The biosynthesis of acetovanillone in tobacco cell-suspension cultures

A soluble enzyme, extracted from tobacco cell-suspension cultures 24 h after treatment with 100 μM methyl jasmonate, has been shown to synthesize acetovanillone (apocynin) from feruloyl-CoA in the presence of NAD. The enzyme displayed Michaelis-Menten kinetics with apparent K_m values of 5.6 μM for feruloyl-CoA and 260 μM for NAD and exhibited very high specificity for its substrates. The increase in acetovanillone synthase activity was followed by an increase in the concentration of both acetovanillone and acetosyringone in the culture medium. No intermediate could be detected when analysing the reaction medium by HPLC during the formation of acetovanillone in cell-free extracts. The apparent molecular mass estimated by gel permeation on an FPLC column was ca. 79 kDa. To our knowledge, this is the first report of an enzymic system catalysing the synthesis of an acetophenone. This work demonstrates that the biosynthesis of acetophenones in tobacco proceeds from hydroxycinnamic acids through a CoA-dependent β-oxidation pathway. Interestingly in methyl jasmonate-treated cells, which synthesize very large amounts of hydroxycinnamoylputrescines, inhibition of the synthesis of these conjugates increased the concentration of acetovanillone and acetosyringone in the culture medium, suggesting that the two metabolic pathways can compete for their common precursors, i.e. hydroxycinnamoyl-CoA thioesters.     

Biotransformation of ferulic acid to acetovanillone using Rhizopus oryzae

Microbial transformation of ferulic acid to acetovanillone was studied using growing cells of Rhizopus oryzae. Ferulic acid was added to the growing medium (0.5 g L^?1) and incubated for 12 days. The progress of formation of metabolites was monitored by GC and GC-MS after extraction with ethyl acetate. The major metabolite was acetovanillone with minor metabolites formed, such as dihydroferulic acid, coniferyl alcohol and dihydroconiferyl alcohol. Traces of metabolites (≤1–3%), such as vanillin, vanillyl alcohol, vanillic acid and phenyl ethyl alcohol, were also produced. Formation of 4-vinyl guaiacol increased from day 1 (12.4%), reaching a maximum on day 4 (31.7%), and reducing to a minimum on day 12 (3.1%). The formation of acetovanillone increased only from day 2 onward, and reached a maximum (49.2%) on day 12. The optimum concentration of ferulic acid to be added into the medium was found to be only 0.5 g L^?1, as any increase in concentration (0.75 and 1.0 g L^?1) precipitated the precursor, resulting in no further degradation.     

Biotransformation of ferulic acid to acetovanillone using Rhizopus oryzae

Microbial transformation of ferulic acid to acetovanillone was studied using growing cells of Rhizopus oryzae. Ferulic acid was added to the growing medium (0.5 g L^-1) and incubated for 12 days. The progress of formation of metabolites was monitored by GC and GC-MS after extraction with ethyl acetate. The major metabolite was acetovanillone with minor metabolites formed, such as dihydroferulic acid, coniferyl alcohol and dihydroconiferyl alcohol. Traces of metabolites (≤1-3%), such as vanillin, vanillyl alcohol, vanillic acid and phenyl ethyl alcohol, were also produced. Formation of 4-vinyl guaiacol increased from day 1 (12.4%), reaching a maximum on day 4 (31.7%), and reducing to a minimum on day 12 (3.1%). The formation of acetovanillone increased only from day 2 onward, and reached a maximum (49.2%) on day 12. The optimum concentration of ferulic acid to be added into the medium was found to be only 0.5 g L^-1, as any increase in concentration (0.75 and 1.0 g L^-1) precipitated the precursor, resulting in no further degradation.     

Relationship of nitrogen to the onset and suppression of ligninolytic activity and secondary metabolism in Phanerochaete chrysosporium

Ligninolytic activity in the white-rot fungus Phanerochaete chrysosporium was previously found not to be induced by lignin, but to develop in cultures in response to nitrogen starvation. Added NH ₄ ⁺ suppressed existing activity. The present study examined amino acid profiles and protein concentrations during onset of ligninolytic activity (synthetic ¹⁴C-lignin¹⁴CO₂) in nitrogen-limited cultures, and defined some characteristics of subsequent suppression by added nutrient nitrogen. During the transition between depletion of medium nitrogen and the onset of ligninolytic activity, total free intracellular amino acids increased, then rapidly decreased; changes in glutamate concentration played a major role. Intracellular protein concentration fluctuated in a manner roughly converse to that of the concentration of free amino acids. Protein turnover was rapid (5–7%/h) during the transition period. Glutamate, glutamine, and histidine were the most effective of 14 nitrogenous compounds in suppressing ligninolytic activity after its onset. The suppressive effect was not mediated through carbon (glucose)-catabolite repression or by alterations in culture pH. Activities responsible for oxidation of lignin and the ligninrelated phenol, 4-hydroxy-3-methoxyacetophenone, responded similarly to added nitrogen. Synthesis of a secondary metabolite, veratryl alcohol, like lignin oxidation, was suppressed quite sharply by glutamate and significantly by NH ₄ ⁺ . Results indicate that nitrogen metabolism affects ligninolytic activity as a part of secondary metabolism, and suggest a role for glutamate metabolism in regulating this phase of culture development.Non-Standard Abbreviations DMS 2,2-dimethylsuccinate - GLC gas-liquid chromatography - TCA trichloroacetic acid