Team:Tokyo-NoKoGen/trehalose production

Revision as of 13:37, 16 October 2014 by Mayumin (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

	Project	Team	Result	Parts	Protocols	Notebook	Safety	Human Practice	Achievement

About otsA and otsB

The trehalose biosynthetic operon – otsA and otsB

Trehalose (α-D-glucopyranosyl-[1,1]-α-D-glucopyranoside) is a non-reducing disaccharide in which two glucose units are linked by an α,α-1,1 bond and is common in nature. In yeast and fungi, trehalose plays an important role in protection against environmental stresses such as desiccation, heat, frost, and high osmolarity. The pathway for trehalose synthesis induced by osmotic stress in E. coli is the same as in other species.

The genes otsA and otsB are required for trehalose production. The otsA gene encodes trehalose-6-phosphate synthase. This enzyme converts UDP-glucose and D-glucose-6-phosphate to trehalose-6-phosphate. The otsB gene encodes trehalose-6-phosphate phosphatase. This enzyme converts trahalose-6-phosphate to trehalose.

The operon otsBA is induced by osmotic stress, extreme heat, extreme cold, desiccation, and entry into stationary phase. Therefore, we determined to culture engineered E. coli under high salt level in order to overexpress the genes.

Reference
(1) Crowe, J. H. et al., Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stabilizing solutes with biomolecules., Cryobiology (1990) Vol. 27 No. 3 pp. 219-231.
(2) Crowe, J. H. et al., Anhydrobiosis., Annu Rev Physiol. 1992;54:579–599.
(3) Kaasen, I. et al., Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by katF (AppR)., J Bacteriol. Feb 1992; 174(3): 889–898.
(4) Giaever, H. M. et al., Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli., J Bacteriol. Jun 1988; 170(6): 2841–2849.
(5) Eastmond, P. J. et al., Trehalose metabolism: a regulatory role for trehalose-6-phosphate?, Current Opinion in Plant Biology 2003, 6:231–235.
(6) Strom, A. R. et al., Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression., Molecular Microbiology (1993) 8(2), 205-210.

Cloning otsA and otsB

1.Construction of Biobrick.

otsA gene and otsB gene were cloned from E.coli K-12 strain. PCR products were digested with EcoRI and PstI and digested products were inserted into pSB1A2.
otsB gene and otsA gene 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.

Evaluation of trehalose production

Optimization of culture condition

It has been reported that trehalase derived from host E. coli was expressed higher when E. coli reach to stationary phase. At first, we monitored the growth of transformants and optimized the growth condition for further experiments.

We added 600 mM NaCl and 2% Glucose to LB medium because it is known that storage of trehalose is increased under osmotic stress and glucose is a precursor of trehalose. Escherichia coli strain TOP10/ pSB1A2-P109-RBS-otsAB (BBa_K1339018) was cultured in LB medium containing 600 mM NaCl and 2% glucose at 150 rpm, 30 ℃

Fig.1 shows the growth curve of transformants. From the result, we found that this transformants reached stationary state after 15 hours cultivation. So, we decided to culture the cell harboring otsBA for12 hours (while growth state) for further evaluation.

Fig. 1 OD660 monitoring of TOP10/pSB1A2-empty and TOP10/pSB1A3-P109-RBS-otsBA-DT

Optimization of trehalose detection by TLC method

We used TLC plate coated with silica gel. As standard sample, 1 m l of 1, 5, 10, 50 and 100 mM glucose and trehalose were spotted onto TLC plates and dried at room temperature. The TLC plates were developed in acetonitrile –water (7:3), dried and dipped into H ₂ SO₄-ethanol (5:95). Fig. 2 showed the result of TLC of glucose and trehalose. The Rf value of glucose is approximately 0.47. And the Rf value of trehalose is approximately 0.35. The detection limit of glucose and trehalose were 5 mM and 10 mM respectively.

Thus, in our experiments, we tried to confirm the production of trehalose by E.coli by this method.

< br>

Fig. 2 the result of TLC of standard samples; glucose and trehalose

Optimization of enzyme reaction time

To measure the concentration of trehalose, trehalose was converted to glucose with trehalase, which was then measured using a glucose dehydrogenase. At first, we investigated the optimal enzyme reaction time. Trehalase was added to 50 mM trehalose solution and incubated for 30 min at 37℃.

This is the result of TLC of three samples (Fig. 3). The Rf value of the trehalose+trehalase, trehalose, and glucose were 0.42, 0.37 and 0.45 respectively. From this result, the time of trehalase reaction was not enough. Thus we dicided to incubate for overnight.

Fig. 3 TLC after trehalase reaction

ここ

3.Confirm the production of trehalose by TLC

Method

Escherichia coli strain TOP10 was transformed with the vectors that constitutively express otsA and otsB under different strength promoter (Table.) Transformants were cultured in Lysogeny Broth medium (LB medium) containing 600 mM NaCl and 2% Glucose) at 150 rpm and 37C for 20 hours. The transformants were harvested and washed with phosphate buffered saline containing 600 mM NaCl and then, resuspended with ultra pure water. Trehalose was extracted by boiling cell pellets at 95C for 5 min and cells were removed by centrifugation at 8,000 g for 15 min.

The supernatant was concentrated by freeze-drying and used for thin-layer chromatography (TLC).
We used TLC plate coated with silica gel. 1 m l of each samples were spotted onto TLC plates and dried at room temperature. The TLC plates were developed in acetonitrile –water (7:3), dried and dipped into H ₂ SO₄-ethanol (5:95). The sugar spots were visualize by heating at 180°C.
The pellet was used for SDS-PAGE.

Result

As standard sample, 1, 5, 10, 50 and 100 mM glucose and trehalose were spotted and analyzed by TLC. Fig. 2 showed the result of TLC of glucose and trehalose. The Rf value of glucose is approximately 0.47. And the Rf value of trehalose is approximately 0.35. The detection limit of glucose and trehalose were 5 mM and 10 mM respectively. Thus, in our experiments, we tried to confirm the production of trehalose by E.coli by this method.

Confirm the production of trehalose by glucose dehydrogenase activity

Then we approached the possibility 2; trehalase reaction time is not enough. This is the result of TLC of three samples (Fig. 5). These are the solution including trehalose and trehalase (trehalose+trehalase)(incubate time for reaction of trehalase : 30 min), trehalose and glucose.

The Rf value of the samples of trehalose+trehalase, trehalose, and glucose were 0.42, 0.37 and 0.45 respectively.
We found that the time of trehalase reaction was not enough. Thus we tried to incubate for overnight.