The Screening Of The Phage-resistant And Optimal Fermentation Conditions Of Gene-engineering Strains Of L-Threonine

As the production of L-threonine, Escherichia coli TDH-6 VIC40, fermented by using genetic engineering strain, is as the parent stain in this dissertation. An anti-bacteriophage engineering strain Escherichia coli FCD13 was screened by UV mutation. The characteristics of FCD13 was shown. The main research contents and results are as follows:1. L-threonine concentration in fermented broth was quantitatively analyzed with the ways of the charge transfer reaction of threonine with tetrachlorobenzoquinon. The experimental conditions for measuring threonine with tetrachlorobenzoquinon method were analyzed. A series of experiments were performed to optimize the colorimetric conditions and validate the described method by single-factor experiment. The results showed that the linear relationship was better in 352 nm when the content of L-threonine was 5ug/mL-30ug/mL. The regression equation was A352nm=0.02414C+0.0597 with R2=0.9949. This method is simple, accurate, rapid, and of good reproducibility, so it can be applied to quick analysis of L-threonine in fermentation industry.2. Based on the means of UV mutations, a L-threonine high-producer FCD13 was successfully screened when the genetic engineering strains Escherichia coli TDH-6 VIC40 is treated by repeatedly selection. The stain FCD13, resistant to the phage, was able to accumulate L-threonine though the fermentation is without being optimized. The mutant trait was inherited stably for five generations in succession.3. In this paper the conditions of shake-flask fermentation of strain FCD13 were studied. The optimum composition of seed medium and fermentation medium were obtained by orthogonal design experiment and single-factor experiment. The fermentation conditions were also studied. Under the best culture condition, the production of L-threonine of the strain FCD13 was to 5.98 g/L in 48 hours, which was more than that of the original stain.4. The stability of the genetic engineering strain FCD13 was studied. The results showed that the stability of the genetic engineering strain FCD13 in fermentation by the single-factor experiment. It improved plasmid stability to add streptomycin to breeds medium.