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

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.

Enzyme fusion for whole-cell biotransformation of long-chain sec-alcohols into esters.

Enzyme fusion was investigated as a strategy to improve productivity of a two-step whole-cell biocatalysis. The biotransformation of long-chain sec-alcohols into esters by an alcohol dehydrogenase (ADH) and Baeyer-Villiger monooxygenases (BVMOs) was used as the model reaction. The recombinant Escherichia coli, expressing the fusion enzymes between the ADH of Micrococcus luteus NCTC2665 and the BVMO of Pseudomonas putida KT2440 or Rhodococcus jostii RHA1, showed significantly greater bioconversion activity with long-chain sec-alcohols (e.g., 12-hydroxyoctadec-9-enoic acid (1a), 13-hydroxyoctadec-9-enoic acid (2a), 14-hydroxyicos-11-enoic acid (4a)) when compared to the recombinant E. coli expressing the ADH and BVMOs independently. For instance, activity of the recombinant E. coli expressing the ADH-Gly-BVMO, in which glycine-rich peptide was used as the linker, with 1a was increased up to 22 µmol g dry cells(-1) min(-1). This value is over 40 % greater than the recombinant E. coli expressing the ADH and BVMO independently. The substantial improvement appeared to be driven by an increase in the functional expression of the BVMOs and/or an increase in mass transport efficiency by localizing two active sites in close proximity.

Isolation and Characterization of Kimchi Starters Leuconostoc mesenteroides PBio03 and Leuconostoc mesenteroides PBio104 for Manufacture of Commercial Kimchi.

This study was focused on developing and obtaining a kimchi starter for use in commercial kimchi production. Kimchi varieties made with selected starters are of high quality, have high levels of mannitol, and extended shelf life. The starters were screened for properties such as mannitol production, low gas/acid production, and acid resistance. Finally, kimchi fermentation testing was performed using selected LAB starters. Kimchi samples were prepared with lactic acid bacteria (LAB) starters, including Leuconostoc mesenteroides PBio03 and Leuconostoc mesenteroides PBio104. The LAB starters are isolated from kimchi and can grow under pH 3.0 and low temperature conditions of 5°C. Four kimchi samples were fermented and stored for 28 days at 5°C. The kimchi samples made with starters (PBio03 and PBio104) had better quality (production of mannitol and maintenance of heterofermentative LAB dominance) than the non-starter kimchi samples. In the starter kimchi, Leu. mesenteroides was the dominant LAB, comprising 80% and 70% of total LAB counts at 7 and 21 days, respectively. Mannitol content of the kimchi with Leu. mesenteroides PBio03 was 1,423 ± 19.1 mg/ 100 g at 28 days, which was higher than that of the non-starter kimchi sample (1,027 ± 12.2 mg/100 g). These results show the possibility of producing kimchi with improved qualities using Leu. mesenteroides PBio03 and PBio104 as starters.