Team:Tokyo-NoKoGen/g3dh

About G3DH

　The enzyme, glucoside 3-dehydrogenase (E.C.1.1.99.13) or glucose-3-dehydrogenase (G3DH) catalyzes the oxidation of the C-3 hydroxyl group of the glucosides and converting them to corresponding 3-ketoglucosides (1). Because G3DH has wide substrate specificity, it can convert not only monosaccharides but disaccharides, including trehalose.

　G3DH is composed of three subunits: catalytic subunit, cytochrome c subunit, and small subunit. The catalytic subunit has a flavin adenine dinucleotide (FAD) as 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, formerly known as Agrobacterium tumefaciens. The enzymatic activity of G3DH in this microorganism was first reported in 1967 (2). The gene encoding this enzyme was found from a putative proteins which is homologous to G3DH derived from Halomonas sp. α-15 (3), which was reported to convert trehalose to 3,3’dkT (see below). The functional expression of R.tumefaciens derived putative enzyme confirmed that the gene encodes the G3DH complex (unpublished data from Sode laboratory, Tokyo univ. of Agric. and Technol.).

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)(3).

Construction of Biobrick

　We amplified G3DH gene from Rhizobium tumefacience by PCR. PCR products were inserted in pSB1C3. Original G3DH gene has two illegal EcoRI restriction sites. In order to remove these restriction sites, the G3DH gene was 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. Then, G3DH (removed illegal restriction sites) were ligated with four promoters and double terminator (BBa_B0010 and BBa_B0012).

Fig 2. Constructed plasmid

　One of those promoters is arabinose inducible, and the others are constitutive promoter.

Table 1. Promoter

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 (3).

This is the method of culturing and extraction of production (Fig. 3).

　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 P_BAD as a promoter.

　After culturing for 20 hours at 30 ℃, we extracted products by boiling and centrifugation. To investigate the inhibition effect of products, 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 P_medium and P_high, P_BAD. Therefore, we confirmed the expression of G3DH (Fig. 2).

Fig. 4 SDS-PAGE analysis

Measurement of glucose dehydrogenase activity

　We measured the oxidase activity of G3DH by PMS-DCIP assay (Fig. 5). On this assay, we measured the decrease in absorbance of DCIP at 600 nm. Substrates were glucose and trehalose. PMS stands for phenazine methosulfate, and DCIP stands for 2,6-dichlorophenol-indophenol.

Fig.5 PMS-DCIP assay

Trehalase inhibition measurement

　This is the result of trehalase inhibition activity assay(Fig. 6). 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 P_high and P_medium activity was lower than that of empty vector.

　We concluded that G3DH which induced by P_high and P_medium expressed G3DH. And G3DH converted trehalose to 3,3’-dkT.

Fig. 6 Trehalose inhibition activity assay

Reference

(1) K Kojima et al., (2001) Cloning and Expression of Glucose 3-Dehydrogenase from Halomonas sp. α-15 in Escherichia coli. Biochem Biophys Res Commun, 282, 21-27

(2) K Hayano et al., (1967) Purification und properties of 3-ketosucrose-forming enzyme from the cells of Agrobacterium tumefaciens. J. Biol. Chem.,　242, 3665-3672

(3) K Sode et al., (2001) Enzymatic synthesis of a novel trehalose derivative, 3,3’-diketotrehalose, and its potential application as the trehalase enzyme inhibitor. FEBS Letters, 489, 42-45

(4) E Arslan et al., (1998) Overproduction of the Bradyrhizobium japonicum c-Type Cytochrome Subunits of the cbb3 Oxidase in Escherichia coli. Biochem Biophys Res Commun, 251, 744-747

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Future work

　 We respectively evaluated trehalose production and 3,3’-dkT. Therefore, we are going to transform E. coli with 3 different plasmids each expresses G3DH, OtsA and OtsB and cytochrome c maturation enzymes at the same time.

　To Exterminator coli eradicate only cockroaches, we are going to introduce it to express some pheromone. And if we want to create the Exterminator coli for other insects, we can engineered it by introducing specific pheromone for each insect.

　On our project, we used OtsA and OtsB to produce trehalose, but E. coli and other microorganism have different kind of pathway to produce trehalose. Therefore we plan to use other kind of enzyme to produce trehalose, and aim the increase of the trehalose producing amount.