Regulatory interventions improve the biosynthesis of limiting amino acids from methanol carbon to improve synthetic methylotrophy inEscherichia coli

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts likeEscherichia colifor industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, that is, the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy inE. coli: the inability of the organism to synthesize several amino acids when grown on methanol. We targeted global and local amino acid regulatory networks. Those include removal of amino acid allosteric feedback inhibition (argA(H15Y),ilvA(L447F),hisG(E271K),leuA(G462D),proB(D107N),thrA(S345F),trpE(S40F)), knockouts of transcriptional repressors (ihfA, metJ); and overexpression of amino acid biosynthetic operons (hisGDCBHAFI, leuABCD, thrABC, trpEDCBA) and transcriptional regulators (crp, purR). Compared to the parent methylotrophicE. colistrain that was unable to synthesize these amino acids from methanol carbon, these strategies resulted in improved biosynthesis of limiting proteinogenic amino acids (histidine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine) from methanol carbon. In several cases, improved amino acid biosynthesis from methanol carbon led to improvements in methylotrophic growth in methanol minimal medium supplemented with a small amount of yeast extract. This study addresses a key limitation currently preventing autonomous methylotrophy inE. coliand possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via global and local regulatory modifications.

Automated summary

This study identified a key limitation of autonomous methylotrophy in E. coli and successfully improved biosynthesis of limiting amino acids from methanol carbon by modifying global and local metabolic networks, leading to enhanced growth in methanol minimal medium.