Dynamic Regulation

System

In our project, we have worked on the insertion of two genes, Pct and PhaC, in Pseudomonas putida for PLA production. Additionally, we have inserted an LDH gene to increase lactate (the precursor) yield. However, in nature, we often observe regulation systems on biological pathways, that allow the cell to optimize resources for the production of compounds.[1] We wondered if we could implement dynamic regulation to our PLA system to optimize the cell metabolism.

Our objective is optimizing the production of PLA by making the cell metabolism more efficient. Thus, we want to express the operon enzymes that produce PLA only when there is the PLA precursor: lactate.

At the same time, we might not need lactate to be constantly produced: we hypothesize that a feedback system that regulates lactate concentration can help the optimization of the ratio PLA produced / enzyme needed. More precisely, the system would increase the yield of PLA first, by controlling the carbon flux of the pathway and toxicity of precursors using "Le Chatelier's principle"; second, optimizing the level of the required enzymes it would avoid the toxic effect of the extra gene expression.

Description

Click to enlarge

Figure 2. Dynamic Regulation system implemented in plasmids. Plasmid pSEVA224 contains genes PhaC and Pct controlled by LldR responsive promoter. Plasmid pSEVA424 contains genes LDH - controlled by McbR repressible promoter -,LldR gene - with J23115 promoter -, and McbR gene - with LldR responsive promoter.

The system is divided in the two plasmids planned to use during basic expression of PLA enzymes: the main change is on the promoters, which are now responsive to transcription factors:

pSEVA224 contains the operon producing PLA, elements PhaC and Pct with their RBSs and the terminator, controlled by a promoter responsive to the TF LldR.

pSEVA424 contains: (1) LDH gene for lactate production, with its RBS and terminator, controlled by a McbR repressible promoter. (2) LldR TF gene, with the J23115 constitutive promoter, its RBS and terminator. (3) mcbR gene controlled also by promoter responsive to LldR. This McbR when expressed will repress McbR repressive promoter.

LldR system

From one side, we have the presence of LldR system. The LldR responsive promoter has been well described in E. coli for regulation of lldPRD operon.[2] It works as a biosensor of lactate. There is the LldR TF codyfing gene, than when expressed produces a TF that has affinity to lactate. When lactate is absent, the TF binds the promoter inhibiting its expression. However, in presence of lactate, it looses affinity to the promoter, allowing transcription.

In our system, we use LldR responsive promoter for expression of PLA operon genes, PhaC and Pct, so they will only be transcribed when there is lactate in the cell, the necessary condition for PLA production. The LldR TF codifying gene has a constitutive promoter so it is always expressed and in basal conditions it represses the promoter. We also have the LldR responsive promoter controlling mcbR gene expression, so when there is lactate, it can express the protein which will eventually inhibit LDH. By this, we are implementing a feedback system that regulates lactate levels.

McbR system

The McbR is part of the TetR-family repressors, widely used in synthetic biology.[3] The repressible promoter is basally active, but when mcbR TF is transcribed, the TF binds the promoter and inhibits it.

We have described mcbR to be useful for regulation the production of lactate. We need lactate for PLA production as precursor, but it does not necessarily mean that lactate has to be constantly increasing: we think an optimized system would autoregulate lactate production, and using the McbR repressible promotor could be an effective feedback system.

Dynamics of responsive elements

Below, we show a schema of the dynamics that the different elements of our system would generate because of their inter-responsiveness:

Click to enlarge

Figure 2. Dynamics of responsive elements. A: Initially, LDH and LldR would be expressed. B. LldR would inhibit LldR responsive promoters. C. Presence of lactate would unbind LldR from LldR responsive promoters, activating transcription. D. There would be gene expression of PhaC-Pct operon and McbR and protein synthesis. E. McbR would inhibit LDH expression by repressing the promoter, by feedback regulation.

The aim of modeling this system is to observe and predict the dynamics generated by the different elements. We would like to study its variations depending on gene expression levels, and find which would be the optimal tunning strategy.

In order to do so, we decided to take two strategies: rule-based modeling using Kappa and differential equations modeling in the system seen as genetic circuit.

References

Venayak, N. et al. Engineering metabolism through dynamic control. Curr opinion Biotech 34: 142-152 (2015)
Aguilera, L., Campos, E., Gimenez, R., Badia, J., Aguilar, J. & Baldoma, L. Dual Role of LldR in Regulation of the lldPRD Operon, Involved in L-Lactate Metabolism in Escherichia coli. J Bact, 190(8): 2997–3005 (2008)
Stanton, B.C., Nielsen, A.A., Tamsir, A., Clancy, K., Peterson, T. & Voigt, C.A. Genomic mining of prokaryotic repressors for orthogonal logic gates. Nat Chem Bio, 10: 99-105 (2014)