Part:BBa_K1465229

Sedoheptulose 1,7-bisphosphatase (glpX) from Bacillus methanolicus under the control of ptac

Sedoheptulose 1,7-bisphosphatase

The SBPase is one of enzymes needed for the Calvin cycle. It catalyzes the reaction from sedoheptulose 1,7-bisphosphate to sedoheptulose 7-phosphate (Figure 1). The enzyme is characteristic for the part of sedoheptulose 7-phosphate regeneration in the Calvin-cycle. It was shown before that oveerexpression of the SBPase in tobacco results in enhanced carbon assimilation and crop yield (Rosenthal et al., 2011). SBPases are homodimers with two identical subunits of 35kD to 38kD. The K_M-value of the SBPase homologue GlpX of Bacillus methanolicus is 14 ± 0.5 µM (Stolzenberger et al., 2013).
E. coli does not have a SBPase homologue which is needed E. coli for enabling the whole cycle.

Figure 1: Reaction of sedoheptulose 1,7-bisphosphatase

Characterization

We decided to use glpX as a target for amplification and transformation because of the shown acitivity (BBa_K1465228). The finished construct of ptac_glpX in pSB1C3 was cultivated in M9 glucose in comparison to the wild type (Figure 2). We performed two biological replicates and two technical replicates.

Figure 2: Cultivation experiment in M9 glucose (two technical and two biological replicates)

The cultivation shows that the modified strain has an extended lag phase. Two hours after IPTG induction of the gene expression, a decrease of growth was observed in comparison to the uninduced strain. There are two possible explanations for this behavior. On the one side IPTG acts as a toxic substance for bacteria which may result in growth decrease and on the other side the production of the protein can result in decreased growth. We exclude IPTG as a reason because earlier cultivations showed that 1 mM IPTG has no effects on the wild type growth. Furthermore the wild type strain shows an one hour reduced lag-phase in comparison to the induced strain. The induced strain also shows a higher OD. By inducing the SBPase in E. coli the substances for glycolysis are deflected towards other pathways. These reactions are reversible which means that the glucose of the M9 medium is not metabolized in another pathway. This time shifted use of glucose results in a higher OD in two biological replicates.
This result shows that the SBPase does not limit the growth maximum of E. coli. The glucose concentration confirms these results. The wild type consumes the glucose faster than the mutant strains.


Figure 3: Cultivation experiment in M9 xylose

The cultivation was repeated with xylose (Figure 3). Xylose could be suitable additional carbon source if the Calvin cycle has a lower efficiency. This second cultivation shows the impact of the induction more clearly. The induced mutant strain shows a lower growth rate in comparison to the uninduced mutant and the wild type. This indicates that the lack of glucose in the medium has a large impact because necessary intermediates for the glycolysis were used by the SBPase.

References

• Rosenthal et al., 2011. Overexpressing the C(3) photosynthesis cycle enzyme sedoheptulose 1,7-bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO(2) fumigation (FACE). BMC Plant Biol., vol. 11, pp. 123
• Stolzenberger et al., 2013. Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphate from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus. Journal of Bacteriology, Vol. 195, pp. 5112-5122







Sequence and Features




Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]