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Glyoxylate cycle

The glyoxylate cycle, also known as the glyoxylate shunt or bypass, is a metabolic pathway that enables certain microorganisms, plants, and fungi to assimilate two-carbon compounds such as acetate or fatty acids into four-carbon intermediates like succinate and malate, thereby supporting gluconeogenesis and biosynthesis by circumventing the carbon-losing decarboxylation steps of the tricarboxylic acid (TCA) cycle.^[1] This pathway operates through two unique enzymes—isocitrate lyase, which cleaves isocitrate into succinate and glyoxylate, and malate synthase, which condenses glyoxylate with another acetyl-CoA to form malate—integrated with shared TCA cycle reactions to net the production of one four-carbon dicarboxylic acid from two acetyl-CoA molecules without CO₂ release.^[2] First identified in 1957 by Hans Kornberg and colleagues in Escherichia coli and germinating castor bean seedlings, the cycle is absent in vertebrates, where acetyl-CoA from such substrates can only be fully oxidized for energy rather than converted to carbohydrates.^[1] In bacteria like E. coli and Mycobacterium tuberculosis, the glyoxylate cycle facilitates growth on acetate as a sole carbon source and plays a critical role in pathogenesis by enabling persistence within host macrophages that rely on fatty acid breakdown.^[1] In plants, particularly oilseed species such as Arabidopsis and sunflower, it is localized to specialized peroxisomes called glyoxysomes in germinating seedlings, where it converts storage lipids into sugars essential for post-germinative growth before photosynthesis begins.^[3] Fungi, including Candida glabrata, utilize the cycle for alternative carbon metabolism under nutrient stress, while some bacteria employ variant pathways lacking canonical enzymes, highlighting evolutionary diversity in C2 assimilation.^[4] Regulation occurs via transcriptional controls and enzyme phosphorylation, such as the E. coli isocitrate dehydrogenase kinase/phosphatase (AceK), ensuring flux diversion to the shunt when TCA activity is suppressed.^[1]

Overview

Definition and Purpose

The glyoxylate cycle is an anabolic variant of the tricarboxylic acid (TCA) cycle that bypasses the decarboxylation steps of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, allowing net synthesis of carbohydrates from two-carbon precursors such as acetate or fatty acids.^[5] This modification enables organisms lacking the ability to directly assimilate two-carbon units to utilize them for biosynthetic purposes, distinguishing it from the catabolic focus of the standard TCA cycle.^[6] The primary purpose of the glyoxylate cycle is to convert acetyl-CoA derived from lipid or acetate breakdown into four-carbon intermediates, such as succinate and malate, which serve as precursors for gluconeogenesis and other anabolic pathways.^[7] By avoiding the carbon loss associated with CO₂ release in the TCA cycle, this pathway conserves carbon atoms, facilitating the net production of glucose or other carbohydrates from non-carbohydrate sources in environments where complex carbon compounds are scarce.^[8] The overall net reaction of the glyoxylate cycle can be summarized as:

$2 \text{[Acetyl-CoA](/page/Acetyl-CoA)} + \text{NAD}^+ + 2 \text{H}_2\text{O} \rightarrow \text{succinate} + 2 \text{[CoA](/page/COA)} + \text{NADH} + 3 \text{H}^+

^[2] This metabolic strategy confers an evolutionary advantage, enabling growth and survival on C₂ substrates like acetate in nutrient-limited conditions where longer-chain carbon sources are unavailable.^[7]

Historical Discovery

The glyoxylate cycle was first identified in the 1950s by Hans Kornberg and his collaborators at the University of Sheffield and Oxford, who were examining the ability of microorganisms to grow using acetate as the sole carbon source. This work revealed that certain bacteria, such as species of Pseudomonas and Escherichia coli, could achieve net synthesis of cellular carbohydrates from two-carbon units, a process incompatible with the standard tricarboxylic acid (TCA) cycle due to its decarboxylative losses. Early studies highlighted the need for an alternative pathway to enable gluconeogenesis from acetate.^[9] Key evidence came from radioisotope labeling experiments using ^{14}C-acetate, which demonstrated the incorporation of labeled carbon into hexose sugars and other carbohydrates in acetate-grown bacteria, confirming net carbon gain beyond what the TCA cycle could support. These findings, conducted in cell suspensions and extracts, indicated a bypass mechanism involving glyoxylate as an intermediate. In 1957, Kornberg and Hans Krebs formalized this in a landmark paper, proposing the glyoxylate cycle as a modified TCA pathway with two additional reactions to circumvent CO_2 release. The term "glyoxylate cycle" was introduced that year, marking its formal naming.^[10]^[11] Confirmation and extension to eukaryotes followed rapidly. Also in 1957, Kornberg collaborated with Harry Beevers to detect the cycle's defining enzymes—isocitrate lyase and malate synthase—in extracts from castor bean (Ricinus communis) endosperm, linking the pathway to fat-to-carbohydrate conversion in germinating oilseeds. By 1960, comprehensive enzyme assays in plants, fungi, and additional bacteria had fully elucidated the cycle's steps and stoichiometry, solidifying its role across organisms.^[11]^[12] Refinements in the 1970s further clarified the cycle's subcellular localization, with studies by Beevers and others associating its enzymes with peroxisomal compartments (glyoxysomes) in plant cells, building on initial fractionation work from the late 1960s. This integration with organelle biology provided deeper insights into the pathway's efficiency and regulation.^[11]^[12]

