Aureobasidium Pullulans Research Articles

Transcriptome Analysis of Aureobasidium pullulans YQ65 Grown on Yeast Extract Peptone Glucose and Potato Dextrose Agar Media and Quantification of Their Effects on Pullulan Production

Pullulan is a high-value polysaccharide produced through the fermentation of Aureobasidium pullulans. It has significant applications in the fields of food, medicine, environmental science, and packaging. However, the yield, molecular weight, and other characteristics of pullulan can vary depending on the fermentation substrate used. Therefore, it is crucial to analyze the underlying causes of these variations at the molecular level. In this study, we first investigated the morphological differences in A. pullulans YQ65 when cultured in YPD and PDA media. The results indicated that different culture media significantly influence the primary cell morphology of A. pullulans YQ65, which in turn affects the synthesis of secondary metabolites. Subsequently, we employed different culture media to ferment pullulan and examined the variations in pullulan yield, molecular weight, and biomass. Moreover, FTIR and thermodynamic stability tests were conducted to analyze the differences among pullulans across different culture media. Finally, transcriptome analysis revealed that A. pullulans YQ65, when cultured in YPD and PDA media, regulates its growth and metabolism through the expression of key genes that are involved in pathways such as the proteasome, oxidative phosphorylation, metabolism of various secondary metabolites, fatty acid anabolism, carbon metabolism, and amino acid metabolism. The transcriptome results were further validated by assessing the expression of specific genes. This study enhances the understanding of the fermentation differences observed with different substrates in A. pullulans and provides valuable insights for optimizing culture substrates. Additionally, it offers guidance for utilizing agricultural and forestry processing waste, as well as food processing by-products, to produce pullulan cost-effectively in the future.

Transcriptome analysis of Aureobasidium pullulans BL06 and identification of key factors affecting pullulan production

Transcriptome analysis of Aureobasidium pullulans BL06 and identification of key factors affecting pullulan production

Enhanced nutritional profile of dehulled peas through fungal fermentation: Impacts on protein, starch, saponins, and total phenolic content

AbstractDehulled peas (DHP) are increasingly popular in food applications, but their integration in food products is limited due to high starch and fiber causing gelling and aggregation during cooking. Additionally, DHP contains saponins (secondary metabolite), contributing to bitter flavors, yet they also hold health benefits. We hypothesize that fungal fermentation could enhance DHP nutritional profile and integration into food products. This study evaluated the effects of six fungal organisms (Aspergillus niger [An], Aspergillus oryzae [Ao], Aureobasidium pullulans [Ap], Neurospora crassa [Nc], Rhizopus microspores var. oligosporus [Ro], Trichoderma reesei [Tr]) on DHP over 120 h of submerged fermentation, evaluating total phenolics, starch, saponins, crude proteins, and overall mass balance. Results from the study demonstrated notable changes post‐fermentation, including increased overall protein content and solubility, decreased starch content, reduced overall mass recovery, and elevated levels of total phenolics and saponins. Filamentous fungi exhibited a significant reduction in starch content, contributing to a substantial reduction in mass recovery (31%–60%) compared to the control. Unexpectedly, saponin concentrations increased (1.5 to 3 folds) during fermentation, possibly attributed to the breakdown of the substrate matrix and release of bound saponins. Total phenolic levels varied among microorganisms, with An and Nc demonstrating the highest increases (6 to 10 folds) as compared to the control. Overall, these findings point to fungal fermentation as a tool for adding value to yellow peas and other crops facing similar processing challenges. Further research is warranted to understand the health impacts and value of these enhancements.

Investigation and determination of CoQ10(H2) and CoQ10(H4) species from black yeast-like fungi and filamentous fungi.

Coenzyme Q (CoQ) or ubiquinone functions as an electron transporter in the electron transport system in both prokaryotes and eukaryotes. The isoprenyl side chain of CoQ is modified in some organisms, especially in fungi, for optimal electron transport performance under various conditions. In this study, we investigated the side chain saturated dihydro CoQ (CoQ10(H2)) in Aureobasidium pullulans EXF-150, Sydowia polyspora NBRC 30562, and naturally isolated Plowrightia sp. A37, all of which are melanized Dothideomycetes species within the Ascomycota, and also in filamentous fungi Aspergillus oryzae and Aspergillus terreus. Plowrightia sp. A37 produced the rarely synthesized tetrahydro type CoQ10(H4), especially in glucose-rich medium, during extended cultivation in contrast to CoQ10(H2) in time-limited cultivation. Using liquid chromatography-mass spectrometry, we identified demethoxyubiquinone-H2 (DMQ(H2)) as an indicative intermediate that suggests that the side chain-saturation of CoQ occurs after the formation of DMQ and not always in the last step as previously considered.

