Title: 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. Here, this studymore » 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.« less

Authors:

Bennett, Robert Kyle  ^[1];

• Search DOE PAGES for author "Bennett, Robert Kyle"
• Search DOE PAGES for ORCID "0000-0002-4715-5443"

Agee, Alec  ^[1];

• Search DOE PAGES for author "Agee, Alec"
• Search DOE PAGES for ORCID "0000-0002-2872-2115"

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

• Search DOE PAGES for author "Antoniewicz, Maciek R."
• Search DOE PAGES for ORCID "0000-0002-0087-2875"

Papoutsakis, Eleftherios T.  ^[1]

• Search DOE PAGES for author "Papoutsakis, Eleftherios T."
• Search DOE PAGES for ORCID "0000-0002-1077-1277"

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+ Show Author Affiliations

1. Univ. of Delaware, Newark, DE (United States)

Publication Date:

Wed Sep 02 00:00:00 EDT 2020

Research Org.:

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

Sponsoring Org.:

USDOE Advanced Research Projects Agency - Energy (ARPA-E)

OSTI Identifier:

1741036

Grant/Contract Number:  

AR0000432

Resource Type:

Accepted Manuscript

Journal Name:

Biotechnology and Bioengineering

Additional Journal Information:

Journal Volume: 118; Journal Issue: 1; Journal ID: ISSN 0006-3592

Publisher:

Wiley

Country of Publication:

United States

Language:

English

Subject:

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