Study On Femtosecond Laser Mutagenesis For Higher-yield Strain Of Micronomicin And Fermentation Kinetics

Micronomicin (MCR) is a kind of aminoglycoside antibiotics produced byM.sagamiensis ver.nor.reducaus which is found by Nara Daka of Kyowa HakkoKogyo Co Ltd in Kanagawa Sagamihara. It has a feature of broad antibacterialspectrum, bactericidal ability, small resistance and large safety. It also has a goodinternal organs and the surrounding tissue distribution, and low ear, kidney toxicity. Itintroduce a methyl at6’-N position of Gentamicin (GM) C1a.So it has antibacterialeffect to6’-N acetyltransferase resistant bacteria, it significantly better than GM. It isa better new aminoglycoside antibiotic salternative to GM.The application of gene manipulation in Micromonospora strains is still impededby the lack of high-level expression vector and receptor plasmid vector system forthese microbes. The improvement of antibiotic-producing strains has beenconventionally achieved through mutation by classical UV, chemical mutagens and soon. It has an important scientific value and commercial value for guiding the rationaltransformation of follow-up strains.First, a titanium-sapphire laser was applied to obtain a mutant MX3004ofMicromonospora sagamiensis ATCC21826for micronomicin (MCR) productionenhancement, under the optimized irradiation conditions of75mW and180s, with amaximum of positive mutation rate of17.8%and the mortality rate of69.2%. A novelhigh-throughput method was established using microplate reader by quantifing theconcentration of MCR for efficient screening of positive mutant from large numbersof mutants. Finally, MX3004displayed the highest MCR production capacity of126mg/L and a stable heredity (ten generations).Second, the optimized fermentation medium was harvested by optimizing theaddition of exogenous precursors and metal inorganic salt. In the optimizedfermentation medium, soluble starch10g/L，soybean5g/L，K2HPO41.2g/L，CaCO34g/L，CoCl20.001g/L，MgSO40.01g/L，FeSO40.015g/L，xylose0.05g/L, theascomycin fermentation titer of MCR was263mg/L，91.9%higher than beforeoptimization (137mg/L). Third, in order to find the underlying high producing mechanism, it is pivotal tocompare the genome differences between the mutant and parent strain. Four basemutations were identified in the Micromonospora sagamiensis gene from MX3004.The base mutation at site492was a synonymous mutation, while the other three basemutations at sites250,362and526resulted in the changes of amino acid residue ofthe protein encoded by this gene (Thr84Ser, Ser121Thr and His176Asp). Thus, thelaser indeed caused gene mutation that might impact the biosynthesis of MCR.Forth, a comprehensive intrinsic kinetics of mutant MX3004and parental strainwere studied and the mathematical models were established, which were used todescribe quantitatively the difference of fermentation behavior of both strains.Moreover, under the optimal fermentation conditions in a7.5liter fermenter, theMCR production of MX3004reached the maximum of263mg/L, which wasincreased by484%compared with the parent strain.