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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., E6BVMOC302L) to become more stable toward oxidative and thermal stresses. The catalytic activity of E6BVMOC302L-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.

SUBMITTER: Woo JM

PROVIDER: S-EPMC6035261 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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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.

Woo Ji-Min JM Jeon Eun-Yeong EY Seo Eun-Ji EJ Seo Joo-Hyun JH Lee Dong-Yup DY Yeon Young Joo YJ Park Jin-Byung JB

Scientific reports 20180706 1

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 ...[more]

PMID: 29980730

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Project description:Baeyer-Villiger monooxygenases (BVMOs) are an emerging class of promising biocatalysts for the oxidation of ketones to prepare corresponding esters or lactones. Although many BVMOs have been reported, the development of highly efficient enzymes for use in industrial applications is desirable. In this work, we identified a BVMO from Rhodococcus pyridinivorans (BVMORp) with a high affinity toward aliphatic methyl ketones (Km < 3.0 μM). The enzyme was highly soluble and relatively stable, with a half-life of 23 h at 30°C and pH 7.5. The most effective substrate discovered so far is 2-hexanone (kcat = 2.1 s-1; Km = 1.5 μM). Furthermore, BVMORp exhibited excellent regioselectivity toward most aliphatic ketones, preferentially forming typical (i.e., normal) products. Using the newly identified BVMORp as the catalyst, a high concentration (26.0 g/liter; 200 mM) of methyl levulinate was completely converted to methyl 3-acetoxypropionate after 4 h, with a space-time yield of 5.4 g liter-1 h-1 Thus, BVMORp is a promising biocatalyst for the synthesis of 3-hydroxypropionate from readily available biobased levulinate to replace the conventional fermentation.IMPORTANCE BVMOs are emerging as a green alternative to traditional oxidants in the BV oxidation of ketones. Although many BVMOs are discovered and used in organic synthesis, few are really applied in industry, especially in the case of aliphatic ketones. Herein, a highly soluble and relatively stable monooxygenase from Rhodococcus pyridinivorans (BVMORp) was identified with high activity and excellent regioselectivity toward most aliphatic ketones. BVMORp possesses unusually high substrate loading during the catalysis of the oxidation of biobased methyl levulinate to 3-hydroxypropionic acid derivatives. This study indicates that the synthesis of 3-hydroxypropionate from readily available biobased levulinate by BVMORp-catalyzed oxidation holds great promise to replace traditional fermentation.

Dataset Information

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.

Publications

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.

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