Gradient radiation breeding and culture domestication of menaquinone producing strains.

By comparing the survival rate and positive mutation rate of the primary mutagenic strain and progeny mutagenic strain under different radiation doses, the results showed that the tolerance of the mutagenic strain to radiation dose increased with the increase of the mutagenic generations. We adopted an improved gradient radiation breeding strategy to improve the breeding efficiency. The strains were treated with radiation in four stages. The first stage was low energy N+ ion implantation (ion energy 15 keV, dose 80 × 2.6 × 1013 cm-2). In the second stage, the energy and dose of N+ ion reached to 20 keV, 90 × 2.6 × 1013 cm-2. In the third stage, 60Co-γ radiation (dose of 1.56 kGy) was used. In the fourth stage, the radiation dose of 60Co-γ increased to 1.82 kGy. After each stage of radiation, the MK (Menaquinone) precursor 1, 4-dihydroxy-2-naphthalate (DHNA) was used as the stress factor to domesticate the mutant strains. By gradually increasing the concentration of DHNA in the culture medium, the substrate tolerance of Flavobacterium sp. was effectively improved. By measuring SOD (superoxide dismutase) activity and malondialdehyde, it showed that the cell damage caused by radiation mutagenesis to the offspring mutant was less than that of the primary mutant. Changes in membrane permeability and membrane potential of the mutant strains were reflected in changes in fluorescence intensity of luciferin diacetate and rhodamine 123, which could explain the enhanced substrate tolerance of strain F-2. After gradient radiation breeding and culture acclimation, the biomass of mutant Strain F-2 was 6.59 g/L, and the MK yield was 9.59 mg/L.