Fig. 5
Key mutation of C. glutamicum WT_EtOH-Evo identified by genomics and proteomics. (A) Overview of ald (cg3096) promoter and regulatory elements. The transcriptional start site (TSS) is indicated with a black arrow and “+1” [30]. The − 10 and − 35 regions as well as the RBS and the translational start site (TLS) are underlined and written in bold letters. Regulator binding sites are shown as colored boxes. The mutation observed in strain WT_EtOH-Evo is indicated by a red arrow. (B) Differentially synthesized proteins of C. glutamicum WT_EtOH-Evo in comparison to the control strain C. glutamicum WT during cultivation in defined CGXII medium and 428 mM ethanol as sole carbon and energy source. Each culture was sampled twice, i.e. during exponential growth (EVO(t1), WT(t1)) and stationary phase (EVO(t2), WT(t2)), respectively. Left: Proteins with significant changes in all four sampled conditions were determined. Right: Volcano plot showing relative protein abundances between WT_EtOH-Evo and WT at first sampling point. Proteins that are significantly up or down regulated and can be associated with ethanol metabolism by C. glutamicum are highlighted in red. (C) Growth performance of the reengineered strain WT::P01-ald in comparison to WT_EtOH-Evo and the non-evolved wild type. Microscale experiments were performed using one FlowerPlate and defined CGXII medium with 428 mM ethanol as sole carbon and energy source. Mean values and standard deviations derived from six replicate cultures are shown as lines and shaded areas, respectively