The Optimization Of KB Overproduction Strain And Research On Synthetic Biology

Aminoglycoside antibiotics (AGAs) are antibiotics with a wide antibacterial spectrum and antibacterial ability, however, there are also series of side effects, and many gram-positive bacterium have been resistance to AGAs in the long-term clinical application. It is an urgent demand to develop a low toxicity and high efficacy AGAs analogues. In recent years, the dibekacin and arbekacin based on kanamycin B are widely used to solve the issue of resistance effectively, but no independent manufacturers have produced such drugs in China and the related market prospect is very broad. Our group uses kanamycin B as raw material to synthesize dibekacin, and arbekacin, which has applied for the Twelfth Five-year National Key Projects for medicine co-operating with Shandong Qilu Tianhe Pharmaceutical Co. Ltd. Now the KB fermentation yield is too low to meet the requirements of industrial, so we studied the breeding of high-yielding kanamycin B strains and genetic modification, which a number of achievements have been made.Firstly, we mutated Streptomyces kanamyceticus using ultraviolet and protoplast combined with resistance to its metabolite respectively to observe kanamycin B yield. Through ultraviolet radiation and couples of training of KB tolerance, a screened strain could reach up to 86.463 mg·L-1 kanamycin B, which is increased by 23.98% than the fermentation medium without added KB and is increased by 52.49% than the original strains. Through protoplast mutagenesis and couples of training of KB tolerance, a screened strain could reach up to 87.500 mg·L-1, which is increased by 8.59% than the fermentation medium without added KB and is increased by 54.32% than the original strains.Secondly, we discussed the genetic engineering methods to obtain high yield of kanamycin B strains. A plasmid with nemD gene is constructed successfully and we have screened two positive transformants, which could produce KB reached up to 94.200 U·mL-1 and KB production capacity increased by 66.14% on average that is increased by 7.66% than the high yield mutant strain. We obtained the high GC sequences successfully through the research on PCR conditions and constructed a plasmid with neo8 fragment after the fusion PCR of the three high GC target fragments.