Relation to TCA Cycle

Shared Components

The glyoxylate cycle shares several foundational enzymes and intermediates with the tricarboxylic acid (TCA) cycle, reflecting their evolutionary and functional relatedness as pathways for acetyl-CoA metabolism.^[13] These overlaps allow the glyoxylate cycle to function as an anaplerotic variant of the TCA cycle, enabling net carbon assimilation for biosynthetic purposes in organisms incapable of glycolysis from two-carbon sources.^[14] Key shared enzymes include citrate synthase, which catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate; aconitase, which isomerizes citrate to isocitrate; and malate dehydrogenase, which oxidizes malate to oxaloacetate while generating NADH.^[7] These enzymes facilitate the initial entry and early processing of acetyl-CoA in both cycles, as well as the regenerative step that closes the cyclic flow.^[14] Common intermediates encompass acetyl-CoA (the primary substrate), citrate, isocitrate, succinate, fumarate, malate, and oxaloacetate, which serve as pivotal points of convergence and divergence between the pathways.^[13] Notably, the shared segments up to isocitrate and from succinate onward provide a scaffold for metabolic flux, bypassing the decarboxylative losses unique to the TCA cycle.^[7] In eukaryotes, the TCA cycle is typically localized to mitochondria, while the glyoxylate cycle operates in peroxisomes (such as glyoxysomes in plants and fungi). In prokaryotes, both pathways occur in the cytosol.^[14] The shared enzymes contribute to NADH and FADH₂ production that links to the electron transport chain for ATP generation via oxidative phosphorylation. This energetic efficiency in the overlapping steps supports cellular respiration while positioning the glyoxylate shunt for net synthesis of four-carbon intermediates.^[13]

Key Modifications

The glyoxylate cycle diverges from the tricarboxylic acid (TCA) cycle by bypassing the two decarboxylation steps that result in carbon loss, specifically the reactions catalyzed by isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. In the standard TCA cycle, isocitrate is oxidized and decarboxylated to α-ketoglutarate with the release of CO₂, followed by the conversion of α-ketoglutarate to succinyl-CoA, which involves another decarboxylation and CO₂ release; these steps collectively eliminate two carbon atoms per acetyl-CoA molecule entering the cycle. By omitting these oxidative decarboxylations, the glyoxylate cycle conserves carbon atoms from acetyl-CoA, enabling net synthesis of four-carbon intermediates rather than complete oxidation to CO₂.^[14] This conservation is achieved through the introduction of a glyoxylate shunt, which replaces the bypassed TCA steps with two unique reactions. First, isocitrate is cleaved into succinate and glyoxylate by the enzyme isocitrate lyase, avoiding the carbon loss associated with isocitrate dehydrogenase. Second, glyoxylate then condenses with a second molecule of acetyl-CoA to form malate, catalyzed by malate synthase; this malate can re-enter the shared TCA pathway components to regenerate oxaloacetate. These shunt reactions effectively redirect the metabolic flow to retain the carbon skeleton from two acetyl-CoA units.^[2] The net effect of these modifications is a carbon-conserving pathway where two molecules of acetyl-CoA are converted to one molecule of succinate (a C4 compound) without net CO₂ release, in contrast to the TCA cycle's catabolic loop that oxidizes acetyl-CoA to two CO₂ molecules. This allows organisms to utilize two-carbon substrates, such as acetate or fatty acids, for biosynthetic purposes, producing succinate or oxaloacetate as precursors for gluconeogenesis while generating reducing equivalents like NADH.^[2] Unlike the mitochondrial localization of the TCA cycle in eukaryotes, the glyoxylate cycle is typically compartmentalized in peroxisomes, where key enzymes like isocitrate lyase and malate synthase reside, facilitating β-oxidation of fatty acids and integration with lipid metabolism. This peroxisomal sequestration separates anabolic carbon conservation from mitochondrial energy production, optimizing resource allocation in nutrient-limited environments.^[15]^[16]

Pathway Mechanisms

Core Reaction Steps

The glyoxylate cycle operates as a modified version of the tricarboxylic acid (TCA) cycle, bypassing the two decarboxylation steps to enable net synthesis of four-carbon compounds from two-carbon units. It begins with the condensation of acetyl-CoA and oxaloacetate to form citrate, catalyzed by citrate synthase, mirroring the initial step of the TCA cycle. This reaction is represented as:

\text{Acetyl-CoA} + \text{oxaloacetate} + \text{H}_2\text{O} \rightarrow \text{citrate} + \text{CoA}

Next, citrate is isomerized to isocitrate via aconitase, which involves dehydration and rehydration to rearrange the molecule without altering its carbon skeleton. The reaction is:

\text{Citrate} \rightleftharpoons \text{cis-aconitate} \rightleftharpoons \text{isocitrate}

The cycle then diverges from the TCA pathway at isocitrate, where isocitrate lyase cleaves it into succinate and glyoxylate in a key shunt reaction that avoids carbon loss as CO₂.^[17] This step is:

\text{Isocitrate} \rightarrow \text{succinate} + \text{glyoxylate}

Subsequently, malate synthase condenses glyoxylate with a second molecule of acetyl-CoA to produce malate, incorporating the second two-carbon unit into a four-carbon intermediate.^[17] The reaction proceeds as:

\text{Glyoxylate} + \text{acetyl-CoA} + \text{H}_2\text{O} \rightarrow \text{malate} + \text{CoA}

Finally, malate dehydrogenase oxidizes malate back to oxaloacetate, regenerating the initial acceptor molecule and producing one molecule of NADH. This closing step is:

\text{Malate} + \text{NAD}^+ \rightarrow \text{oxaloacetate} + \text{NADH} + \text{H}^+

The cyclic nature of the pathway allows succinate to exit for use in gluconeogenesis or other biosynthetic processes, while oxaloacetate is recycled; the net input of two acetyl-CoA molecules thus yields one succinate without net consumption of TCA intermediates.^[17] Overall, each turn of the cycle produces one NADH, providing reducing power but yielding less energy than the full TCA cycle, which generates multiple reduced cofactors per acetyl-CoA.^[17] The net reaction can be summarized as:

$2 \text{ acetyl-CoA} + \text{NAD}^+ + 2 \text{ H}_2\text{O} \rightarrow \text{succinate} + 2 \text{ CoA} + \text{NADH} + 2 \text{ H}^+

^[18]

Essential Enzymes

Isocitrate lyase (ICL) is the primary enzyme unique to the glyoxylate cycle, functioning as a tetrameric protein that catalyzes the reversible aldol cleavage of isocitrate into glyoxylate, a two-carbon compound, and succinate, a four-carbon compound.^[19] The enzyme's active site coordinates a Mg²⁺ cofactor, which binds to the substrate isocitrate to form the true reactive complex, facilitating the lyase reaction through a mechanism involving proton abstraction and C-C bond breakage.^[20] In organisms like Paracoccidioides brasiliensis, ICL activity is regulated by phosphorylation, which inactivates the enzyme during growth on glucose, while dephosphorylation by phosphatases restores catalytic function upon shifting to alternative carbon sources.^[21] Kinetic studies indicate a Michaelis constant (K_m) for isocitrate of approximately 0.2–0.3 mM in bacterial species such as Escherichia coli and Corynebacterium glutamicum, reflecting efficient substrate affinity under physiological conditions.^[22] Malate synthase (MS) serves as the second key enzyme in the cycle, catalyzing the irreversible condensation of glyoxylate with acetyl-CoA to form L-malate and coenzyme A.^[23] The reaction proceeds via a Claisen-like mechanism where the enolate of acetyl-CoA, generated by deprotonation, attacks the carbonyl of glyoxylate to yield malyl-CoA, followed by hydrolysis of the high-energy thioester bond, which drives the overall irreversibility and prevents reversal.^[23] In certain bacteria, including Mycobacterium tuberculosis, MS is often fused to ICL at the C-terminus, creating a bifunctional enzyme (e.g., Icl2 isoform) that enhances pathway efficiency by localizing both activities within a single polypeptide.^[24] Supporting enzymes in the glyoxylate cycle include peroxisomal isoforms of citrate synthase, which initiates the pathway by condensing acetyl-CoA with oxaloacetate, and malate dehydrogenase, which interconverts malate and oxaloacetate while reoxidizing NADH from β-oxidation.^[25] In plants like Arabidopsis thaliana, the peroxisomal malate dehydrogenase isoform specifically supports fatty acid catabolism by maintaining redox balance, though it does not directly participate in glyoxylate cycle carbon flow.^[26] The genes encoding ICL and MS exhibit coordinated regulation; for example, the aceA gene for ICL in E. coli is part of the aceBAK operon and is induced more than 10-fold when acetate serves as the carbon source, enabling adaptation to C2 substrates.^[27] This acetate-dependent induction ensures high expression of both enzymes during growth on two-carbon compounds, optimizing flux through the cycle.^[27]

Biological Functions

Role in Plants

In plants, the glyoxylate cycle primarily functions within glyoxysomes, specialized subtypes of peroxisomes located in the cotyledons and other storage tissues of germinating seeds. These organelles house the key enzymes of the cycle, facilitating the coordination with β-oxidation of stored lipids to generate acetyl-CoA. Glyoxysomes are particularly abundant during early seedling development, enabling efficient metabolic conversion before the onset of photosynthesis.^[28]^[29] During seed germination in oilseed plants such as Arabidopsis thaliana, the glyoxylate cycle plays a critical role in mobilizing storage lipids to support heterotrophic growth. Stored triacylglycerols are broken down via β-oxidation in glyoxysomes to produce acetyl-CoA, which enters the cycle to form succinate and malate, bypassing the decarboxylation steps of the TCA cycle. These four-carbon intermediates are exported to the cytosol and mitochondria for gluconeogenesis, ultimately yielding sucrose that fuels seedling expansion and root development. This lipid-to-sugar conversion is indispensable for etiolated seedlings reliant on seed reserves, as it provides both energy and biosynthetic precursors in the absence of external carbon sources.^[30]^[31]^[32] Genetic evidence underscores the cycle's essentiality in plant germination. In Arabidopsis mutants deficient in isocitrate lyase (ICL), a pivotal enzyme that cleaves isocitrate to succinate and glyoxylate, seedlings fail to effectively convert lipids to carbohydrates, resulting in arrested growth and inability to establish on media with acetate as the sole carbon source. These icl mutants exhibit normal initial germination but succumb to carbon starvation during post-germinative stages, particularly under prolonged dark conditions that mimic soil burial. Such phenotypes confirm the cycle's non-redundant role in sustaining seedling vigor in oil-rich species.^[31]^[33] The activity of the glyoxylate cycle is tightly regulated during plant development to align with metabolic transitions. It is highly active in etiolated (dark-grown) seedlings, where lipid catabolism predominates, but becomes repressed upon exposure to light, which promotes photosynthetic competence and shifts carbon acquisition. Phytochrome-mediated light signaling accelerates lipid mobilization initially but downregulates cycle enzymes as chloroplasts develop. Additionally, accumulating sucrose post-germination represses the synthesis of key enzymes like ICL and malate synthase through feedback mechanisms, preventing unnecessary activity once exogenous or photosynthetically derived sugars are available. This developmental control ensures resource allocation matches the seedling's changing needs.^[34]^[35]^[36] Beyond germination, the glyoxylate cycle intersects with photorespiration in leaf peroxisomes, where it can help mitigate carbon losses under stress. Photorespiration generates glyoxylate from glycolate oxidation, and in conditions of pathway perturbation, upregulation of cycle enzymes like ICL and malate synthase allows glyoxylate to condense with acetyl-CoA, forming malate that re-enters central metabolism and recycles carbon more efficiently. This compensatory mechanism reduces the accumulation of toxic intermediates and enhances photosynthetic recovery, particularly in high-light environments where photorespiratory flux is elevated. Additionally, recent research has identified a cytosolic glyoxylate shunt that complements the canonical peroxisomal pathway, further reducing carbon loss during photorespiration.^[37]^[38]

Role in Microorganisms

The glyoxylate cycle enables bacteria such as Escherichia coli to assimilate acetate as a carbon source during periods of nutrient limitation, such as fasting conditions, by bypassing the decarboxylation steps of the tricarboxylic acid (TCA) cycle to generate four-carbon intermediates for biosynthesis.^[2] In pathogens like Mycobacterium tuberculosis, the cycle is upregulated during infection to utilize acetate and fatty acids derived from host lipids, supporting persistence within macrophages and contributing to chronic infection.^[39] This pathway is essential for M. tuberculosis virulence, as mutants lacking isocitrate lyase (ICL), a key enzyme, exhibit severely attenuated survival in mouse models of tuberculosis.^[40] In fungi, the glyoxylate cycle supports pathogens like Candida albicans and Aspergillus species in colonizing host tissues by enabling the metabolism of fatty acids and acetate abundant in nutrient-poor environments, such as during phagocytosis.^[41] For C. albicans, the cycle facilitates adaptation to lipid-rich niches, including the formation of biofilms on fatty acid substrates within the host, enhancing persistence and dissemination.^[42] The pathogenic role of the glyoxylate cycle in microorganisms involves bypassing the TCA cycle's energy-generating but carbon-losing steps, allowing net synthesis of carbohydrates from two-carbon units for survival in hostile environments like macrophages.^[39] In fungal pathogens, ICL knockout strains demonstrate reduced virulence in mouse models of systemic infection, with C. albicans Δicl1 mutants showing markedly lower fungal burden and prolonged host survival compared to wild-type strains. Beyond pathogenesis, the glyoxylate cycle is critical for environmental adaptation in soil bacteria and fungi, enabling the utilization of plant exudates and organic compounds from decaying matter as carbon sources in carbon-limited rhizosphere niches.^[43] For instance, in biocontrol fungi like Trichoderma species, the cycle supports growth on acetate-derived exudates, contributing to antagonism against plant pathogens and promotion of plant health.^[44] Microorganisms with an active glyoxylate cycle exhibit significantly higher growth yields on ethanol as a carbon source compared to mutants lacking the pathway; this enhanced yield supports gluconeogenesis, providing precursors for essential cellular components during alternative carbon metabolism.^[7]

Regulation and Inhibition

Regulatory Mechanisms

The glyoxylate cycle is subject to multifaceted regulatory mechanisms that ensure its activation under conditions favoring growth on two-carbon sources like acetate, while repression occurs during glucose abundance to prioritize efficient energy production via glycolysis and the TCA cycle. In bacteria such as Escherichia coli, transcriptional control plays a central role, primarily through the repressors IclR and FadR acting on the aceBAK operon, which encodes key enzymes isocitrate lyase (ICL), malate synthase (MS), and isocitrate dehydrogenase kinase/phosphatase (AceK). IclR directly binds to the aceBAK promoter to repress transcription, while FadR, a fatty acid-responsive regulator, activates iclR expression, thereby indirectly enhancing repression of the operon under non-inducing conditions.^[45]^[46] Induction by acetate occurs via the cAMP-CRP complex, which binds the promoter to counteract repression when glucose levels are low and cAMP is elevated, thereby promoting aceBAK expression during acetate utilization.^[47]^[48] In eukaryotes like yeast (Saccharomyces cerevisiae), post-translational regulation contributes to rapid adaptation, particularly through glucose-mediated catabolite inactivation of ICL during shifts to fermentable carbon sources. This inactivation involves a reversible process triggered by glucose-induced signaling via the cAMP-PKA pathway, leading to intracellular acidification and subsequent enzyme modification or sequestration, which inhibits ICL activity and favors TCA cycle flux over the glyoxylate bypass.^[49]^[50] The PKA-mediated response ensures that glyoxylate cycle enzymes are swiftly downregulated upon glucose availability, preventing unnecessary anaplerotic activity.^[50] Allosteric regulation fine-tunes enzyme activities to match metabolic demands and avoid futile cycling. These mechanisms integrate with shared TCA enzymes, where oxaloacetate levels modulate citrate synthase activity through conformational changes that influence its association with other proteins, thereby balancing entry into the glyoxylate versus TCA pathways.^[51] In eukaryotic cells, compartmental signals regulate the glyoxylate cycle by controlling enzyme localization to peroxisomes, where ICL and MS reside. Peroxisome biogenesis factors encoded by PEX genes, such as PEX5 and PEX7, facilitate the import of peroxisomal targeting signal-bearing enzymes, ensuring proper assembly of the cycle machinery in response to lipid or acetate metabolism cues.^[52]^[53] Disruption of PEX function impairs enzyme import, thereby repressing cycle activity and linking peroxisome dynamics to overall metabolic regulation.^[54]

Pharmacological Inhibitors

The glyoxylate cycle is a promising target for pharmacological intervention in microbial pathogens that rely on it for persistence and virulence, particularly through inhibition of its key enzymes, isocitrate lyase (ICL) and malate synthase (MS). These inhibitors disrupt carbon assimilation from two-carbon sources like fatty acids, forcing reliance on less favorable metabolic routes and impairing growth in host environments. Itaconate, an endogenous antimicrobial metabolite produced by activated macrophages during immune responses, serves as a potent ICL inhibitor by forming a covalent adduct with a conserved active-site cysteine residue, competitively blocking isocitrate cleavage into succinate and glyoxylate. This mechanism effectively halts glyoxylate shunt activity and bacterial proliferation on acetate or lipids.^[55] Synthetic analogs, such as 3-nitropropionate, act as mechanism-based inactivators of ICL across bacterial and fungal species; as a succinate analog and reaction intermediate mimic, it undergoes nitroalkane activation to form a stable thiohydroximate with the catalytic cysteine, leading to irreversible enzyme blockade and accumulation of toxic isocitrate.^[56] Inhibition of MS, the enzyme condensing glyoxylate and acetyl-CoA to form malate, is less directly targeted but achieved indirectly via fluoroacetate derivatives. These compounds are metabolized to fluorocitrate, which mimics isocitrate and potently inhibits aconitase in the upstream TCA cycle, disrupting carbon flux into the glyoxylate shunt, elevating glyoxylate toxicity, and blocking net malate production essential for gluconeogenesis in pathogens.^[57] Therapeutically, ICL-targeted inhibitors hold potential against glyoxylate-dependent infections, including fungal aspergillosis, where cycle disruption impairs Aspergillus fumigatus nutrient scavenging in host tissues, and bacterial tuberculosis, with ICL essential for M. tuberculosis latency and persistence on fatty acids. As of 2025, studies have further elucidated itaconate's mechanism in M. tuberculosis, showing interference with central carbon metabolism beyond ICL inhibition.^[58] Research in the 2020s has advanced preclinical candidates like optimized itaconate derivatives and novel ICL chemotypes for antitubercular therapy, though challenges in potency and isoform coverage have delayed clinical trials.^[59]^[60] A major hurdle in developing these inhibitors is achieving selectivity, as many compounds cross-react with mammalian TCA cycle enzymes—such as 3-nitropropionate's inhibition of succinate dehydrogenase—risking host cytotoxicity; this is mitigated by exploiting pathogen-specific ICL/MS structural features absent in humans, who lack the full glyoxylate cycle. In vitro efficacy underscores their antimicrobial promise: itaconate reduces Candida albicans growth by approximately 50% at physiologically relevant concentrations (1–5 mM) by suppressing ICL-dependent acetate utilization, sensitizing the fungus to host immune clearance.^[61]

