Fig. 8: Characterization of synthetic methylotrophic E. coli.

a Complementation of mutated genes with BAC harboring a wild-type copy of the indicated genes in SM7n showed that gltA, ihfA, and rhlB mutations were important to the high growth rate of SM7. Growth rates of various strains with and without BAC complementation were measured in 400 mM methanol (n = 4 for SM7n strains with ihfA, ompR, cra, and gadW expression, n = 3 for SM7n strains with gltA, rhlB, gtrS, quuD, ylcG, gatY and rusA expression; All are biological repeats; data are presented as mean values ± SD; significance was tested by 2-side Welsh-test; *P < 0.05, **P < 0.01). b High-cell-density cultivation of SM8 (SM7 with WT mutS restored) in 500 mM methanol performed in a 2.0 L bioreactor or a 50 mL Falcon tube. The strain was cultivated in a fed-batch mode with continuous supply of methanol and NH₄OH. The M9 and MOPS media have been modified with an adjustment of an initial NH₄Cl concentration of 25 mM. SM8 reached 20 g DCW L⁻¹ within 77 h in the bioreactor with M9-methanol medium. The orange line represents SM8 grown in the bioreactor with M9-methanol medium, the blue line represents growth in the bioreactor with MOPS-methanol medium, and the red line represents growth in the Falcon tube with MOPS-methanol medium. c Growth of SM8 in a logarithmic scale. The doubling time of SM8 grown in a 50 mL falcon tube with modified-MOPS minimal medium containing 400 mM methanol reached 3.6 h. d The profile of fermentation products of SM8 in the bioreactor with the modified M9 methanol medium. (n = 3, sampling repeats; data are presented as mesan values ± SD) (e) Growth of SM7 in Falcon tubes containing MOPS minimal medium with 400 mM methanol (red line), 1% glucose (dark yellow line), 0.1% glucose (blue line), 400 mM methanol plus 0.1% glucose (purple line). All data were done with n = 3, biological repeats. Source data are provided as a Source Data file.