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- Title
Escherichia coli Protoplast Fusion and Screening of High L-Lysine-Producing Strain.
- Authors
Zirri WANG; Junqing WANG
- Abstract
[Objectives] Protoplast fusion of two parentat strains with weak L-lysinc production abilite was earned out te obtain new fusion strains with strong L-lysinc production ability. [Metiods]Thc effect of bacteial age, lysozyme concentration, enzymolysis temperature and time on the protoplast formation rate and reaeneration rate were investioated by singie factor experiments. On this basis, wii the protoplast formation rate as an index, the protoplast preparation process was optimized by an orthogoni experiment. [Results] Bacterial age and enzymolysis I otic had a greater impacC on the protoplast formaton rate, followed by enzymolysis temperature and lysozyme concentration. The optirnal process ior Wc preparation of L-lysine-producing Escherichia coli protoplasts was to prepare parental protoplasW from bacteriai cells cultured for 15 h in the late togaithmic growth phase bs enzymolysis with 0. 8 mg/ml lysozyme at 37 a for 180 min and promote fusion with PEG6000. In order to facilitate the screening of fusion protoplasts, the empty plasmiVs pET-28a and pET-Duet were transformed into L-lysine-producing E. coii, a-spectively, and strains pET-28a-lys01 and pET-Duet-lys01 were obtWned. Fusion swains were then obtWned through protoplast fusion. Double-resistancc KA1-10 were screened on plates containing kanamycin and ampicillin, and a high-yielding fusion strain KA8, which produced L-lysine, was screened by fermentation exper-irnents I'inrilly. [Conclusions] The results of this study proWe a referencc method for iurWer improving the yield of L-lysinc and other amino acid strains.
- Subjects
PLANT protoplasts; ESCHERICHIA coli; LYSOZYMES; PROTOPLASTS; CELL culture; KANAMYCIN; CORYNEBACTERIUM glutamicum
- Publication
Agricultural Biotechnology (2164-4993), 2022, Vol 11, Issue 3, p127
- ISSN
2164-4993
- Publication type
Article