Applications in Biotechnology

Metabolic Engineering

Metabolic engineering of the glyoxylate cycle involves introducing or enhancing its key enzymes in non-native organisms to redirect carbon flux toward valuable industrial products, particularly in bacteria and yeast lacking a complete native shunt. In Escherichia coli, engineering via derepression of native isocitrate lyase (ICL) and malate synthase (MS), such as through iclR knockout, bypasses the decarboxylation steps of the tricarboxylic acid cycle, enabling efficient succinate production from glucose under anaerobic conditions. This strategy activates the glyoxylate shunt, allowing the conversion of isocitrate to succinate and malate without CO₂ loss, thereby improving theoretical yields to near 1 mol succinate per mol glucose. For instance, engineered E. coli strains with iclR deletion and blocks in competing pathways like lactate dehydrogenase achieved succinate titers of up to 40 g/L with a yield of 1.6 mol/mol glucose after 96 hours of fermentation.^[17] Flux optimization strategies further refine glyoxylate cycle activity by modulating enzyme expression and localization. In Saccharomyces cerevisiae, overexpression of the native genes aceA (encoding ICL) and aceB (encoding MS) enhances acetate assimilation, redirecting flux from ethanol toward succinate production during mixed-substrate fermentations. This approach couples the glyoxylate shunt with gluconeogenesis, converting acetyl-CoA from ethanol breakdown into C4 intermediates for succinate. Additionally, targeting ICL and MS to peroxisomes, the native compartment for the cycle in yeast, improves enzyme stability and flux efficiency in acetate-limited conditions. These modifications alleviate catabolite repression and boost overall carbon recovery.^[17] Industrial applications leverage the engineered glyoxylate cycle for biofuel and bioproduct synthesis. In bacterial hosts like E. coli, glyoxylate shunt knockout has been used to improve 1-butanol production by addressing CoA imbalance, achieving titers of 18.3 g/L by enhancing branched-chain pathways. In oleaginous algae, such as Chlorella vulgaris, the native glyoxylate cycle aids lipid accumulation from acetate-rich waste streams under nitrogen limitation, supporting biodiesel production. These applications capitalize on the cycle's role in assimilating C2 feedstocks like acetate from industrial effluents.^[62]^[63] Key challenges in glyoxylate cycle engineering include maintaining redox balance, as the shunt generates an NADH surplus that can inhibit downstream pathways and reduce yields. Strategies like co-overexpression of NADH oxidases or transhydrogenases address this by regenerating NAD⁺, improving succinate productivity in E. coli. Reviews describe bacterial chassis with constitutive glyoxylate shunt activation via iclR knockout, enabling robust acetate utilization for succinate.^[64] A notable case study involves Saccharomyces cerevisiae modified for waste acetate utilization, where activation of the glyoxylate cycle through aceA/aceB overexpression and acetyl-CoA synthetase enhancement improves biomass yields on lignocellulosic hydrolysates compared to wild-type strains. This engineering increases growth rates by improving C2 assimilation and reduces ethanol inhibition, demonstrating scalability for bioethanol coproduction.^[17]

Therapeutic Targeting

The glyoxylate cycle is an attractive therapeutic target for antimicrobial drugs due to its absence in humans, who lack the key enzymes isocitrate lyase (ICL) and malate synthase (MS), enabling selective inhibition of pathogens that rely on it for survival without host toxicity.^[40] This selectivity is particularly relevant for pathogens like Mycobacterium tuberculosis (Mtb), which activates the cycle during latency and persistence within host macrophages to metabolize fatty acids as carbon sources.^[65] Drug development efforts have focused on ICL inhibitors for tuberculosis treatment, with several compounds advancing in preclinical pipelines. For instance, mechanism-based inactivators targeting both ICL1 and ICL2 isoforms have shown potent inhibition of Mtb growth in vitro, providing a foundation for novel anti-TB agents.^[66] In agriculture, MS inhibitors are explored as fungicides against plant pathogens like Candida albicans and Paracoccidioides spp., where alkaloids and other small molecules disrupt the cycle to impair fungal virulence on lipid-rich substrates.^[67]^[42] Clinical progress remains preclinical, with ICL and MS inhibitors demonstrating synergy in combination therapies alongside standard antifungals or anti-TB drugs. In vivo studies using ICL-deficient Mtb mutants in mouse models revealed a greater than 1.5 log reduction in lung bacterial burden by 16 weeks post-infection compared to wild-type strains, highlighting the cycle's role in chronic persistence and supporting inhibitor efficacy in reducing pathogen load.^[65] Derivatives of known pharmacological inhibitors have shown favorable pharmacokinetics and bactericidal effects in acute infection models.^[68] Emerging host-directed therapies leverage itaconate, an endogenous immune metabolite produced by activated macrophages, to block the glyoxylate shunt in invading pathogens. Itaconate inhibits ICL activity in Mtb, disrupting methylcitrate and glyoxylate cycles during infection, and dimethyl itaconate has exhibited antimicrobial effects against both tuberculous and nontuberculous mycobacteria in cellular models.^[69]^[70] As of 2025, future outlooks emphasize high-throughput screening for allosteric ICL binders to identify leads with improved potency and reduced resistance potential, informed by computational phytochemical studies and structural analyses, including 2024 investigations of natural compounds targeting ICL for latent TB inhibition.^[71] These efforts aim to translate preclinical successes into viable therapeutics for latent infections and fungal diseases.

