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.
Highlights
Since the Baeyer–Villiger monooxygenases (BVMOs, EC 1.14.13.x) were first isolated in 19761, the enzymes have been intensively studied for oxygenation of a variety of ketone substrates[2,3,4,5,6]
The engineered enzyme E6BVMO15, which was produced via fusion of the EthA
The reaction intermediate (2) concentration began to increase up to over 5 mM at t > 5 h while the ester (3) concentration remained rather unchanged. This result indicated that the catalytic activity of E6BVMO might be reduced during the biotransformation
Summary
Since the Baeyer–Villiger monooxygenases (BVMOs, EC 1.14.13.x) were first isolated in 19761, the enzymes have been intensively studied for oxygenation of a variety of ketone substrates[2,3,4,5,6]. Since the BVMO from P. putida KT2440 was unstable and difficult to express in a functional form in whole-cells (e.g., Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae), the enzyme was engineered to improve its stability and functional expression level under biotransformation conditions[14,15,37]. The major goal of the present study was to identify the factors that influence stability of the BVMO (i.e., E6BVMO) from P. putida KT2440 during E. coli-based biotransformation of fatty acids (i.e., ricinoleic acid) (Scheme S1). Another goal was to characterize the factors to improve catalytic stability of the BVMO-based E. coli biocatalysts. Use of the newly engineered enzyme (E6BVMOC302L) and feeding of carbon and energy source into the reaction medium allowed us to produce the ester (3) to final a concentration of 132 mM (41 g/L) in the cultivation medium without applying any in situ product recovery system
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