Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica

Abstract

Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 °C) in a reaction medium containing 0.5% v/v limonene and 10 g/L of stationary phase cells (dry weight). The best results, corresponding to 564 mg/L of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 °C for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mg/L, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry.

Synthesis of the Prospective Anticancer Molecule Perillic Acid from Orange Essential Oil by the Yeast <i>Yarrowia lipolytica</i>

“…The handling of volatile limonene to enhance its bioavailability to the yeast cells in a stirred-process of bioconversion, without using emulsifiers, turned out to be quite challenging. Thus, six limonene additions to the reaction medium had been done over a period of 48 h in our previous work [21]. In the present study however, only two additions were needed, which meant an operational gain, without decreasing the final product concentration.…”

“…The optimized conditions found to the bioconversion of high-purity limonene with Yarrowia lipolytica were replicated by using orange essential oil containing Table 4 compares published data from our laboratory [21] and data from the present work regarding the main biotransformation process parameters in terms of reaction conditions, perillic acid final concentration and perillic acid molar yield. The handling of volatile limonene to enhance its bioavailability to the yeast cells in a stirred-process of bioconversion, without using emulsifiers, turned out to be quite challenging.…”

“…The fungus Aspergillus cellulosae [16] and the non-conventional yeasts Arxula adeninivorans and Yarrowia lipolytica [20] have also been approached to converting limonene to perillic derivatives. Recently, our research group successfully studied the bio-oxidation of R-(+)-limonene into R-(+)-perillic acid by Y. lipolytica ATCC 18942 [21], which is recognized as safe and suitable yeast to be used in processes aiming at products for human consumption [22] [23]. As a continuation of the previous study, the present work aimed to increase the perillic acid yield from high-purity limonene, by applying statistical design of experiments to the bioreaction parameters.…”

Effectiveness of recombinant Escherichia coli on the production of (R)-(+)-perillyl alcohol

“…Perillyl alcohol can be synthesized in Escherichia coli [12,13], Pseudomonas putida [5,[14][15][16], Yarrowia lipolytica [17,18], Mortierella minutissima [19], Fusarium verticilloides [20], Aspergillus strain [21,22] from the biotransformation of limonene to perillyl alcohol. The maximum yield of (R)-(+)-perillyl alcohol was 258.1 mg/L for 3 days using Mortierella minutissima cultivation containing 0.5% (R)-(+)limonene at 15 °C [19].…”

Effectiveness of recombinant Escherichia coli for the production of (R)-(+)-perillyl alcohol 

“…effective synthesis procedures are however required. While (R)-(+)-perillyl alcohol can be synthesized in Escherichia coli [12,13], Pseudomonas putida [5,[14][15][16][17][18], Yarrowia lipolytica [17,19], Mortierella minutissima [20], Fusarium verticilloides [21], Aspergillus strain [22,23]. All of these microorganisms can biotransform limonene to perillyl alcohol.…”