E coli is commonly used for the production of recombinant protei

E. coli is commonly used for the production of recombinant proteins and other

valuable products, and the corresponding cultures are usually grown at high growth rates. BTK inhibitor High consumption of glucose is often associated with the excretion of acetate that inhibits recombinant protein production [44, 45]. The findings presented here can provide a better understanding of the strategies involved in metabolizing glucose (as the only carbon-source component of the medium) and acetate that is subsequently produced during glucose utilization, and thus contribute to the development of new strategies for improving growth of industrial strains. Methods Bacterial strains All E.coli K-12 MG1655 [50] strains with reporter plasmids used in this study are listed in Table  4. The strain ARRY-438162 manufacturer containing the plasmid with the reporter Pacs-gfp was constructed as follows. A 858 bp-long intergenic region (comprising the region between acs and nrfA and the parts of the open reading frames) was amplified from the MG1655 chromosome using buy SB202190 the primers Fwd_Pacs_XhoI 5’-CCGCTCGAGTAAGCTGAAGATACGGCGTGC-3’

and Rev_Pacs_BamHI 5’-CGGGATCCCCATCGGCATATAAATCGCCACC-3’ (italic parts of sequences are the restriction sites). The construct was cloned via XhoI/BamHI restriction into the plasmid containing the PptsG-gfp reporter [30] (thus swapping the existing ptsG promoter) and transformed into MG1655. Table 4 List of E. coli strains and plasmids Strain name Characteristics Source MG1655 Wild-type

E.coli K-12 F-, λ-, ilvG-, rfb-50, rph-1 Lab collection, [50] DH5α Strain for plasmid propagation F-, glnV44(AS), λ-, deoR481, rfbC1?, gyrA96(NalR), recA1, endA1, thiE1, hsdR17 Lab collection MG1655 PptsG-gfp ptsG reporter Plasmid library [30] MG1655 PmglB-gfp mglB reporter Plasmid library [30] MG1655 PrpsM-gfp rpsM reporter Plasmid library [30] MG1655 Ppck-gfp pck reporter L-gulonolactone oxidase Plasmid library [30] MG1655 pUA66 Promoterless plasmid in MG1655 Plasmid library [30] MG1655 Pacs-gfp acs reporter This study Growth media The growth conditions are listed in Table  5. Briefly, E.coli strains were grown in minimal media supplemented with carbon source(s) in mini-chemostats [33] or in batch cultures at 37 °C. Table 5 Growth conditions Experiment Batch or chemostat Supplemented carbon source Glucose environments Chemostat, D = 0.15 h-1 0.56 mM Glc   Batch 0.56 mM Glc   Chemostat, D = 0.3 h-1 0.56 mM Glc   Chemostat, D = 0.15 h-1 5.6 mM Glc   Batch 5.6 mM Glc Acetate environments Chemostat, D = 0.15 h-1 0.56 mM Ac   Batch 0.56 mM Ac   Chemostat, D = 0.15 h-1 5.6 mM Ac   Batch 5.6 mM Ac Mixed-substrate environments Chemostat, D = 0.15 h-1 2.8 mM Glc, 2.8 mM Ac   Batch 2.8 mM Glc, 2.8 mM Ac   Chemostat, D = 0.15 h-1 0.28 mM Glc, 0.28 mM Ac   Batch 0.

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