Periodic Reporting for period 1 - ADVANC1-METH (ADVANced engineering of C1 metabolism towards METHanol-based sustainable biotechnology)
Berichtszeitraum: 2021-11-01 bis 2023-10-31
The project addresses pressing issues associated with climate change and fossil-fuel dependency. This challenge has compelled the EU to promptly swing into developing a biotechnological manufacturing platform to produce chemical commodities in a more sustainable manner. However, the major challenge for the development of bio-manufacturing is the need for sustainable, cheap, abundantly available feedstocks for microbial bio-production, which do not compete for food production. Bio-manufacturing platforms currently base their production mostly on plant-derived sugars or cellulosic material, which demand land use and thereby causing undesirable pressure on food security and biodiversity and hence, making EU framework unrealistic. Methanol is a promising, alternative one-carbon (C1) feedstock, which can be sustainably produced from;(i) waste resources or (ii) CO2 and renewable hydrogen. Being a liquid that can be easily transported, stored and utilized as a substrate for microbial production of valuable chemicals and fuels, utilizing methanol for bio-manufacturing could alleviate pressure on agricultural land, environmental concerns, and fossil-resource dependency, and hence, meet the EU policies on climate and energy framework of 2030 and beyond. Although the biological conversion of methanol to valuable products by natural methylotrophic bacteria and yeast is being studied. However, these natural methylotrophs still face various limitations for economically feasible bio-manufacturing, such as energy-inefficient methanol assimilation pathways, restricted product spectra as well as low product yields and rates, while their limited genetic toolboxes further prohibit their adoption for bio-manufacturing. Hence, the engineering of well-known biotechnological production microorganisms, such as the bacterium Escherichia coli, towards growth on methanol poses a pressing issue being addressed in this study. Through engineering of two synthetic methylotrophic routes in E. coli, namely, the Serine Threonine Cycle and the Reductive Glycine Pathway, the project developed an improved methanol assimilation in the system and hence, comprehensively supplement the wealth of knowledge towards methanol bio-economy.
1. Investigative catalytic degradation of methylamines for 5,10-Methylene-THF synthesis
Methanol metabolism begins with its oxidation to highly toxic intermediate formaldehyde and since the enzymatic oxidation of methanol dehydrogenases is low due to unfavorable thermodynamics, addressing formaldehyde toxicity is essential. Addressing formaldehyde could be addressed by feeding formaldehyde to the cell, however, feeding it to the cell is hard as only 1 mM is workable above, above which the cell dies. To address this challenge, an intracellular formaldehyde generation that does not form a kinetic or thermodynamic bottleneck in E. coli, namely trimethyl-amine N-oxide (TMAO) demethylase and its derivatives were proposed.
Summary: Studies have indicated the synthesis of formaldehyde in the degradation of Methylamine (MAs). The reactive toxicity was also speculated to be addressed through substrate channeling with 5,10-methylene-THF (Figure 1) as mechanism of MAs demethylation by oxidative enzymes. Although, the secretion of 5,10-methylene-THF by these oxidative enzymes is yet to be demonstrated, owing to difficulties in their quantification through in vivo assay. Inspired by this, we unravel the role of these oxidative enzymes in the secretion of this metabolite. We showed this by using different bio-selection strains, whose auxotrophy could be relieved only when either formaldehyde and/or 5,10-methylene-THF is secreted. We clearly identified that not all Trimethylamine N-oxide demethylase enzymes secrete 5,10-methylene-THF, during demethylation processes. We equally affirmed the three subunits of dimethyl monooxygenase (dmmABC) enzymes required the last sub-unit (dmmD) to drive the cell to growth in the demethylation of Dimethylamine. Our study has demonstrated the role of these oxidative enzymes in the secretion of 5,10-methylene-THF.
2.Topic: Towards synthetic methylotrophic growth via Reductive Glycine Pathway in E. coli (rGlyP)
Summary
The present study aims to deliver the methanol oxidation in E. coli via synthetic reductive glycine pathway route (Figure 2). Following the delivery of this pathway for formatotrophic growth by Kim 2020, we sought to engineer this pathway for efficient methylotrophic growth. Hence we created this pathway in the E. coli system and to enable the strain grow on methanol, three variants of methanol dehydrogenases (MDHs) were selected. Investigative formaldehyde assimilating enzymes, the native glutathione formaldehyde dehydrogenase (EcfrmAB), Pichia pastoris NAD-dependent formaldehyde dehydrogenase (PpFaLDH), Pseudomonas putida NAD-dependent formaldehyde dehydrogenase (PpFaLDH), and Pseudomonas aeruginosa NAD-dependent Formaldehyde dehydrogenase (PaFaLDH) were codon optimized into the reductive glycine (K4) strain. Lastly energy balancing was investigated by fine-tuning the counterproductive NAD/NADH of the formaldehyde metabolism. Through these stepwise evaluation and assay, methanol assimilation with approximately 15 h doubling time was realized as against the 55 h from previous study, which makes the pathway favorably compared to the native RumP pathway as recently reported by other scholars and thereby represent the first methylotrophic synthetic pathway to achieve this milestone.
3.Topic: Advancing Synthetic Methylotrophic Growth through the Serine Threonine Cycle (STC).
