Part:BBa_K2349002
SUC2
Invertase for sucrose hydrolysis into glucose and fructose; this enzyme is usually secreted. Invertase for sucrose hydrolysis into glucose and fructose; this enzyme is secreted into the medium
.Introduction
EBY 4000 strain had more than 20 hexose transporters knocked out which made it unable to transport hexose sugars. As a result of these deletions, the strain is only able to use ethanol (produced by the IMU051 strain) as its main carbon source. Since this strain is derivate from the CEN.PK 2-1C strain it has a SUC2 gene (encodes for a secreted invertase which breaks down sucrose into glucose and fructose) integrated into the genome, whose expressions is highly dependent on the glucose concentration in the medium: induced by low glucose concentration, but repressed by high concentrations of the same sugar. We have transformed the EBY 4000 with TEF2+SUC2+CYC1 BioBrick in order to overexpress the SUC2 gene. This will also relieve the glucose-dependency of SUC2 expression. The EBY 4000 TEF2+SUC2+CYC1 (hereafter the strain B) was used for the characterization of the SUC2 part. In this experiment, we looked further into the growth kinetics of our strain in different concentrations of ethanol along with measuring the sucrose conversion rate by the strain B compared to the one of EBY 4000, which was used as a control.
Material and methods
The strains EBY 4000, EBY 4000 TEF2 SUC2 CYC1 were inoculated into 50 ml of CSM medium supplemented with 10 g/L ethanol and incubated overnight at 220 rpm and 30 ºC. The overnight cultures were used as inoculum to 25 ml of CSM medium supplemented with 10 g/L of sucrose and different concentrations of ethanol (2.5, 5.0, and 8.0 g/L). In order to measure growth, ethanol consumption and sucrose conversion into glucose and fructose 1 ml samples were taken at different time points. Cell growth was estimated by measuring OD at 600 nm followed by centrifugation at 3000 g for five minutes and dry cell biomass (DCW) was calculated based on the calibration curve DCW (g/L)=0.3726*(OD600)-0.0522. Ethanol consumption was measured by HPLC (sulfuric acid at 5 mM at a flow rate of 0.6 ml/min as mobile phase, HPX-87H was used as stationary phase at 45 ºC). Sucrose conversion was measured in terms of reducing sugars by the method of Miller et al. 1959 [1]
.Results
Figure 1 shows that in the medium containing the strain B, all sucrose has been converted into hexose sugars already 90 minutes after the start of the experiment. On the other hand, the control strain needed more than 14 h to break down all sucrose. This strongly suggests that the SUC2 submitted part, central to our project, is working and is more efficient than the native gene. Furthermore, the overexpression of SUC2 did not have a negative influence on the growth of strain B, as their growth kinetics were similar up to 6 h (Figure 2) followed by the overgrowth of the B strain (1.7 AU compared to 1 AU reached by the control strain).
FIGURE 1. Kinetics of sucrose breakdown in 5 g/L ethanol medium.
FIGURE 2. Growth kinetics in 5 g/L ethanol medium.
[1.] Miller, G. L. (1959). Modified DNS method for reducing sugars. Anal. Chem, 31(3), 426-428.
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