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Screemomg, expression, and characterization of Baeyer‐Villiger Monooxygenase for the biotransformation of ricinoleic acid
Author(s) -
Yun Joohyun,
Choi KwonYoung
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.796.31
Subject(s) - biotransformation , biochemistry , monooxygenase , ricinoleic acid , chemistry , oleic acid , enzyme , hexanoic acid , escherichia coli , cytochrome p450 , castor oil , gene
Here, the biotransformation of ricinoleic acid into of 9‐(nonanoyloxy)nonanoic acidusing Baeyer‐Villiger monooxygenases (BVMOs) was investigated. The whole cell biotransformation of oleic acid includes OhyA (hydratase), ADH (alcohol dehydrogenase), and BVMO Enzymes consecutively. BVMOs are known to catalyze regiospecific Baeyer‐Villiger oxygenation of a variety of cyclic and linear ketones to generate the corresponding lactones and esters, respectively. 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 this fusion enzyme resulted in 3 to 5‐fold enhancement in product formation compared with the non‐fusion counterpart. Final productivity up to 105.3 mgL −1 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. Support or Funding Information This work was supported by the Industrial Strategic Technology Development program (No.10044604), funded by the Ministry of Trade, Industry & Energy (MI, Korea) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .