Is the TCA cycle malate dehydrogenase-citrate synthase metabolon an illusion?

This review discusses the intriguing yet controversial concept of metabolons, focusing on the malate dehydrogenase-citrate synthase (MDH-CISY) metabolon as a model. Metabolons are multienzyme complexes composed of enzymes that catalyze sequential reactions in metabolic pathways. Metabolons have been proposed to enhance metabolic pathway efficiency by facilitating substrate channeling. However, there is skepticism about the presence of metabolons and their functionality in physiological conditions in vivo. We address the skepticism by reviewing compelling evidence supporting the existence of the MDH-CISY metabolon and highlighting its potential functions in cellular metabolism. The electrostatic interaction between MDH and CISY and the intermediate oxaloacetate, channeled within the metabolon, has been demonstrated using various experimental techniques, including protein-protein interaction assays, isotope dilution studies, and enzyme coupling assays. Regardless of the wealth of in vitro evidence, further validation is required to elucidate the functionality of MDH-CISY metabolons in living systems using advanced structural and spatial analysis techniques.

Physiology of malate dehydrogenase and how dysregulation leads to disease.

Malate dehydrogenase (MDH) is pivotal in mammalian tissue metabolism, participating in various pathways beyond its classical roles and highlighting its adaptability to cellular demands. This enzyme is involved in maintaining redox balance, lipid synthesis, and glutamine metabolism and supports rapidly proliferating cells' energetic and biosynthetic needs. The involvement of MDH in glutamine metabolism underlines its significance in cell physiology. In contrast, its contribution to lipid metabolism highlights its role in essential biosynthetic processes necessary for cell maintenance and proliferation. The enzyme's regulatory mechanisms, such as post-translational modifications, underscore its complexity and importance in metabolic regulation, positioning MDH as a potential target in metabolic dysregulation. Furthermore, the association of MDH with various pathologies, including cancer and neurological disorders, suggests its involvement in disease progression. The overexpression of MDH isoforms MDH1 and MDH2 in cancers like breast, prostate, and pancreatic ductal adenocarcinoma, alongside structural modifications, implies their critical role in the metabolic adaptation of tumor cells. Additionally, mutations in MDH2 linked to pheochromocytomas, paragangliomas, and other metabolic diseases emphasize MDH's role in metabolic homeostasis. This review spotlights MDH's potential as a biomarker and therapeutic target, advocating for further research into its multifunctional roles and regulatory mechanisms in health and disease.

Acetylation, ADP-ribosylation and methylation of malate dehydrogenase.

Metabolism within an organism is regulated by various processes, including post-translational modifications (PTMs). These types of chemical modifications alter the molecular, biochemical, and cellular properties of proteins and allow the organism to respond quickly to different environments, energy states, and stresses. Malate dehydrogenase (MDH) is a metabolic enzyme that is conserved in all domains of life and is extensively modified post-translationally. Due to the central role of MDH, its modification can alter metabolic flux, including the Krebs cycle, glycolysis, and lipid and amino acid metabolism. Despite the importance of both MDH and its extensively post-translationally modified landscape, comprehensive characterization of MDH PTMs, and their effects on MDH structure, function, and metabolic flux remains underexplored. Here, we review three types of MDH PTMs - acetylation, ADP-ribosylation, and methylation - and explore what is known in the literature and how these PTMs potentially affect the 3D structure, enzymatic activity, and interactome of MDH. Finally, we briefly discuss the potential involvement of PTMs in the dynamics of metabolons that include MDH.

Innovate and empower: the malate dehydrogenase course-based undergraduate research experiences and community of practice.

College science programs exhibit high rates of student attrition, especially among Students of Color, women, members of the LGBTQ+ community, and those with disabilities. Many of the reasons students choose to leave or feel pushed out of science can be mitigated through participation in faculty-mentored research. However, faculty resources are limited, and not every student has access to faculty mentoring due to systemic or structural barriers. By bringing authentic scientific research into the classroom context, course-based undergraduate research experiences (CUREs) expand the number of students who participate in research and provide benefits similar to faculty-mentored research. Instructors also benefit from teaching CUREs. Using a systematic review of 14 manuscripts concerning the Malate Dehydrogenase CUREs Community (MCC) and malate dehydrogenase (MDH) CUREs, we demonstrate that CUREs can be implemented flexibly, are authentic research experiences, generate new scientific discoveries, and improve student outcomes. Additionally, CURE communities offer substantial advantages to faculty wishing to implement CUREs.

