Efficient Synthesis of (R)-(+)-Perillyl Alcohol From (R)-(+)-Limonene Using Engineered Escherichia coli Whole Cell Biocatalyst

Abstract

(R)-(+)-perillyl alcohol is a much valued supplemental compound with a wide range of agricultural and pharmacological characteristics. The aim of this study was to improve (R)-(+)-perillyl alcohol production using a whole-cell catalytic formula. In this study, we employed plasmids with varying copy numbers to identify an appropriate strain, strain 03. We demonstrated that low levels of alKL provided maximal biocatalyst stability. Upon determination of the optimal conditions, the (R)-(+)-perillyl alcohol yield reached 130 mg/L. For cofactor regeneration, we constructed strain 10, expressing FDH from Candida boidinii, and achieved (R)-(+)-perillyl alcohol production of 230 mg/L. As a result, 1.23 g/L (R)-(+)-perillyl alcohol was transformed in a 5 L fermenter. Our proposed method facilitates an alternative approach to the economical biosynthesis of (R)-(+)-perillyl alcohol.

Keywords: (R)-(+)-perillyl alcohol; NADH regeneration; alkL; escherichia coli; whole cell catalysis.

FIGURE 1

(R)-(+)-perillyl alcohol synthesis using whole cell biocatalysts. (A) (R)-(+)-perillyl alcohol biosynthesis from (R)-(+)-limonene, using a whole cell system. (B) (R)-(+)-perillyl alcohol production with different plasmids. Reaction conditions (20 ml): whole cell catalysts (OD₆₀₀ = 30), 25 μl (R)-(+)- limonene, 8.0 ml dioctyl phthalate (DINP), and PBS buffer (50 mM, pH 7.4) at 30°C for 6 h. Data expressed as mean ± s.d. (n = 3). Abbreviations: CymAa, p-cymene monoxygenase hydroxylase, CymAb, p-cymene monoxygenase reductase, CymA, CymAa and CymAb, FDH, formate dehydrogenase.

FIGURE 2

Effect of alkL protein expression on (R)-(+)-perillyl alcohol production. (A) Strains constructed by different promotors. (B) Whole-cell activity assays related to alkL expression. (C) Comparison of (R)-(+)-perillyl alcohol production in each strain. Reaction conditions (20 ml): whole cell catalysts (OD₆₀₀ = 30), 25 μl (R)-(+)- limonene, 8.0 ml dioctyl phthalate (DINP), and PBS buffer (50 mM, pH 7.4) at 30°C for 6 h. Data expressed as mean ± s.d. (n = 3).

FIGURE 3

Various concentrations of (R)-(+)-perillyl alcohol production by different E. coli strains. Reaction conditions (20 ml): whole cell catalysts (OD₆₀₀ = 30), 25 μl (R)-(+)- limonene, 8.0 ml dioctyl phthalate (DINP), and PBS buffer (50 mM, pH 7.4) at 30°C for 6 h. Data expressed as mean ± s.d. (n = 3).

FIGURE 4

Reaction condition optimization for (R)-(+)-perillyl alcohol synthesis. The effect of (A) varying pHs and (B) temperatures on (R)-(+)-perillyl alcohol synthesis. Reaction conditions (20 ml) for different pHs: whole cell catalysts (OD₆₀₀ = 30), 25 μl (R)-(+)- limonene, 8.0 ml dioctyl phthalate (DINP), and PBS buffer (50 mM, pH 5.8–8.0) at 30°C for 6 h. Reaction conditions (20 ml) for different temperatures: whole cell catalysts (OD₆₀₀ = 30), 25 μl (R)-(+)-limonene, 8.0 ml dioctyl phthalate (DINP), and PBS buffer (50 mM, pH 7.4) at 16–37°C for 6 h. (C) Effects of NADH addition on (R)-(+)-perillyl alcohol production. Reaction conditions (20 ml): whole cell catalysts (OD₆₀₀ = 30), 25 μl (R)-(+)-limonene, 8.0 ml dioctyl phthalate (DINP), NADH (0–2 mM), and PBS buffer (50 mM, pH 7.4) at 30°C for 6 h. (D) Effect of biocatalysts concentrations on (R)-(+)-perillyl alcohol production. Reaction conditions (20 ml): whole cell catalysts (OD₆₀₀ = 10, 30, 50, 70), 25 μl (R)-(+)-limonene, 8.0 ml dioctyl phthalate (DINP), and PBS buffer (50 mM, pH 7.4) at 30°C for 6 h. (E) Whole cell biotransformation of (R)-(+)-perillyl alcohol at high (R)-(+)-limonene concentration. Reaction conditions (20 ml): whole cell catalysts (OD₆₀₀ = 50), (R)-(+)-limonene (1, 5, 10, 20, 40 g/L), 8.0 ml dioctyl phthalate (DINP), 2 mM NADH, and PBS buffer (50 mM, pH 7.4) at 30°C for 6 h. (F) (R)-(+)-perillyl alcohol production with different ammonium formate concentrations. Reaction conditions (20 ml): whole-cell catalysts (OD₆₀₀ = 50), 500 μl (R)-(+)-limonene, 8.0 ml dioctyl phthalate (DINP), ammonium formate (0, 10, 40, 70, 100 g/L), and PBS buffer (50 mM, pH 7.4) at 30°C for 6 h. Data expressed as mean ± s.d. (n = 3).

FIGURE 5

Time course of (R)-(+)-limonene hydroxylation using whole cell of strain 10. Reaction conditions (1 L): whole cell catalysts (OD₆₀₀ = 50), 25 ml (R)-(+)-limonene, 40 g ammonium formate, 400 ml dioctyl phthalate (DINP), and PBS buffer (50 mM, pH 7.4) at 20°C for 24 h.