Machine Learning-Based Optimization for Enhanced Pullulan Recovery from Aureobasidium pullulans

Machine Learning-Based Optimization for Enhanced Pullulan Recovery from <i>Aureobasidium pullulans</i>

Engineering the by-products pathway in Aureobasidium pullulans for highly purified polymalic acid fermentation with concurrent recovery of l-malic acid

The fermentation of polymalic acid (PMA) by Aureobasidium pullulans, followed by acid hydrolysis to release the monomer l-malic acid (l-MA), has emerged as a promising process for the bio-based production of l-MA. However, the presence of specific by-products significantly affects the quality of the final products. In this study, chassis strains harboring an overexpressed endogenous malate dehydrogenase gene (ApMDH2) were engineered to delete key genes involved in the pullulan, melanin, and liamocin biosynthetic pathways. Furthermore, to enhance PMA synthesis productivity and prevent intracellular NADPH accumulation, an irreversible trans-hydrogenase transformation system was designed to efficiently convert NADPH to NADH. In fed-batch fermentation, the engineered strain produced the highest PMA titer (194.3 ± 1.1 g/L) and l-MA yield (0.89 ± 0.01 g/g) with an increased productivity (1.45 ± 0.06 g/L∙h). Moreover, a total of 86.19 % l-MA, with a purity of 99.7 %, was successfully extracted from fermentation broth.

Chemical transformation of the multibudding yeast, Aureobasidium pullulans.

Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists for its unconventional mode of proliferation in which multinucleate yeast cells make multiple buds within a single cell cycle. Here, we combine a chemical transformation method with genome-targeted homologous recombination to yield ∼60 transformants/μg of DNA in just 3 days. This protocol is simple, inexpensive, and requires no specialized equipment. We also describe vectors with codon-optimized green and red fluorescent proteins for A. pullulans and use these tools to explore novel cell biology. Quantitative imaging of a strain expressing cytosolic and nuclear markers showed that although the nuclear number varies considerably among cells of similar volume, total nuclear volume scales with cell volume over an impressive 70-fold size range. The protocols and tools described here expand the toolkit for A. pullulans biologists and will help researchers address the many other puzzles posed by this polyextremotolerant and morphologically plastic organism.

Investigation into the Production of Melanin from By-Products of Huangjiu Brewing.

Melanin is a high value bioproduct generated through the fermentation of Aureobasidium pullulans, playing a crucial role in various fields, including food, medicine, environmental protection, and materials science. However, its high production costs and low synthetic yields significantly limit its applications. Therefore, it is essential to identify high-yield strains, reduce production costs, and optimize fermentation strategies. In this study, a high melanin-yielding Aureobasidium pullulans 53LC7 was screened and identified, and the fermentation process was optimized based on melanin yield, color value, and pullulan yield. The results indicated that the melanin yield peaked at an initial pH of 6.0, temperature of 27 °C, fermentation time of 6.5 d, and inoculation quantity of 2.5%, achieving a melanin yield of 16.33 g/L. Subsequently, huangjiu lees, a byproduct of huangjiu production, was incorporated into the fermentation medium, resulting in a melanin yield of 5.91 g/L. This suggests that the Aureobasidium pullulans was not effectively utilizing huangjiu lees. To address this, we employed an adaptive evolution strategy, which increased the melanin yield to 8.72 g/L. The enhanced production was correlated with the expression of key genes, including FKS, PKS, and Cmr1. Finally, cellulase was utilized to convert the crude fibers in huangjiu lees, which were difficult to utilize, into usable substrates, while pullulanase was employed to minimize byproduct formation in the fermentation system, resulting in a melanin yield of 19.07 g/L. This study not only provides promising strains for further research but also offers valuable insights for resource production technologies.

Melanin Synthesized by the Endophytic Aureobasidium Pullulans AKW: A Multifaceted Biomolecule with Antioxidant, Wound Healing, and Selective Anti-Cancer Activity.