SlMDH3 Interacts with Autophagy Receptor Protein SlATI1 and Positively Regulates Tomato Heat Tolerance.

Autophagy, a highly conserved protein degradation system, plays an important role in protecting cells from adverse environmental conditions. ATG8-INTERACTING PROTEIN1 (ATI1) acts as an autophagy receptor, but its functional mechanisms in plants' heat stress tolerance remain unclear. In this study, using LC-MS/MS, we identified malate dehydrogenase (SlMDH3) as a SlATI1-interacting protein. Further studies showed that heat stress induced the expression of SlMDH3 and SlMDH3 co-localized with SlATI1 under both 22 °C and 42 °C heat treatment conditions. Moreover, silencing of SlMDH3 increased the sensitivity of tomato to heat stress, as evidenced by exacerbated degradation of chlorophyll; accumulation of MDA, H2O2, and dead cells; increased relative conductivity; and inhibition of stress-related gene expression. Conversely, overexpression of SlMDH3 improved tomato's heat tolerance, leading to opposite effects on physiological indicators and gene expression compared to SlMDH3 silencing. Taken together, our study suggests that SlMDH3 interacts with SlATI1 and positively regulates tomato heat tolerance.

Roles of the oncometabolite enantiomers of 2-hydroxyglutarate and their metabolism by diverse dehydrogenases.

2-Hydroxyglutarate (2HG) is an oncometabolite that can contribute to tumor progression. Two enantiomer forms, L-2HG and D-2HG, arise from independent pathways starting from the precursor α-ketoglutarate (αKG). L-2HG production occurs through the promiscuous activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) under acidic and/or hypoxic conditions. D-2HG frequently accumulates by gain-of-function mutations in the genes encoding two isoforms of isocitrate dehydrogenase (IDH1 and IDH2). Cognate metabolite repair enzymes, L- and D-2-hydroxyglutarate dehydrogenases, oxidize the enantiomers and cause abnormally high 2HG accumulation and disease when mutated. Elevated levels of either oncometabolite affect redox homeostasis, metabolism, and immune system functioning. Moreover, the oncometabolites inhibit several α-ketoglutarate-dependent dioxygenases resulting in epigenetic changes such as DNA and histone hypermethylation as well as deficiencies in DNA repair. L-2HG, and D-2HG in some cases, inhibit degradation of hypoxia-inducible factor (HIF1α), a transcription factor that alters gene expression to adapt to hypoxic conditions, favoring tumorigenesis. Patients with the rare disease 2-hydroxyglutaric aciduria (2HGA) have exceedingly high levels of 2HG, which is neurotoxic, causing developmental delays and brain abnormalities. D-2HG also has specific effects on collagen production and NADPH pools. Recently, D-2HG has been targeted in new chemotherapies aimed at disrupting the gain-of-function IDH1 and IDH2 mutants, resulting in successful clinical trials for several cancers.

Mammalian target of differentiation-inducing factor-1 is mitochondrial malate dehydrogenase for activation of AMP-activated protein kinase and induction of mitochondrial fission.

AIMS: Differentiation-inducing factor-1 (DIF-1) is a polyketide produced by Dictyostelium discoideum that inhibits growth and migration, while promoting the differentiation of Dictyostelium stalk cells through unknown mechanisms. DIF-1 localizes in stalk mitochondria. In addition to its effect on Dictyostelium, DIF-1 also inhibits growth and migration, and induces mitochondrial fission followed by mitophagy in mammalian cells, at least in part by activating AMP-activated protein kinase (AMPK). In a previous study, we found that DIF-1 binds to mitochondrial malate dehydrogenase (MDH2) and inhibits its activity in HeLa cells. In the present study, we investigated whether MDH2 serves as a pharmacological target of DIF-1 in mammalian cells. MAIN METHODS: To examine the enzymatic activity of MDH, mitochondrial morphology, and molecular mechanisms of DIF-1 action, we conducted an MDH reverse reaction assay, immunofluorescence staining, western blotting, and RNA interference using mammalian cells such as human umbilical vein endothelial cells, human cervical cancer cells, mouse endothelial cells, and mouse breast cancer cells. KEY FINDINGS: DIF-1 inhibited mitochondrial but not cytoplasmic MDH activity. Similar to DIF-1, LW6, an authentic MDH2 inhibitor, induced phosphorylation of AMPK, resulting in the phosphorylation of acetyl-CoA carboxylase (ACC) and the dephosphorylation of p70 S6 kinase with approximately the same potency. DIF-1 and LW6 induced mitochondrial fission. Furthermore, MDH2 knockdown using siRNA reproduced the DIF-1 action on the AMPK signaling and mitochondrial morphology. Conversely, an AMPK inhibitor prevented DIF-1-induced mitochondrial fission. SIGNIFICANCE: We propose that MDH2 is a mammalian target of DIF-1 for the activation of AMPK and induction of mitochondrial fission.

