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P_gltAB

This sequence includes the promoter(forward) of gltA/B(glutamate synthase) and the promoter(backward) of gltC(LysR family transcriptional regulator), GltC can bind specific DNA site on it and upregulate the expression of downstream proteins, and GltC is repressed by high level glutamate.

Sequence and Features

Introduction

Bacillus amyloliquefaciens LL3

In our whole project, we chose the B. amyloliquefaciens LL3 as our genetically engineered microorganism (GEM), which is a glutamic acid- independent poly-γ-glutamic acid (γ-PGA)-producing strain isolated from traditional fermented food. According to previous studies, its pathway of synthesizing γ-PGA through pgsBCA did not rely on the exogenous glutamate, allowing it to synthesize γ-PGA normally on glutamate-free medium, where the glutamate- dependent ones can’t synthesize γ-PGA even with a high intracellular glutamate concentration.

The regulation of GltAB

gltA and gltB genes are organized as an operon, encoding glutamate synthase, a heterodimeric protein. Since glutamate is the most abundant anion in the cell, the expression of gltA with gltB is thought to be subject to nutritional regulation, it's no wonder that their Regulatory mechanism has been attracting more and more attention.
Scientists have found TnrA and GltC as regulators of GltAB. TnrA is active only under conditions of nitrogen limitation and inactive after interaction with a complex of glutamine synthetase and glutamine, it can repress gltAB expression by binding to the promoter region of gltA, however, even without TnrA, there is little expression of the GltAB operon unless GltC is active. GltC is a member of the LysR family of bacterial transcription factors which can activate transcription of GltA and GltB.

Figure 1. An effective way for biosynthesis being more suitable for large-scale industrial production. Red squares represent high producers, yellow ones represent moderate producers, white ones represent low producers. With our effective system, high producers occupy a larger proportion in the population.

In our work, promoter P_gltAB (BBa_K2705000), promoter P_grac(BBa_K2705002), lacI(BBa_K2705001) gene and tetA(BBa_K2705003) gene were composed to build up the system. (See Figure 2.)

Figure 2. High glutamate concentration ensures the individual to survive in tetracycline condition with PopQC system. High-producers will synthesize enough amount of tetracycline efflux pumps to maintain alive while low-producers are not be able to survive in tetracycline condiction.

In Bacillus amyloliquefaciens LL3 exists the glt operon, which is responsible for intracellular glutamate synthesis (See BBa_K2705000 for more details about P_gltAB). TetA is a tetracycline resistance protein[TetA(C) inner-membrane-associated protein] (See BBa_K2705007 and BBa_K2705003 for more details about TetA).
With a specific extracellular tetracycline concentration, when intracellular glutamate-precursor of γ-PGA-concentration of the individual is low, GltC level will go up, which activates the P_gltAB to express lacI. LacI furthermore represses P_grac and as a result, represses tetA expression. On the contrary, for high-producers, the concentration of intracellular GltC will go down, which represses the P_gltAB to express lacI, and the tetA expression is not affected. Therefore, high-producers will synthesize enough amount of tetracycline efflux pumps to maintain alive while low-producers won’t be able to survive. Consequently, the average intracellular glutamate concentration among the population is enhanced, which will finally lead to γ-PGA yield enhancement in LL3.

Proof of Function

Vector P_gltAB-LacI-P_grac-TetA was converted into LL3, and correct transformants were fermented in M9 culture medium with different extracellular glutamate concentrations (0, 2.5, 5 and 7.5 g/L). From the 6^th hour, we tested bacteria with several assays every 3 hours. We chose LL3 δBAM strain as control system.

Expression level of tetA by microplate assay

To test the expression of tetA, we tagged it with the fluorescent reporter GFP-coding gene (BBa_K2705004), whose expression can be detected by microplate assay (395nm\509nm). The intracellular glutamate concentration and bacteria concentration (OD₆₀₀) were also examined, respectively. It can be concluded that with the increasing glutamate, tetA of PopQC was upregulated to express. (See Figure 3.) The results sugguests that the system can help individuals with higher intracellular glutamate concentration express more TetA, so that survive in tetracycline condition.

Figure 3. The relationship of CIGA and GFP in Plateau stage.（CIGA: The concentration of intracellular glutamic acid；A.U.: Arbitrary Unit）With the increasing glutamate, tetA of PopQC was upregulated to express, which supports individuals with high glutamate concentration to survive in tetracycline condition. More than 5ml fermented liquid was analyzed per treatment. Error bars indicate and can show significant tendency. (P value < 0.005).

Transcription level of gltAB, gltC, lacI by qPCR

Together with the microplate assay, total RNA of the bateria was extracted every 3 hours from the 6^th hour. The transcription levels of gltAB, gltC and lacI were tested by qPCR assays, and the relationship between intracellular glutamate concentration and transcription of these genes are analyzed. Xxx was chosen as the internal reference of qPCR assay. Primers used in the assays are listed in Table 1.

Table 1. Primers used in qPCR essays.

Figure 4a,c,e indicated that with higher intracellular glutamate concentration, less gltAB, gltC and lacI of the circuit were expressed. Figure 4b,d also shows that in LL3 ΔBAM strain, gltC and gltAB were affected by diverse intracellular glutamate concentration, similar to LL3 ΔBAM strain with PopQC system. (See BBa_K2705000 for more details about P_gltAB functions)

Figure 4. With higher intracellular glutamate concentration, transcription of gltC, gltAB and lacI are downregulated in logarithmic and plateau phases. a, c, e illustrate the transcription level of gltC, gltAB and lacI in LL3 ΔBAM strain with PopQC system. b, d show the effect of intracellular glutamate concentration on gltC and gltAB transcription. Records are from the logarithmic and plateau phases. R squares are shown on the graphs.

Measurement of γ-PGA yield

After 32 hours fermentation, the γ-PGA yield was tested. With PopQC system, LL3 ΔBAM strain produced more γ-PGA, which approved the system function. See Figure 5.

Figure 5. γ-PGA yield is enhanced in LL3 ΔBAM strain with PopQC system comparing with the LL3 ΔBAM strain without it. 32 hours fermentation production of both strains is tested, PopQC system makes sense in yield enhancement.

References

[1] Weitao G, Mingfeng C, Cunjiang S et al. Complete Genome Sequence of Bacillus amyloliquefaciens LL3, Which Exhibits Glutamic Acid-Independent Production of Poly-γ-Glutamic Acid. J Bacteriol. 2011 Jul; 193(13): 3393–3394.

[2] Picossi S, Belitsky B R, Sonenshein A L. Molecular mechanism of the regulation of Bacillus subtilis gltAB expression by GltC[J]. J Mol Biol., 2007, 365(5):1298-1313.

[3] Commichau FM, Herzberg C, Tripal P et al. A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC. Mol Microbiol. 2007 Aug;65(3):642-654.

[4] Bohannon D E and Sonenshein A L. Positive regulation of glutamate biosynthesis in Bacillus subtilis. J Bacteriol. 1989 Sep; 171(9): 4718–4727.