미생물 생산 개선을 위한 트랜스포터 고처리능 선별 방법 개발

Abstract

Biorefinery refers to a sustainable process of converting biomass into valuable chemicals such as natural products, polymers, biofuels, or novel chemicals. The process typically uses microorganisms, which natural biological systems have evolved metabolisms suitable for cell survival and reproduction. Therefore, it is necessary to manipulate the overall metabolism of microbes to produce large amounts of specific chemicals. Comprehensive engineering of microbial metabolism to improve the productivity of microbial cell factories mainly employs two strategies. The first strategy focuses on the analysis and modification of key enzymes, the construction of novel synthetic pathways and optimization of carbon flux, and metabolic adaptation. The other strategy focuses on the molecular transfer of substrates, intermediates, and end products between the extracellular and membrane-enclosed intracellular space. Transporting phenotype engineering can improve substrate utilization, concentrate the metabolic flux from intermediates to end products, mitigate cytotoxicity, and simplify the purification process. Although regulating molecular transfer is an effective strategy in biotechnology, transporting phenotype engineering has not been widely used due to the limited genetic information. Transport is generally known to use membrane protein transporters. However, in the case of non-native chemicals, which are often produced in biological processes, the engineering is limited because of lack of knowledge about transport route. To improve the efficiency of biological processes rapidly, finding transporting phenotype-related genes is necessary without prior knowledge. This can be achieved through a library with different transporting phenotypes and high-throughput methods for screening strains with improved transporting phenotypes in the library. To find a genotype that changes the transporting phenotype in the production strain, a knockout library or an endogenous random gene overexpression library of the genomic DNA of the production strain can be used. In the knockout library, phenotypic changes may not be caused by other genes with similar functions. A library that randomly overexpresses endogenous genes can be constructed by fragmenting the genomic DNA of the production strain, ligating it to a high-copy-plasmid, and transforming the production strain. A biosensor is a genetic circuit that detects the concentration of a target chemical in a specific and sensitive manner and converts it into easy-to-measure gene expressions such as fluorescent protein-encoding genes or antibiotic resistance genes. Since riboswitch and transcription factor-based biosensors bind to chemicals in the cytosol to regulate gene expression, they can detect only intracellular chemical concentrations that are distinct from those outside the cell and convert them into fluorescent proteins. Differences in transporting phenotypes between strains can be detected and used for screening in a high-throughput manner using the characteristics of biosensors. In this study, high-throughput screening methods of strains-enhanced importing and exporting phenotypes were developed, respectively. Different methodologies were employed but briefly summarized as follows. First, a library with changes in transporting phenotype was constructed through endogenous random gene overexpression. The library was constructed by fragmenting genomic genes and attaching them to high-copy-number plasmids. Second, the concentrations of the transported chemicals were converted into fluorescence through the biosensor. Finally, differences in fluorescence between cells were detected, and strains with enhanced transporting phenotypes were selected through flow cytometry.

The followings are summarized contents by chapters.

In chapter 1, microbial-based chemical production and the necessity of transporting phenotype engineering were introduced. In addition, recent efforts in transporting phenotype engineering and previous transport-related gene identification methods were described. In chapter 2, the screening process of strains with enhanced importing phenotype was developed and the 3-hydroxypropionic acid (3-HP) import-related gene was identified. Among the random gene overexpression library, strains overexpressing the 3-HP import-related genes exhibited high fluorescence by the biosensor, and the strains were sorted through fluorescence-activated cell sorting (FACS). Following sequence analysis of the gene fragment, narQ was identified as the gene that improves 3-HP uptake. In chapter 3, a system to screen strains with enhanced exporting phenotype was established by distinguishing the amount of the desired substrate secreted out of individual cells. Depending on the amount of the substrate imported into the cell, the intracellular concentration varies among individual cells and can be easily compared through a biosensor. However, the amount of the substrate secreted out of the individual cell is difficult to distinguish because all cells export the substrate to a shared medium. By compartmentalizing cells into individualized droplets using a microfluidic chip, the amount of substrate secreted by each cell can be distinguished from the others. Each random gene overexpression library strain and the biosensor-introduced cells were co-cultured in a droplet to convert the corresponding secreted amount into fluorescence intensity by the biosensor. In this platform, the strains with enhanced exporting phenotypes can be screened through the process of the production and secretion of chemicals into droplets by library cells and the detection and conversion to fluorescence by sensor cells. In chapter 4, with the application of previously developed cell-to-cell communication-based strains with exporting phenotype screening method, two strains with improved 3-HP secretion were found. Overexpressed gene fragments in the strains were identified through sequencing and individual evaluation of several gene fragments. The result showed that the overexpression of setA and yjcO improved 3-HP efflux. In addition, overexpressing the corresponding exporters, respectively, enhanced 3-HP titers by 39% and 36%, compared to the parental strain.