Team:XJTU-China/Proof_Of_Concept

Proof of Concept

Design

Overview

In our project, we plan to realize the efficiently production of tryptophan in E.coli. Based on the biosynthesis pathway of tryptophan, aroG(encoding 3-deoxy-7-phosphoheptulonate synthase) and trpBA (encoding tryptophan synthase) were used to improve the yield of tryptophan (Fig.1.1). aroG can divert the intermediate products of glycolysis into the chorismate synthesis pathway, while trpBA can synthesize the precursor chorismate into tryptophan.[1-4])

Fig.1.1

We consider that excessive tryptophan production could interfere the proliferation of E.coli, for over-expressed aroG will competitive inhibit the glycolysis pathway while large amount of ATP and NADPH are consumed during the synthesis. Therefore, a toggle-switch circuit are used to reconciling the contradiction between cell proliferation and tryptophan production. In one of the bistable state, over expression of pykA(encoding pyruvate kinase II) is used to eliminate the competitive inhibition of aroG, reducing the production process and enabling a rapid cell proliferation. When cells reach a high density, they can be induced into another state where aroG and trpBA are expressed to product tryptophan efficiently (Fig.1.2).

Here two types of inducible promoter, Pλ promoter and lacUV5 promoter ,are used, which can be induced by heat (>42℃) and IPTG respectively. When lacUV5 promoter activated by IPTG, the cells will enter the "proliferation" state; while Pλ promoter is activated by heat, they will turn into the "production" state for expression of aroG and trpBA.

Fig.1.2 Toggle-switch circuit with tryptophan production Realizing the bistable states of "Proliferation" and "Production"

In order to test whether each part are functional individually, our working circuit is divided into several devives.

Fig. 1.3(a) AroG testing circuit(aroG), BBa_K3832008

AroG catalyzes the key branching reaction of the glycolysis and shikimate pathways. Expression of aroG can lead to more substrate into the shikimate pathway, which can improve the yield of downstream products as tryptophan, phenylalanine, tyrosine and benzazole etc. An inducible circuit of aroG-S211F (a mutant of wild-type aroG which can improve its catalyzing ability [5]) is constructed. In this circuit, aroG-S211F is under the control of lavUV5 promoter, and can be induced by IPTG. In this way, we can verify the function of aroG to produce tryptophan, and the effect on cell proliferation.

Fig. 1.3(b) Tryptophan synthesis circuit (aroG-trpBA)

This circuit can achieve the “Production” state. Containing the trpBA which encodes tryptophan synthase following aroG-S211F, this circuit can be used to further verify the performance of tryptophan production in presence of both aroG-S211F and trpBA.

Fig. 1.3(c) Proliferation circuit (pykA)

This circuit can achieve the “Proliferation” state. Over-expression of pykA has an competitive inhibition effect on aroG, which enable cells to spend more substrate and energy on their proliferation.

Fig. 1.3(d) Toggle-switch circuit with GFP and RFP, BBa_K3832007

This circuit is used to verify the feasibility of our toggle-switch design. Reporter genes as sfGFP and mRFP are contained to monitor the two states of circuit.
With induction of IPTG, the downstream genes of lacUV5, that is, cI and mRFP will expressed, while those in the downstream of lambda promoter (lacI and sfGFP) will be repressed. Even without IPTG induction after several hours, the lack of LacI expression will result in the stability of red fluorescence. At temperatures above 42 ℃, gene expression will be flipped into another state, the stable expression of lacI and sfGFP, and the state will maintain even without heat.
The GFP and RFP can be altered with other functional genes such as tryptophan synthetic genes to achieve the bistable expression and synthesis of tryptophan.

In order to realize the family application of our project and the automatic control of production conditions, we have designed a cultivation device. It contains controlling, detecting and cultivating modules. The equipment can monitor the cell growth and tryptophan production while conducting fermentation culture, and control the induction culture conditions through singlechip at different stages with this signal, so as to activate the expression of specific genes in the gene circuit, controlling the cells into "proliferation/production" state.

Considering it is difficult to realize real-time detection of tryptophan concentration by chemical method in hardware, we also have designed a detecting circuit which can sense the concentration of tryptophan and report green fluorescence of different light intensities (inversely proportional to the concentration of tryptophan) (Fig.1.4). By introducing this circuit into the engineered bacteria, the concentration of tryptophan in culture medium can be converted into light intensity that can be detected by hardware.

Fig.1.4

Through the interaction of hardware circuit and gene circuit, we can achieve both the automatic control and the high production, giving a full play to the advantages and potential of Synthetic Biology.

Reference

[1] SHEN T，LIU Q，XIE X，et al． Improved production of tryptophan in genetically engineered Escherichia coli with TktA and PpsA overexpression[J]．J Biomed Biotechnol，2012 (11) : 605219．
[2] CHEN L，ZENG A P．Rational design and metabolic analysis of Escherichia coli for effective production of L -tryptophan at high concentration[J]. Applied Microbiology and Biotechnology，2017，101( 2) : 559－568．
[3] Zhan JJ,Du LH. Progress of metabolic engineering modification of Escherichia coli for L-tryptophan production[J]. Shandong Chemical Industry,2021,50(01):85-87+89.
[4] Hu,Changyun.Study on the structure and function of 3-deoxy-D-arabinoheptulose-7-phosphate synthase AroG[D]. Fudan University, 2003.
[5] HAO Dali et al. Site-mutation of AroG Gene and Co-expression with TrpBA Gene in Escherichia coli. Chinese Journal of Applied and Environmental Biology 19, 817-821 (2013).

