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Engineering Escherichia coli to grow on methanol as the sole carbon source

  • Author(s): Chen, Yu-Hsiao
  • Advisor(s): Liao, James C
  • et al.
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Abstract

Electron-rich and potentially renewable, methanol is a promising alternative feedstock for chemical production. Methanol bioconversion further benefits by boasting mild reaction conditions and higher specificity. Though methylotrophs can utilize methanol natively, engineering on these organisms had been challenging due to limited genome editing tools, sensitivity and comparatively low growth. Thus, many has attempted to enable methanol utilization in industrial-friendly organisms. Unfortunately, converting a non-methylotrophic organism to a synthetic methylotroph that can grow to a high cell density has been strenuous, despite the fact that typical sugar metabolism, namely glycolysis (the Embden–Meyerhof–Parnas pathway), and the ribulose monophosphate (RuMP) cycle which methylotrophs use for methanol assimilation differs by only three enzymes.

Here, our first aim is to reprogram an Escherichia coli to be dependent on methanol for growth. We sought to design an E. coli that survives only when it is able to co-utilize methanol with other carbon sources. We selected to disrupt two genes from the pentose phosphate pathway, ribose-5-phosphate isomerase (rpi) and ribulose-phosphate 3-epimerase (rpe) to construct two different strains that can grow on methanol with xylose and ribose respectively. We then used adaptive evolution and eventually isolated a stable ΔrpiAB auxotroph that can co-utilize xylose with methanol in a 1 to 1 molar ratio and grow to OD600 4.0 in less than 30 hours. This strain was also characterized by genomic sequencing and subsequently engineered to produce ethanol and n-butanol, demonstrating the usefulness of the strain.

Our next aim is establishing completely methylotrophy, and engineering an E. coli to become a synthetic methylotroph using methanol as the sole carbon source. We began from adding the rpi gene back into the auxotroph strain, and took advice from an in-house theoretical flux prediction program, ensemble modeling robustness analysis (EMRA), to determine two enzymes in glycolysis should be tuned down, namely phosphofructokinase (PFK) and glceraldehyde-3phosphte dehydrogenase (GAP). After extensive adaptive evolution, a strain achieved to utilize methanol as the sole carbon source, reaching an OD600 2.0 in 30 hours. This synthetic methylotroph SM1 then revealed a major hurdle that was expected but underestimated: DNA-protein crosslinking (DPC). SM1 eventually overcome this formaldehyde-induced toxicity by metabolic flux balancing through mutations and insertion sequence (IS) mediated copy number variations (CNV). Not only SM1 can grow at comparable rates against natural methylotrophs, but also in a wide-range of different methanol concentrations. While demonstrating how rational-based genome editing and adaptive evolution can steer a major tropism shift, this synthetic methylotroph also expands the horizon of C1 bioconversion.

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This item is under embargo until September 10, 2022.