References

[1]
https://www.annualreviews.org/doi/10.1146/annurev-micro-090817-062257
[2]
Revisiting the glyoxylate cycle: alternate pathways for microbial ...
Jun 15, 2006 · The glyoxylate cycle, identified by Kornberg et al. in 1957, provides a simple and efficient strategy for converting acetyl-CoA into anapleurotic and ...
[3]
https://www.cell.com/trends/plant-science/fulltext/S1360-1385(00)01835-5
[4]
The glyoxylate cycle and alternative carbon metabolism as ...
Jul 13, 2019 · This review summarizes the current knowledge of the glyoxylate cycle as an alternative carbon metabolic pathway of C. glabrata.
[5]
Glyoxylate Cycle - an overview | ScienceDirect Topics
The glyoxylate cycle provides the means to convert C2-units to C4-precursors for biosynthesis, allowing growth on fatty acids and C2-compounds. The conventional ...
[6]
Bacterial Metabolism - Medical Microbiology - NCBI Bookshelf - NIH
The glyoxylate cycle converts oxaloacetate either to pyruvate and CO2 (catalyzed by pyruvate carboxylase) or to phosphoenolpyruvate and CO2 (catalyzed by the ...Missing: net | Show results with:net
[7]
The glyoxylate cycle and alternative carbon metabolism as ...
Jul 13, 2019 · The primary function of the glyoxylate cycle is to allow growth when glucose is not available and two-carbon compounds, such as ethanol and ...
[8]
Glyoxylate Cycle - an overview | ScienceDirect Topics
The glyoxylate cycle is defined as a metabolic pathway that enables the conversion of acetyl-CoA into malate, occurring in peroxisomes and involving enzymes ...
[9]
Annual Reviews
This began several years of study at Purdue of the glyoxylate cycle and its role in plants, the distribution and development of the key enzymes and their ...
[10]
None
Nothing is retrieved...<|control11|><|separator|>
[11]
The Discovery of Glyoxysomes: the Work of Harry Beevers - PMC - NIH
Working together, Beevers and Kornberg showed that malate synthase and isocitrate lyase, the two enzymes that characterize the glyoxylate cycle, were present ...
[12]
Harry Beevers | Biographical Memoirs: Volume 86
This was the time when the glyoxylate cycle was being formulated in bacteria. Hans (Kornberg) and Harry then collaborated to demonstrate that the two enzymes ...
[13]
Role of Glyoxylate Shunt in Oxidative Stress Response - PMC
The glyoxylate cycle is a variant of the tricarboxylic acid cycle and shares five of the eight enzymes; the glyoxylate cycle bypasses the carbon dioxide ...Results · Enhanced Biofilm Formation... · Intracellular Nadh Level...<|control11|><|separator|>
[14]
Evolution of glyoxylate cycle enzymes in Metazoa - Biology Direct
Oct 23, 2006 · The glyoxylate cycle is thought to be present in bacteria, protists, plants, fungi, and nematodes, but not in other Metazoa.Missing: timeline | Show results with:timeline
[15]
A central role for the peroxisomal membrane in glyoxylate cycle ...
The glyoxylate cycle provides the means to convert C2-units to C4-precursors for biosynthesis, allowing growth on fatty acids and C2-compounds.
[16]
Targeting of malate synthase 1 to the peroxisomes of ... - FEBS Press
Feb 14, 2002 · The eukaryotic glyoxylate cycle has been previously hypothesized to occur in the peroxisomal compartment, which in the yeast Saccharomyces ...
[17]
Engineering the glyoxylate cycle for chemical bioproduction - Frontiers
The glyoxylate cycle, also known as glyoxylate shunt (GS), was identified by Kornberg and Krebs in 1957, explaining how organisms could grow on acetate as the ...Missing: discovery radioisotope
[18]
The structure and domain organization of Escherichia coli isocitrate ...
Isocitrate lyase catalyses the first committed step in this pathway and the structure of this tetrameric enzyme from Escherichia coli has been determined at 2.1 ...
[19]
Isocitrate Lyase - an overview | ScienceDirect Topics
The true substrate for these enzymes is the Mg2+−isocitrate complex. The enzyme has four active sites which have affinities for free magnesium of about Kd = 6 ...
[20]
Phosphorylation is the major mechanism regulating isocitrate lyase ...
May 3, 2011 · Phosphorylation is the major mechanism regulating isocitrate lyase ... Since inhibition of alkaline phosphatase by o-vanadate did not lead ...
[21]
Characterization of the isocitrate lyase gene from Corynebacterium ...
between the tricarboxylic acid cycle and the glyoxylate cycle, the affinity of ICL towards isocitrate as well as its inhibition by different effectors was ...Missing: cofactor | Show results with:cofactor
[22]
Kinetic and Chemical Mechanism of Malate Synthase from ...
Malate synthase catalyzes the Claisen-like condensation of acetyl-coenzyme A and glyoxylate in the glyoxylate shunt of the citric acid cycle.Missing: high | Show results with:high
[23]
Acetyl-CoA-mediated activation of Mycobacterium tuberculosis ...
Oct 11, 2019 · Isocitrate lyase is important for lipid utilisation by Mycobacterium tuberculosis but its ICL2 isoform is poorly understood.
[24]
Permeability of the peroxisomal membrane - PubMed Central - NIH
In many organisms glyoxylate is fed into the glyoxylate cycle. Enzymes participating in this metabolism are located on both sides of the peroxisomal membrane.
[25]
Arabidopsis peroxisomal malate dehydrogenase functions in β ...
Mar 21, 2007 · We conclude that PMDH serves to reoxidize NADH produced from fatty acid β-oxidation and does not participate directly in the glyoxylate cycle.
[26]
https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2007.03055.x
[27]
Glyoxysome - an overview | ScienceDirect Topics
At the beginning of germination, the glyoxysomes function to convert fat to carbohydrate via β-oxidation and the glyoxylate cycle. However, after greening, the ...
