Biosynthesis of p-hydroxyphenyl-2-butanone and its correlation to primary metabolism in cell suspension cultures of Rubus idaeus

The principle objective of this research was to investigate carbon substrate metabolism in the biosynthesis of p-hydroxyphenyl-2-butanone (p-HPB₂) in Rubus idaeus (raspberry). This was achieved by feeding various precursors and through detailed stoichiometric modeling of the biosynthetic pathway. The typical cell culture profiles for the flavor component (p-HPB₂), its intermediate precursor p-hydroxyphenylbut-3-ene-2-one (p-HPB ₁) and a distant precursor p-coumaric acid (p-CA) were measured and related to carbohydrate consumption and cell proliferation. The transient production of these metabolites and lack of sequential changes in concentration along the pathway indicated a complex regulatory process controlling their production. The signal coupler methyl jasmonate was found to induce the synthesis of p-HPB₂ and p-HPB ₁. Its effect was a function of elicitor concentration, exposure time and time of addition.; The link between primary and secondary metabolism was investigated by metabolic modeling and by feeding precursors involved in p-HPB ₂ formation. Exogenous addition of precursors (sucrose, L-phenylalanine, p-CA and p-HPB ₁) indicated a possible limitation in the phenylpropanoid pathway. Sucrose fed cultures exhibited negligible change in sucrose consumption rates from control cultures, thus implying a self-regulatory step in sucrose uptake and further, that sucrose is not a limiting substrate for p-HPB₂ synthesis. Lack of p-CA increase in sucrose fed cultures, suggested a preferential channeling of the fed sucrose via the glycolysis into p-HPB₂ formation. A synergistic effect of combination feeds on metabolite levels demonstrated the requirement of primary and secondary metabolites for p-HPB₂ synthesis. The rapid utilization of fed precursors without any intracellular accumulation established that they are not stable end products. Modeling the biosynthetic pathway via metabolic flux analysis was carried out to generate a stoichiometric equation that indicates a maximum theoretical yield of p-HPB₂ from sucrose of 0.46 mol/mol. Metabolic modeling further demonstrated that majority of the carbon flux (60%–70%) is channelled via glycolysis, thus confirming the experimental observation of preferential channeling of carbon substrate.