Yeasts volatile organic compounds (VOCs) as potential growth enhancers and molds biocontrol agents of mushrooms mycelia.

Volatile organic compounds (VOCs) produced by yeasts can positively affect crops, acting as antifungals or biostimulants. In this study, Aureobasidium pullulans and Metschnikowia pulcherrima were evaluated as potential antagonists of Trichoderma spp., common fungal pathogen in mushroom cultivation. To assess the biocontrol ability and biostimulant properties of the selected yeast species, in vitro co-culture and VOCs exposure assays were conducted. In both assays, VOCs produced by Aureobasidium spp. showed the stronger antifungal activity with a growth inhibition up to 30 %. This result was further confirmed by the higher volatilome alcohol content revealed by solid phase microextraction-gas chromatography mass spectrometry (SPME/GC-MS). Overall, Aureobasidium strains can be potentially used as biocontrol agent in Pleorotus ostreatus and Cyclocybe cylindracea mycelial growth, without affecting their development as demonstrated by VOCs exposure assay and Fourier-transform infrared spectroscopy (FT-IR). Conversely, M. pulcherrima was characterized by a lower or absent antifungal properties and by a volatilome composition rich in isobutyl acetate, an ester often recognized as plant growth promoter. As confirmed by FT-IR, Lentinula mycelia exposed to M. pulcherrima VOCs showed a higher content of proteins and lipids, suggesting an improvement of some biochemical properties. Our study emphasizes that VOCs produced by specific yeast strains are potentially powerful alternative to synthetic fungicide in the vegetative growth of mushroom-forming fungi and also able to modify their biochemical composition.

Water-soluble biopolymers calcium polymalate derived from fermentation broth of Aureobasidium pullulans markedly alleviates osteoporosis and fatigue.

Osteoporosis is a prevalent condition characterized by bone loss and decreased skeletal strength, resulting in an elevated risk of fractures. Calcium plays a crucial role in preventing and managing osteoporosis. However, traditional calcium supplements have limited bioavailability, poor solubility, and adverse effects. In this study, we isolated a natural soluble biopolymer, calcium polymalate (PMACa), from the fermentation broth of the fungus Aureobasidium pullulans, to investigate its potential as an anti-osteoporosis therapeutic agent. Characterization revealed that linear PMA-Ca chains juxtaposed to form a porous, rod-like state, in the presence of Ca2+. In vivo mouse models demonstrated that PMA-Ca significantly promoted the conversion of serum calcium into bone calcium, and stimulated bone growth and osteogenesis. Additionally, PMA-Ca alleviated exercise fatigue in mice by facilitating the removal of essential metabolites, such as serum lactate (BLA) and blood urea nitrogen (BUN), from their bloodstream. In vitro studies further showed that PMA-Ca strengthened osteoblast cell activity, proliferation, and mineralization. And PMA-Ca upregulated the expression of some genes involved in osteoblast differentiation, indicating a potential correlation between bone formation and PMACa. These findings indicate that soluble PMA-Ca has the potential to be a novel biopolymer-based calcium supplement with sustainable production sourced from the fermentation industry.

Optimization of pullulan production by Aureobasidium pullulans using semi-solid-state fermentation and artificial neural networks: Characterization and antibacterial activity of pullulan impregnated with Ag-TiO2 nanocomposite.

This study presents a novel and efficient approach for pullulan production using artificial neural networks (ANNs) to optimize semi-solid-state fermentation (S-SSF) on faba bean biomass (FBB). This method achieved a record-breaking pullulan yield of 36.81 mg/g within 10.82 days, significantly exceeding previous results. Furthermore, the study goes beyond yield optimization by characterizing the purified pullulan, revealing its unique properties including thermal stability, amorphous structure, and antioxidant activity. Energy-dispersive X-ray spectroscopy and scanning electron microscopy confirmed its chemical composition and distinct morphology. This research introduces a groundbreaking combination of ANNs and comprehensive characterization, paving the way for sustainable and cost-effective pullulan production on FBB under S-SSF conditions. Additionally, the study demonstrates the successful integration of pullulan with Ag@TiO2 nanoparticles during synthesis using Fusarium oxysporum. This novel approach significantly enhances the stability and efficacy of the nanoparticles by modifying their surface properties, leading to remarkably improved antibacterial activity against various human pathogens. These findings showcase the low-cost production medium, and extensive potential of pullulan not only for its intrinsic properties but also for its ability to significantly improve the performance of nanomaterials. This breakthrough opens doors to diverse applications in various fields.

NsdD, a GATA-type transcription factor is involved in regulation and biosynthesis of macromolecules melanin, pullulan, and polymalate in Aureobasidium melanogenum.

In this study, an NSDD gene, which encoded a GATA-type transcription factor involved in the regulation and biosynthesis of melanin, pullulan, and polymalate (PMA) in Aureobasidium melanogenum, was characterized. After the NSDD gene was completely removed, melanin production by the Δnsd mutants was enhanced, while pullulan and polymalate production was significantly reduced. Transcription levels of the genes involved in melanin biosynthesis were up-regulated while expression levels of the genes responsible for pullulan and PMA biosynthesis were down-regulated in the Δnsdd mutants. In contrast, the complementation of the NSDD gene in the Δnsdd mutants made the overexpressing mutants restore melanin production and transcription levels of the genes responsible for melanin biosynthesis. Inversely, the complementation strains, compared to the wild type strains, showed enhanced pullulan and PMA yields. These results demonstrated that the NsdD was not only a negative regulator for melanin biosynthesis, but also a key positive regulator for pullulan and PMA biosynthesis in A. melanogenum. It was proposed how the same transcriptional factor could play a negative role in melanin biosynthesis and a positive role in pullulan and PMA biosynthesis. This study provided novel insights into the regulatory mechanisms of multiple A. melanogenum metabolites and the possibility for improving its yields of some industrial products through genetic approaches.

Tools for live-cell imaging of cytoskeletal and nuclear behavior in the unconventional yeast, Aureobasidium pullulans.

Aureobasidium pullulans is a ubiquitous fungus with a wide variety of morphologies and growth modes including "typical" single-budding yeast, and interestingly, larger multinucleate yeast than can make multiple buds in a single cell cycle. The study of A. pullulans promises to uncover novel cell biology, but currently tools are lacking to achieve this goal. Here, we describe initial components of a cell biology toolkit for A. pullulans, which is used to express and image fluorescent probes for nuclei as well as components of the cytoskeleton. These tools allowed live-cell imaging of the multinucleate and multibudding cycles, revealing highly synchronous mitoses in multinucleate yeast that occur in a semiopen manner with an intact but permeable nuclear envelope. These findings open the door to using this ubiquitous polyextremotolerant fungus as a model for evolutionary cell biology.

Microbial fermentation-based synthesis of nano-curcumin suggesting the role of pullulan in nano-formulation.

Curcumin is a multitargeting nutraceutical with numerous health benefits, however, its efficacy is limited due to poor aqueous solubility and reduced bioavailability. While nano-formulation has emerged as an alternative to encounter such issues, it often involves use of toxic solvents. Microbial synthesis may be an innovative solution to address this lacuna. Present study, for the first time, reports exploitation of Aureobasidium pullulans RBF4A3 for production of nano-curcumin. For this purpose, Aureobasidium pullulans RBF4A3 was inoculated in YPD media along with curcumin (0.1 mg/mL) and incubated for 24 h, 48 h, and 72 h. Subsequently, residual sugar, biomass, EPS concentration, curcumin concentration, and curcumin nanoparticle size were measured. As a result, nano-curcumin with an average particle size of 31.63 nm and enhanced aqueous solubility was obtained after 72 h. Further, investigations suggested that pullulan, a reducing polysaccharide, played a significant role in curcumin nano-formulation. Pullulan-mediated nano-curcumin formulation, with an average particle size of 24 nm was achieved with conversion rate of around 59.19 %, suggesting improved aqueous solubility. Additionally, the anti-oxidant assay of the resulting nano-curcumin was around 53.7 % per µg. Moreover, kinetics and thermodynamic studies of pullulan-based nano-curcumin revealed that it followed first-order kinetics and was favored by elevated temperature for efficient bio-conversion. Also, various physico-chemical investigations like FT-IR, NMR, and XRD reveal that pullulan backbone remains intact while forming curcumin nanoparticle. This study may open up new avenues for synthesizing nano-polyphenols through a completely green and solvent free process with plausible diverse applications.

Adsorption of Zearalenone by Aureobasidium pullulans Autolyzed Biomass Preparation and Its Detoxification Properties in Cultures of Saccharomyces cerevisiae Yeast.