This study explores the potential of the endophytic fungus Aureobasidium pullulans AKW for melanin production and its anticancer activity. We report a significant achievement: A. pullulans AKW synthesized 4.89 g/l of melanin in a simple fermentation medium devoid of tyrosine, a precursor typically required for melanin biosynthesis. This suggests a potentially novel pathway for melanin production compared to previous studies relying on complex media and tyrosine. Furthermore, the isolated and characterized melanin exhibited promising selectivity as an anti-cancer agent. It triggered apoptosis in A431 cancer cells, demonstrating some selectivity compared to normal cells. This selectivity was confirmed by IC50 values and further supported by gene expression changes in A431 cells. Melanin treatment downregulated the anti-apoptotic Bcl2 gene while upregulating pro-apoptotic Bax and p53 genes, indicating its ability to induce programmed cell death in cancer cells. Our results demonstrate that A. pullulans AKW-derived melanin exhibits cytotoxic effects against A431, HEPG2, and MCF7 cell lines. Interestingly, the present fungal strain synthesized melanin in a simple medium without requiring precursors. The selective activity of the current melanin towards cancer cells, its ability to induce apoptosis, and its relatively low toxicity towards normal cells warrant further investigation for its development as a novel therapeutic option.

Efficient production of pullulan by Aureobasidium pullulans using a multi-objective optimization strategy with orthogonal experimental design coupling artificial neural network and genetic algorithm

Efficient pullulan production has long been a central research focus. This study used maltodextrin as the carbon source for pullulan production by Aureobasidium pullulans fermentation. A hybrid optimization approach, integrating orthogonal experimental design (OED), backpropagation artificial neural network (BP-ANN), and elite strategy non-dominated sequential genetic algorithm-II (NSGA-II), was developed. Range analysis based on OED revealed that MgSO4·7H2O significantly affects production but less impacts molecular weight, while pH notably influences molecular weight with a lesser effect on production, underscoring the need for multi-objective optimization. The BP-ANN model showed strong predictive capabilities, with goodness-of-fit values of 0.984 and 0.980 for production and molecular weight, respectively. Using this model as the fitness function for the optimization algorithm enhanced efficiency. Taking cost factors into account, the BP-ANN-NSGA-II algorithm identified the optimal fermentation medium conditions, resulting in a 6.89 % increase in production, a 368.97 % increase in molecular weight, and a 42.49 % reduction in cost. The maximum comprehensive optimization efficiency is 63.73 %, and the multi-objective optimization is better than the single objective optimization. This method significantly guides the improvement of pullulan fermentation optimization efficiency.

Etiologies of Infectious Keratitis in Malawi.

Infectious keratitis is a leading cause of corneal blindness worldwide with little information known about causative etiologies in Malawi, Africa. This area is resource-limited with ophthalmologist and microbiology services. The Department of Ophthalmology at the Kamuzu College of Health Sciences in Blantyre, Malawi, is a participating site of an international corneal ulcer consortium, capriCORN (Comprehensive Analysis of Pathogens, Resistomes, and Inflammatory-markers in the CORNea). In this study, 50 patients with corneal ulcers were swabbed for pathogen identification using RNA-sequencing. Corneal trauma was reported in 41% and 19% of the patients worked in agriculture. A pathogen was identified in 58% of the cases. Fungal pathogens predominated, followed by viruses and bacteria. Aspergillus, Fusarium, HSV-1, and Gardnerella were the most common pathogens detected. 50% of patients reported treatment with an antibiotic before presentation. Pathogens unusual for infectious keratitis, such as Subramaniula asteroids, Aureobasidium pullulans, and Gardnerella vaginalis, were also detected.

Enhancing lower respiratory tract infection diagnosis: implementation and clinical assessment of multiplex PCR-based and hybrid capture-based targeted next-generation sequencing

