Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli -Expressed Cyclopentanone 1,2-Monooxygenase

Abstract

54 -dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-␤-thio-D-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.Pseudomonas sp. strain NCIMB 9872, which is capable of growth on 0.1% cyclopentanol as sole carbon source, was isolated from a freshwater stream in Illinois by P. J. Chapman some three decades ago. Trudgill and coworkers (27) used this strain as a prototype organism to establish the biochemical pathway of cyclopentanol metabolism shown in Fig. 1. For reason of utility as a Baeyer-Villiger (BV) biocatalyst and its mechanistic aspects (2,8,13,58,71), the second biochemical step catalyzed by cyclopentanone 1,2-monooxygenase (CPMO) has been most extensively studied in this organism. The conversion of cyclopentanone to 5-valerolactone is NADPH and flavin adenine dinucleotide (FAD) dependent, and CPMO has been purified to near homogeneity. It is a tetramer of subunit molecular weight 50,000 to 54,000 (8,66). Assay of the FAD/ protein ratio gave values of 2 to 4 molecules of FAD bound to each tetrameric enzyme molecule (28,66). An N-terminal 29-amino-acid sequence of CPMO has been determined (8).A chemical BV oxidation is the transformation of ketones into esters or of cyclic ketones into lactones by peracids, such as 3-chloroperbenzoic acid. This century-old reaction (for reviews, see references 54 and 63) continues to attract interest not only in broadening the spectrum of applications (ranging from the synthesis of steroids, antibiotics, and pheromones to the synthesis of monomers for polymerization, etc.) but also in developing oxidants that are more chemoselective and efficient and thus result in more product than waste (20).The biological BV reactions catalyzed by BV monooxygenases (BVMOs) offer the prospect of eco-efficient chemical transformations in whole cells or improved expression systems via cloning. Moreover, BVMOs can provide products in optically active form which are difficult to obtain by other strategies (for reviews, see references 55, 61, 71, and 74). Notable ste-* Corresponding author. Mailing address:

Self‐Sufficient Baeyer–Villiger Monooxygenases: Effective Coenzyme Regeneration for Biooxygenation by Fusion Engineering

“…In time samples were taken, extracted by EtOAc supplemented with an internal standard (1 mM of methyl ester of benzoic acid), and analyzed by GC. [4] 48 (+) 100 46(+) 68 [5] 2 OH [b] 12 (-) 100 10 (-) 61 [6] 51 (+) 100 85(+) 73 [7] 3 Cl 93 (-) 100 95 (-) 56 [7] 34 (+) 100 34(+) 64 [ 7] 4 Br 97 (-) 100 97 (-) 63 [8] 70(+) 100 64 (+) 70 [7] 5 I 99 (-) 100 97 (-) 60 [8] 90 (+) 100 82(+) 65 [7] 6 COOEt 96 (-) 50 93 (-) 15 [7] 65. (+) 100 64 (+) 83 [7] 7 Me, Me' n.a.…”

Characterization of CpdC, a Large-Ring Lactone-Hydrolyzing Enzyme from Pseudomonas sp. Strain HI-70, and Its Use as a Fusion Tag Facilitating Overproduction of Proteins in Escherichia coli

“…strain NCIMB 9872. There, the respective hydrolase (chnC or cpnC) and the BVMO-encoding genes (chnB or cpnB) are not only distally located but divergently transcribed (5,6,12). In at least two organisms, Brevibacterium sp.…”

“…In contrast to the ring-expansion reaction of BVMOs, C-C breakage of the lactones by a hydrolase that effectively opens the alicyclic ring to produce a hydroxy acid (5-hydroxyvaleric acid, 6-hydroxyhexanoic acid, or 12-hydroxylauric acid in the case of 5-valerolactone, caprolactone, or lauryl lactone, respectively) is hardly studied beyond the initial gene localization and identification (3,6,12). These hydrolases are members of the ␣/␤-hydrolase fold superfamily (13,14).…”

“…A common theme that runs through the metabolic pathway of the degradation of alicyclic alcohols of various carbon-ring sizes (C 5 to C 15 ) by both Gram-negative and Gram-positive bacteria is a five-step biochemical reaction that transforms an alicyclic alcohol to a linear dicarboxylic acid before undergoing ␤-oxidation to generate the energy for growth (1)(2)(3)(4)(5)(6)(7)(8). In these pathways, the most-studied biochemical step is the conversion of a cyclic ketone to a lactone catalyzed by an NADPH-dependent flavoprotein, better known as a type 1 Baeyer-Villiger monooxygenase (BVMO).…”

Isolation and Genetic Characterization of Phenol-Utilizing Marine Bacteria and Their Phenol Degradation Pathway

“…16S rRNA gene sequences were determined as previously described [8]. Phylogenetic trees were generated by the neighbor-joining method in MEGA version 6.0 [9].…”