Team:Tartu TUIT/Project/Results
Project: Yeasthylene
The demand on ethylene has only been increasing during the last decade. It is used as an essential building block in many chemical compounds.The main aim of our project is to find an alternative and biological way of producing ethylene. That is why we have decided to genetically engineer yeast cells to produce ethylene from sucrose.
Results
Experiment for measuring ethanol production of IMU051 strain
(strain A) under different glucose concentrations.
(strain A) under different glucose concentrations.
Introduction
In our project, we are going to use two co-cultured yeast strains with totally dissimilar roles. As our strain A is responsible for the conversion of hexose sugars into ethanol, we decided to conduct the following experiment in order to measure the amount of ethanol produced from different concentrations of glucose.
Materials and methods
The strain IMU051 was inoculated in 25 ml of YPD medium overnight at 220 rpm and 30 ºC. Then the overnight culture was inoculated into CSM medium supplemented with different concentration of glucose (1.0, 2.5, 5.0 and 8.0 g/L) to an initial OD600 of 0.02. One milliliter samples were taken every two hours and the growth was estimated by measuring OD at 600 nm followed by centrifugation at 3000 g for five minutes and ethanol and glucose concentrations in the supernatant were 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). Dry cell biomass (DCW) was calculated based on the calibration curve DCW (g/L)=0.3726*(OD600)-0.0522
Results
As it can be seen from the Figures 1 and 2, the growth and ethanol production kinetics were dependent on the glucose concentration, with the maximum growth and production being obtained with 8 g/L of glucose (the maximum OD value of 6.41, 2.3 g/L of dry biomass, and 3.2 g/L of ethanol). After the depletion of glucose (Figure 3) cells started using ethanol as their main carbon source, therefore, as suggested by our modelling, a continuous or fed-batch system would be more appropriate for our project. This approach would assure that most of ethanol would be available for strain B. Another possible solution would be to delete the ADH2 gene which encodes for the alcohol dehydrogenase 2 that catalyses the conversion of ethanol into acetaldehyde.
Figure 1. Growth kinetics of IMU051 strain using different concentrations of glucose
Figure 2. Ethanol production kinetics of IMU051 strain using different concentrations of glucose
Figure 3. Glucose consumption kinetics of IMU051 strain using different concentrations of glucose
Experiment for growth and sucrose conversion of strain B
under different ethanol concentrations
under different ethanol concentrations
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 derived 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, the expression of which 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 over-express 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].
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
As it can be seen from the Figures 4 and 5, both EBY 4000 and our strain B were consuming ethanol very slowly over a long period of time. There was an error in ethanol consumption reading of the strain B in 5 g/L ethanol medium, which was probably caused by pipetting and dilution errors.
Figure 6 shows that in the medium containing 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 over-expression 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 7) followed by the overgrowth of the B strain (1.7 AU compared to 1 AU reached by the control strain).
Figure 4. Kinetics of ethanol consumption by EBY 4000 strain
Figure 5. Kinetics of ethanol consumption by EBY 4000:: TEF2+SUC2+CYC1 strain
Figure 6. Kinetics of sucrose breakdown in 5 g/L ethanol medium
Figure 7. Growth kinetics in 5 g/L ethanol medium
