Since the selection of spontaneous mutations during metabolic evolution has been a major component of the development of ethanologenic E. coli, many of the underlying changes contributing to ethanologenesis remain unidentified. Identification of these changes will aid in the development of biocatalysts with desired properties for production of other products.

3.1

Physiological Differences Conferring Ethanol Resistance to LY01

LY01 is a derivative of KO11 that was selected in rich medium for increased ethanol tolerance and yield [18]. As described above, LY01 had greater than 80% survival from brief exposure to 100 gL-1 ethanol, compared to only 10% survival for KO11 [18]. The transcriptomes of these two strains were compared in LB with glucose or xylose and with 0, 10, or 20 gL-1 ethanol [72]. Some 205 genes were differentially expressed in LY01 rela­tive to KO11, as determined by the student’s f-test; 49 of these genes were greater than twofold different in each comparison. Functional groups related to amino acid biosynthesis, cell processes, cell structure, central intermediary metabolism, and energy metabolism contained a high percentage of differ­entially expressed genes. Additionally, many stress-related genes, including those related to acid and osmotic stress, were differentially expressed.

Three major physiological differences between LY01 and KO11 were sug­gested by transcriptome data and supported by further analysis: increased glycine degradation, increased expression of genes related to betaine syn­thesis and uptake of protective osmolytes, and lack of FNR regulatory func­tion [72]. Normally, FNR regulates the expression of genes required for fer­mentation and anaerobic respiration (reviewed in [73]). Glycine metabolism and expression of FNR-regulated genes both impact the availability and dis­tribution of pyruvate. It is interesting to note that betaine synthesis genes are affected by FNR via ArcA [74,75]. Thus, the increased ethanol tolerance of LY01 seems to be a combination of several physiological factors, particularly those related to pyruvate partitioning and osmotic protection.

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