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Highly efficient bioproduction from gaseous substrates (e.g., hydrogen and carbon oxides) will require systematic optimization of the host microbes. To date, the rational redesign of gas-fermenting bacteria is still in its infancy, due in part to the lack of quantitative and precise metabolic knowledge that can direct strain engineering.

The Wood-Ljungdahl pathway (WLP) is a natural carbon fixation pathway capable of converting one-carbon (C1) compounds (CO2, CO, formate) to two-carbon (C2) metabolite acetyl-CoA or coordinating with canonical glycolysis to convert sugar feedstocks to acetyl-CoA with high carbon yield. The catalytic inefficiency and engineering difficulty in key enzymes, however, limit the biosynthetic potential of this pathway. Here we design a synthetic acetyl-CoA bi-cycle to synergize the WLP for efficient C2 metabolite synthesis. This pathway produces an acetyl-CoA by fixation of two CO2 equivalents via three functional modules acting in series: carbon fixation, gluconeogenesis, and non-oxidative glycolysis. We examine the pathway through comprehensive in silico thermodynamic and kinetic analyses. The prototypic pathway is implemented in a syngas-fermenting Clostridium ljungdahlii DSM 13528 by expressing a heterologous phosphoketolase and coordinating with native enzymes in the host acetogen. We demonstrated the effectiveness of this synthetic pathway in carbon conversion under various growth conditions, which complements the WLP for valorization of syngas as well as sugar feedstocks with high catalytic efficiency. This study underscores the reductive acetyl-CoA bi-cycle as a practical strategy to improve carbon conversion and redox homeostasis in the acetogenic host.

Metabolic flux analysis represents an essential perspective to understand cellular physiology and offers quantitative information to guide pathway engineering. A valuable approach for experimental elucidation of metabolic flux is dynamic flux analysis, which estimates the relative or absolute flow rates through a series of metabolic intermediates in a given pathway. It is based on kinetic isotope labeling experiments, liquid chromatography-mass spectrometry (LC-MS), and computational analysis that relate kinetic isotope trajectories of metabolites to pathway activity. Herein, we illustrate the mathematic principles underlying the dynamic flux analysis and mainly focus on describing the experimental procedures for data generation. This protocol is exemplified using cyanobacterial metabolism as an example, for which reliable labeling data for central carbon metabolites can be acquired quantitatively. This protocol is applicable to other microbial systems as well and can be readily adapted to address different metabolic processes.

Cellulolytic bacteria have the potential to perform lignocellulose hydrolysis and fermentation simultaneously. The metabolic pathways of these bacteria, therefore, require more comprehensive and quantitative understanding. Using isotope tracer, gas chromatography-mass spectrometry, and metabolic flux modeling, we decipher the metabolic network of Clostridium thermocellum, a model cellulolytic bacterium which represents as an attractive platform for conversion of lignocellulose to dedicated products. We uncover that the Embden–Meyerhof–Parnas (EMP) pathway is the predominant glycolytic route whereas the Entner–Doudoroff (ED) pathway and oxidative pentose phosphate pathway are inactive. We also observe that C. thermocellum's TCA cycle is initiated by both Si- and Re-citrate synthase, and it is disconnected between 2-oxoglutarate and oxaloacetate in the oxidative direction; C. thermocellum uses a citramalate shunt to synthesize isoleucine; and both the one-carbon pathway and the malate shunt are highly active in this bacterium. To gain a quantitative understanding, we further formulate a fluxome map to quantify the metabolic fluxes through central metabolic pathways. This work represents the first global in vivo investigation of the principal carbon metabolism of C. thermocellum. Our results elucidate the unique structure of metabolic network in this cellulolytic bacterium and demonstrate the capability of isotope-assisted metabolite studies in understanding microbial metabolism of industrial interests.