Difference between revisions of "Part:BBa K2448033"

Revision as of 19:04, 1 November 2017

D-Psicose 3-epimerase (Dpe) from Clostridium cellulolyticum under the control of pTacI promoter

This part is an E. coli codon optimized version of the D-Psicose 3-epimerase (Dpe) from Clostridium cellulolyticum str. ATCC 35319 (Uniprot B8I944, BBa_K2448021) equipped with a strong RBS (BBa_K2448019) and placed under the control of the pTacI promoter (BBa_K864400).

Usage and Biology

D-Psicose 3-epimerase (EC: 5.1.3.30) catalyses the reversible epimerization of D-fructose into D-psicose, D-psicose being the carbon-3 (C3) epimer of D-fructose.

The C. cellulolyticum Dpe is a 33.034 kDa protein of 293 amino acids.

This enzyme is highly specific for D-psicose, but it is also able to catalyse with very low activity the epimerization of D-tagatose [1]. For optimal activity, it requires cofactors such as Mn2+ or Co2+, although it has a low basal activity without ions. The native Dpe shows a tetrameric arrangement of 132 kDa, and each subunit has 293 amino acids with a molecular weight of 33 kDa. The topology of each subunit is a TIM-barrel fold with a cluster of eight β-strands surrounded by twelve α-helices [2].

Bioproduction of Psicose in Escherichia coli

To bioproduce psicose from fructose we had to choose from many bioproduction methods. Since our goal was to test if we were able to produce psicose with this part we chose the fastest and cheapest method: Batch cultures for a whole cell production. Indeed, this technique requires little material and offers strong resistance to environmental perturbations. It also limits the purification steps compared to in vitro or cell lysate. This is essential for us since we want to directly measure our psicose with an HPLC. This method has still some limitations such as culture conditions that do not match those of the enzyme, therefore our yields may be limited.

Study of the strains and culture conditions

We first investigated in which strain we would perform our psicose bioproduction. We were interested in two strain of E. coli: Top10, a strain often used for cloning and biosensor characterization, with a fast growth and a high plasmid replication; and BL21-AI, a strain used for expression of recombinant proteins. We compared the growth of these strains. The cultures were made in Synthetic Medium to ensure its exact composition and avoid column deterioration in further HPLC measurements. We also wanted to investigate the impact of the carbon source on the growth of these two strains. We used four kinds of carbon sources: Glucose, Fructose, Glucose + Fructose and Glycerol + Fructose. The BL21-AI strains had high growth rates regardless of the sugar, with a stationary phase reached in five hours. The carbon source has no impact on this strain. The growth of the Top10 strain is slower compared to BL21-AI. The sugar used in the medium has a great impact on the fitness of the strain, with a maximal optical density for the fructose condition. Therefore, we decided to do all further bioproduction assays in BL21-AI.

Impact of the concentration of fructose on the psicose production

Knowing that the sugar used had no impact we decided to use a standard concentration of 2 g/L of glucose for every culture and vary the fructose concentration. To test whether or not our strains were producing psicose we prepared a negative control, a BL21-AI strain transformed with pSB1C3 containing BBa_J04450, an RFP under the control of a constitutive promoter (BL21-AI-RFP). Cultures have been made for 24 hours, with IPTG induction when the culture reached an OD600 of 0.6. Samples were taken throughout the bioprocess and centrifuged to purify the samples. Then the psicose contained in each fraction was measured with an HPLC previously calibrated to quantify sugars such as glucose, fructose, glycerol and psicose.

As shown in figure 2, we had similar growths for every strain except for BL21AI-DPE with 2 g/L of fructose which has a delayed growth. The HPLC analysis showed us that BL21-AI-DPE is able to produce up to 9 g/L of psicose from 50 g/L and 80 g/L of fructose in the medium and that there is a proportional relationship between the fructose concentration and the psicose measured. No psicose was produced with our negative control which was expressing RFP.

In these results we did not reach saturation of the enzyme with 80 g/L of fructose. Therefore, we wanted to test the highest concentration of fructose and see if the yield could be enhanced. So, we used higher concentrations of fructose and we observed that only the cultures with 50 g/L of fructose showed a high growth rate. The increase in the fructose concentration is inversely proportional to the growth of this strain. The high concentrations might create an osmotic stress on the cultures. We decided to use a concentration of 50 g/L of fructose to ensure high levels of growth and psicose production.

Figure 2. Growth curves and psicose production of BL21-AI transformed with this part and BL21-AI transformed with (BBa_J04450), depending on the fructose concentration: 2 g/L, 20 g/L, 50 g/L or 80 g/L. A- Growth curves of cultures in Synthetic Medium with varying fructose concentrations. B- Psicose production. BL21-AI-RFP is used as a negative control with no Dpe.

Sequence and Features