Biosynthesis enables renewable and environment-friendly production of various compounds. However, present biosynthetic performances still await improvements to be cost competitive with petroleum-based chemical synthesis and suitable for large-scale industrial production. In order to achieve this goal, many approaches have been created, among which the Population Quality Control (PopQC) system is proved efficient. In our project, a PopQC system was developed as a plasmid based gene circuit in Bacillus amyloliquefaciens LL3 to continuously select high-performing cells in order to improve the yield of target metabolite, glutamate. In the presence of our PopQC system, high-producers stayed alive while low-producers were unable to survive. Consequently, the average intracellular concentration as well as the yield of glutamate among the population was enhanced, which finally led to the yield enhancement of poly-γ-glutamate, a high-value-added secondary metabolite.
Artificial biosynthetic pathways have enabled renewable, environment-friendly production of a variety of significant products ranging from simple fuels (such as ethanol, butanol and fatty acid derivatives) to intricate natural products (such as artemisinin, strictosidine, erythromycin, and so on). However, these biosynthetic processes are always criticized for being uneconomical for large-scale industrial production because of their relatively lower yield compared to petroleum-based chemical synthesis. Therefore, it’s urgent and important to create new approaches to enhance biosynthetic performance.
2. Our Choice—PopQC
PopQC, which is the abbreviation for population quality control, is a new approach designed for biosynthesis yield enhancement based on non-genetic cell-to-cell variation. Because of some non-genetic differences, different cells in a single colony will have considerable variations in protein and metabolite concentrations. Therefore, in cell cultures there will be both high- and low-producers, and the intrinsic low-producers might cause suboptimal ensemble biosynthesis. The elimination of low-producers can lead to the efficient utilization of substrates and high yield of target products. Based on this, in our project PopQC was developed as a plasmid-based gene circuit, which continuously selects high-producers to optimize the biosynthetic performance for large-scale industrial production.
3. Introduction of Chassis Organism
In our project, Bacillus amyloliquefaciens LL3 was chose to be the host of the plasmid of PopQC. Bacillus amyloliquefaciens LL3, which is a gram-positive bacterium, was originally isolated from traditional fermented foods by Dr. Cunjiang Song’s lab to produce poly-γ-glutamate(γ-PGA). γ-PGA is a high value-added product which has good hydroscopicity and can be chemically modified. It has the potential to be applied into cosmetic, food, drug carrier and other fields. Bacillus amyloliquefaciens LL3 has a clear genetic background as our lab has already finished its genome sequencing in 2011. Its synthesis of poly-γ-glutamate (γ-PGA) is independent of exogenous glutamate, which can reduce the cost of production.
4. Our Design
Here is the basic mechanism of PopQC we designed. In specific, we use the promoter gltAB, the promoter grac, lacI gene and tetA gene to construct our circuit. In Bacillus amyloliquefaciens LL3, there exists the Glt operon which is responsible for intracellular glutamate synthesis. With a specific extracellular tetracycline concentration, if the intracellular glutamate concentration of this individual is low, the concentration of GltC protein will go up, which activates the promoter gltAB to express lacI. LacI protein furthermore repress the promoter grac and as a result, repress tetA expression. On the contrary, for high-producers, the concentration of intracellular GltC will go down, which represses the promoter gltAB to express lacI. Meanwhile, the tetA expression pathway 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 concentration as well as the yield of glutamate among the population was enhanced, which will finally lead to the yield enhancement of γ-PGA in Bacillus amyloliquefaciens LL3.
Our experiments are consisted with 4 parts.
Part 0 is to enhance the yield of glutamate by traditional metabolic engineering. We expected to enhance the promoter of icd and gltAB genes by constructing promoters in tandem to overexpress these 2 genes.
Part 1 is to validate whether the glutamate can regulate the gltAB promoter through GltC and to map glutamate concentration to tetracycline resistance. We chose GFP as our signal and will test the intracellular glutamate concentration, the transcription level of GltC, GFP, and the florescence intensity with different extracellular glutamate concentration. We chose GFP as our signal and will test the intracellular glutamate concentration, the transcription level of GltC, GFP, and the florescence intensity with different extracellular glutamate concentration.
Part 2 is to test the inhibition effect to the promoter grac with different concentrations of LacI. So we use different promoters to regulate lacI to see the tolerance of tetracycline of our chassis.