The cofactor challenge in synthetic methylotrophy: bioengineering and industrial applications.

Methanol is a promising feedstock for industrial bioproduction: it can be produced renewably and has high solubility and limited microbial toxicity. One of the key challenges for its bio-industrial application is the first enzymatic oxidation step to formaldehyde. This reaction is catalysed by methanol dehydrogenases (MDH) that can use NAD+, O2 or pyrroloquinoline quinone (PQQ) as an electron acceptor. While NAD-dependent MDH are simple to express and have the highest energetic efficiency, they exhibit mediocre kinetics and poor thermodynamics at ambient temperatures. O2-dependent methanol oxidases require high oxygen concentrations, do not conserve energy and thus produce excessive heat as well as toxic H2O2. PQQ-dependent MDH provide a good compromise between energy efficiency and good kinetics that support fast growth rates without any drawbacks for process engineering. Therefore, we argue that this enzyme class represents a promising solution for industry and outline engineering strategies for the implementation of these complex systems in heterologous hosts.

Renewable methanol and formate as microbial feedstocks.

Methanol and formate are attractive microbial feedstocks as they can be sustainably produced from CO2 and renewable energy, are completely miscible, and are easy to store and transport. Here, we provide a biochemical perspective on microbial growth and bioproduction using these compounds. We show that anaerobic growth of acetogens on methanol and formate is more efficient than on H2/CO2 or CO. We analyze the aerobic C1 assimilation pathways and suggest that new-to-nature routes could outperform their natural counterparts. We further discuss practical bioprocessing aspects related to growth on methanol and formate, including feedstock toxicity. While challenges in realizing sustainable production from methanol and formate still exist, the utilization of these feedstocks paves the way towards a truly circular carbon economy.

Underground isoleucine biosynthesis pathways in E. coli.

The promiscuous activities of enzymes provide fertile ground for the evolution of new metabolic pathways. Here, we systematically explore the ability of E. coli to harness underground metabolism to compensate for the deletion of an essential biosynthetic pathway. By deleting all threonine deaminases, we generated a strain in which isoleucine biosynthesis was interrupted at the level of 2-ketobutyrate. Incubation of this strain under aerobic conditions resulted in the emergence of a novel 2-ketobutyrate biosynthesis pathway based upon the promiscuous cleavage of O-succinyl-L-homoserine by cystathionine γ-synthase (MetB). Under anaerobic conditions, pyruvate formate-lyase enabled 2-ketobutyrate biosynthesis from propionyl-CoA and formate. Surprisingly, we found this anaerobic route to provide a substantial fraction of isoleucine in a wild-type strain when propionate is available in the medium. This study demonstrates the selective advantage underground metabolism offers, providing metabolic redundancy and flexibility which allow for the best use of environmental carbon sources.

Reinforcing carbon fixation: CO2 reduction replacing and supporting carboxylation.

Carbon dioxide enters the biosphere via one of two mechanisms: carboxylation, in which CO2 is attached to an existing metabolite, or reduction, in which CO2 is converted to formate or carbon monoxide before further assimilation. Here, we focus on the latter mechanism which usually receives less attention. To better understand the possible advantages of the 'reduction-first' approach, we compare the two general strategies according to the kinetics of the CO2-capturing enzymes, and the resource consumption of the subsequent pathways. We show that the best CO2 reducing enzymes can compete with the best carboxylases. We further demonstrate that pathways that fix CO2 by first reducing it to formate could have an advantage over the majority of their carboxylation-only counterparts in terms of ATP-efficiency and hence biomass yield. We discuss and elaborate on the challenges of implementing 'reduction-first' pathways, including the thermodynamic barrier of CO2 reduction. We believe that pathways based on CO2 reduction are a valuable addition to nature's arsenal for capturing inorganic carbon and could provide promising metabolic solutions that have been previously overlooked.