Summary
As earlier discussed, atmospheric CO2 is the only carbon source that is scalable enough to establish a circular carbon economy. Accordingly, technologies to capture and convert CO2 to reduced one-carbon (C1) molecules (e.g. formate) using renewable energy are improving fast. In a bid to create sustainable bioproduction platforms engineering unnatural methanol utilization pathways into industrially relevant microbes is key. Although engineering synthetic methanol pathways in living host is challenging. The third aspect of this study engineered the autocatalytic serine threonine cycle (Fig 3), which was recently developed for formatotrophic growth in Escherichia coli. Following this delivery for formatotrophy we opined that the autocatalytic nature of this cycle could achieve synthetic methylotrophic growth more efficiently. With expression of methanol dehydrogenase enzyme, being the only gene lacking in the cycle and through upstream and downstream optimizations a 10 h doubling time was established using methanol as sole carbon source. Through optimization of the methanol dehdyrogenase enzyme in the Serine threonine cycle and stepwise downstream optimization a methylotrophic growth was developed, which we believe will open this pathway for further methylotrophic study.
Methanol metabolism begins with its oxidation to highly toxic intermediate formaldehyde and since the enzymatic oxidation of methanol dehydrogenases is low due to unfavorable thermodynamics, addressing formaldehyde toxicity is essential. Addressing formaldehyde could be addressed by feeding formaldehyde to the cell, however, feeding it to the cell is hard as only 1 mM is workable above, above which the cell dies. To address this challenge, an intracellular formaldehyde generation that does not form a kinetic or thermodynamic bottleneck in E. coli, namely trimethyl-amine N-oxide (TMAO) demethylase and its derivatives were proposed.
Summary: Studies have indicated the synthesis of formaldehyde in the degradation of Methylamine (MAs). The reactive toxicity was also speculated to be addressed through substrate channeling with 5,10-methylene-THF (Figure 1) as mechanism of MAs demethylation by oxidative enzymes. Although, the secretion of 5,10-methylene-THF by these oxidative enzymes is yet to be demonstrated, owing to difficulties in their quantification through in vivo assay. Inspired by this, we unravel the role of these oxidative enzymes in the secretion of this metabolite. We showed this by using different bio-selection strains, whose auxotrophy could be relieved only when either formaldehyde and/or 5,10-methylene-THF is secreted. We clearly identified that not all Trimethylamine N-oxide demethylase enzymes secrete 5,10-methylene-THF, during demethylation processes. We equally affirmed the three subunits of dimethyl monooxygenase (dmmABC) enzymes required the last sub-unit (dmmD) to drive the cell to growth in the demethylation of Dimethylamine. Our study has demonstrated the role of these oxidative enzymes in the secretion of 5,10-methylene-THF.
2.Topic: Towards synthetic methylotrophic growth via Reductive Glycine Pathway in E. coli (rGlyP)
Summary
The present study aims to deliver the methanol oxidation in E. coli via synthetic reductive glycine pathway route (Figure 2). Following the delivery of this pathway for formatotrophic growth by Kim 2020, we sought to engineer this pathway for efficient methylotrophic growth. Hence we created this pathway in the E. coli system and to enable the strain grow on methanol, three variants of methanol dehydrogenases (MDHs) were selected. Investigative formaldehyde assimilating enzymes, the native glutathione formaldehyde dehydrogenase (EcfrmAB), Pichia pastoris NAD-dependent formaldehyde dehydrogenase (PpFaLDH), Pseudomonas putida NAD-dependent formaldehyde dehydrogenase (PpFaLDH), and Pseudomonas aeruginosa NAD-dependent Formaldehyde dehydrogenase (PaFaLDH) were codon optimized into the reductive glycine (K4) strain. Lastly energy balancing was investigated by fine-tuning the counterproductive NAD/NADH of the formaldehyde metabolism. Through these stepwise evaluation and assay, methanol assimilation with approximately 15 h doubling time was realized as against the 55 h from previous study, which makes the pathway favorably compared to the native RumP pathway as recently reported by other scholars and thereby represent the first methylotrophic synthetic pathway to achieve this milestone.
3.Topic: Advancing Synthetic Methylotrophic Growth through the Serine Threonine Cycle (STC).
Summary
As earlier discussed, atmospheric CO2 is the only carbon source that is scalable enough to establish a circular carbon economy. Accordingly, technologies to capture and convert CO2 to reduced one-carbon (C1) molecules (e.g. formate) using renewable energy are improving fast. In a bid to create sustainable bioproduction platforms engineering unnatural methanol utilization pathways into industrially relevant microbes is key. Although engineering synthetic methanol pathways in living host is challenging. The third aspect of this study engineered the autocatalytic serine threonine cycle (Fig 3), which was recently developed for formatotrophic growth in Escherichia coli. Following this delivery for formatotrophy we opined that the autocatalytic nature of this cycle could achieve synthetic methylotrophic growth more efficiently. With expression of methanol dehydrogenase enzyme, being the only gene lacking in the cycle and through upstream and downstream optimizations a 10 h doubling time was established using methanol as sole carbon source. Through optimization of the methanol dehdyrogenase enzyme in the Serine threonine cycle and stepwise downstream optimization a methylotrophic growth was developed, which we believe will open this pathway for further methylotrophic study.
This study systematically generated an approximately 15 h and 10 h doubling time through the reductive glycine pathway and Serine threonine cycle respectively, as against the 55 h (Kim et al 2020) doubling time ever achieved for E. coli growth on sole methanol.