Breeding High-yield Strains Of Vitamin K₂and Research On Expression Lever Of Related Synthetic Gene

Vitamin K2is a kind of physiological activity material which has importantroles not only in blood coagulation but also in bone metabolism. In recent years, ithas curative effect in antineoplastic and hepatocellular carcinoma resistance, so itis widely used in pharmaceutical industry. The traditional chemical synthesis is toonumerous to low-yield. This study was conducted to investigate the research onhigh-yield strain of vitamin K2(MK) with protoplast electrofusion, and thehigh-yield mechanism of fusion strain was investigated.(1) The experiment select natto bacillus BS-53as the ancestral strain, whichwas continuously treated with mutagenesis3generation using ultraviolet and NTGcombination of mutation processing. We obtained a high-yield of MK mutationBS-53strain after flat screen and shakeing screen, whose yield increased90.5%andarrived at19.85mg/L, the output is about1.9times as the original strains. With the25times pass down experiment, the result showed that strain BS-53had the geneticstability. The optimization fermentation liquid medium of BS-53was:5%glycerin,soybean extract3%, yeast powder0.6g/L and K2HPO40.3mol/L, CaCl2·2H2O0.1g/L, MgS04·7H2O0.3g/L.(2) In the experiment, we make some break through in the factors of effectformation of protoplast and regeneration. The outcome proves that enzyme’schroma, the time of disposal by enzyme, the optimal temperature, pH osmoticpressure, the choice of steady matter, penicillin pretreatment and synchronousculture had a great effect in the formation of protoplasts and regeneration. On thebase of every effect factors, we do single-factor experiment, obtained the optimalcondition in the formation of protoplasts and regeneration of mutant BS-53is4℃preservation2h synchronous culture,0.6U/ml of penicillin pretreatment2h,centrifugal collect bacteria in pH7.5the mannitol solution, add1.4mg/ml lysozyme37℃enzyme solution40min, the conditions for the formation of protoplasts was97.6%, the regeneration of protoplasts was26.3%. the optimal condition in theformation of protoplasts and regeneration of CICC10262is4℃preservation2hsynchronous culture,2.0U/ml of penicillin pretreatment2h, centrifugal collectbacteria in pH7.5the mannitol solution, add1.0mg/ml lysozyme37℃enzymesolution60min, the conditions for the formation of protoplasts was98.3%, the regeneration of protoplasts was24.3%.(3) The experiment used parents inactivated protoplast electro fusion to breedMK high yield, explores the inactivated strains and the electro fusion conditions.The results showed that through thermal inactivated and UV inactivated, and thenunder the condition of the fusion of frequency2MHz, voltage32V, fusion voltage500V,40μs, pulse number six, get a good screening plant BKU-6, the strains ofvitamin K2reach39.6mg/L, much higher than the low yield of parents CICC10262strains.(4) In order to analyze gene expression changes related to the genes involvedin the biosynthesis of MK，such as menD、menB、menA were investigated byreverse transcription PCR(RT-PCR)between the high-yield strain BKU-6and theirancestral strain BS-53．The results showed the persistent expression of menD andmenB involved in the biosynthesis of MK respectively in the BKU-6had a closecorrelation to the increase of the MK production．It might imply that menD was akey gene for the biosynthesis of MK, while menB was another key gene for the MKbiosynthesis. The other gene menA displayed no obvious changes at this timecourse of fermentation between the high-yield strain and the ancestor. Therefore,menA had no direct link with the increase of the MK production, and the specificmechianism needed to be futher explored.