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.

Production of raspberry ketone by redirecting the metabolic flux to the phenylpropanoid pathway in tobacco plants

“…There have been several attempts to synthesize raspberry ketone in heterologous-expression systems by expressing plant BAS or RZS genes in several hosts ( Suzuki et al, 2014 ). Thus, for example, overexpression of RiRZS1 in tobacco hairy roots and various plant cell suspensions has allowed for raspberry ketone to be successfully produced with precursor feeding (5.5 mg/L) by bioconversion, using either 4-hydroxybenzalacetone or rhododenol as substrates ( Hakkinen et al, 2015 ). Furthermore, previous work showed that engineered E. coli and S. cerevisiae successfully resulted in heterologous production of raspberry ketone; namely, 2.8 mg/L for de novo synthesis and 5–90 mg/L with precursor feeding ( Lee et al, 2016 ; Wang et al, 2019 ).…”

Raspberry Ketone Accumulation in Nicotiana benthamiana and Saccharomyces cerevisiae by Expression of Fused Pathway Genes

“…Our observation of rhododendrol accumulation in N. benthamiana is aligned with recent work on stable production of raspberry ketone in N. tabacum. 44 Our work suggests that the reduction of 4-hydroxybenzalacetone to raspberry ketone and similarly the reduction of raspberry ketone 9,28 to rhododendrol are achieved by nonspecific reductases functioning at least in N. tabacum 44 and in human liver microsomes. 47 Phloretic acid is another unique compound produced by our de novo raspberry ketone yeast strains, and the fact that it was not detected in the parental p-HCA-producing yeast strain nor the control suggested that p-coumaric acid is not a precursor for the compound.…”

“…Bioconversion has been used by Hakkinen et al as a route to convert betuligenol and 4hydroxybenzalacetone to raspberry ketone utilizing hairy root cultures and plant cell cultures, producing up to 29 μg/g (dry weight) in Catharanthus hairy root cultures. 28 However, this procedure requires time-consuming laboratory setups. Stable transformation of N. tabacum with multigene expression and upregulation of the phenylpropanoid pathway with PAP1 transcription factor was utilized by Koeduka et al to produce up to 2.2 μg/g (fresh weight) of raspberry ketone glucoside, but no aglycone was produced in tobacco leaves.…”

Potential metabolic activities of raspberry ketone

“…Recently, by overexpressing BSA from R. palmatum and rzs1 from raspberry, the content of RK production reaches 91 mg/L when using E. coli BL21(DE3) as host strains (Wang et al., 2019). Besides, using betuligenol, p ‐coumaric acid, benzoic acid, or benzaldehyde as a precursor, RK has been synthesized by different hosts including bacteria, yeast, and plant cells (Figure 1) (Hakkinen et al., 2015).…”