Phosphorylation of mammalian cytosolic and mitochondrial malate dehydrogenase: insights into regulation.

Malate dehydrogenase (MDH) is a key enzyme in mammalian metabolic pathways in cytosolic and mitochondrial compartments. Regulation of MDH through phosphorylation remains an underexplored area. In this review we consolidate evidence supporting the potential role of phosphorylation in modulating the function of mammalian MDH. Parallels are drawn with the phosphorylation of lactate dehydrogenase, a homologous enzyme, to reveal its regulatory significance and to suggest a similar regulatory strategy for MDH. Comprehensive mining of phosphorylation databases, provides substantial experimental (primarily mass spectrometry) evidence of MDH phosphorylation in mammalian cells. Experimentally identified phosphorylation sites are overlaid with MDH's functional domains, offering perspective on how these modifications could influence enzyme activity. Preliminary results are presented from phosphomimetic mutations (serine/threonine residues changed to aspartate) generated in recombinant MDH proteins serving as a proof of concept for the regulatory impact of phosphorylation. We also examine and highlight several approaches to probe the structural and cellular impact of phosphorylation. This review highlights the need to explore the dynamic nature of MDH phosphorylation and calls for identifying the responsible kinases and the physiological conditions underpinning this modification. The synthesis of current evidence and experimental data aims to provide insights for future research on understanding MDH regulation, offering new avenues for therapeutic interventions in metabolic disorders and cancer.

Malate dehydrogenase (MDH) in cancer: a promiscuous enzyme, a redox regulator, and a metabolic co-conspirator.

Malate dehydrogenase (MDH) is an essential enzyme in the tricarboxylic acid cycle that functions in cellular respiration and redox homeostasis. Recent studies indicate that MDH facilitates metabolic plasticity in tumor cells, catalyzing the formation of an oncometabolite, contributing to altered epigenetics, and maintaining redox capacity to support the rewired energy metabolism and biosynthesis that enables cancer progression. This minireview summarizes current findings on the unique supporting roles played by MDH in human cancers and provides an update on targeting MDH in cancer chemotherapy.

Exploring the uncharted territory of the potential protein-protein interactions of cytosolic malate dehydrogenase.

In this review, we examine the protein-protein interactions of cytosolic malate dehydrogenase (MDH), an under-studied area in cellular metabolism. We provide a comprehensive overview of MDH involvement in metabolism, especially its interactions with metabolic partners and dynamics of changing metabolism. We present an analysis of the biophysical nature of these interactions and the current methods used to study them. Our review includes an assessment of computational docking studies, which offer initial hypotheses about potential MDH interaction partners. Furthermore, we provide a summary of the sparse yet insightful experimental evidence available, establishing a foundation for future research. By integrating biophysical analysis and methodological advancements, this paper aims to illuminate the intricate network of interactions involving cytosolic MDH and their metabolic implications. This work not only contributes to our understanding of MDH's role in metabolism but also highlights the potential impact of these interactions in metabolic disorders.

Malate dehydrogenase in plants: evolution, structure, and a myriad of functions.

Malate dehydrogenase (MDH) catalyzes the interconversion of oxaloacetate and malate coupled to the oxidation/reduction of coenzymes NAD(P)H/NAD(P)+. While most animals have two isoforms of MDH located in the cytosol and mitochondria, all major groups of land plants have at least six MDHs localized to the cytosol, mitochondria, plastids, and peroxisomes. This family of enzymes participates in important reactions in plant cells including photosynthesis, photorespiration, lipid metabolism, and NH4+ metabolism. MDH also helps to regulate the energy balance in the cell and may help the plant cope with various environmental stresses. Despite their functional diversity, all of the plant MDH enzymes share a similar structural fold and act as dimers. In this review, we will introduce readers to our current understanding of the plant MDHs, including their evolution, structure, and function. The focus will be on the MDH enzymes of the model plant Arabidopsis thaliana.