Different preventive strategies are needed to minimize the intake risks of mycotoxins, including zearalenone (ZEN). The aim of this study was to determine the ZEN adsorption ability of an autolyzed biomass preparation of polymorphic yeast Aureobasidium pullulans A.p.-3. The evaluation of the antitoxic properties of the preparation was also performed in relation to Saccharomyces cerevisiae yeast (ATCC 2366, ATCC 7090 and ATCC 9763) used as a model cell exposed to a toxic ZEN dose. The preparation at a dose of 5 mg/mL showed the adsorption of ZEN present in model systems at concentrations between 1 µg/mL to 100 µg/mL. The highest degree of adsorption was established for ZEN concentrations of 1 µg/mL and 5 µg/mL, becoming limited at higher doses of the toxin. Based on the Langmuir model of adsorption isotherms, the predicted maximum ZEN adsorption was approx. 190 µg/mL, regardless of pH. The growth of three strains of S. cerevisiae yeast cells in the medium with ZEN at concentrations within the range of 1.56 µg/mL-100 µg/mL was analyzed to determine the minimum inhibitory concentration. The growth of all tested strains was especially limited by high doses of ZEN, i.e., 50 and 100 µg/mL. The protective effect of the tested preparation was noted in relation to yeast cells exposed to toxic 100 µg/mL ZEN doses. The highest yeast cell growth (app. 36% percentage) was noted for a S. cerevisiae ATCC 9763 strain compared to the medium with ZEN but without preparation. More detailed tests determining the antitoxic mechanisms of the A. pullulans preparation are planned in the future, including cell culture bioassays and animal digestive tract models.

Characteristic analysis and fermentation optimization of a novel Aureobasidium pullulans RM1603 with high pullulan yield.

A high-yielding microbial polysaccharide-producing strain, named RM1603, was isolated from rhizosphere soil and identified by morphological and phylogenetic analysis. The extracellular polysaccharides (EPS) were identified by thin-layer chromatography and infrared spectroscopy. The fermentation conditions were optimized by single factor experiments in shake flasks and a 5-L fermentor. The results of morphological and phylogenetic tree analysis showed that RM1603 was a strain of Aureobasidium pullulans. Its microbial polysaccharide was identified as pullulan, and the EPS production capacity reached 33.07 ± 1.03 g L-1 in shake flasks. The fermentation conditions were optimized in a 5-L fermentor, and were found to encompass an initial pH of 6.5, aeration rate of 2 vvm, rotor speed of 600 rpm, and inoculum size of 2 %. Under these conditions, the pullulan yield of RM1603 reached 62.52 ± 0.24 g L-1. Thus, this study contributes RM1603 as a new isolation with high-yielding pullulan and potential application value in biotechnology.

Use of Aureobasidium in a sustainable economy.

Aureobasidium is omnipresent and can be isolated from air, water bodies, soil, wood, and other plant materials, as well as inorganic materials such as rocks and marble. A total of 32 species of this fungal genus have been identified at the level of DNA, of which Aureobasidium pullulans is best known. Aureobasidium is of interest for a sustainable economy because it can be used to produce a wide variety of compounds, including enzymes, polysaccharides, and biosurfactants. Moreover, it can be used to promote plant growth and protect wood and crops. To this end, Aureobasidium cells adhere to wood or plants by producing extracellular polysaccharides, thereby forming a biofilm. This biofilm provides a sustainable alternative to petrol-based coatings and toxic chemicals. This and the fact that Aureobasidium biofilms have the potential of self-repair make them a potential engineered living material avant la lettre. KEY POINTS: •Aureobasidium produces products of interest to the industry •Aureobasidium can stimulate plant growth and protect crops •Biofinish of A. pullulans is a sustainable alternative to petrol-based coatings •Aureobasidium biofilms have the potential to function as engineered living materials.

Efficient Production of Melanin by Aureobasidium Melanogenum Using a Simplified Medium and pH-Controlled Fermentation Strategy with the Cell Morphology Analysis.

Natural melanin is a biopolymer with wide application prospects in medicine, food, cosmetics, environmental protection, agriculture, and so on. Microbial fermentation is an important and effective way to produce melanin. In this study, Aureobasidium melanogenum, known as black yeast with cellular pleomorphism, was used for the production of melanin. Based on the characteristic of A. melanogenum secreting melanin under oligotrophic stress, a simple medium containing only glucose, MgSO4·7H2O, and KCl was constructed for the production of melanin. The melanin titer of 6.64 ± 0.22 g/L was obtained after 20 days of fermentation without pH control. The cell morphological changes of A. melanogenum during the production of melanin were recorded, and the results showed that chlamydospore might be the most favorable cell morphology for melanin synthesis. Then, different fermentation strategies with cell morphology analysis were developed to further improve the production of melanin in a 5-L fermenter. Results showed that the maximum titer of melanin reached 18.50 g/L by using the fermentation strategy integrating pH control, ammonium salt addition, and H2O2 stimulation, which increased by 178.6% than that of the strategy without pH control. Furthermore, the melanin obtained from the fermentation broth was characterized as eumelanin containing an indole structure. This study provided a potentially feasible fermentation strategy for the industrial production of melanin.

