Part:BBa_K5000001
introduce new pathway to produce isoprenoid in ecoli
This part comprise three genes, including SccK,AtiPK,idi.They work together to consecutively catalyze prenol and isoprenol to form DMAPP and IPP, which are the precursor of isoprenoid.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 3473
Illegal SpeI site found at 3467 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3473
Illegal SpeI site found at 3467
Illegal NotI site found at 3498 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3473
Illegal BamHI site found at 1904
Illegal XhoI site found at 3507 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3473
Illegal SpeI site found at 3467 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3473
Illegal SpeI site found at 3467 - 1000COMPATIBLE WITH RFC[1000]
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 part comprise three genes (IUP module), including SccK,AtiPK,idi.They work together to consecutively catalyze prenol and isoprenol to form MDAPP and IPP, which are the precursor of isoprenoid.
Figure 2. The construction of the part.
Results
Strain A is a blank control, Strain B contains MEP module and steviol module, strain C contains IUP 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.
Figure 3. Results for miR-223 RT-qPCR.
Through experimentation, we discovered that increasing the precursor process can effectively improve the yield of steviol glycosides. Introduction of IUP pathway is better than overexpression of the MEP pathway (BBa_K5000000) due to the influence of E. coli's inherent metabolic regulation. We can see the expression of steviol in Escherichia coli, the production of steviol is 60 relative abundance, the concentration of strain B is 1.5 times that of strain A.
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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