Shotgun metagenomic next-generation sequencing (mNGS) is widely used to detect pathogens in bronchoalveolar lavage fluid (BALF). However, mNGS is complex and expensive. This study explored the feasibility of targeted next-generation sequencing (tNGS) in distinguishing lower respiratory tract infections in clinical practice. We used 229 retrospective BALF samples to establish thresholds and diagnostic values in a prospective cohort of 251 patients. After target pathogen selection, primer and probe design, optimization experiments, and bioinformatics analysis, multiplex PCR-based tNGS (mp-tNGS) and hybrid capture-based tNGS (hc-tNGS), targeting 198 and 3060 pathogens (DNA and RNA co-detection workflow) were established and performed. mp-tNGS and hc-tNGS took 10.3 and 16h, respectively, with low sequencing data sizes of 0.1M and 1M reads, and test costs reduced to a quarter and half of mNGS. The LoDs of mp-tNGS and hc-tNGS were 50-450CFU/mL. mp-tNGS and hc-tNGS were highly accurate, with 86.5% and 87.3% (vs. 85.5% for mNGS) sensitivities and 90.0% and 88.0% (vs. 92.1% for mNGS) specificities. tNGS detection rates for casual pathogens were 84.3% and 89.5% (vs. 88.5% for mNGS), significantly higher than conventional microbiological tests (P<0.001). In seven samples, tNGS detected Pneumocystis jirovecii, a fungus not detected by mNGS. Whereas mNGS detected six samples with filamentous fungi (Rhizopus oryzae, Aureobasidium pullulans, Aspergillus niger complex, etc.) which missed by tNGS. The anaerobic bacteria as pathogen in eight samples was failed to detect by mp-tNGS. tNGS may offer a new, broad-spectrum, rapid, accurate and cost-effective approach to diagnosing respiratory infections. National Natural Science Foundation of China (81625014 and 82202535).

Efficacy of Postharvest Application of Aureobasidium pullulans to Control White Haze on Apples and Effect on the Fruit Mycobiome

White haze, an emerging disorder caused by extensive fungal growth on the apple surface, results in a compromised fruit quality and decreased marketability. The use of biological control agents could be an interesting tool to reduce its development. This work aimed to test the efficacy of two Aureobasidium pullulans strains (AP2 and PL5) in controlling white haze on stored apples. An in vivo trial was conducted by inoculating fruits with white haze causal agents (Entyloma belangeri, Golubevia pallescens, Tilletiopsis washingtonensis) and treating them with the antagonistic yeasts. Three commercial biofungicides were also included in the trial. Both A. pullulans strains, along with the Metschnikowia fructicola-based product, reduced white haze incidence after 110 days of storage at 1 ± 1 °C and after 7 days of shelf life. Furthermore, the effect of A. pullulans application on the fruit fungal microbiome was assessed. A significant impact of apple matrix and treatment on the mycobiome composition was observed. Analyses showed a good colonization of A. pullulans on the treated apples, both epiphytically and endophytically. A decrease in white haze-related fungi abundance was observed in the treated fruits. Additionally, a reduction of Ramularia spp. and modifications in the abundance of other fungal genera were detected after storage and shelf life.

Role of AplaeA in the regulation of spore production and poly(malic acid) synthesis in Aureobasidium pullulans

Poly(malic acid) (PMLA) as one of the important metabolites in Aureobasidium pullulans has great application in a variety of fields. LaeA, a global regulator capable of controlling fungal secondary metabolites, has been identified in A. pullulans. The involvement of LaeA in the biosynthesis of PMLA through direct or indirect means and its role in A. pullulans is currently unknown. Here we extracted and characterized the AplaeA gene from A. pullulans HIT-LCY3T and resolved it by bioinformatics. The function of AplaeA was subsequently characterized by Rnai-LaeA and OEX-LaeA mutants. Based on phenotypic observations of the mutant strains, it was found that the gene had a large effect on the morphology and melanin production aspects of the strain, and that overexpression of the AplaeA gene resulted in a substantial increase in spore numbers. RT-qPCR combined with protein interactions results analyzed that ApLaeA positively regulates PMLA biosynthesis by controlling the expression of core genes of PMLA biosynthesis gene cluster and other related regulatory factors. Finally, OEX-LaeA was used as the starting strain to obtain the optimal fermentation conditions. These findings illustrate the regulatory mechanism of a hypothesized global regulator, LaeA, in A. pullulans HIT-LCY3T, suggesting its potential application in industrial-scale PMLA biosynthesis.

