초록
<P>Baeyer-Villiger monooxygenases (BVMOs) are able 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 usually difficult to express in a functional form in microbial cells and are rather unstable under process conditions hindering their large-scale applications. Thereby, we investigated engineering of the BVMO from <I>Pseudomonas putida</I> KT2440 and the gene expression system to improve its activity and stability for large-scale biotransformation of ricinoleic acid (<B>1</B>) into the ester (i.e., (<I>Z</I>)-11-(heptanoyloxy)undec-9-enoic acid) (<B>3</B>), which can be hydrolyzed into 11-hydroxyundec-9-enoic acid (<B>5</B>) (i.e., a precursor of polyamide-11) and <I>n</I>-heptanoic acid (<B>4</B>). The polyionic tag-based fusion engineering of the BVMO and the use of a synthetic promoter for constitutive enzyme expression allowed the recombinant <I>Escherichia coli</I> expressing the BVMO and the secondary alcohol dehydrogenase of <I>Micrococcus luteus</I> to produce the ester (<B>3</B>) to 85 mM (26.6 g/L) within 5 h. The 5 L scale biotransformation process was then successfully scaled up to a 70 L bioreactor; <B>3</B> was produced to over 70 mM (21.9 g/L) in the culture medium 6 h after biotransformation. This study demonstrated that the BVMO-based whole-cell reactions can be applied for large-scale biotransformations.</P>