Hee-Kyoung Ryu

Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli

The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for beta-carotene production in recombinant Escherichia coli harboring beta-carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of beta-carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and beta-carotene synthesis genes produced high amount of beta-carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains - MG1655, DH5alpha, S17-1, XL1-Blue and BL21 - the DH5alpha was found to be the best beta-carotene producer. Using glycerol as the carbon source for beta-carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5alpha harboring the whole MVA pathway and beta-carotene synthesis genes produced beta-carotene of 465mg/L at glycerol concentration of 2% (w/v).

Enhanced Vanillin Production from Recombinant E.coli Using NTG Mutagenesis and Adsorbent Resin

Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl‐CoA synthase) and ech (enoyl‐CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5α harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin‐resistant mutant of NTG‐VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD‐2 resin absorption. The vanillin production of NTG‐VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild‐type strain. When 50% (w/v) of XAD‐2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG‐VR1 was 2.9 g/L, which was 2‐fold higher than that obtained with no use of the resin.