Intracellular fluxes shape all cellular functions, and understanding how they are shaped by the joint effects of enzyme abundances and metabolite concentrations in vivo currently requires gathering matched quantitative proteomic and metabolomic data sets from resource-intensive experiments. Here, we present KineFlux, a hybrid approach that combines machine learning with enzyme-constrained metabolic models to accurately predict steady-state flux distributions using only quantitative proteomic data. KineFlux builds machine learning models for metabolite concentration effects on reaction fluxes, obtained by using fluxomics and proteomics data from a training set of experiments. Using fluxomic and proteomic data sets of Escherichia coli and Saccharomyces cerevisiae, we show that the steady-state flux distributions predicted by KineFlux are in line with fluxes estimated by classical approaches. We also demonstrate that the machine learning models embedded in KineFlux are transferrable at marginal loss of accuracy using independent testing data from E. coli. Therefore, KineFlux expands the usability of enzyme-constrained models towards accurate prediction of genome-scale flux distributions compatible with metabolite concentration effects without knowledge of enzyme kinetics.

Metabolites acting as substrates and regulators of all biochemical reactions play an important role in maintaining the functionality of cellular metabolism. Despite advances in the constraint-based framework for genome-scale metabolic modeling, we lack reliable proxies for metabolite concentrations that can be efficiently determined and that allow us to investigate the relationship between metabolite concentrations in specific metabolic states in the absence of measurements. Here, we introduce a constraint-based approach, the flux-sum coupling analysis (FSCA), which facilitates the study of the interdependencies between metabolite concentrations by determining coupling relationships based on the flux-sum of metabolites. Application of FSCA on metabolic models of Escherichia coli, Saccharomyces cerevisiae, and Arabidopsis thaliana showed that the three coupling relationships are present in all models and pinpointed similarities in coupled metabolite pairs. Using the available concentration measurements of E. coli metabolites, we demonstrated that the coupling relationships identified by FSCA can capture the qualitative associations between metabolite concentrations and that flux-sum is a reliable proxy for metabolite concentration. Therefore, FSCA provides a novel tool for exploring and understanding the intricate interdependencies between the metabolite concentrations, advancing the understanding of metabolic regulation, and improving flux-centered systems biology approaches.

The increasing availability of enzyme turnover number measurements from experiments and of turnover number predictions from deep learning models prompts the use of these enzyme parameters in precise metabolic engineering. Yet, there is no computational approach that allows the prediction of metabolic engineering strategies that rely on the modification of turnover numbers. It is also unclear if modifications of turnover numbers without alterations in the host's transcriptional regulatory machinery suffice to increase the production of chemicals of interest. Here, we present a constraint-based modeling approach, termed Overcoming Kinetic rate Obstacles (OKO), that uses enzyme-constrained metabolic models to predict in silico strategies to increase the production of a given chemical, while ensuring specified cell growth. We demonstrate that the application of OKO to enzyme-constrained metabolic models of Escherichia coli and Saccharomyces cerevisiae results in strategies that can at least double the production of over 40 compounds with little penalty to growth. Interestingly, we show that the overproduction of compounds of interest does not entail only an increase in the values of turnover numbers. Lastly, we demonstrate that a refinement of OKO, allowing also for manipulation of enzyme abundance, facilitates the usage of the available compendia and deep learning models of turnover numbers in the design of precise metabolic engineering strategies. Our results expand the usage of genome-scale metabolic models toward the identification of targets for protein engineering, allowing their direct usage in the generation of innovative metabolic engineering designs for various biotechnological applications.

Intracellular fluxes represent a joint outcome of cellular transcription and translation and reflect the availability and usage of nutrients from the environment. While approaches from the constraint-based metabolic framework can accurately predict cellular phenotypes, such as growth and exchange rates with the environment, accurate prediction of intracellular fluxes remains a pressing problem. Parsimonious flux balance analysis (pFBA) has become an approach of choice to predict intracellular fluxes by employing the principle of efficient usage of protein resources. Nevertheless, comparative analyses of intracellular flux predictions from pFBA against fluxes estimated from labeling experiments remain scarce. Here, we posited that steady-state flux distributions derived from the principle of maximizing multi-reaction dependencies are of improved accuracy and precision than those resulting from pFBA. To this end, we designed a constraint-based approach, termed complex-balanced FBA (cbFBA), to predict steady-state flux distributions that support the given specific growth rate and exchange fluxes. We showed that the steady-state flux distributions resulting from cbFBA in comparison to pFBA show better agreement with experimentally measured fluxes from 17 Escherichia coli strains and are more precise, due to the smaller space of alternative solutions. We also showed that the same principle holds in eukaryotes by comparing the predictions of pFBA and cbFBA against experimentally derived steady-state flux distributions from 26 knock-out mutants of Saccharomyces cerevisiae. Furthermore, our results showed that intracellular fluxes predicted by cbFBA provide better support for the principle of minimizing metabolic adjustment between mutants and wild types. Together, our findings point that other principles that consider the dynamics and coordination of steady states may govern the distribution of intracellular fluxes.

MOTIVATION: Large-scale metabolic models are widely used to design metabolic engineering strategies for diverse biotechnological applications. However, the existing computational approaches focus on alteration of reaction fluxes and often neglect the manipulations of gene expression to implement these strategies. RESULTS: Here, we find that the association of genes with multiple reactions leads to infeasibility of engineering strategies at the flux level, since they require contradicting manipulations of gene expression. Moreover, we identify that all of the existing approaches to design gene knockout strategies do not ensure that the resulting design may also require other gene alterations, such as up- or downregulations, to match the desired flux distribution. To address these issues, we propose a constraint-based approach, termed GeneReg, that facilitates the design of feasible metabolic engineering strategies at the gene level and that is readily applicable to large-scale metabolic networks. We show that GeneReg can identify feasible strategies to overproduce ethanol in Escherichia coli and lactate in Saccharomyces cerevisiae, but overproduction of the TCA cycle intermediates is not feasible in five organisms used as cell factories under default growth conditions. Therefore, GeneReg points at the need to couple gene regulation and metabolism to design rational metabolic engineering strategies. AVAILABILITY AND IMPLEMENTATION: https://github.com/MonaRazaghi/GeneReg. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Constraint-based modeling approaches allow the estimation of maximal in vivo enzyme catalytic rates that can serve as proxies for enzyme turnover numbers. Yet, genome-scale flux profiling remains a challenge in deploying these approaches to catalogue proxies for enzyme catalytic rates across organisms. RESULTS: Here, we formulate a constraint-based approach, termed NIDLE-flux, to estimate fluxes at a genome-scale level by using the principle of efficient usage of expressed enzymes. Using proteomics data from Escherichia coli, we show that the fluxes estimated by NIDLE-flux and the existing approaches are in excellent qualitative agreement (Pearson correlation > 0.9). We also find that the maximal in vivo catalytic rates estimated by NIDLE-flux exhibits a Pearson correlation of 0.74 with in vitro enzyme turnover numbers. However, NIDLE-flux results in a 1.4-fold increase in the size of the estimated maximal in vivo catalytic rates in comparison to the contenders. Integration of the maximum in vivo catalytic rates with publically available proteomics and metabolomics data provide a better match to fluxes estimated by NIDLE-flux. Therefore, NIDLE-flux facilitates more effective usage of proteomics data to estimate proxies for kcatomes. AVAILABILITY AND IMPLEMENTATION: https://github.com/Rudan-X/NIDLE-flux-code. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.