Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria

Schrader, Jens; Schilling, M.; Holtmann, Dirk; Sell, Denilson; Filho, Murillo Villela; Marx, Achim; Vorholt, Julia A.

doi:10.1016/j.tibtech.2008.10.009

Cited by 246 publications

(185 citation statements)

References 68 publications

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“…Biofuels and bio-based chemicals have gained tremendous traction over the past decade as a means to produce both an alternative energy source as well as to replace bulk chemical production that is heavily reliant upon petrochemicals. Methanol, which can be produced inexpensively from natural gas or renewably through reduction of carbon dioxide by hydrogen (2,4), can serve as a feedstock to produce biofuels, amino acids, and polymers (5,6). Biological conversion of methanol to value-added chemicals may provide more favorable, economic operating conditions (i.e., lower temperature and pressure) than more traditional catalytic conversions.…”

mentioning

confidence: 99%

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Price

Chen

Whitaker

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

100

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Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3-ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an "NADH Sink" was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol. methane | methylotophs | supramolcular | scaffold | substrate channeling

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mentioning

confidence: 99%

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Price

Chen

Whitaker

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

100

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“…They also represent promising organisms in biotechnology for the conversion of one-carbon (C1) substrates to value-added products (1). The elucidation of the mechanisms enabling growth on reduced C1 compounds of Methylobacterium extorquens AM1, one of the most studied methylotrophs, has been a longstanding goal, and although great progress has been made (2)(3)(4)(5), it is still not fully achieved.…”

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confidence: 99%

Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

Peyraud

Kiefer

Christen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The assimilation of one-carbon (C1) compounds, such as methanol, by serine cycle methylotrophs requires the continuous regeneration of glyoxylate. Instead of the glyoxylate cycle, this process is achieved by a not yet established pathway where CoA thioesters are known to play a key role. We applied state-of-the-art metabolomics and 13 C metabolomics strategies to demonstrate how glyoxylate is generated during methylotrophic growth in the isocitrate lyase-negative methylotroph Methylobacterium extorquens AM1. High-resolution mass spectrometry showed the presence of CoA thioesters specific to the recently proposed ethylmalonyl-CoA pathway. The operation of this pathway was demonstrated by short-term 13 C-labeling experiments, which allowed determination of the sequence of reactions from the order of label incorporation into the different CoA derivatives. Analysis of 13 C positional enrichment in glycine by NMR was consistent with the predicted labeling pattern as a result of the operation of the ethylmalonyl-CoA pathway and the unique operation of the latter for glyoxylate generation during growth on methanol. The results also revealed that 2 molecules of glyoxylate were regenerated in this process. This work provides a complete pathway for methanol assimilation in the model methylotroph M. extorquens AM1 and represents an important step toward the determination of the overall topology of its metabolic network. The operation of the ethylmalonyl-CoA pathway in M. extorquens AM1 has major implications for the physiology of these methylotrophs and their role in nature, and it also provides a common ground for C1 and C2 compound assimilation in isocitrate lyase-negative bacteria.13 C labeling ͉ CoA ester ͉ methylotroph ͉ one-carbon metabolism ͉ glyoxylate regeneration M ethylotrophic bacteria are organisms capable of using reduced carbon compounds, such as methanol or methane, as sole sources of carbon and energy, and they play a key role in carbon cycling in their environment. They also represent promising organisms in biotechnology for the conversion of one-carbon (C1) substrates to value-added products (1). The elucidation of the mechanisms enabling growth on reduced C1 compounds of Methylobacterium extorquens AM1, one of the most studied methylotrophs, has been a longstanding goal, and although great progress has been made (2-5), it is still not fully achieved. A key point has been to understand how the bacterium incorporates C1 units into cell material. The serine cycle was elucidated in this organism during the early 1960s by Quayle and coworkers (6-9). The assimilation of C1 units by this pathway requires continuous regeneration of glyoxylate from acetyl-CoA and can be achieved, in principle, via the well-known glyoxylate cycle (10). However, Dunstan and coworkers (11)(12)(13)(14) showed in 1972 and 1973 that M. extorquens AM1 lacks the key enzyme of the glyoxylate cycle, isocitrate lyase, but has an alternative route involving oxidation of acetate to glyoxylate that functions during growth on both C1 and C2 co...

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“…6,7 Important advances have been made in understanding methylotrophic metabolism. 8 Research into metabolic activities of Methylobacterium extorquens regarding C1 compounds and, last but not least, the recent success in sequencing the genome of three M. extorquens strains have helped to understand how these microorganisms cope with methanol and what they are capable of producing from it.…”

Section: Genomic Potential Of Methylobacterium Extorquens To Functionmentioning

confidence: 99%

Introducing a new Bioengineered Bug:Methylobacterium extorquenstuned as a microbial bioplastic factory

Höfer¹,

Vermette²,

Groleau³

2011

Bioengineered Bugs

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Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria

Cited by 246 publications

References 68 publications

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

Introducing a new Bioengineered Bug:Methylobacterium extorquenstuned as a microbial bioplastic factory

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Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria

Cited by 246 publications

References 68 publications

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Demonstration of the ethylmalonyl-CoA pathway by using 13 C metabolomics

Introducing a new Bioengineered Bug:Methylobacterium extorquenstuned as a microbial bioplastic factory

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Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics