Biotechnology and Bioprocess Engineering 2017; 22(6): 717-724  
Screening, Expression, and Characterization of Baeyer-Villiger Monooxygenases for the Production of 9-(nonanoyloxy)nonanoic Acid from Oleic Acid
Pamidimarri D. V. N. Sudheer, Joohyun Yun, Sushma Chauhan, Taek Jin Kang, and Kwon-Young Choi*
Department of Environmental Engineering, College of Engineering, Ajou University, Suwon 16499, Korea
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

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Abstract
In this study, the production of 9-(nonanoyloxy) nonanoic acid from oleic acid was investigated. The whole cell biotransformation of oleic acid includes OhyA (hydratase), ADH (alcohol dehydrdogenase), and BVMO (Baeyer-Villiger Monooxygenase) enzymes consecutively. BVMOs are known to catalyze oxidative cleavage of long chain aliphatic ketones (e.g., 2-decanone, 10-ketooctadecanoic acid). However, the enzymes are difficult to overexpress in a soluble form in microorganisms. Thereby, this study has focused on screening and functional expression of the BVMOs in Escherichia coli. Initially BVMOs were selected by protein sequence analysis and were examined for their ability to express in soluble and active form to generate 9-(nonanoyloxy)nonanoic acid from oleic acid. Secondly various optimization strategies of inducer concentrations, co-expression with molecular chaperones, and different media conditions were investigated. Among the 9 BVMOs screened, three BVMOs were found to produce the target product and among these, Di_BVMO3 isolated from Dietzia sp. D5 was found to be best. Further, the soluble expression of Di_BVMO3 was enhanced by adding phosphoglycerate kinase as N-terminal fusion tag. The whole cell biotransformation with fusion enzyme resulted in 3 ~ 5-fold enhancement in product formation compared with the non-fusion counterpart. Final productivity up to 105.3 mg/L was achieved. Besides Di-BVMO3, other two new BVMOs of Rh_BVMO4 from Rhodococcus sp. RHA1 and AFL838 from Aspergillus flavus NRRL3357 were screened for production of 9-(nonanoyloxy)nonanoic acid and could be used for whole cell biotransformation reaction of other long chain ketones.
Keywords: Baeyer-Villiger monooxygenases, soluble expression, whole-cell bio transformation and 9-(nonanoyloxy) nonanoic acid


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