Description

0. Abstract

Synthetic biology has been used to produce various compounds, yet its’ yield still await improvements to become economical. It has been revealed that in a single colony, different cells have considerably variations in protein and metabolite concentrations due to some nongenetic differences, suggesting the existence of both high- and low-performance variants in all cultures. Our project is aimed to design an in vivo population quality control system(PopQc) to continuously select high-performing, nongenetic variants and in that improving the yield of target products. In the presence of PopQc, we could accurately select cells producing glutamate above a settled level by changing the concentration of tetracycline, which could give a notable improvement to the yield of glutamate.

1. Motivation

By means of gene manipulation, microorganisms, especially bacteria, have been widely used as bio-factories to synthesize various kinds of significant products ranging from simple fuels (for example, ethanol, butanol and fatty acid derivatives) to intricate natural products (artemisinin, strictosidine, erythromycin, and so on). However, one great weakness of biosynthesis compared to petroleum-based chemical synthesis is that biosynthesis often tends to be uneconomical. Its’ relatively low productivity and yield have suppressed it future development. So, it’s necessary to create new approaches to enhance biosynthetic performance.

Nongenetic variation emerging in microbial cultures is known to be attributed to naturally inherent factors including uneven cell division, epigenetic modifications and variations in gene copy numbers, random gene expression and variant mRNA stabilities and protein activities. The overall effect of these factors, no matter in plasmid-based or in chromosome-based gene expression, could generate a considerably range of variation in protein and metabolite concentrations, suggesting a potential approach to improve biosynthetic yields. So based on that, we developed in vivo PopQc system.

2. PopQc

What is PopQc? PopQc is the logogram of population quality control system. This system selects high-performing cells by the product-mediated regulation of transcription of antibiotic resistance gene. In the presence of antibiotic, while high-performing cells could produce antibiotic-resistant proteins properly and maintain alive, low-performing cells due to the low concentration of target product in vivo, are suppressed to express antibiotic-resistant proteins and ends in death. In our assays, we choose Bacillus amyloliquefaciens LL3 as the host to be engineered, and our aim is to select cells in which the concentration of intracellular glutamate is high.

3. Introduction of Bacillus amyloliquefaciens LL3

Bacillus amyloliquefaciens LL3, which is a gram-positive bacterium, was originally separated from traditional fermented foods by prof. Song’s lab. Here are the main characteristics making it suitable to be the host. First, it has a clear genetic background, because our lab has already finished its’ genome sequencing in 2011. Second, its’ synthesis of poly-γ-glutamate (γ-PGA) is independent of exogenous glutamate.

4. Our design

To achieve the selection of high-performing cells, our project is aiming to apply PopQc in Bacillus amyloliquefaciens LL3. Here is the basic mechanism of PopQc we designed. Transcription factor GltC, positively regulated by α-ketoglutarate and negatively regulated by glutamate, could enhance the promoter PgltAB, which is followed by lacI, the gene of protein LacI. LacI could bind promoter PlacO，then suppress the expression of the following tetA, gene of tetracycline discharge pump. So, assuming the outer tetracycline concentration is at an even level, when the glutamate concentration in the cell achieves a certain level, the cell will have the ability to pump out tetracycline to stay alive. On the contrast, in low-perform cells, the suppression of tetA expression would lead these cells to death. After designing this system, we also need to quantify it. So, we designed some further assays to examine.