Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

Bennett, R. Kyle; Dillon, Michael O.; Har, Jie Ren Gerald; Agee, Alec; Hagel, Bryan von; Rohlhill, Julia; Antoniewicz, Maciek R.; Papoutsakis, Eleftherios T.

doi:10.1016/j.ymben.2020.03.003

Cited by 35 publications

(37 citation statements)

References 43 publications

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“…The Δ frmA Δ fbp and Δ frmA Δ tpiA strains described here depend on methanol for growth and maintain the potential to rely exclusively on methanol as the carbon and energy source. Especially the for a long-term evolution promising strain Δ frmA Δ tpiA shows significant methanol incorporation into RuMP cycle intermediates that exceeds previously reported strains under steady-state conditions 21 , 22 , 28 , 29 . A future long-term evolution with the Δ frmA Δ tpiA or any of the other strains predicted here is a highly promising strategy to achieve exclusive growth on methanol and represents an important step towards a biotechnologically applicable methanol to product conversion.…”

Section: Discussioncontrasting

confidence: 55%

“…1b), and can thus evolve in a methanol co-substrate growth regime. This excluded metabolic makeups 21,22,28,29 that lacked the potential for pure methylotrophic growth due to a compromised RuMP cycle (Fig. 1c).…”

Section: Resultsmentioning

confidence: 99%

“…This engineering challenge of creating a synthetic methylotrophic organism has attracted considerable interest in recent years, with the focus mostly on the naturally occurring ribulose monophosphate (RuMP) cycle due to its energy efficiency [18][19][20][21][22][23][24][25][26][27][28][29][30][31] . The construction of the RuMP cycle in E. coli theoretically requires the expression of only three genes, i.e., a methanol dehydrogenase (mdh), a 3-hexulose 6-phosphate synthase (hps) and a 6-phospho 3hexuloisomerase (phi).…”

mentioning

confidence: 99%

“…Gleizer et al 32,33 recently applied a similar approach to create a fully autotrophic E. coli, highlighting the power of the approach. Methanol-dependent strains have been described in literature 21,22,28,29 , which-due to a compromised RuMP cycle-lacked the potential for evolution towards growth on methanol alone.…”

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confidence: 99%

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Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle

et al. 2020

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Methanol is a biotechnologically promising substitute for food and feed substrates since it can be produced renewably from electricity, water and CO2. Although progress has been made towards establishing Escherichia coli as a platform organism for methanol conversion via the energy efficient ribulose monophosphate (RuMP) cycle, engineering strains that rely solely on methanol as a carbon source remains challenging. Here, we apply flux balance analysis to comprehensively identify methanol-dependent strains with high potential for adaptive laboratory evolution. We further investigate two out of 1200 candidate strains, one with a deletion of fructose-1,6-bisphosphatase (fbp) and another with triosephosphate isomerase (tpiA) deleted. In contrast to previous reported methanol-dependent strains, both feature a complete RuMP cycle and incorporate methanol to a high degree, with up to 31 and 99% fractional incorporation into RuMP cycle metabolites. These strains represent ideal starting points for evolution towards a fully methylotrophic lifestyle.

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Section: Discussioncontrasting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle

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“…These plasmids are compatible with pUD9 and were transformed into E. coli BW25113 ΔfrmA containing pUD9 to generate dual-plasmid strains. The deletion of frmA (formaldehyde dehydrogenase) has previously been shown to improve 13 C-methanol assimilation by eliminating complete formaldehyde oxidation to CO 2 , thus conserving formaldehyde carbon for assimilation via the synthetic RuMP pathway (Muller et al, 2015;Whitaker et al, 2017), and we have routinely used E. coli BW25113 strains in our lab for synthetic methylotrophy (Bennett, Dillon, et al, 2020;Bennett, Gonzalez, et al, 2018;Gonzalez et al, 2018;Rohlhill et al, 2020;Whitaker et al, 2017 Figure 3a). We hypothesized that by channeling methanol-carbon flux through E4P and PEP toward tryptophan, the cells would experience improved 13 C-methanol assimilation in the upper central carbon metabolism (i.e., glycolysis and PPP), which we also hypothesized would improve the production of Ru5P, an essential intermediate for methanol utilization when using the RuMP pathway (Bennett, Gonzalez, et al, 2018).…”

Section: Overexpression Of Dysregulated Amino Acid Biosynthetic Opementioning

confidence: 99%

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

Bennett

Agee

Har

et al. 2020

Biotech & Bioengineering

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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.

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Developing Synthetic Methylotrophs by Metabolic Engineering-Guided Adaptive Laboratory Evolution

Wang

Zheng

Sun

2022

One-Carbon Feedstocks for Sustainable Bioproduction

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Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

Cited by 35 publications

References 43 publications

Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle

Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle

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

Developing Synthetic Methylotrophs by Metabolic Engineering-Guided Adaptive Laboratory Evolution

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