Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production.

Methanol is a potentially attractive substrate for bioproduction of chemicals because of the abundance of natural gas and biogas-derived methane. To move towards utilizing methanol as a sole carbon source, here we engineer an Escherichia coli strain to couple methanol utilization with growth on five-carbon (C5) sugars. By deleting essential genes in the pentose phosphate pathway for pentose utilization and expressing heterologous enzymes from the ribulose-monophosphate (RuMP) pathway, we constructed a strain that cannot grow on xylose or ribose minimal media unless methanol is utilized, creating a phenotype termed "synthetic methanol auxotrophy".

Construction and evolution of an strain relying on nonoxidative glycolysis for sugar catabolism.

The Embden-Meyerhoff-Parnas (EMP) pathway, commonly known as glycolysis, represents the fundamental biochemical infrastructure for sugar catabolism in almost all organisms, as it provides key components for biosynthesis, energy metabolism, and global regulation. EMP-based metabolism synthesizes three-carbon (C3) metabolites before two-carbon (C2) metabolites and must emit one CO in the synthesis of the C2 building block, acetyl-CoA, a precursor for many industrially important products. Using rational design, genome editing, and evolution, here we replaced the native glycolytic pathways in with the previously designed nonoxidative glycolysis (NOG), which bypasses initial C3 formation and directly generates stoichiometric amounts of C2 metabolites.