Catalytic mechanism and kinetics of malate dehydrogenase.

Malate dehydrogenase (MDH) is a ubiquitous and central enzyme in cellular metabolism, found in all kingdoms of life, where it plays vital roles in the cytoplasm and various organelles. It catalyzes the reversible NAD+-dependent reduction of L-malate to oxaloacetate. This review describes the reaction mechanism for MDH and the effects of mutations in and around the active site on catalytic activity and substrate specificity, with a particular focus on the loop that encloses the active site after the substrates have bound. While MDH exhibits selectivity for its preferred substrates, mutations can alter the specificity of MDH for each cosubstrate. The kinetic characteristics and similarities of a variety of MDH isozymes are summarized, and they illustrate that the KM values are consistent with the relative concentrations of the substrates in cells. As a result of its existence in different cellular environments, MDH properties vary, making it an attractive model enzyme for studying enzyme activity and structure under different conditions.

Malate dehydrogenase as a multi-purpose target for drug discovery.

Malate dehydrogenase (MDH) enzymes play critical roles in cellular metabolism, facilitating the reversible conversion of malate to oxaloacetate using NAD+/NADH as a cofactor. The two human isoforms of MDH have roles in the citric acid cycle and the malate-aspartate shuttle, and thus both are key enzymes in aerobic respiration as well as regenerating the pool of NAD+ used in glycolysis. This review highlights the potential of MDH as a therapeutic drug target in various diseases, including metabolic and neurological disorders, cancer, and infectious diseases. The most promising molecules for targeting MDH have been examined in the context of human malignancies, where MDH is frequently overexpressed. Recent studies have led to the identification of several antagonists, some of which are broad MDH inhibitors while others have selectivity for either of the two human MDH isoforms. Other promising compounds have been studied in the context of parasitic MDH, as inhibiting the function of the enzyme could selectively kill the parasite. Research is ongoing with these chemical scaffolds to develop more effective small-molecule drug leads that would have great potential for clinical applications.

Activity-based protein profiling technology reveals malate dehydrogenase as the target protein of cinnamaldehyde against Aspergillus niger.

Cinnamaldehyde displays strong antifungal activity against fungi such as Aspergillus niger, but its precise molecular mechanisms of antifungal action remain inadequately understood. In this investigation, we applied chemoproteomics and bioinformatic analysis to unveil the target proteins of cinnamaldehyde in Aspergillus niger cells. Additionally, our study encompassed the examination of cinnamaldehyde's effects on cell membranes, mitochondrial malate dehydrogenase activity, and intracellular ATP levels in Aspergillus niger cells. Our findings suggest that malate dehydrogenase could potentially serve as an inhibitory target of cinnamaldehyde in Aspergillus niger cells. By disrupting the activity of malate dehydrogenase, cinnamaldehyde interferes with the mitochondrial tricarboxylic acid (TCA) cycle, leading to a significant decrease in intracellular ATP levels. Following treatment with cinnamaldehyde at a concentration of 1 MIC, the inhibition rate of MDH activity was 74.90 %, accompanied by an 84.5 % decrease in intracellular ATP content. Furthermore, cinnamaldehyde disrupts cell membrane integrity, resulting in the release of cellular contents and subsequent cell demise. This study endeavors to unveil the molecular-level antifungal mechanism of cinnamaldehyde via a chemoproteomics approach, thereby offering valuable insights for further development and utilization of cinnamaldehyde in preventing and mitigating food spoilage.

A novel HDAC6 inhibitor attenuate APAP-induced liver injury by regulating MDH1-mediated oxidative stress.

