The engineering of "Beauty G. hansenii" is the central goal of our project, which involves the construction and assembly of the three different modules (See Design for details). Hereby, we demonstrate the engineering success in the construction of the GshF expression and GSH production module with two “DBTL” cycles.
The biosynthetic pathway of GSH usually contains two ATP-dependent consecutive reactions that are catalyzed by γ-glutamylcysteine (γ-GC) synthetase (γ-GCS or GSH I) and GSH synthetase (GS or GSH II). However, it is difficult to accumulate high levels of intracellular GSH since the enzymatic activity of the native GSH synthetases (the above mentioned GSH I and GSH II) would be strongly inhibited by the GSH. To overcome this inhibition effect of the end-product, the gene encoding a bifunctional glutathione synthetase GshF originated from Streptococcus thermophilus, which is less sensitive to GSH, is selected and codon optimized for expressing in G. hansenii.
In this section, our goal is to construct the gshF expression system using the vector pSEVA331. The recombinant plasmid pSEVA331-J23102-RBS003422-gshF-T0 is referred to herein as "GF22". Firstly, the CDS region of gshF was amplified by high-fidelity PCR, purified and ligated to the linearized vector pSEVA331. Next, competent cells of E. coli TOP10 were transformed with the newly constructed plasmid and spread on selective agar plates. Finally, a few of monoclonal colonies formed by the E. coli TOP10 transformants were individually selected for examining the presence of the inserted gshF sequence using PCR. The size of PCR products (2306 bp) was determined using agarose gel electrophoresis. In this fashion, we screened for correct builds and obtained the expected plasmid.
Characterization of GshF: Western blot
Constitutive expression of our protein of interest, GshF labelled with His-Tag, was enabled by the expression system. Then the bacterial growth was launched in liquid broth to ensure to have sufficient biomass, and cell pellets were collected by centrifugation and further disrupted by ultrasonics. Raw protein matrix was then obtained from the supernatant after centrifugation. The protein was purified by the immobilized metal affinity chromatography (IMAC) loaded with high affinity Ni-NTA Resins. The molecular weight (approximately 85 kDa) of the purified protein products was finally determined using immunoblotting, as shown in Figure 4.
Detection of GSH production content: Colorimetry
The GSH content in the cell lysate of wild typeE. coli TOP10 and E. coli TOP10 containing GF22 were determined using Glutathione Colorimetric Detection Kit (Beyotime). The results shows that the GSH production of E. coli TOP10 containing GF22 is much higher (~92fold) than that of wild-type E. coli TOP10.
Our experimental results were within our expectation, indicating that our design is feasible in E. coli. This promotes us to expect that a high level of GSH in G. hansenii ATCC53582 can also be achieved if this plasmid was introduced into our final chassis.
The recombinant plasmid GF22 was firstly extracted from E. coli, and then introduced into our bacterial chassis using electroporation. Figure 6 showed that the size of the DNA fragments amplified from G. hansenii, thus confirming the successful incorporation of the plasmid.
G. hansenii containing the plasmid GF22 exhibited enhanced GSH biosynthsis, as evidenced by colorimetric analysis of glutathione (Figure 7).
To summarize, the GshF expression and GSH production module (BBa_K4325015) functions well in our final chassis G. hansenii. We plan to improve the system in the future to achieve higher GSH production.
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[2] Li, Wei , et al. "Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli." journal of biotechnology 154.4(2011):261-268.
[3] Dezheng, et al. "Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase." Journal of Industrial Microbiology & Biotechnology (2016).
[4] Xiong, Z.-Q. et al. Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus. J. Dairy Sci. 101, 6937-6945 (2018).
[5] Cui, X. et al. Efficient glutathione production in metabolically engineered Escherichia coli strains using constitutive promoters. J. Biotechnol. 289, 39-45 (2019).
[6] https://2018.igem.org/Team:H14Z1_Hangzhou/Team
