Team:NKU CHINA/Project/Description

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Project Description

Abstract

Biosynthesis enables renewable and environmental-friendly production of various compounds. However, present biosynthetic performance still awaits improvements to be cost competitive with petroleum-based chemical synthesis and to be suitable for large-scale industrial production. In order to achieve this goal, many approaches have been created, among which PopQC (Population Quality Control) is proved to be efficient. In our project, PopQC was developed in Bacillus amyloliquefaciens LL3 and Bacillus subtilis 168 to continuously select high-performing cells in order to improve the yield of target metabolite—glutamate. In the presence of PopQC, 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 poly-γ-glutamate yield enhancement.

Motivation

Artificial biosynthetic pathways have enabled renewable, environmental-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.

Our choice—PopQC

PopQC, which is the abbreviation for population quality control, is a new approach designed for biosynthesis yield enhancement based on the non-genetic cell-to-cell variation. Because of some nongenetic 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 realize the efficient utilization of substrates and high yield of target products. Based on this, PopQC was designed as a plasmid-based gene circuit, which continuously selects high-producers to optimize the biosynthetic performance.

Introduction of chassis organism

In our project, Bacillus amyloliquefaciens LL3 and Bacillus subtilis 168 were chose to be the host of the plasmid of PopQC. Bacillus subtilis 168 is a Type Strain of Bacillus, which was constructed for easier genegene-manipulation. 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). Poly-γ-glutamate is a high-value 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.

Our design

Here is the basic mechanism of PopQC we designed. In Bacillus amyloliquefaciens LL3 and Bacillus subtilis 168, glutamate synthase, a major enzyme of nitrogen metabolism, is encoded by the gltAB operon. High level expression of this operon requires a LysR-family protein, GltC, encoded by the divergently transcribed gene. GltC regulated gltAB transcription through binding to three dyad-symmetry elements, Box I, Box II and Box III, located in the intergenic region of gltC and gltA. GltC binds almost exclusively to Box I and only marginally activated gltAB transcription. Glutamate-bound GltC binds to Box I and Box III, and repressed gltAB transcription. In the presence of α-ketoglutarate, GltC bound to BoxI and Box II, stabilized binding of RNA polymerase to the gltA promoter, and activates gltAB transcription.

To achieve our goal, plasmid pHT01-PopQC (lacI gene under glt promoter control) was constructed, with the mechanism of gltAB regulation applied, and transformed into Bacillus amyloliquefaciens LL3 and Bacillus subtilis 168. Transformants were cultured in M9 medium with tetracycline at a certain concentration. In high-producers, due to the high concentration of glutamate, gltAB promoter was repressed by Glutamate-bound GltC. Therefore, the expression of Lac I, which binds P_lacO and repress it transcription, was repressed. Without Lac I binding to P_lacO, the expression of Tet R (tetracycline resistance gene) was not repressed, so these cells synthesized enough amount of tetracycline efflux pumps to maintain alive. In contrast, in low-producers the repression of TetR caused inability to pump out tetracycline and put cells to death.

To make this PopQC more practical, the quantitative relationship in it was determined through three parts of assays. In part I, plasmid pHT01-P-eGFP (gfp gene under glt promoter control) was constructed and transformed into Bacillus amyloliquefaciens LL3 and Bacillus subtilis 168. Transformants were cultured in M9 mediums with different concentration of glutamate, which will consequently affect the transcription level of eGFP. By determining the fluorescence density, the effect of Glu-GltC-P_gltAB regulation was determined.

In part II, a group of plasmids named pHT01-Tet (lacI gene under promoters varing in strengths) was constructed by altering lacI promoter and transformed into hosts. Different promoters finally led to discrepant resistance to tetracycline in different transformants. By plate counting, the repression effect of LacI to tetA expression was determined.

In part III, plasmid pHT01-PopQC was constructed and transformed into hosts. By plate counting and testing the concentration of glutamate, the overall effect of PopQC was determined.