Reverse Engineering of Improved Strains

Our dependence on evolutionary methods of strain improvement, such as those described in Section 18.2.4, demonstrates Orgel s second rule that evolution is cleverer than you are. When we lack the information needed for rational design, we can build improved strains by evolution and selection of expression libraries. But in order to learn from these improved strains, we must invest the effort in identifying and understanding the key mutations, so that these new design strategies can be implemented into other strains. 

The identification of mutations within . coli s LPD andS. cerevisiae s SPT15 that enable improved ethanol production has been described above, as has the identification of increased expression of otsA as a means of improving ethanol production in E. coli. Unfortunately, many of the evolutionarily acquired improvements in ethanol production remained unexplained. Here we briefly describe a few of the tools that are becoming available to aid in this reverse engineering. 

In addition to engineering KOll for improved ethanol production, ethanol tolerance, and substrate utilization, efforts have also focused on identifying the mutations acquired by KOll during the evolution process and understanding role in the ethanologenic phenotype. Specifically, KOll was subjected to 

Extensive characterization of an evolved ethanol-tolerant R coli strain through both genome and transcriptome analysis recently identified three mutations that counteract the detrimental effect of ethanol on transcription and translation machinery [144]. Specifically, Haft etal. identified mutations within a ribosomal protein RpsQ, methionine synthesis regulatory MetJ, and the transcription termination factor Rho that improved ethanol tolerance, though at the time of this writing the effect of these mutations on ethanol production has not yet been publically described. 

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