Sinapic acid-pullulan based inflammation responsive nanomicelles for the local treatment of experimental inflammatory arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disorder of joints. It is one of the major causes of disability and morbidity worldwide. Administration of conventional drugs through the systemic route restricts the bioavailability of drugs, systemic toxicity, and reduced efficacy. We have introduced Rebamipide (Reb)-loaded Sinapic acid (SA)-Pullulan (PL) nanomicelles (Reb@SA-PL NMs), a nanotechnology based drug delivery system for the treatment of inflammatory arthritis. PL is a polysaccharide obtained from the fungus Aureobasidium pullulans, and SA is a bioactive polyphenol found in various plants. Both are classified by US-FDA Generally Recognised as Safe (GRAS) materials. Reb@SA-PL NMs found to be cytocompatible. Subsequently, intra-articular administration of Reb@SA-PL NMs enhances the anti-arthritic potential compared to free Reb drug in collagen-induced experimental inflammatory arthritis rat model. Reb@SA-PL NMs reduced the expression of RANKL receptor and Nf-κB. Reb@SA-PL NMs reverses the breakdown of type II collagen, MMP-13, and inhibits the pro-inflammatory markers. Reb@SA-PL NMs prevented bone erosion, cartilage degradation, joint oedema, and synovial inflammation. The results of the study demonstrated that Reb@SA-PL NMs, an enzyme-responsive drug delivery system, has excellent potential for alleviating inflammatory arthritis by blocking MMP-13 and RANKL.

Biotechnological application of Aureobasidium spp. as a promising chassis for biosynthesis of ornithine-urea cycle-derived bioproducts.

The ornithine-urea cycle (OUC) in fungal cells has biotechnological importance and many physiological functions and is closely related to the acetyl glutamate cycle (AGC). Fumarate can be released from argininosuccinate under the catalysis of argininosuccinate lyase in OUC which is regulated by the Ca2+ signaling pathway and over 93.9 ± 0.8 g/L fumarate can be yielded by the engineered strain of Aureobasidium pullulans var. aubasidani in the presence of CaCO3. Furthermore, 2.1 ± 0.02 mg of L-ornithine (L-Orn)/mg of the protein also can be synthesized via OUC by the engineered strains of Aureobasidum melanogenum. Fumarate can be transformed into many drugs and amino acids and L-Orn can be converted into siderophores (1.7 g/L), putrescine (33.4 g/L) and L-piperazic acid (L-Piz) (3.0 g/L), by different recombinant strains of A. melanogenum. All the fumarate, L-Orn, siderophore, putrescine and L-Piz have many applications. As the yeast-like fungi and the promising chassis, Aureobasidium spp, have many advantages over any other fungal strains. Further genetic manipulation and bioengineering will enhance the biosynthesis of fumarate and L-Orn and their derivates.

ARTP mutagenesis of Aureobasidium pullulans RM1603 for high pullulan production and transcriptome analysis of mutants.

Pullulan is a microbial exopolysaccharide produced by Aureobasidium spp. with excellent physical and chemical properties, resulting in great application value. In this study, a novel strain RM1603 of Aureobasidium pullulans with high pullulan production of 51.0 ± 1.0g·L- 1 isolated from rhizosphere soil was subjected to atmospheric and room temperature plasma (ARTP) mutagenesis, followed by selection of mutants to obtain pullulan high-producing strains. Finally, two mutants Mu0816 and Mu1519 were obtained, with polysaccharide productions of 58.7 ± 0.8 and 60.0 ± 0.8g∙L- 1 after 72-h fermentation, representing 15.1 and 17.6% increases compared with the original strain, respectively. Transcriptome analysis of the two mutants and the original strain revealed that the high expression of α/β-hydrolase (ABHD), α-amylase (AMY1), and sugar porter family MFS transporters (SPF-MFS) in the mutants may be related to the synthesis and secretion of pullulan. These results demonstrated the effectiveness of ARTP mutagenesis in A. pullulans, providing a basis for the investigation of genes related to pullulan synthesis and secretion.

Effects of Aureobasidium Pullulans produced β-1,3-1,6-glucan on CA19-9, sCD44, IgA and sCD209 in patients undergoing surgical resection of malignant pancreatic tumors.

