Part:BBa_K5000000
overexpression dxs, ispA, idi under the control of T7 promoter
This plasmid is used to enhance the production of isoprenoid precursor in E.coli by overexpression three key enzyme in the MEP pathway under the control of T7 promoter, the three genes are 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphatesynthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E.coli.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 3483
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 953
Illegal SpeI site found at 3483 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 3489
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 3483
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 3483
Illegal AgeI site found at 249 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1758
Background
Our research aims to heterologously express steviol glycosides in Escherichia coli. We divided the project into three modules: the DMAPP/IPP module, the steviol module, and the glycosyltransferase module. We synthesized the genes for each module and assembled them using a method like NEB BioBrick assembly. These modules were then inserted into vectors pACYCduet-1, PET21a, and PET28a. Under induction with IPTG, we expect to detect the product of rebaudiosides.
Figure 1. The passway of steviol glycosides in Escherichia coli.
Design
This plasmid is used to enhance the production of isoprenoid precursor in E.coli by overexpression three key enzyme in the MEP pathway under the control of T7 promoter, the three genes are 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphatesynthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E.coli.
Figure 2. The construction of the part.
Results
Strain A is a blank control, Strain B contains MEP module and steviol module. After induction by 0.05 mM IPTG, these strains were cultivated in shake flask at 28℃ for 3 days. The culture was extracted by equivalent volume of n-butanol. After extraction, the organic phase was collected and dried under vacuum. Then they were dissolved by dimethyl sulfoxide and filtered. The samples were analyzed using ThermoScientific Q Exactive Plus Mass Spectrometer. We can see the expression of steviol in Escherichia coli, the production of steviol is 40 relative abundance.
Figure 3. Results for Strain contains MEP module and steviol module.
References
Kong, M., Kang, H. J., Kim, J. H., Oh, S. H., & Lee, P. C. (2015). Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli. Journal of Biotechnology, 214, 95–102. https://doi.org/10.1016/j.jbiotec.2015.09.016
Chatzivasileiou, A. O., Ward, V. C., Edgar, S., & Stephanopoulos, G. (2018). Two-step pathway for isoprenoid synthesis. Proceedings of the National Academy of Sciences of the United States of America, 116(2), 506–511. https://doi.org/10.1073/pnas.1812935116
Wang, J., Li, S., Xiong, Z., & Wang, Y. (2015). Pathway mining-based integration of critical enzyme parts for de novo biosynthesis of steviolglycosides sweetener in Escherichia coli. Cell Research, 26(2), 258–261. https://doi.org/10.1038/cr.2015.111
Lin, M., Wang, F., & Zhu, Y. (2020). Modeled structure-based computational redesign of a glycosyltransferase for the synthesis of rebaudioside D from rebaudioside A. Biochemical Engineering Journal, 159, 107626. https://doi.org/10.1016/j.bej.2020.107626
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