Improvement

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 list in Fig. 1.1

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.

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

2.1 Tryptophan can be easily determined by modified PDAB chromogenic method

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.

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. 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.

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.

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. 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.

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 gene overexpression) and tryptophan production (aroG-S211F overexpression) was constructed in the two arms of toggle-switch. (View our design on Team:XJTU-China/Design).