Carbon fixation represents the fundamental process that bridges the biotic and abiotic spheres. Orchestrated by plant organisms on Earth, these processes sequester approximately tenfold the quantity of carbon dioxide emitted through human activities. Notably, the bio-products sector, encompassing bulk chemicals and biofuels, confronts limitations imposed by the finite nature of global carbon fixation, primarily reliant on plant-derived substrates. The imperative for sustenance further accentuates this challenge, as humans necessitate crops for nourishment. In light of the relatively stable global carbon fixation scenario, traditional bio-production methods that hinge on sugars as substrates pose a dilemma, as they directly compete with our food sources. Consequently, there is an ongoing exploration and development of novel pathways aimed at assimilating carbon dioxide into biomass.

In alignment with the global commitment to carbon-neutral production, a paradigm shift is witnessed in the form of the emerging concept of one-carbon compound assimilation. This concept swiftly evolves into a prospective solution for the feedstock requirements of future bioproducts. One-carbon compounds, exemplified by methane, methanol, formaldehyde, and formic acid, are directly captured from the atmosphere utilizing renewable energy. In contrast to the conventional approach of fixing CO2 from the atmosphere, the assimilation of one-carbon compounds offers several distinct advantages: 1) the substrate is in a reduced and more active state; 2) there is a broader range of enzymatic options available; and 3) the system operates without the need for an additional external energy source. Notably, formaldehyde stands out as the most reactive among the one-carbon compounds. To establish a novel gateway for the integration of formaldehyde into biomass, an enticing synthetic pathway has been meticulously designed in silico. This design ensures the highest energy efficiency comparing to existing pathways in terms of energy cost.

In conventional textbooks, enzymes are traditionally defined as highly efficient catalysts with a specific biochemical function. However, the reality presents a nuanced perspective, as many enzymes exhibit promiscuity, thereby challenging the notion of singular specificity. For instance, alcohol dehydrogenase, which typically catalyzes the hydroxyl group rather than specifically towards ethanol alone. The inherent diversity within the enzyme realm provides an opportunity to explore varied combinations, enable the connection of all metabolic reactions in vivo through the application of synthetic biology. The emulation of a vast library of known enzymes emerges as a pivotal strategy in the endeavor to craft novel metabolic pathways. This mimicking process naturally becomes a key technique for the creation of innovative pathways within the realm of synthetic biology.

The EuMP (erythrulose monophosphate) cycle, developed herein, represents a novel formaldehyde assimilation pathway founded on the utilization of a promiscuous dihydroxyacetone phosphate-dependent aldolase as the key enzyme. This comprehensive cycle is delineated into four distinct modules: the formaldehyde assimilation module, the erythrulose phosphate isomerization module, the carbon rearrangement module and the C6-sugar cleavage module. Each module was meticulously tested in isolation to validate its functionality. Following the successful implementation of individual modules, including the integration of erythrulose monophosphate isomerase from Mycolicibacterium, we have successfully constructed the EuMP pathway as a functional pathway for the assimilation of formaldehyde.

Upon in vivo assembly of the EuMP cycle, we subjected it to testing in various auxotroph strains under different selective pressures. Initially, the EuMP pathway exhibited functionality only under the least demanding selection pressure conditions. To enhance the flux of the EuMP pathway, we employed adaptive laboratory evolution (ALE), a potent tool for global adaptation. Subsequent whole-genome sequencing after evolution revealed substantial deletions in significant portions and mutations in tpi and lerI in two distinct bio-replicates.

In the first evolved strain, FUI, the complete deletion of the colonic acid synthesis pathway was observed, effectively reducing the fructose-6P requirements for growth and redirecting carbon flux towards the EuMP pathway. Conversely, the second evolved candidate, FUII, manifested mutations directly associated with the EuMP pathway. Both genome modifications facilitated cell growth under higher selection pressure strains. Subsequently, reverse engineering experiments were conducted to validate the functional significance of these mutations.

https://doi.org/10.1038/s41467-023-44247-2