RESUMO
Engineering microorganisms to grow on alternative feedstocks is crucial not just because of the indisputable biotechnological applications but also to deepen our understanding of microbial metabolism. One-carbon (C1) substrate metabolism has been the focus of extensive research for the prominent role of C1 compounds in establishing a circular bioeconomy. Methanol in particular holds great promise as it can be produced directly from greenhouse gases methane and carbon dioxide using renewable resources. Synthetic methylotrophy, i.e. introducing a non-native methanol utilization pathway into a model host, has therefore been the focus of long-time efforts and is perhaps the pinnacle of metabolic engineering. It entails completely changing a microorganism's lifestyle, from breaking up multi-carbon nutrients for growth to building C-C bonds from a single-carbon molecule to obtain all metabolites necessary to biomass formation as well as energy. The frontiers of synthetic methylotrophy have been pushed further than ever before and in this review, we outline the advances that paved the way for the more recent accomplishments. These include optimizing the host's metabolism, "copy and pasting" naturally existing methylotrophic pathways, "mixing and matching" enzymes to build new pathways, and even creating novel enzymatic functions to obtain strains that are able to grow solely on methanol. Finally, new approaches are contemplated to further advance the field and succeed in obtaining a strain that efficiently grows on methanol and allows C1-based production of added-value compounds.
RESUMO
In this work, we shed light on the metabolism of dihydroxyacetone (DHA), a versatile, ubiquitous, and important intermediate for various chemicals in industry, by analyzing its metabolism at the system level in Escherichia coli Using constraint-based modeling, we show that the growth of E. coli on DHA is suboptimal and identify the potential causes. Nuclear magnetic resonance analysis shows that DHA is degraded nonenzymatically into substrates known to be unfavorable to high growth rates. Transcriptomic analysis reveals that DHA promotes genes involved in biofilm formation, which may reduce the bacterial growth rate. Functional analysis of the genes involved in DHA metabolism proves that under the aerobic conditions used in this study, DHA is mainly assimilated via the dihydroxyacetone kinase pathway. In addition, these results show that the alternative routes of DHA assimilation (i.e., the glycerol and fructose-6-phosphate aldolase pathways) are not fully activated under our conditions because of anaerobically mediated hierarchical control. These pathways are therefore certainly unable to sustain fluxes as high as the ones predicted in silico for optimal aerobic growth on DHA. Overexpressing some of the genes in these pathways releases these constraints and restores the predicted optimal growth on DHA.IMPORTANCE DHA is an attractive triose molecule with a wide range of applications, notably in cosmetics and the food and pharmaceutical industries. DHA is found in many species, from microorganisms to humans, and can be used by Escherichia coli as a growth substrate. However, knowledge about the mechanisms and regulation of this process is currently lacking, motivating our investigation of DHA metabolism in E. coli We show that under aerobic conditions, E. coli growth on DHA is far from optimal and is hindered by chemical, hierarchical, and possibly allosteric constraints. We show that optimal growth on DHA can be restored by releasing the hierarchical constraint. These results improve our understanding of DHA metabolism and are likely to help unlock biotechnological applications involving DHA as an intermediate, such as the bioconversion of glycerol or C1 substrates into value-added chemicals.
