Fig. 4
Probing and alleviating the potential metabolic bottlenecks of the upstream or downstream modules of EcJW204 by varying the strength of promoters and/or ribosome binding sites. a Design of synthetic operons for the upstream and downstream modules. For the upstream module, the T7 promoter in MCS2 and the RBS between T7 promoter in MCS2 and the start codon of pdc were replaced with the combination of PAY1 or PAY3 promoter and 0.3 or 0.03 a.u. RBS. For the downstream module, the RBS between T7 promoter in MCS1 and the start codon of pct gene and the RBS between T7 promoter in MCS2 and the start codon of VAAT gene were replaced with the combination of 90, 9000, or 90000 a.u. RBS and 90, 9000, or 90000au RBS, respectively. Production of ethyl lactate in high cell density cultures of b EcJW209-212 and c EcJW213-221. Green rectangle: low copy number plasmid (10); P15A: origin of pACYCDuet-1; red rectangle: high copy number plasmid (100); RSF1030: origin of pRSFDuet-1; PT7: T7 promoter; TT7: T7 terminator. All of the strains were induced at 0 h with 0.01 mM IPTG. Each error bar represents 1 s.d. (n = 3)