OBJECTIVE To solve the bottleneck of plasmid instability during microbial fermentation of L-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase. RESULTS The tyrosine phenol lyase from Fusobacterium nucleatum… Click to show full abstract
OBJECTIVE To solve the bottleneck of plasmid instability during microbial fermentation of L-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase. RESULTS The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed. Efficient strategies including replacing the original ampicillin resistance gene, as well as inserting cer site that is active for resolving plasmid multimers were applied. As a result, the plasmid stability was increased. The co-use of cer site on plasmid and kanamycin in culture medium resulted in proportion of plasmid containing cells maintained at 100% after fermentation for 35 h. The specific activity of tyrosine phenol lyase reached 1493 U/g dcw, while the volumetric activity increased from 2943 to 14,408 U/L for L-DOPA biosynthesis. CONCLUSIONS The established strategies for plasmid stability is not only promoted the applicability of the recombinant cells for L-DOPA production, but also provides important guidance for industrial fermentation with improved microbial productivity.
               
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