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 (Fig.1.3). 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.1.5

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).