Team:Tokyo-NoKoGen/trehalose production

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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(1, 2). The pathway for trehalose synthesis induced by osmotic stress in E. coli is the same as in other species(3, 4).

　The genes otsA and otsB are required for trehalose production in E. coli. 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 trehalose-6-phosphate to trehalose.

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

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_B0015)(Fig.1).

Fig.1 Constructed plasmids

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

Table.1 Promoter

Evaluation of trehalose production

1. 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 (1). 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 (2). Escherichia coli strain TOP10/ pSB1A2-P_low-RBS-otsBA (BBa_K1339018) was cultured in LB medium containing 600 mM NaCl and 2% glucose at 150 rpm, 30 ℃.

　Fig.2 shows the growth curve of transformants. From the result, we found that this transformant reached stationary state after 15 hours cultivation. So, we decided to culture the cells harboring the cell harboring otsBA for 12 hours (while exponential growth) for further evaluation.

Fig.2 OD₆₆₀ monitoring of empty vector and P_low-RBS-otsBA-DT

2. Optimization of trehalose detection by thin-layer chromatography (TLC) method

　We used TLC plate coated with silica gel. As standard sample, 1 μL 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. 3 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.

Fig.3 The result of TLC of standard samples; glucose and trehalose

3. 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 ℃.

　 Fig.4 is the result of TLC. 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 decided to incubate for overnight.

Fig.4 TLC after trehalase reaction

4. Evaluation of trehalose production in Escherichia coli

　Escherichia coli strain TOP10 was transformed with the vectors that constitutively express otsA and otsB under different strength promoter (Table.). Transformants were cultured in LB medium containing 600 mM NaCl and 2% Glucose) at 150 rpm and 37 ℃ for 12 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 ℃ 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 TLC. 1 μL 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 visualized by heating at 180 ℃ (Fig.5).

Fig.5 Flowchart of experiment

TLC

The spot of a transformant harboring otsBA under constitutive promoter Pmedium was almost the same height as the standard sample of trehalose. On the other hand, the spot of a transformant harboring empty vector was not detected (Fig.6). This result suggested that trehalose was produced by OtsB and OtsA in E. coli.

Fig.6 The result of TLC (OtsB+OtsA)

Improving a BioBrick part - OtsB

BBa_K200017

　In iGEM 2009, Imperial College London team submitted OtsB part (BBa_K200017). However, evaluation data was not available in the parts registry, so we tried to evaluate the BBa_K200017 and compared with our BioBrick part encoding OtsB (BBa_K1339001). Promoter (medium constitutive promoter or low constitutive promoter) and double terminator was ligated to otsB, and inserted into pSB1A2 vector by 3A or standard assembly.

　Escherichia coli strain TOP10 transformed with the constructed vectors were cultured in LB medium containing 600 mM NaCl and 2% Glucose at 150 rpm and 37 ℃ for 20 hours. The transformants were harvested and washed with phosphate buffered saline containing 600 mM NaCl and then, resuspended with SPB buffer. Cells were fractured by sonication and then centrifugated at 8,000 g for 15 min. Trehalose 6-phosphate, which is a precursor of trehalose was added to the supernatant and incubated at 37℃ overnight. The supernatant was concentrated by freeze-drying and subjected to thin-layer chromatography (TLC) to detect trehalose (Fig. 7). Moreover, the expression of otsB was confirmed by SDS-PAGE using the cell pellet (Fig. 8).

TLC

This is the result of TLC. As standard sample, trehalose and trehalose-6-hosphate were spotted. We confirmed that the spot of E. coli/P_medium -otsB was located almost the same height as standard sample of trehalose. So, we concluded that trehalose was produced from trehalose-6-hosphate by OtsB.

Fig.7 The result of TLC (OtsB)

SDS-PAGE

OtsB's molecular weight is about 30 kDa. We confirmed the expression of OtsB.

Fig.8 The result of SDS-PAGE

Reference
(1) Crowe, J. H. et al., (1990) Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stabilizing solutes with biomolecules., Cryobiology. 27, 219-231.
(2) Crowe, J. H. et al., (1992) Anhydrobiosis., Annu Rev Physio. 54, 579–599.
(3) Kaasen, I. et al., (1992) 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. 174, 889–898.
(4) Giaever, H. M. et al., (1988) Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli., J Bacteriol. 170, 2841–2849.