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AroG (3-deoxy-7-phosphoheptulonate synthase, EC 2.5.1.54, BBa_K1060000), catalyzes the following reaction:
phosphoenolpyruvate(PEP) + D-erythrose-4-phosphate(E4P) + H2O = 3-deoxy-D-arabino-hept-2-ulosonate 7-phosphate (DAHP) + phosphate

The reaction is a key branching point 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.[1-4]

AroG-S211F, in which the serine at 211 was replaced by phenylalanine, has also been reported to be able to increase the production of downstream product in shikimate pathway[5]. However the structural mechanism is unclear. And also it is not sure whether it can increase the production of our tryptophan. So In our project, aroG-S211F was overexpressed, attempted to improve the production of tryptophan.

An inducible circuit BBa_K3832008 containing lacUV5-controlled aroG-S211F were constructed to characterize and measure the function of AroG-S211F in E.coli DH5alpha(Fig. 1.1). Firstly the yield of tryptophan of mutant aroG and the native one respectively were detected by PDAB method modified by ourselves. Secondly, considering that the over-expression of aroG will significantly reduce the amount of substrate (glucose) entering the glycolysis pathway, in turn affecting the normal cell proliferation, 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.

1. Construction and Verification of aroG circuit

aroG-S211F gene was chemically synthetized by Genewiz and cloned into pET28a+ backbone by Golden Gate assembly (BsaI). After transformed into E.coli DH5alpha, plasmid extraction and electrophoresis, PCR amplification and sequencing were conducted to confirm its correctness. The results are listed in Fig. 1.1

Fig. 1.1 The DNA agarose gel electrophoresis result of AroG-S211F circuit, plasmid and PCR product. (a) The length of the circuit is 2503bp (b) The length of the plasmid is 4738bp. And the two discrete bands are thought as either open-coiled or super-coiled plasmids (c)The amplicon is expected to be 2526bp.

Meanwhile, the quantitatively assay by RT-qPCR was also performed to verified its mRNA level. As shown in Fig. 1.2, the transcriptional level was increased about two folds after IPTG induction, indicating the circuit was successfully constructed with functional AroG mutant. The basal expression of aroG without IPTG induction can be observed due to one copy of native aroG in E.coli genome.

Fig. 1.2 The relative mRNA level of aroG-S211F in DH5alpha strain with Part:BBa_K3832008 inserted in pET28a+ vector.

2. Characterization the effect of AroG-S211F on tryptophan production

2.1 Tryptophan can be easily determined by modified PDAB chromogenic method

Freeze-thaw bacterial culture medium with suspension cells for over 3 times.
Add 100 ul medium into 400 ul PDAB (p-dimethylaminobezaldehyde) solution (3 mg/ml in 9 M solution of sulfuric acid). Then keep at 60℃ for 20 min.
Add 3 ul 0.5% (w/w) solution of sodium nitrite. Then keep at 60℃ for 15min.
Measure absorption under 590 nm wavelength (OD590).

2.2 The yield of tryptophan was significantly improved in AroG-S211F strain compared to native AroG

As shown in Fig. 2.1, compared with the E.coli harboring the blank vector and native aroG gene (BBa_K1060000), the yield of tryptophan in the engineered E.coli with aroG-S211F induced by 1 mM IPTG continuously increased in the 30 h cultivation (green triangle), reaching a maximal productivity of 160 mg/ml per OD, while the blank controls slowly increased and maintained its production at about 1200 min, arriving about 80 mg/ml per OD, half of the previous one (circle and square). It is the same case in absent of IPTG (blue triangle), indicating the low leaky expression of our circuit. In all, our circuit containing AroG-S211F can efficiently produce tryptophan with the highest productivity of 160 mg/ml per OD, which can be further improved under the control of toggle-switch.

Fig. 2.1 The tryptophan production curve of the engineering E.coli with aroG-S211F and E.coli with native aroG.

2.3 The structural mechanisms was elucidated by protein structure modeling

To explain the concrete mechanisms of the promotion effect by AroG-S211F comparing wild-type AroG, protein structure modeling is used to analyze the thermodynamics and structure of them.

From an energy perspective, our modeling results show that the mutant protein exhibits lower binding free energy with the catalytic substrate in the presence of the inhibitor (Phe), that is, it is able to bind more tightly and stably to the substrate, thus improving catalytic efficiency.

Fig. 2.2 Affinity of docking

On the other hand, structural analysis also reflected that the binding tightness between the mutated site and the inhibitor was reduced, which weakened its inhibitory effect.

Fig. 2.3 Differences between the Phe docking site

By the modeling result, the mutation (S211 to F211) in AroG is proposed to eliminate the allosteric inhibition of phenylalanine, thus increasing the catalytic rate and downstream product yield.

For more detail of our protein modelling, see also our Protein Modelling

3. Characterization the effect of aroG-S211F on cell proliferation

The over-expression of aroG inhibits the glycolysis pathway, thus definitely affecting the cell growth. So the effect of aroG-S211F on cell proliferation was also detected. The OD600 of engineered E.coli and blank strain were continuously monitored, as shown in Fig. 3.1. The Logistic equation was used to fit the growth curve, the obvious inhibitory effect of aroG expression on cell proliferation was observed, especially with IPTG induction. The growth parameters K (environmental capacity) and r (intrinsic growth rate) of different experimental groups was also obtained from the fitting Logistic curve, and the parameter r decreased dramatically in E.coli with aroG-S211F induced by IPTG, indicating the increased doubling time of the cell.

Fig. 3.1 (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).

4. Conclusions:

A lacUV5-controlled aroG-S211F gene circuit was successfully constructed, and the overexpression of aroG-S211F significantly improved the tryptophan production, with a highest productivity of 160 mg/ml per OD. Protein structure modeling elucidate that the improvement may attribute to the elimination of the allosteric inhibition of phenylalanine, thus increasing the catalytic rate and downstream product yield. However, because of the inhibition on the glycolysis pathway of AroG, the cell growth was obviously inhibited. The results 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 overexpression) and tryptophan production (aroG-S211F overexpression) was constructed in the two arms of toggle-switch. (View our design on Team:XJTU-China/Design).

5. 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).

Team:XJTU-China/Improve