Screening And Breeding Of High L-glutamine-producing Strains By Genome Shuffling

L-glutamine（L-Gln） is one of the20essential basicl amino acids constituting proteins. Asthe energy substance and amino carrier,L-glutamine plays an important role in nutrition andpharmacological action,which is supposed to be one of the most important amino acid andconditionally essential amino acid. L-glutamine is clinically mainly used in the treatment ofpeptic ulcer and cranial nerve diseases, is a very promising new drugs and the dosage of it athome and abroad is very impressive. At present L-glutamine is mainly produced through thefermentation process of microbes,but fermentation production of L-glutamine in China has yetto realize large-scale industrialized production.The reason mainly is that there are still somegaps between the domestic production of with which of developed countries such as Japan.According to the above present situation, the paper used genome shuffling to improve theperformance of Brevibacterium flavum B7and breed a excellent L-glutamine-producing strainwhich was high-yield, short growth cycle and stable. The specific results were as follows:1. Brevibacterium flavum B7（L-glutamine yield was37.27g/L）was used as the originalstrain. Experiments used UV,DES and both of them combined mutagenesis to get fourstrains（No. U-86, U-107, D-97, DU-12）which had higher yield,stable genetic traits andmethionine sulfoxide （MSO） resistance. Their yield were41.78g/L,42.62g/L,41.38g/L,42.23g/L,which were14.05%to17.47%higher than the starting strain.All of them were used asthe parents of genome shuffling.2. The way of Brevibacterium flavum protoplast formation, regeneration and fusion wasestablished through exploreing the influencing factors of Brevibacterium flavum protoplastpreparation, regeneration and fusion. The optimal conditions of protoplast preparation,regeneration and fusion were as follows：pretreated the Brevibacterium flavum at the early time of exponential phase, the optimum concentration of penicillin was0.4u/mL, glycine was2%,preconditioning time was2h, enzyme concentration was1mg/mL, enzymolysis time was12h,hydrolysis temperature was37℃,PEG6000was40%,fusion temperature was32℃,fusion timewas20min. Under the optimal conditions, the protoplast formation rate reached99.71%,theregeneration rate reached16.52%,the fusion rate reached20.69×10^-5%.3. The four mutants were used as the original strains and after three rounds of genomeshuffling we got a recombinant strain F3-37which yield L-glutamine highly. The productionwas48.61g/L, the production increased30.42%than the original strain B7,increased from14.76%to17.07%than the parent strains. After continuous passaging experiments we provedthat its high yield genetic characteristics were relatively stable, and the yield characteristics ofthe recombinant strains were caused by genome shuffling, rather than the protoplastsmutagenesis occured in the preparation and regeneration process by control experiments.4. The fermentation medium and fermentation conditions of recombinant strain F3-37were optimized by single factor experiment and orthogonal experiment.The optimumfermentation medium compositions were determined as follows: glucose140g/L, KH₂PO₄2.5g/L, MgSO₄·7H₂O0.5g/L, MnSO₄·H₂O10mg/L, ZnSO₄·7H₂O1.0mg/L, CuSO₄·5H₂O1.0mg/L, Thiamine1.0mg/L, corn steep liquor20mL/L、（NH₄）₂SO₄80g/L、CaCO₃60g/L；Theoptimal fermentation conditions were as follows: temperature32℃, the initial pH value7.0,and inoculation quantity12%, volume shake flask25mL/250mL, reciprocating shaker110r/min.Under the optimal conditions, the fermentation cycle was44h, The production reached53.24g/L, which was9.52%higher than before.