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P_gltAB-LacI-P_grac-TetA

LacI is an inhibitor of P_grac, TetA is a tetracycline resistance protein. The LacI expression is downregulated by glutamate because P_gltAB is upregulated by GltC, which is repressed by cellular glutamate level; P_grac is down regulated by LacI. As a result, higher the cellular gultamate level is, more TetA expressed, better the bacteria can survive under tetracycline circumstance.

Sequence and Features

Introduction

Bacillus amyloliquefaciens LL3 and poly-γ-glutamate（γ-PGA）

B. amyloliquefaciens LL3 is one of most prevalent Gram-positive aerobic spore-forming bacteria. It was isolated from fermented food with ability to produce poly-γ-glutamic acid in a glutamic acid-independent way.

γ-PGA is a commercially available biopolymer made of D- or L-glutamate units connected by amide linkages, which is nontoxic, edible, degradable and absorbent. It can be used as food and cosmetic additives as well as flocculants for sewage disposal.

Here, we chose B. amyloliquefaciens LL3 as our genetically engineered microorganism (GEM) and γ-PGA as the target product. We intended to enhance the yield of γ-PGA in LL3 with our PopQC system. Besides, the PopQC system also has great potential to be utilized in other bacterial strains.

Principle of Population Quality Control (PopQC) system

Population Quality Control (PopQC) system is a new approach designed for biosynthesis yield enhancement, based on non-genetic cell-to-cell variation, which includes unequal cell division, different gene copy numbers, epigenetic modifications, random gene expression, volatile RNA stability, protein activity, etc. Because of those 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. Thus, it has been proved to be an efficient way for biosynthesis being more suitable for large-scale industrial production. In our project, PopQC was designed as a plasmid-based gene circuit in Bacillus amyloliquefaciens LL3, which continuously selects high-performing cells in order to further improve the yield of target metabolite—glutamate, and then the secondary metabolites—γ-PGA. (See Figure 1.)

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.