Improving catalytic activity of the Baeyer-Villiger monooxygenase-based Escherichia coli biocatalysts for the overproduction of (Z)-11-(heptanoyloxy)undec-9-enoic acid from ricinoleic acid

Abstract

Baeyer-Villiger monooxygenases (BVMOs) can be used for the biosynthesis of lactones and esters from ketones. However, the BVMO-based biocatalysts are not so stable under process conditions. Thereby, this study focused on enhancing stability of the BVMO-based biocatalysts. The biotransformation of ricinoleic acid into (Z)-11-(heptanoyloxy)undec-9-enoic acid by the recombinant Escherichia coli expressing the BVMO from Pseudomonas putida and an alcohol dehydrogenase from Micrococcus luteus was used as a model system. After thorough investigation of the key factors to influence stability of the BVMO, Cys302 was identified as an engineering target. The substitution of Cys302 to Leu enabled the engineered enzyme (i.e., E6BVMO_C302L) to become more stable toward oxidative and thermal stresses. The catalytic activity of E6BVMO_C302L-based E. coli biocatalysts was also greater than the E6BVMO-based biocatalysts. Another factor to influence biocatalytic performance of the BVMO-based whole-cell biocatalysts was availability of carbon and energy source during biotransformations. Glucose feeding into the reaction medium led to a marked increase of final product concentrations. Overall, the bioprocess engineering to improve metabolic stability of host cells in addition to the BVMO engineering allowed us to produce (Z)-11-(heptanoyloxy)undec-9-enoic acid to a concentration of 132 mM (41 g/L) from 150 mM ricinoleic acid within 8 h.

Figure 1

Time course of the biotransformation of ricinoleic acid by the recombinant E. coli BL21(DE3) pAPTm-E6BVMO-ADH, expressing an alcohol dehydrogenase (ADH) of Micrococcus luteus and the engineered Baeyer-Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440 (i.e., E6BVMO). The biotransformation was initiated by adding 15 mM ricinoleic acid and 0.5 g/L Tween80 at t = 0 into the recombinant E. coli culture broth (cell density: 3 g cell dry weight (CDW)/L). Ricinoleic acid was added once more to a concentration of 15 mM at t = 5 h. Symbols indicate the concentrations of ricinoleic acid (1) (●), 12-keto-octadec-9-enoic acid (2) (△), and the ester (3) (■). The experiments were performed in triplicate. The error bars indicate standard deviations.

Figure 2

The in vitro activity of E6BVMO, which was prepared from the cells taken out at t = 0, 4, and 6 h during the biotransformation shown in Fig. 1. The second and the third bars indicated the in vitro activities of E6BVMOs, which were prepared during the biotransformations without the reaction substrate under aerobic condition and without the reaction substrate under oxygen-deprived condition, respectively. The E6BVMO activity at t = 0 was 0.056 U/mg proteins. The experiments were performed in triplicate. The error bars indicate standard deviations.

Figure 3

The sequence (A) and the three-dimensional structure (B) alignments of the BVMO of P. putida KT2440 with the phenyl acetone monooxygenase (PAMO) from Thermobifida fusca and the cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. strain HI-31. The previously reported homology model of the P. putida KT2440 BVMO and the crystal structures of the PAMO (PDB code 1W4X) and the CHMO (PDB code 3GWD and 3GWF) were used for the study. The cyan, grey and yellow colors indicated the structure of P. putida KT2440 BVMO, PAMO, and CHMO, respectively.

Figure 4

The stabilities of E6BVMO and E6BVMO_C302L to oxidative and thermal stress. The oxidative stabilities of the enzymes (black bars) were measured 10 min after incubation at the Tris-HCl buffer (pH 8) containing 0.2 M hydrogen peroxide on ice. The thermal stabilities of the enzymes (grey bars) were evaluated 10 min after incubation at the Tris-HCl buffer at 30 °C. The enzymes, which were purified via affinity chromatography on a Ni-NTA gel matrix, were used. The initial activity of E6BVMO and E6BVMO_C302L was 0.9 and 1.0 U/mg proteins, respectively. The experiments were performed in triplicate. The error bars indicate standard deviations.

Figure 5

Time course of the biotransformation of ricinoleic acid by the recombinant E. coli BL21(DE3) pAPTm-E6BVMO_C302L-ADH, expressing the ADH of M. luteus and the engineered E6BVMO (i.e., E6BVMO_C302L) (A) and the in vitro activity of E6BVMO_C302L, which was prepared from the cells at t = 0, 4, and 6 h during the biotransformation shown in (A,B). The whole-cell biotransformation was conducted at the same condition as the biotransformation shown in Fig. 1. The in vitro activity of E6BVMO_C302L at t = 0 was 0.050 U/mg proteins. Symbols indicate the concentrations of ricinoleic acid (1) (●), 12-keto-octadec-9-enoic acid (2) (△), and the ester (3) (■). The experiments were performed in triplicate. The error bars indicate standard deviations.

Figure 6

Time course of the biotransformation of ricinoleic acid by the recombinant E. coli BL21(DE3) pAPTm- E6BVMO_C302L-ADH, pACYC-FadL expressing the long chain fatty acid transporter FadL in the outer membrane in addition to the ADH and E6BVMO_C302L. The biotransformation was initiated by adding 150 mM ricinoleic acid and 0.5 g/L Tween80 into the recombinant E. coli culture broth (cell density: 25 g CDW/L). Glucose was added into the cultivation medium to a rate of 5 g/L/h to facilitate regeneration of the nicotinamide cofactors in E. coli cells. Symbols indicate the concentrations of ricinoleic acid (1) (●), 12-keto-octadec-9-enoic acid (2) (△), and the ester (3) (■). The experiments were performed in triplicate. The error bars indicate standard deviations.