Glutathione (GSH) depletion, mitochondrial damage, and oxidative stress have been implicated in the pathogenesis of acetaminophen (APAP) hepatotoxicity. Here, we demonstrated that the expression of histone deacetylase 6 (HDAC6) is highly elevated, whereas malate dehydrogenase 1 (MDH1) is downregulated in liver tissues and AML-12 cells induced by APAP. The therapeutic benefits of LT-630, a novel HDAC6 inhibitor on APAP-induced liver injury, were also substantiated. On this basis, we demonstrated that LT-630 improved the protein expression and acetylation level of MDH1. Furthermore, after overexpression of MDH1, an upregulated NADPH/NADP+ ratio and GSH level and decreased cell apoptosis were observed in APAP-stimulated AML-12 cells. Importantly, MDH1 siRNA clearly reversed the protection of LT-630 on APAP-stimulated AML-12 cells. In conclusion, LT-630 could ameliorate liver injury by modulating MDH1-mediated oxidative stress induced by APAP.

Induced pluripotent stem cell-derived hepatocytes reveal TCA cycle disruption and the potential basis for triheptanoin treatment for malate dehydrogenase 2 deficiency.

Mitochondrial malate dehydrogenase 2 (MDH2) is crucial to cellular energy generation through direct participation in the tricarboxylic acid (TCA) cycle and the malate aspartate shuttle (MAS). Inherited MDH2 deficiency is an ultra-rare metabolic disease caused by bi-allelic pathogenic variants in the MDH2 gene, resulting in early-onset encephalopathy, psychomotor delay, muscular hypotonia and frequent seizures. Currently, there is no cure for this devastating disease. We recently reported symptomatic improvement of a three-year-old girl with MDH2 deficiency following treatment with the triglyceride triheptanoin. Here, we aimed to better characterize this disease and improve our understanding of the potential utility of triheptanoin treatment. Using fibroblasts derived from this patient, we generated induced pluripotent stem cells (hiPSCs) and differentiated them into hepatocytes (hiPSC-Heps). Characterization of patient-derived hiPSCs and hiPSC-Heps revealed significantly reduced MDH2 protein expression. Untargeted proteotyping of hiPSC-Heps revealed global dysregulation of mitochondrial proteins, including upregulation of TCA cycle and fatty acid oxidation enzymes. Metabolomic profiling confirmed TCA cycle and MAS dysregulation, and demonstrated normalization of malate, fumarate and aspartate following treatment with the triheptanoin components glycerol and heptanoate. Taken together, our results provide the first patient-derived hiPSC-Hep-based model of MDH2 deficiency, confirm altered TCA cycle function, and provide further evidence for the implementation of triheptanoin therapy for this ultra-rare disease. Synopsis: This study reveals altered expression of mitochondrial pathways including the tricarboxylic acid cycle and changes in metabolite profiles in malate dehydrogenase 2 deficiency and provides the molecular basis for triheptanoin treatment in this ultra-rare disease.

Effects of Aerobic Treadmill Training on Oxidative Stress Parameters, Metabolic Enzymes, and Histomorphometric Changes in Colon of Rats with Experimentally Induced Hyperhomocysteinemia.

The aim of this study was to investigate the effects of aerobic treadmill training regimen of four weeks duration on oxidative stress parameters, metabolic enzymes, and histomorphometric changes in the colon of hyperhomocysteinemic rats. Male Wistar albino rats were divided into four groups (n = 10, per group): C, 0.9% NaCl 0.2 mL/day subcutaneous injection (s.c.) 2x/day; H, homocysteine 0.45 µmol/g b.w./day s.c. 2x/day; CPA, saline (0.9% NaCl 0.2 mL/day s.c. 2x/day) and an aerobic treadmill training program; and HPA, homocysteine (0.45 µmol/g b.w./day s.c. 2x/day) and an aerobic treadmill training program. The HPA group had an increased level of malondialdehyde (5.568 ± 0.872 µmol/mg protein, p = 0.0128 vs. CPA (3.080 ± 0.887 µmol/mg protein)), catalase activity (3.195 ± 0.533 U/mg protein, p < 0.0001 vs. C (1.467 ± 0.501 U/mg protein), p = 0.0012 vs. H (1.955 ± 0.293 U/mg protein), and p = 0.0003 vs. CPA (1.789 ± 0.256 U/mg protein)), and total superoxide dismutase activity (9.857 ± 1.566 U/mg protein, p < 0.0001 vs. C (6.738 ± 0.339 U/mg protein), p < 0.0001 vs. H (6.015 ± 0.424 U/mg protein), and p < 0.0001 vs. CPA (5.172 ± 0.284 U/mg protein)) were detected in the rat colon. In the HPA group, higher activities of lactate dehydrogenase (2.675 ± 1.364 mU/mg protein) were detected in comparison to the CPA group (1.198 ± 0.217 mU/mg protein, p = 0.0234) and higher activities of malate dehydrogenase (9.962 (5.752-10.220) mU/mg protein) were detected in comparison to the CPA group (4.727 (4.562-5.299) mU/mg protein, p = 0.0385). Subchronic treadmill training in the rats with hyperhomocysteinemia triggers the colon tissue antioxidant response (by increasing the activities of superoxide dismutase and catalase) and elicits an increase in metabolic enzyme activities (lactate dehydrogenase and malate dehydrogenase). This study offers a comprehensive assessment of the effects of aerobic exercise on colonic tissues in a rat model of hyperhomocysteinemia, evaluating a range of biological indicators including antioxidant enzyme activity, metabolic enzyme activity, and morphometric parameters, which suggested that exercise may confer protective effects at both the physiological and morphological levels.

