase gene, using the plasmids pSUP5011 and pBBR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by omega Km or omega Gm, and the functional vdh gene was replaced by vdh omega Km or vdh omega Gm in Pseudomonas sp. strain HR199 by homologous recombination. Cells of the mutant Pseudomonas sp. strain HR vdh omega Km, pregrown on gluconate, accumulated up to 2.9 mM vanillin during incubation in mineral medium with 6.5 mM eugenol. As a result of another vanillin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characterization of the purified VDH-II revealed the identify of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase.

Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of Escherichia coli

“…However, during further incubation vanillin was reduced to vanillyl alcohol and no more ferulic acid was converted to vanillin. The reduction of vanillin represents a detoxification mechanism, which was also observed with E. coli XL1-Blue cells during incubation with vanillin (17 …”

“…Eugenol is a cheap "natural" substrate that can be isolated from the essential oil of the clove tree Syzygium aromaticum on an industrial scale. The genes encoding proteins of the oxidative eugenol catabolism pathway have been investigated in detail in different Pseudomonas strains (1,5,13,15,16,17,18,20,21). The genes ech and fcs of Pseudomonas sp.…”

“…Subsequently, washed cells of a 50-ml culture of E. coli XL1-Blue(pSKechE/Hfcs), which had been grown to the late exponential growth phase and induced by the addition of IPTG, were added to the culture. In order to inactivate the calB gene product, which besides coniferyl aldehyde dehydrogenase also possesses vanillin dehydrogenase activity (17), the temperature was raised to 37°C. Further incubation of this mixed culture and analysis of culture supernatants via HPLC revealed a production of 0.3 g of vanillin liter…”

Obtenção de vanilina: oportunidade biotecnológica

“…HR199 produzindo a enzima protocatecóico 3,4-dioxigenase foram caracterizados molecularmente 39,40 . Posteriormente, o catabolismo de ácido ferúlico em Pseudomonas sp.…”

Characterization of Two Isozymes of Coniferyl Alcohol Dehydrogenase fromStreptomycessp. NL15-2K

“…Bacterial degradation pathways for lignin-related aromatic compounds have been characterized extensively for gram-negative bacteria, particularly Pseudomonas spp. [1][2][3] and Sphingomonas paucimobilis SYK-6. 4) Most of these compounds are degraded to protocatechuic acid or catechol, and further broken down via specific ring cleavage pathways.…”