High-throughput Screening And Genome Shuffling Of Streptomyces Virginiae For Improved Virginiamycin Production

Virginiamycin,produced by Streptomyces virginiae,is an animal feed additive composing two completely different components with a narrow inhibition range.Virginiamycin has strong antibacterial activity against gram-positive bacteria,including Sarcina lutea,Bacillus subtilis,Staphylococcus aureus.It is recognized in the industry as a safe and non-toxic special antibiotic for livestock and poultry.Virginiamycin has been commercially produced in overseas and has not been accomplished in China currently.Therefore,it is very valuable to investigate and develop virginiamycin further as a new generation of antibiotic.In this dissertation,the conditions of preparation,regeneration and fusion of protoplast were studied,and the production of virginiamycin from S.virginiae was improved by genome shuffling and ribosome engineering companied with a high-throughput screening method integrating deep-well cultivation and the cylinder-plate detecting.The main research contents and results are as follows.A HPLC method for virginiamycin analysisin fermentation broth was developed.The method is accurate and reliable for the determination of virginiamycin.A high-throughput screening method combining deep-well microtiter-plates cultivation with cylinder-plate method using Micrococcus luteus CMCC(B)28001 as test organism was established,which makes the screening procedure more efficiently and the expensive and tedious HPLC analysis can be reduced to the minimum.The new high-throughput procedure is versatile to screen high-yield mutants for antibiotics production systems.S.virginiae CICC 11013 were used as the starting strains.After treated with ion implantation,ultraviolet(UV)and microwave(MW)mutagenesis,four strains,strain UV1150,UV1154,MW158,and MW1243 with the highest VGM production were selected by using the medium with selective pressure of streptomycin sulfate.VGM yields of the four strains were 61.2 ± 5 mg/L,53.3±4 mg/L,58.2 ± 5 mg/L,and 50.7± 3 mg/L,respectively.The conditions for preparation and regeneration of the protoplast,including enzyme concentration,hydrolysis time,and mycelia age,were optimized by a L9(33)orthogonal experimental design.The conditions for protoplast fusion were also studied.The optimal conditions for protoplast regeneration were chosen that 30 h mycelia of S.virginiae CICC 11013 were treated with 3 mg/mL lysozyme for 60 min.The protoplasts were inactivated by UV and heat respectively and then fusion between biparental inactivated protoplasts was carried out.A 10 min UV irradiation using a 30-W UV lamp above the plate and heat treatment at 65 ? for 20 min both gave an inactivation efficiency of 99.9%when a fusant probability of 98%was observed.40%PEG 1000 and 30 ? was chosen as the optimal concentration and temperature for the fusion of protoplasts.Genome shuffling combined streptomycin resistance screening technique was carried out with the four mutants(UV1150,UV1154,MW158 and MW1243)as the starting strain.After five rounds of successive protoplast fusion,a 100 ug/mL streptomycin resistant recombinant,G 5-103,was obtained.VGM yield reached 251 mg/L,which was 3.1-fold higher than that of the highest mutant strain UV1150.