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

Abstract

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for 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 in E. 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 methylotrophic E. coli strain 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 in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via global and local regulatory modifications.

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

“…Methanol was measured via high performance liquid chromatography (HPLC) (Whitaker et al, 2017). Extraction of metabolites and proteinogenic amino acids was performed as previously described and analyzed for 13 C-labeling using gas chromatography-mass spectrometry (Whitaker et al, 2017;Bennett et al, 2018aBennett et al, , 2020aLong and Antoniewicz, 2019). 13 C-labeling was determined from the measured mass isotopomer data (Whitaker et al, 2017;Long and Antoniewicz, 2019).…”

“…E. coli BW25113 DfrmA was obtained from the Keio collection and used for growth characterization (Baba et al, 2006). Deletion of ihfA was performed as previously described via the lambda red recombineering system (Datsenko and Wanner, 2000;Bennett et al, 2020a). Methanol assimilation genes were cloned into pETM6 (Xu et al, 2012) for episomal expression as previously described to produce the pUD9 plasmid (Bennett et al, 2018a).…”

“…Methanol assimilation genes were cloned into pETM6 (Xu et al, 2012) for episomal expression as previously described to produce the pUD9 plasmid (Bennett et al, 2018a). Overexpression of crp was achieved as previously described (Bennett et al, 2020a).…”

“…We previously examined mutants of several transcriptional regulators and found that deletion of integration host factor subunit α (ihfA), which is known to repress multiple amino acid metabolic pathways (Goosen and van de Putte, 1995;Karp et al, 2018), resulted in an improved methylotrophic phenotype, as indicated by increased 13 C-labeling of intracellular metabolites from 13 C-methanol (Bennett et al, 2020a). Overexpression of cAMP-receptor protein (crp), a known activator of multiple amino acid metabolic pathways (Karp et al, 2018), also resulted in an improved methylotrophic phenotype (Bennett et al, 2020a). Given the success of ALE in improving the methanol tolerance of the synthetic E. coli methylotroph described above (E. coli BW25113 frmA + pUD9), we sought to also improve the methanol tolerance of these transcriptional regulator mutant strains.…”

Transcriptome analysis reveals the roles of nitrogen metabolism and sedoheptulose bisphosphatase pathway in methanol‐dependent growth of Corynebacterium glutamicum

“…The same group further investigated the effects of activating amino acid biosynthetic pathways on methanol assimilation and methylotrophic growth of E. coli . Based on the 13 C‐methanol labelling data, several amino acids including l ‐histidine, l ‐isoleucine, l ‐leucine, l ‐lysine, l ‐methionine, l ‐phenylalanine, l ‐threonine, l ‐tyrosine and l ‐valine were identified as the limiting amino acids for E. coli growth on methanol (Bennett et al ., 2020a; Bennett et al ., 2021; Har et al ., 2021). Although much progress has been made in studying the biosynthesis of limiting amino acids from methanol–carbon in methylotrophic E. coli , few studies focus on that in methylotrophic C. glutamicum .…”