Research On Transcription Factor And Multiplex Genome Evolution For Improved Performance In E.coli

E, coli is one of the most commonly used industrial strain because of its clear genetic background, easy culturing and rapid growth. Transcriptional engineering and multiplex genome engineering could manipulate multiple genetic locus or the transcription strength of multiple gene at the same time, providing an alternative method for strain improvement.Firstly, this thesis focuses on transcriptional engineering, which regulate the genome-wide scale transcription level directly or indirectly, to achieve the purpose of strain improvement. Global transcription factor CRP is evolved for improving lycopene production in E. coli, the mutant CRP harboring A26T(D8V) mutation improve the lycopene production by 25% in 10 L fermentation process. Microchip-based method reveal that 396 genes are differentially expressed, some key genes related with strain traits and lycopene synthesis are also found. In order to explore the capacity of other global transcription factor, FNR is evolved for E. coli to improve tolerance towards ethanol and butanol, which is enhanced by 30% and 28%, respectively. Thus, the feasibility of strain improvement through transcriptional engineering is fully verified.Secondly, this thesis analyzes the mechanism and procedure of multiplex genome engineering technology, setting up a set of automation platform, automating the building of E. coli mutants library. The device fills the domestic research of genome evolution automation instrument, using automation platform to manipulate multiple genetic locus at the same time instead of manual operation. Bacteria culturing, competent cell preparation and transformation of exogenous DNA are completed through multi-axis manipulator and multiple components, which are the three important steps in multiplex genome engineering.Finally, oligonucleotide-mediated protein evolution in situ is carried out. Two oligos are mismatched in the pqqgdh gene on the chromosome in E. coli through multiplex genome engineering, aiming to gain better thermal stability and substrate specificity. The mutants are selected through high-throughput screen, the half-life of variants protein are improved 4-fold after 55℃ treatment, also the substrate specificity are improved about 0.5-fold. To lower the cost of oligo synthesis, double-strand DNA prepared by error-prone PCR is introduced. Double-strand DNA are mismatched in the dxs gene on the chromosome in E. coli through multiplex genome engineering, aiming to enhance lycopene production. The mutants are selected through color-based high-throughput screen, the highest lycopene production of mutant is more than 5-fold compared to the original strain’s. The enzyme activity of DXS variants protein are measured through HPLC-based method, which confirm that the improved lycopene production is caused by enhanced DXS activity. Thus, the strategy of double-strand DNA mediated protein evolution in situ is established. In order to improve the efficiency of homologous recombination, research is further carried out on preparation of single-strand DNA. The efficiency of homologous recombination is improved to 10% with single-strand DNA through a two-step PCR method. The single-strand DNA is further treated with DNase I, recover 90 bp DNA as mutant library, which makes the efficiency of homologous recombination reach 15%. The dxs gene on the chromosome is evolved through this 90 bp single-strand mediated strategy, improving the lycopene production ability by about 50%, which establish a commonly used multiplex genome evolution strategy in situ in E. coli.In short, this thesis devotes to strain improvement through manipulating multiple genetic locus, setting up two strategies:multiplex genome engineering and transcriptional engineering. The research results are expected to play an important role in metabolic engineering of industrial stains.