Design principles of autocatalytic cycles constrain enzyme kinetics and force low substrate saturation at flux branch points

Abstract

A set of chemical reactions that require a metabolite to synthesize more of that metabolite is an autocatalytic cycle. Here, we show that most of the reactions in the core of central carbon metabolism are part of compact autocatalytic cycles. Such metabolic designs must meet specific conditions to support stable fluxes, hence avoiding depletion of intermediate metabolites.As such, they are subjected to constraints that may seem counter-intuitive: the enzymes of branch reactions out of the cycle must be overexpressed and the affinity of these enzymes to their substrates must be relatively weak. We use recent quantitative proteomics and fluxomics measurements to show that the above conditions hold for functioning cycles in central carbon metabolism of E. coli. This work demonstrates that the topology of a metabolic network can shape kinetic parameters of enzymes and lead to seemingly wasteful enzyme usage.

Conversion of Escherichia coli to Generate All Biomass Carbon from CO2

“…The first category consists of genes encoding enzymes with a direct metabolic link to the function of the Calvin cycle. In line with previous analysis showing the need to balance the flux branch (bifurcation) points from autocatalytic cycles to ensure stable biomass production (Barenholz et al., 2017), we found a mutation in prs (I171T), the main flux branch point of the Calvin cycle in clone 6. This gene, which encodes ribose-phosphate-diphosphokinase, diverts ribose-phosphate toward biomass.…”

“…To enable a complete transition to autotrophy, the host must (1) operate CO 2 fixation machinery in a pathway where the carbon input is comprised solely of CO 2 , while the outputs are organic molecules that enter central carbon metabolism and supply all 12 essential biomass precursors of the cell (Nielsen and Keasling, 2016); (2) express enzymatic machinery to obtain reducing power, either by harvesting non-chemical energy (light, electricity, etc.) or by oxidizing a reduced chemical compound that does not serve as a carbon source; and (3) regulate and coordinate the energy-harvesting and CO 2 -fixation pathways so that they together support steady-state growth with CO 2 as the sole source of carbon (Barenholz et al., 2017). Previous attempts (Mattozzi et al., 2013, Antonovsky et al., 2016, Schada von Borzyskowski et al., 2018) to establish autocatalytic CO 2 fixation cycles in model heterotrophs required the addition of multi-carbon organic compounds, which served, at least partially, as a carbon source, in order to achieve stable growth.…”

Recent Advances in Developing Artificial Autotrophic Microorganism for Reinforcing CO2 Fixation

“…This study shows that the functionality of the CBB cycle depends not only on heterologous enzymes (Rubisco and PRK) but also on the endogenous components that interact with them, particularly metabolic enzymes in the circulating carbon pool (Antonovsky et al, 2016). Proper balance of kinetic properties in enzymes at branch point is imperative to maintain a stable metabolism in vivo (Barenholz et al, 2017). In this work, three molecules of CO 2 were fixed to one molecule of pyruvate via CBB cycle.…”

Awakening a latent carbon fixation cycle inEscherichia coli

“…However, to our knowledge, the current study is the first one in which the capacity for net carbon fixation was explored in vivo using only endogenous enzymes of a heterotrophic host, thus shedding light on the emergence of novel carbon fixation pathways. Importantly, the establishment of the RuBP cycle in E. coli required long-term adaptive evolution of the microbe under selective conditions, which modulated the partitioning of metabolic fluxes between carbon fixation and biosynthetic pathways 23,63 . We expect that autotrophic growth via the GED cycle can be achieved in a similar manner.…”

“…1). Similarly to the RuBP cycle, the GED cycle is autocatalytic and any one of its intermediates can be used as a product to be diverted towards biosynthesis of cellular building blocks 23 . Production of pyruvate, a key biosynthetic building block, is more ATP-efficient via the GED cycle than via the RuBP cycle: while the former pathway requires 6 ATP molecules to generate pyruvate, the latter pathway needs 7 ATP molecules (not accounting for further losses due to Rubisco's oxygenation reaction and the resulting photorespiration).…”