[28]
Aconitase: To Be or not to Be Inside Plant Glyoxysomes, That ... - NIH
Jul 12, 2020 · Thus, the glyoxylate cycle is fundamental to the germination of seeds rich in lipid reserves, and for the mobilization of lipids during ...
[29]
Re-examining the role of the glyoxylate cycle in oilseeds - PubMed
The glyoxylate cycle is instrumental in this metabolic process. It allows acetyl-CoA derived from the breakdown of storage lipids to be used for the synthesis ...
[30]
Postgerminative growth and lipid catabolism in oilseeds lacking the ...
The aim of this study was to investigate the role of the glyoxylate cycle in the postgerminative growth of the oilseed Arabidopsis thaliana.
[31]
Specific elements of the glyoxylate pathway play a significant role in ...
Dec 19, 2007 · The glyoxylate cycle has been associated with the mobilization and utilization of lipids during germination in several plant species ...
[32]
Postgerminative growth and lipid catabolism in oilseeds lacking the ...
May 9, 2000 · The icl mutants also demonstrate that the glyoxylate cycle is important for seedling survival and recovery after prolonged dark conditions that ...
[33]
Regulation of Sugar and Storage Oil Metabolism by Phytochrome ...
Irradiation of the etiolated seedlings with R and FR light dramatically accelerated oil body mobilization in a phytochrome-dependent manner. Glyoxylate cycle- ...
[34]
Metabolic regulation of glyoxylate-cycle enzyme synthesis in ...
Previous work (Graham et al, 1992, Plant Cell 3, 349–357) has suggested that sucrose or other carbohydrates can repress the synthesis of MS and ICL.
[35]
Expression regulation of MALATE SYNTHASE involved in glyoxylate ...
Jun 22, 2020 · In germinating oilseeds, glyoxylate cycle is located in the glyoxysome surrounded by a specialized single membrane. In this paper, we found a ...
[36]
Photorespiratory glycolate–glyoxylate metabolism - Oxford Academic
Mar 19, 2016 · Here we review our current knowledge concerning the enzymes that carry out the glycolate–glyoxylate metabolic steps of photorespiration.Abstract · Introduction · Proteins involved in glycolate... · Regulation of glycolate...
[37]
Life and Death in a Macrophage: Role of the Glyoxylate Cycle in ...
A recent report suggests that the glyoxylate cycle is also important for the virulence of phytopathogens. An insertional mutagenesis screen for avirulence in ...
[38]
M. tuberculosis isocitrate lyases 1 and 2 are jointly required for in ...
We show that prokaryotic- and eukaryotic-like isoforms of the glyoxylate cycle enzyme isocitrate lyase (ICL) are jointly required for fatty acid catabolism and ...
[39]
The glyoxylate cycle is required for fungal virulence - PubMed
In C. albicans, phagocytosis also upregulates the principal enzymes of the glyoxylate cycle, isocitrate lyase (ICL1) and malate synthase (MLS1). Candida ...
[40]
Inhibitors of the Glyoxylate Cycle Enzyme ICL1 in Candida albicans ...
Apr 29, 2014 · The glyoxylate cycle, which enables C. albicans to survive in nutrient-limited host niches and its. Key enzymes (e.g., isocitrate lyase (ICL1), ...<|separator|>
[41]
The Glyoxylate Cycle in an Arbuscular Mycorrhizal Fungus. Carbon ...
Aug 6, 2025 · PDF | The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil.
[42]
The glyoxylate cycle is involved in pleotropic phenotypes ...
The glyoxylate cycle is involved in pleotropic phenotypes, antagonism and induction of plant defence responses in the fungal biocontrol agent Trichoderma ...
[43]
Precise tuning of the glyoxylate cycle in Escherichia coli for efficient ...
Mar 19, 2019 · In this study, we demonstrated an efficient method of converting acetate to tyrosine via precise tuning of the glyoxylate cycle in Escherichia ...
[44]
Regulated expression of a repressor protein: FadR activates iclR - NIH
The control of the glyoxylate bypass operon (aceBAK) of Escherichia coli is mediated by two regulatory proteins, IclMR and FadR.Missing: cAMP- CRP
[45]
Members of the IclR family of bacterial transcriptional regulators ...
The implication of these findings is that control of the glyoxylate bypass operon ( aceBAK ) in E. coli is mediated by two regulatory proteins, IclR and FadR.
[46]
Large‐scale 13C‐flux analysis reveals distinct transcriptional control ...
Mar 29, 2011 · The PEP‐glyoxylate cycle is controlled by cAMP‐Crp in a hexose uptake rate‐dependent manner.Missing: FadR IclR
[47]
Effect of iclR and arcA knockouts on biomass formation and ...
Apr 11, 2011 · IclR clearly represses transcription of glyoxylate pathway genes under glucose abundance, a condition in which Crp activation is absent. Under ...
[48]
Catabolite inactivation of isocitrate lyase from Saccharomyces ...
A reversible carbon catabolite inactivation step is described for isocitrate lyase from Saccharomyces cerevisiae. This reversible inactivation step of isoc.Missing: PKA | Show results with:PKA
[49]
The Evolutionary Rewiring of Ubiquitination Targets Has ...
Dec 11, 2012 · Like Fbp1, the glyoxylate cycle enzyme, isocitrate lyase (Icl1) is also subject to catabolite inactivation in S. cerevisiae (24). This tight ...
[50]
The early steps of glucose signalling in yeast - Oxford Academic
This review focuses on the early elements in glucose signalling and discusses their relevance for the regulation of specific processes.
[51]
Feedback Inhibition of an Allosteric Triphosphopyridine Nucleotide ...
Since isocitrate can either enter the glyoxylate cycle or be metabolized via the tricarboxylic acid cycle, it is believed that the inhibition of this enzyme by ...
[52]
