Abstract
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 Pseudomonas putida KT2440 and the gene expression system to improve its activity and stability for large-scale biotransformation of ricinoleic acid (1) into the ester (i.e., (Z)-11-(heptanoyloxy)undec-9-enoic acid) (3), which can be hydrolyzed into 11-hydroxyundec-9-enoic acid (5) (i.e., a precursor of polyamide-11) and n-heptanoic acid (4). The polyionic tag-based fusion engineering of the BVMO and the use of a synthetic promoter for constitutive enzyme expression allowed the recombinant Escherichia coli expressing the BVMO and the secondary alcohol dehydrogenase of Micrococcus luteus to produce the ester (3) 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; 3 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.
Highlights
In order to improve functional expression and structural stability of the oxygenases (e.g., EthA of P. putida KT2440)[12] in microbial cells, a variety of approaches have been explored
The BVMO from P. putida KT2440 12 was engineered to enhance its functional expression and stability in E. coli BL21(DE3) by assuming that negatively charged residues in the N- or C-terminal are of great importance to thermal stability of proteins
After construction of E6, K6, Ub-BVMO fusion genes, they were expressed in E. coli BL21(DE3) at 20 °C
Summary
In order to improve functional expression and structural stability of the oxygenases (e.g., EthA of P. putida KT2440)[12] in microbial cells, a variety of approaches have been explored. Introduction of molecular chaperones[15,16], the protein fusion with soluble peptides and proteins[17,18], introduction of disulfide bonds[19,20], and other protein engineering methods (e.g., directed evolution)[21,22] have been intensively examined to increase functional expression and stability of the oxygenases in microbial cells. These approaches are not so satisfactory enough for large scale biotransformations. The E. coli-based biocatalyst expressing the engineered enzyme by using the constitutive promoter was applied for ricinoleic acid biotransformation at high cell density culture in a 5 L and 70 L scale bioreactor
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