| Directed evolution enhances biological phenotypes with wide-ranging applications for pharmaceuticals and biotechnology. In the laboratory, scientists regularly engineer organisms for specific purposes, such as the biosynthesis of biologics. This type of purpose-driven engineering typically involves targeted manipulations of known cellular processes (metabolic engineering) or the creation and subsequent screening of mutant libraries (strain improvement).; We created a library of E. coli mutants with transposon insertions and screened this library for increased titers of 6-deoxyerythronolide B (6dEB), a precursor of the antibiotic erythromycin. The screen of over two thousand mutants identified a strain that showed around a two-fold increase of 6dEB titers. The transposon disrupted the treC gene, which encodes the enzyme trehalose-6-phosphate hydrolase. The parental strain showed a surprising decrease in 6dEB titers, beginning at 72 hours post-inoculation. We hypothesized that this decrease in 6dEB titers is due to the hydrolysis of the ester group of 6dEB, resulting in ring-opening and giving the molecule a net negative charge that prevents the molecule from being extracted by ethyl acetate. Subsequent mass spectrometry investigations appear to support this hypothesis, with the detection of a molecular mass that corresponds to the predicted molecular mass of this 6dEB-derived hydroxy acid. The rate of this ring-opening reaction appears to correlate with the alkalinity of the culture; the parental strain reaches a maximum pH of 9.39 while the mutant strain reaches a maximum pH of 8.61. Furthermore, the alkalinity of the culture appears to correlate with the growth rate of the culture. By growing slower due to the inability to metabolize trehalose (a component of yeast extract that is used in LB medium), the mutant strain may maintain a more neutral pH, leading to slower base-catalyzed ring-opening of 6dEB.; This work illustrates insights and applications that can arise from applying directed evolution to laboratory biosynthesis of small molecules. The identification of a mutation created by a transposon led to a hypothesis that involved growth rates, alkalinity of LB cultures, and base-catalyzed hydrolysis of a macrolactone, which further experiments supported. Furthermore, properties of the mutant strain suggest various ways to increase yields of pharmaceuticals from laboratory processes. |
