Fig. 5

Reductions in n-butanol-derived byproducts and screening of various E. coli strains for n-butanol production. Escherichia coli DH5α cells containing pAB-HCTA(Cam^R) or pABA-HCTA(Amp^R) were grown in TB medium supplemented with 20 g/L glycerol at 37 °C for 48 h. a, b Metabolic products, including n-butanol and its derivatives, were determined by GC. The fold increase of n-butanol production was calculated as \(n{\text{-}}butanol\; (fold) = \frac{{BtOH_{amp} }}{{BtOH_{cam} }} \times 100,\) where BtOH is n-butanol concentration. The subscript amp designates the tested cells harboring the pABA-HCTA, whereas cam indicates a control with the pAB-HCTA plasmid. c To screen the best n-butanol producer among various E. coli strains, three different E. coli strains (DH5α, MG1655, and BW25113) were examined. Two plasmids, pSECRi-PFLA carrying a quadruple sgRNA array and pABA-HCTA encoding heterologous n-butanol-pathway genes, were introduced into each E. coli strain, which was grown in TB medium supplemented with 20 g/L glycerol and 4 mM l-rhamnose at 37 °C for 48 h. n-Butanol levels were determined by GC. The fold increase of n-butanol production was calculated as \(n{\text{-}}butanol \;(fold) = \frac{{BtOH_{xv} }}{{BtOH_{dh} }}\; \times \;100,\) where BtOH is n-butanol concentration. The subscript xv designates the tested cells harboring the pABA-HCTA and pSECRi-PFLA, whereas dh indicates E. coli DH5α cells with the pABA-HCTA and pSEVA221. Data represent the averages of three biological cultures, and error bars show the standard deviation (SD)