Abstract
3′-Untranslated region (3′UTR) engineering was investigated to improve solubility of heterologous proteins (e.g., Baeyer-Villiger monooxygenases (BVMOs)) in Escherichia coli. Insertion of gene fragments containing putative RNase E recognition sites into the 3′UTR of the BVMO genes led to the reduction of mRNA levels in E. coli. Importantly, the amounts of soluble BVMOs were remarkably enhanced resulting in a proportional increase of in vivo catalytic activities. Notably, this increase in biocatalytic activity correlated to the number of putative RNase E endonucleolytic cleavage sites in the 3′UTR. For instance, the biotransformation activity of the BVMO BmoF1 (from Pseudomonas fluorescens DSM50106) in E. coli was linear to the number of RNase E cleavage sites in the 3′UTR. In summary, 3′UTR engineering can be used to improve the soluble expression of heterologous enzymes, thereby fine-tuning the enzyme activity in microbial cells.
Highlights
Mostly produced in an insoluble form in E. coli
According to a recent study, stability of Salmonella enterica hilD mRNA was markedly influenced by the presence or absence of its 3′UTR, which consists of 310 nucleotides including fourteen putative RNase E cleavage sites (Fig. S2A)[30]
When ricinoleic acid was added into the reaction medium containing E. coli pCOLA-alcohol dehydrogenase (ADH) and pET22b-BmoF1-3′UTRhilD, the BmoF1 reaction product (3) was produced at a specific formation rate and bioconversion yield, which were significantly higher than using the recombinant E. coli pCOLA-ADH and pET22b-BmoF1 with 3′UTRnative (Fig. 1C,D). These results suggested that insertion of the 3′UTRhilD sequence might result in a decrease of the stability of the bmoF1 mRNA, which might in turn reduce the BmoF1 expression level, but overall improve the gene expression in a functional form in E. coli BL21(DE3)
Summary
Mostly produced in an insoluble form in E. coli. The 3′UTR engineering allowed a substantial increase of biotransformation activities of the E. coli cells expressing the BVMO enzymes. After construction of E. coli BL21(DE3) pET22b-BmoF1-3′UTRCAT to contain the full-length CAT coding sequence in the 3′UTR, the bmoF1 mRNA level was compared with E. coli BL21(DE3) pET22b-BmoF1 with 3′UTRnative, which contains 5 putative RNase E recognition sites, by RT-PCR (Fig. S3B) and qRT-PCR (Fig. 2A).
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