Functional investigations of cell wall alterations in chemical-evolved E. coli strains

Project Details

Description

One of the largest barriers to achieving economical bio-based production of bulk chemicals such as biofuels and polymer precursors is poor tolerance of microbial production hosts toward high concentrations of excreted product. These concentrations are often in excess of 100 g/L in order to minimize capital and downstream purification costs. However virtually all chemicals at these levels result in stresses and poor growth in the majority of microbial hosts, which can decrease product yields and productivities. To help address this issue, we utilized a robotic platform to evolve parallel populations of Escherichia coli K-12 MG1655 for enhanced growth in the presence of toxic concentrations of 11 chemicals representing diverse functional classes that are of interest as biofuels or their precursors, polymer precursors, and other bulk chemicals and intermediates. Resequencing of over 200 strains and subsequent reconstruction of sets of mutations has provided unparalleled insight on the genomic basis of tolerance. In addition to more specific mechanisms for individual chemicals or classes of chemicals, many broader mechanisms of tolerance have been putatively identified that recur in strains evolved on different chemicals.
One class of common mutations across chemical conditions are coding mutations in genes related to cell wall biogenesis, maintenance, and recycling. It is suspected that many of the strains harboring these mutations feature altered cell morphologies and altered membrane protein and lipid compositions. In order to understand the connection between genotype and phenotype for cell wall mutations, it is proposed to conduct work at EMSL to further characterize the phenotype of a subset of evolved strains with confirmed morphological changes. The proposed tests include using cryogenic transmission electron microscopy and helium ion microscopy to observe cross-sectional and surface modifications of single cells, and performing differential membrane proteomics and lipidomics analyses.
The data obtained from this study will be used to develop further targeted tests on strains with cell wall mutations, and will ultimately be integrated together with other datasets concerning other types of mutations to develop predictive models of chemical and stress tolerance. The direct effect of cell wall mutations on endogenous production and excretion of relevant chemicals will also be tested by employing them directly in engineered production host strains.
StatusFinished
Effective start/end date01/10/201530/09/2016