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Team:Tokyo-NoKoGen/g3dh
From 2014.igem.org
About G3DH
The enzyme glucose-3-dehydrogenase (G3DH) converts trehalose to 3,3’-diketotrehalose (3,3’-dkT), a trehalase inhibitor. This enzyme can oxidize the 3rd hydroxyl group of pyranose (1). Because G3DH has wide substrate specificity, it can convert not only monosaccharides but disaccharides.
G3DH is composed of three subunits: a catalytic subunit, a cytochrome c subunit, and a small subunit. The catalytic subunit has a flavin adenine dinucleotide (FAD) cofactor, and the cytochrome c subunit is bound to the cytoplasmic membrane in the periplasm. In our project, we used G3DH derived from Rhizobium tumefaciens EHA101. This enzyme is a homolog of G3DH derived from Halomonas sp. α-15, which was reported to convert trehalose to 3,3’dkT.
About 3,3'-diketotrehalose (3,3'-dkT)
3,3’-dkT is a novel trehalose derivative in which the third hydroxyl group of both glucose moieties are oxidized. It was already reported that 3,3-dkT showed an inhibitory effect toward the trehalose from pig-kidney and Bombyx mori (silkworm).(2)
Reference
(1) K Kojima et al., (2001) Cloning and Expression of Glucose 3-Dehydrogenase from Halomonas sp. α-15 in Escherichia coli. 282, 21-27
(2) K Sode et al., (2001) Enzymatic synthesis of a novel trehalose derivative, 3,3’-diketotrehalose, and its potential application as the trehalase enzyme inhibitor. 489, 42-45.
1.Construction of Biobrick
G3DH gene were cloned from pTrc99a-G3DH which originated from Agrobacterium tumefaciens. PCR products were inserted pSB1C3. Original G3DH gene has two illegal restriction sites. In order to remove these restriction sites, the G3DH gene were amplified by overlap extension PCR. We designed two primer sets for overlap extension PCR.
Fig.1. Remove illegal restriction sites
G3DH gene fragments were amplified and three PCR products were connected.
G3DH (removed illegal restriction sites) were ligated with four promoters and double terminator (BBa_B0010 and BBa_B0012).
One of those promoters is arabinose inducible, and the others are constitutive promoter.
Table 1. Promoter
Fig 2. Constructed plasmid
2. Evaluation
This G3DH needs the maturation of cytochrome c subunit to have catalytic activity. Therefore, we transformed G3DH with the plasmid, pEC86; which expresses cytochrome c maturation (CCM) enzymes (1).
This is the method of culturing and extraction of production (Fig. 3).
Culture condition and evaluation method
We cultured E. coli TOP10 transformed with two plasmids; G3DH expression vector, and cytochrome c maturation enzymes expression vector (pEC86) in LB medium containing 20 mM Trehalose. For expression of G3DH, we used four different promoters.
When OD660 achieves 0.6, 0.2 % arabinose was added to the medium for induction in the case we used PBAD as a promoter.
After culturing for 20 hours at 37C, we extracted products by boiling and centrifugation.
0.007U trehalase and 20 mM trehalose mixed sample and incubated 30h.
First, we investigated expression of G3DH by SDS-PAGE analysis. Second, we measured the glucose dehydrogenase activity of G3DH. Finally we tried to detect 3,3’-dkT by thin-layer chromatography (TLC).
Fig. 3 evaluation of G3DH
SDS-PAGE
By SDS-PAGE analysis, there was the band which showed about 68 kDa on G3DH under the constitutive promoter Pmedium and Phigh, PBAD. Therefore, we confirmed the expression of G3DH (Fig. 2).
Fig. 4 SDS-PAGE analysis
Trehalase inhibition measurement (Fig. 3)
Fig. 3 Trehalose inhibition activity assay
This is the result of trehalase inhibition activity assay. Each value of activity was normalized at the value of activity of empty vector which were not induced by arabinose. Samples of G3DH which induced by Phigh and Pmedium activity was lower than that of empty vector.
We concluded that G3DH which induced by Phigh and Pmedium expressed G3DH. And G3DH converted trehalose to 3,3’-dkT.
Reference
(3) E Arslan et al., (1998) Overproduction of the Bradyrhizobium japonicum c-Type Cytochrome Subunits of the cbb3 Oxidase in Escherichia coli. 251, 744-747