96 Background: Pancreatic cancer is a highly refractory tumor with poor prognosis, which aggressively progresses by suppressing the immunity. Surgical procedure related stress further weakens the immunity and aids cancer stem cell metastases, warranting immune enhancement strategies. AFO-202 strain of Aureobasidium Pullulans produced β-1,3-1,6 glucan (Nichi BRITE), having yielded immune enhancement in earlier studies, we evaluated its effects in patients undergoing surgical resection of malignant pancreatic tumors. Methods: 30 subjects in two groups were enrolled; control arm (n=15, "P" group) received a placebo, and treatment arm (n=15, "G" group) received Nichi BRITE 250mg/day, in three divided doses, for 22 days from the pre-operative day. Serum beta glucan specific IgA2 (BG-IgA), total IgA (IgA), sCD209, Serum Amyloid A (SAA), sCD44 and CA19-9 were evaluated at base line and at periodic intervals. Results: sCD44 significantly decreased 48.94% in “G” group and only 4.58% in “P” group (p = &lt; 0.0001) on day 21. sCD209 on day 21 in “G” group increased by 54.68% (p = 0.04) against 15.03% in “P”. BG-IgA at day 10 increased around 150% in “G”, and only by 6% in “P”. Total IgA, increased by 20.60% in “G” and only 5.73% in “P”. SAA increased to 251.32% in “G” on day 21 and in “P” it decreased by 6.11%. Decreased sCD44 when correlated against Neutrophil to Lymphocyte Ratio (NLR), a significant positive correlation was in “G” (r statistic= 0.758; p-value =0.006; 95% CI= 0.2907~0.9335), but a negative correlation in “P”. Pancreatic cancer marker CA19-9 decreased significantly in three months follow up in “G” (-86.02 U/ml) whereas it increased in “P” by +5.91 U/ml. Mean survival of Nichi BRITE group was 25.9 months against 22.3 months in “P” group. Conclusions: Administration of Nichi BRITE glucans to patients undergoing surgical resection of malignant pancreatic tumor was safe and it enhanced biomarkers of innate and adaptive immunity. Nichi BRITE significantly decreased the circulating cancer stem cell marker sCD44 implying a lesser recurrence risk, especially in patients with high NLR ratio who have a poor prognosis, and significantly reduced the CA 19-9 levels implying a better prognosis, making us recommend it as an adjuvant to patients undergoing surgeries for malignant tumors, and be included in the guidelines of clinical nutrition.

Investigating the Immune-Stimulating Potential of β-Glucan from Aureobasidium pullulans in Cancer Immunotherapy.

β-glucan, a polysaccharide found in various sources, exhibits unique physicochemical properties, yet its high polymerization limits clinical applications because of its solubility. Addressing this limitation, we introduce PPTEE-glycan, a highly purified soluble β-1,3/1,6-glucan derived from Aureobasidium pullulans. The refined PPTEE-glycan demonstrated robust immune stimulation in vitro, activated dendritic cells, and enhanced co-stimulatory markers, cytokines, and cross-presentation. Formulated as a PPTEE + microemulsion (ME), it elevated immune responses in vivo, promoting antigen-specific antibodies and CD8+ T cell proliferation. Intratumoral administration of PPTEE + ME in tumor-bearing mice induced notable tumor regression, which was linked to the activation of immunosuppressive cells. This study highlights the potential of high-purity Aureobasidium pullulans-derived β-glucan, particularly PPTEE, as promising immune adjuvants, offering novel avenues for advancing cancer immunotherapy.

The role of the acetyl-glutamate cycle in fumarate biosynthesis through L-ornithine supply via the ornithine-urea cycle in Aureobasidium pullulans var. aubasidani

In Aureobasidium pullulans var. Aubasidani, the ornithine-urea cycle (OUC) plays a role in fumarate biosynthesis with L-ornithine serving as a significant intermediate. The acetyl-glutamate cycle (AGC) is also responsible for synthesizing L-ornithine. But it remains unclear whether AGC can assist OUC in fumarate biosynthesis. N-acetyl-glutamate kinase and N-acetyl-gamma-glutamyl-phosphate reductase present in AGC were both encoded by the ARGBC gene in A. pullulans var. Aubasidani and N-acetyl-glutamate kinase was observed to be insensitive to feedback inhibition by L-arginine from OUC. This observation could be attributed to the presence of conserved residues, specifically L-threonine (T340) and L-arginine (R336), in the N-acetyl-glutamate kinase as opposed to the conserved L-threonine (T340) and L-lysine (K336) residues that could interact with L-arginine. Hence, AGC remained active and consistently produced L-ornithine for OUC. The deletion of the ARGBC gene in the parent glucose oxidase-deficient mutant (Δgox) led to decreased L-ornithine accumulation and reduced fumarate production in the disruptant Δgox Δargbc while complementation of the ARGBC gene enhanced accumulation of L-ornithine and restored fumarate production by the complementing strain ARGBC-H. The introduction of exogenous L-arginine clearly impacted fumarate production and cell growth in the disruptant strain Δgox Δargbc. It was concluded that achieving elevated fumarate levels through OUC, maintaining the activity of AGC for high L-ornithine accumulation was crucial. The produced fumarate had many applications.