Bioconversion to Raspberry Ketone is Achieved by Several Non-related Plant Cell Cultures

Abstract

Bioconversion, i.e., the use of biological systems to perform chemical changes in synthetic or natural compounds in mild conditions, is an attractive tool for the production of novel active or high-value compounds. Plant cells exhibit a vast biochemical potential, being able to transform a range of substances, including pharmaceutical ingredients and industrial by-products, via enzymatic processes. The use of plant cell cultures offers possibilities for contained and optimized production processes which can be applied in industrial scale. Raspberry ketone [4-(4-hydroxyphenyl)butan-2-one] is among the most interesting natural flavor compounds, due to its high demand and significant market value. The biosynthesis of this industrially relevant flavor compound is relatively well characterized, involving the condensation of 4-coumaryl-CoA and malonyl-CoA by Type III polyketide synthase to form a diketide, and the subsequent reduction catalyzed by an NADPH-dependent reductase. Raspberry ketone has been successfully produced by bioconversion using different hosts and precursors to establish more efficient and economical processes. In this work, we studied the effect of overexpressed RiZS1 in tobacco on precursor bioconversion to raspberry ketone. In addition, various wild type plant cell cultures were studied for their capacity to carry out the bioconversion to raspberry ketone using either 4-hydroxybenzalacetone or betuligenol as a substrate. Apparently plant cells possess rather widely distributed reductase activity capable of performing the bioconversion to raspberry ketone using cheap and readily available precursors.

Keywords: 4-hydroxybenzalacetone; betuligenol; bioconversion; plant cell culture; raspberry ketone.

FIGURE 1

Conversion of 4-hydroxybenzalacetone (1) and betuligenol (2) into raspberry ketone (3).

FIGURE 2

Accumulation of raspberry ketone in Nicotiana benthamiana leaves after feeding with 1 mM 4-OHBA. Agrobacterium tumefaciens LBA4404 carrying either p19 silencing suppressor or 35S-RiZS1 was infiltrated alone or in combination (in 1:1 or in 1:4 v:v ratio). (A) extracellular (mg/l), (B) intracellular (mg/g FW) levels. Error bars represent SD of biological replicates (N = 3) where each sample consisted of 20 infiltrated leaf disks (see Transient Expression in N. benthamiana).

FIGURE 3

Formation of raspberry ketone (mg/l) in the extracellular space of N. tabacum hairy roots expressing 35S-RiZS1 with different concentrations of fed 4-OHBA. Feeding was performed after 9 days of cultivation and samples were taken 1 d after feeding. Error bars represent the standard deviation of three biological replicates. The experiment was repeated twice. WT, wild type. Asterisks indicate the significance level obtained by Student’s t-test: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 4

Accumulation of raspberry ketone in culture medium (μg/l) (A) and in intracellular space (μg/g DW) (B) in N. tabacum BY-2 cell suspension culture after feeding with either 4-OHBA or betuligenol.