The Neuroprotective Effects of Curcumin Nanoparticles on The Cerebral Ischemia-Reperfusion Injury in The Rats-The Roles of The Protein Kinase RNA-Like ER Kinase/Extracellular Signal-Regulated Kinase and Transcription Factor EB proteins.

OBJECTIVE: Reduction of cerebral ischemia-reperfusion injury (IRI)/re-oxygenation injury, is defined as the paradoxical exacerbation of the cellular dysfunction and death, following restoration of the blood flow to previously ischemic tissues. The re-establishment of blood flow is essential to salvage the ischemic tissues. As a result, the treatment of IRI with novel therapies, which have fewer side effects, are of great importance. Therefore, this study aimed to investigate the effects of curcumin nanoparticle (CN) pre-treatment on the cerebral I/R rat model. MATERIALS AND METHODS: In this experimental study, CN was administered to rats orally five days before the bilateral common carotid artery occlusion (BCCAO) and continued for three days. The intensity of oxidative stress, the activities of antioxidant enzymes, glutathione (GSH) content, the activity of mitochondrial enzymes, including succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), curcumin bioavailability, pERK/ERK expression ratio and TFEB protein were studied. Data analysis was performed using Graphpad Prism V.8 software, one-way analysis of variance (ANOVA) with the statistical package for the social sciences (SPSS V.26 software). RESULTS: Cerebral IRI-damage significantly increased the oxidative stress (P=0.0008) and decreased the activity of the antioxidant enzymes including catalase (CAT) (P<0.001), super oxide dismutase (SOD) (P<0.001), reduced GSH (P<0.001), mitochondrial enzymes, pERK/ERK expression ratio (P=0.002) and TEFB protein (P=0.005) in rats' brains. In addition, the pre-treatment of the rats with CN resulted in a decrease in the reactive oxygen species (ROS), and an increase in the activities of antioxidants and mitochondrial enzymes. This in turn up-regulated the pERK/ERK expression ratio and TEFB expression. CONCLUSION: CN has neuroprotective effects on the cerebral IRI condition due to its antioxidant properties and is able to overexpress the pERK and TFEB proteins; thus, it can be considered as a suitable treatment option during and after the incidence of stroke.

Enhancing l-Malic Acid Production in Aspergillus niger via Natural Activation of sthA Gene Expression.

The efficient production of l-malic acid using Aspergillus niger requires overcoming challenges in synthesis efficiency and excessive byproduct buildup. This study addresses these hurdles, improving the activity of NADH-dependent malate dehydrogenase (Mdh) in the early stages of the fermentation process. By employing a constitutive promoter to express the Escherichia coli sthA responsible for the transfer of reducing equivalents between NAD(H) and NADP(H) in A. niger, the l-malic acid production was significantly elevated. However, this resulted in conidiation defects of A. niger, limiting industrial viability. To mitigate this, we discovered and utilized the PmfsA promoter, enabling the specific expression of sthA during the fermentation stage. This conditional expression strain showed similar phenotypes to its parent strain while exhibiting exceptional performance in a 5 L fermenter. Notably, it achieved a 65.5% increase in productivity, reduced fermentation cycle by 1.5 days, and lowered succinic acid by 76.2%. This work marks a promising advancement in industrial l-malic acid synthesis via biological fermentation, showcasing the potential of synthetic biology in A. niger for broader applications.