Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer–Villiger monooxygenase

Abstract

We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts.

3′-UTR engineering to improve soluble expression and fine-tuning of activity of cascade enzymes in Escherichia coli

“…Not only engineering of gene expression systems and expression conditions, but also the introduction of molecular chaperones and directed evolution of the bmoF1 did not allow satisfactory functional expression in E. coli cells 12 24 . 5′UTR engineering, which was used to regulate expression level of the soluble proteins (e.g., green fluorescent protein 16 , phosphoenolpyruvate synthase 29 , γ-prefoldin and recombinant thermosome 20 , was also not so useful to improve soluble expression of the insoluble enzyme (data not shown).…”

“…Optimization of not only the induction conditions for gene expression (e.g., cultivation temperature, type and concentration of inducer), but also the gene expression systems including the promoters, ribosome binding sites (RBSs), 5′-untranslated region (5′UTR), and codon usage have been largely investigated to enhance soluble expression of enzymes and proteins 14 15 16 17 18 . In addition, introduction of molecular chaperones 19 20 , the fusion of proteins with soluble peptides and proteins 21 22 , and other protein engineering methods (e.g., directed evolution) 23 24 often allowed or improved functional expression of foreign proteins and enzymes in bacterial host cells.…”

Fatty acid hydration activity of a recombinant Escherichia coli‐based biocatalyst is improved through targeting the oleate hydratase into the periplasm

“…Lactones, flavors, surfactants, and plasticizers are also manufactured from hydroxy fatty acids [1–3]. In addition, C9–C13 ω‐hydroxycarboxylic acids, α, ω‐dicarboxylic acids, and ω‐aminocarboxylic acids are produced from long chain hydroxy fatty acids via multistep whole‐cell biocatalysis [4–11].…”

Engineering of Baeyer-Villiger monooxygenase-based Escherichia coli biocatalyst for large scale biotransformation of ricinoleic acid into (Z)-11-(heptanoyloxy)undec-9-enoic acid

“…Not only the induction conditions for gene expression (e.g., cultivation temperature, inducer type and concentration), but also the gene expression systems including the promoters, ribosome binding sites (RBSs), 5′-untranslated region (5′UTR), and codon usage have been largely investigated to enhance soluble expression of the enzymes11121314. In addition, introduction of molecular chaperones1516, the protein fusion with soluble peptides and proteins1718, introduction of disulfide bonds1920, and other protein engineering methods (e.g., directed evolution)2122 have also been intensively examined to increase functional expression and stability of the oxygenases in microbial cells. However, these approaches are not so satisfactory enough for large scale biotransformations.…”