Protein Modelling Result

To investigate the mechanism of allosteric inhibition on AroG by Phe and the alleviation in S211F mutant, it is proposed to be quantified and visualized using PyMOL, Gaussian16.0W, GaussView6.0, Swiss, AutoDockTools software. To see the result of our protein model, plese check our wiki of Protein Modelling.

View: Protein Modelling

Experiment Result

Until now, we have successfully constructed AroG test circuit(BBa_K3832008) and Toggle switch with GFP/RFP(BBa_K3832007).

And we have demonstrated that, the overexpression of aroG-S211F mutant has significantly improved the tryptophan production with a highest productivity of 160 mg/ml per OD. The detailed structural mechanism of the improvement has also been elucidated by modeling.

In addition, the toggle-switch circuit with GFP/RFP was successfully constructed, and demonstrated to be functional by RT-qPCR and fluorescence measurement. The circuit achieved the bistable expression of sfGFP and mRFP under different induction conditions, and can be fast switched by the change of inducers.

Verification of aroG-S211F (BBa_K3832000) ——Functional to improve the tryptophan production

AroG located in the branching point of the glycolysis and shikimate pathways, and overexpression of AroG has also been reported to be able to increase the production of downstream product in shikimate pathway. In our project, we constructed its mutant, aroG-S211F (Part:BBa_K3832000) and demonstrated its positive effect on the improvement of tryptophan production.

lacUV5 controlled-aroG S211F were constructed to characterize and measure the function of AroG-S211F in E.coli DH5alpha. Firstly the yield of tryptophan of mutant aroG and the native one respectively were measured by PDAB method (Fig 3.1). Secondly, the effect of aroG-S211F on the cell proliferation was also tested by the comparison of growth rate of the wild-type E.coli and the engineered E.coli with aroG-S211F(Fig 3.3). The structural mechanism was elucidated by protein structure modeling (Fig 3.2).

Fig. 3.1 The relationship between tryptophan concentration in culture medium and culture time. The concentration of tryptophan is measured by PDAB chromogenic method.

More details for the experiment design and result of aroG:
Improvement: aroG-S211F

Fig. 3.2 (a) The population density of E.coli was measured at 600nm by colorimetry. The scatter represents the result of the measurement. The Logistic equation was used to fit the growth curve, and the fitting results were shown in the curve. (b) shows the growth parameters K (environmental capacity) and r (intrinsic growth rate) of different experimental groups obtained from the fitting results in (a).

Verification of toggle-switch circuit with GFP and GFP (BBa_K3832007) —Functional to achieve the separated bistability and fast switch by two inducers.

In this stage, GFP and RFP are used to help us detect and check whether our design of toggle-switch circuit will work properly (Fig. 1.3(d)). Each promoter downstream follows another promoter's repressor gene and a fluorescent protein in different color. By detecting the relative fluorescence intensity under different inducing conditions, we can get information about the intensity of each promoter in different states and whether they can function as we designed.

Our experiment result have confirmed the feasibility of our design. As shown in Fig. 3.3, we successfully achieved bistable protein expression under different induction conditions. The relative expression levels of GFP and RFP were significantly reversed with the change of inducting conditions, indicating that the circuit design can be used to switch cell states between “proliferation” and “production”

Fig.3.3 Relative fluorescence intensity of sfGFP and mRFP in toggle-switch circuit under different inducing conditions

More details for the experiment design and result of toggle-switch circuit:
Engineering Success: toggle-switch circuit

Conclusion

The results of function of aroG and toggle-Switch circuits confirmed the feasibility of our general design. The expression of aroG-S211F can significantly promote the production of tryptophan and observed its inhibitory effect on cell proliferation. Meanwhile, for the control circuit, two groups of promoter-repressor systems and RBS with corresponding strength we used can achieve separated bistable state and fast switch under different inducers.

The results also confirmed our hypothesis that cell proliferation and tryptophan production should be separated, and it has been designed to be strictly controlled by toggle-switch circuit, in which cell proliferation (pykA gene overexpression) and tryptophan production (aroG-S211F overexpression) will be constructed in the two arms of toggle-switch. The one arm of toggle switch will be used for the expression of aroG-S211F and trpBA for tryptophan synthesis (production state), and another arm for the expression of pykA gene for cell growth (proliferation state), thus the cell growth and tryptophan synthesis can be will regulated and balanced.

Although the toggle-switch with aroG-S211F, trpBA and pykA genes for tryptophan synthesis was not verified experimentally, we confirmed its feasibility by modeling based on the data obtained in the above experiment (Fig. 4.1, also see results on our Modelling page). Furthermore, we predict the best time to change the induction condition from IPTG to 42℃ heat when the tryptophan production reaches the maximum (about 1,170min), providing suitable parameters for automatic control of hardware and our further optimizations.

Fig. 4.1

Team:XJTU-China/Proof Of Concept