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

Bennett, Robert Kyle; Agee, Alec; Har, Jie R. G.; Hagel, Bryan von; Antoniewicz, Maciek R.; Papoutsakis, Eleftherios T.

doi:10.1002/bit.27549

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

Journal Article · Wed Sep 02 00:00:00 EDT 2020 · Biotechnology and Bioengineering

DOI:https://doi.org/10.1002/bit.27549· OSTI ID:1741036

^[1]; ^[2]; Har, Jie R. G. ^[2]; Hagel, Bryan von ^[2]; ^[2]; ^[2]

Univ. of Delaware, Newark, DE (United States); University of Delaware
Univ. of Delaware, Newark, DE (United States)

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. Here, 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.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of Delaware, Newark, DE (United States)

Sponsoring Organization:: USDOE Advanced Research Projects Agency - Energy (ARPA-E)

Grant/Contract Number:: AR0000432

OSTI ID:: 1741036

Journal Information:: Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Journal Issue: 1 Vol. 118; ISSN 0006-3592

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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60 APPLIED LIFE SCIENCES
Escherichia coli
allosteric feedback inhibition
amino acid biosynthesis
methanol
synthetic methylotrophy
transcriptional regulation

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

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References (42)

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