Fig. 8: Application of ReaL-MGE 2.0 in the construction of S. brevitalea DSM7029 malonyl-CoA chassis cells.

a FACS sorting for GFP expression from the FapR biosensor. SSC - side scatter, FSC - forward scatter. b Intracellular malonyl-CoA concentrations were determined for 150 candidates leading to the choice of clone 21# (S. brevitalea DSM7029Δglb.C21) for further work. c Whole genome sequencing of S. brevitalea DSM7029Δglb.C21 showing mutated sites. Green arrows, malonyl-CoA metabolic pathway promoter replacements; purple arrows, LVA insertion; orange blocks, transposase/prophage deletions. d Evaluation of malonyl-CoA in S. brevitalea DSM7029Δglb and S. brevitalea DSM7029Δglb.C21. e Selection frequency of the 22 metabolic targets and 9 deletions in the 150 selected S. brevitalea DSM7029Δglb clones. f FACS analysis using side and forward scatter during growth of S. brevitalea DSM7029Δglb and S. brevitalea DSM7029Δglb.C21 to evaluate cell death at 24, 48, and 72 h in batch culture. g Images of cells from the growth experiment of (f). The experiment was repeated twice independently with similar results. h Scheme of ReaL-MGE 1.0 to replace the promoters of the epoA-epoF BGC with artificial promoters. P11, P12, P13, P16, P35 and P37 are described in previous study⁷². i Quantitation of epothilone C/D yields obtained from S. brevitalea DSM7029Δglb and S. brevitalea DSM7029Δglb.C21 using the epo BGC BAC with or without the accA1/pccB/tRNA/epi/matB cassette and/or the six-promoter exchange as indicated. Values are means of the biological replicates, and the error bars indicate the standard deviations of all (n = 5 for d and n = 3 for i) biological replicates. P-values were obtained using the two-tailed Student’s t test: ****P < 0.0001.