Part:BBa_K817033:Experience
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Improvement
Designed by NTHU_Taiwan 2019. (See the part at BBa_K3040007)
Background
This has an improvement on the natural acyl-CoA responsive promoter pfadBA submitted by iGEM12_NTU-Taida (BBa_K817033). However, according to their result, this promoter has a massive leakage, which has very high value of downstream reporter gene baseline expression. Besides, the fold change after 0.04% of oleic acid induction has only achieved 1.67-fold, considered no significant signal rise. Thus, we coupled an endogenous thioesterase coding gene with this promoter. By doing so, we are able to perform a tunable and dynamic gene expression.
Mechanism
Tes A is a heterologous thioesterase, which is able to hydrolyze fatty acyl-CoAs to free fatty acids. Here, we combine Tes A with pFadBA (wild type) together as a new promoter, Tes A pFadBA, to see if the sensitivity range of fatty acid concentration lowers due to the increase of endogenous fatty acid.
In the microbial, carbon source such as glucose or fatty acid will be metabolized to acetyl-CoA. When fatty acid is needed to be catabolized into other macromolecules, the acetyl-CoA will be converted into Acyl-ACP and finally formed free fatty acid. The following is the detailed mechanism of the biosynthetic and degradation pathway of fatty acid. [1]
Nevertheless, the accumulation of Acyl-ACP will negatively inhibit the conversion of malonyl-CoA to Acyl-ACP and thus repress biosynthesis of free fatty acid. Thus, endogenous TesA gene which encodes a thioesterase will hydrolyze these Acyl-ACP and subsequently produce a significant level of free fatty acid.
Concept of design
As you know, high concentration of fatty acid will promote β-oxidation but not synthesis of fatty acid. However, overexpression of TesA can help the E. coli to deplete the Acyl-ACP and thus rescue the production of free fatty acid. The more fatty acid present, the more acyl-CoA can be converted and thus the higher transcription rate can pfadBA can achieved. The following was the mechanism proposed.
Some previous literature has reported that the strain carries TesA overexpression is capable of showing 10 to 25-fold of fluorescence than the native promoter [2]. This result matched our proposed model.
Result
As we predicted that the fluorescence fold change after induction of fatty acid will be greater as tesA has produce more fatty acid. The result shows that the fold change of fluorescence after 16 hours 5mM oleic acid induction can come up to 9-fold. This has greatly improved the native strength of the promoter since it can only increase to about 2-fold. This modification helped us to control the strength of promoter more precisely compared to the native pfadBA since the induced-transcription range of the promoter has been broaden.
Future work
According to the previous research, the fold change should be able to reached 10 to 25-fold. We deduce that the problem is we used a weaker promoter placed before TesA, thus the fatty acid produced endogenously is fewer than previously reported. Hence, we proposed to insert the TesA sequence directly at the downstream of pfadBA promoter and made it regulated by this promoter. Once we have added the fatty acid, then pfadBA promoter will be activated, RFP and tesA will be produced. Tes A will further catalyze the formation of fatty acid through the dissociation of Acyl-ACP. The more fatty acid, the more pfadBA will be activated and more tesA protein will be produced. This will form a positive feedback loop and thus the fold change will become higher.
Reference
[1] Janßen, H. J., & Steinbüchel, A. (2014). Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnology for biofuels, 7(1), 7.
[2] Zhang, F., Carothers, J. M., & Keasling, J. D. (2012). Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature biotechnology, 30(4), 354.
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