Article The Ubiquitin Ligase SCF Ucc1 Acts as a Metabolic Switch ...
Jul 2, 2015 · The glyoxylate cycle was first described as a “modified” tricarboxylic acid (TCA) cycle (also known as the Krebs cycle or the citric acid ...
[53]
CITRATE SYNTHASE: Structure, Control, and Mechanism
Complex formation of citrate synthase and m-MDH is enhanced by palmitoyl-Coenzyme A. In contrast, oxaloacetate inhibits the citrate synthase m-MDH association.
[54]
Peroxisome Biogenesis and Function - BioOne
Sep 1, 2009 · During the transition of seedling peroxisomes to leaf peroxisomes, mutants of these peroxins have stabilized glyoxylate cycle enzymes, i.e., ICL ...<|separator|>
[55]
Peroxisomal biogenesis is genetically and biochemically linked to ...
Jun 22, 2017 · Peroxisomes are formed by the action of 14 peroxins encoded by PEX genes, the majority of which are involved in translocation of peroxisomal ...
[56]
Peroxisome biogenesis, protein targeting mechanisms and PEX ...
Peroxisomes use 2 types of targeting signal for matrix protein import. The receptors bind at the peroxisome membrane, deliver their cargo and are recycled.
[57]
Itaconate is a covalent inhibitor of the Mycobacterium tuberculosis ...
Oct 19, 2020 · Itaconate is a mammalian antimicrobial metabolite that inhibits the isocitrate lyases (ICLs) of Mycobacterium tuberculosis.
[58]
Inhibition of isocitrate lyase by 3-nitropropionate, a reaction ...
Pyruvate kinase catalyzed phosphorylation of 3-nitro-2-hydroxypropionate by ATP and concomitant inactivation of the enzyme. Archives of Biochemistry and ...
[59]
Fluoroacetic Acid Is a Potent and Specific Inhibitor of Reproduction ...
Fluoroacetic acid is known to lead to inhibition of aconitase and block both the Krebs and glyoxylate cycles. In this study, we discovered it to be a potent ...Missing: fluoroacetate | Show results with:fluoroacetate
[60]
Targeting isocitrate lyase for the treatment of latent tuberculosis
ICL is important for the growth and survival of M. tuberculosis during latent infection. ICL is not present in humans and is therefore a potential therapeutic ...
[61]
Aspergillus fumigatus and Aspergillosis in 2019 - ASM Journals
Nov 13, 2019 · Inhibitors of the glyoxylate cycle also look promising. The ... fungal infections: opportunities and challenges for therapeutic development.
[62]
Food Fight: Role of Itaconate and Other Metabolites in Anti-Microbial ...
Indeed, growth of MTB is significantly inhibited by addition of itaconate to growth medium (Michelucci et al., 2013). In vitro bacterial growth inhibition by ...
[63]
https://www.sciencedirect.com/science/article/abs/pii/S0960852422012986
[64]
Engineering the glyoxylate cycle for chemical bioproduction - PMC
As outlined in this review, the biochemistry and regulation of glyoxylate cycle was demonstrated in two industrial microorganisms (E. coli and C. glutamicum) ...
[65]
https://www.researchgate.net/publication/232802207_Persistence_of_Mycobacterium_tuberculosis_in_macrophages_and_mice_requires_the_glyoxylate_shunt_enzyme_isocitrate_lyase
[66]
Metabolic engineering of lipid catabolism increases microalgal ... - NIH
We report that disrupting lipid catabolism is a practical approach to increase lipid yields in microalgae without affecting growth or biomass.Missing: glyoxylate isobutanol
[67]
Biosynthetic Pathway and Metabolic Engineering of Succinic Acid
3.2 Balancing the Redox Ratio (NADH/NAD+). The intracellular redox ratio is closely related to metabolite profiles, membrane transport, microbial fitness and ...
[68]
US8877466B2 - Bacterial cells having a glyoxylate shunt for the ...
The present invention is concerned with bacteria for the production of succinic acid. Specifically, the invention relates to a bacterial cell of the genus ...Missing: chassis | Show results with:chassis
[69]
Increasing Anaerobic Acetate Consumption and Ethanol Yields in ...
However, succinate dehydrogenase, a central enzyme in the glyoxylate cycle, is part of the respiratory chain and does not function anaerobically. Unfavorable ...
[70]
Persistence of Mycobacterium tuberculosis in macrophages and ...
Aug 9, 2025 · Here we report that persistence of M. tuberculosis in mice is facilitated by isocitrate lyase (ICL), an enzyme essential for the metabolism of ...
[71]
Mechanism-based inactivator of isocitrate lyases 1 and 2 ... - PNAS
Jul 5, 2017 · Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (Mtb) ...
[72]
Alkaloids as Inhibitors of Malate Synthase from Paracoccidioides spp.
Aug 14, 2015 · 2009. The malate synthase of Paracoccidioides brasiliensis Pb01 is required in the glyoxylate cycle and in the allantoin degradation pathway.Missing: fungicides | Show results with:fungicides
[73]
Identification of a novel inhibitor of isocitrate lyase as a potent ...
In this study, we screened a large number of compounds to discover new ICL inhibitors which can be potentially served as potent anti-TB agents to combat M.
[74]
Dimethyl itaconate is effective in host-directed antimicrobial ...
Mar 8, 2023 · Itaconate, a crucial immunometabolite, plays a critical role in linking immune and metabolic functions to influence host defense and ...
[75]
Itaconate, Arginine, and Gamma-Aminobutyric Acid: A Host ...
Feb 3, 2022 · In Mtb, itaconate has an antimicrobial activity for methyl citrate lyase (MCL) in the methyl citrate cycle (MCC) and isocitrate lyase (ICL) in ...
[76]
Computational Investigation of Phytochemicals Targeting Isocitrate ...
Jun 16, 2025 · Isocitrate lyase (ICL) has been shown as a potential target that plays a role in the la-tent/dormant stage of M. tuberculosis. Several ...Missing: 2022-2025 | Show results with:2022-2025