Biosynthesis of poly(ß-L-malic acid) from rubberwood enzymatic hydrolysates in co-fermentation by Aureobasidium pullulans.

Co-fermentation of multiple substrates has emerged as the most effective method to improve the yield of bioproducts. Herein, sustainable rubberwood enzymatic hydrolysates (RWH) were co-fermented by Aureobasidium pullulans to produce poly(ß-L-malic acid) (PMA), and RWH + glucose/xylose was also investigated as co-substrates. Owing to low inhibitor concentration and abundant natural nitrogen source content of RWH, a high PMA yield of 0.45 g/g and a productivity of 0.32 g/L/h were obtained by RWH substrate fermentation. After optimization, PMA yields following the fermentation of RWH + glucose and RWH + xylose reached 59.92 g/L and 53.71 g/L, respectively, which were 52 % and 36 % higher than that after the fermentation of RWH. RWH + glucose more significantly affected the correlation between PMA yield and substrate concentration than RWH + xylose. The results demonstrated that the co-fermentation of RWH co-substrate is a promising method for the synthesis of bioproducts.

Engineering the reductive tricarboxylic acid pathway in Aureobasidium pullulans for enhanced biosynthesis of poly-L-malic acid.

Aureobasidium pullulans produced poly-L-malic acid (PMA) as the main metabolite in fermentation but with relatively low productivity and yield limiting its industrial application. In this study, A. pullulans ZX-10 was engineered to overexpress cytosolic malate dehydrogenase (MDH) and pyruvate carboxylase (PYC) and PMA synthetase (PMS) using a high-copy yeast episomal plasmid with the gpdA promoter from Aspergillus nidulans. Overexpressing endogenous PMS and heterologous MDH and PYC from Aspergillus oryzae respectively increased PMA production by 19 % - 37 % (0.64 - 0.74 g/g vs. 0.54 g/g for wild type) in shake-flask fermentations, demonstrating the importance of the reductive tricarboxylic acid (rTCA) pathway in PMA biosynthesis. A. pullulans co-expressing MDH and PYC produced 96.7 g/L PMA at 0.90 g/L∙h and 0.68 g/g glucose in fed-batch fermentation, which were among the highest yield and productivity reported. The engineered A. pullulans with enhanced rTCA pathway is advantageous and promising for PMA production.

Liamocin biosynthesis is induced by an autogenous host acid activation in Aureobasidium melanogenum.

It has been known that maximal liamocin production must be carried out at low environmental pH (around 3.0). In this study, it was found that the low pH was mainly caused by the secreted citric acid which is one precursor of acetyl-CoA for liamocin biosynthesis. Determination of citric acid in the culture, deletion, complementation and overexpression of the CEXA gene encoding specific citrate exporter demonstrated that the low pH was indeed caused by the secreted citric acid. Deletion, complementation and overexpression of the ACL gene encoding ATP-citric acid lyase and effects of different initial pHs and added citric acid showed that the low pH in the presence of citric acid was suitable for lysis of intracellular citric acid, liamocin production and expression of the PACC gene encoding the pH signaling transcription factor PacC. This meant that the PACC gene was an acid-expression gene. Deletion, complementation and overexpression of the PACC gene indicated that expression of the key gene cluster GAL1-EST1-PKS1 for liamocin biosynthesis was driven by the pH signaling transcription factor PacC and there was weak nitrogen catabolite repression on liamocin biosynthesis at the low pH. That was why liamocin biosynthesis was induced at a low pH in the presence of citric acid. The mechanisms of the enhanced liamocin biosynthesis by the autogenous host acid activation, together with the pH signaling pathway, were proposed.

Green process for isolation and purification of poly(ß-L-malic acid) from Aureobasidium spp. by an integrated ion exchange and membrane separation.

Poly (ß-L-malic acid) (PMLA) is a biopolymer used in food and medical fields. However, the industrial processes are susceptible to the pollution of CaSO4 waste and organic solvent owing to the heavy use of CaCO3 in fermentation process and organic solvents in isolation process. This study developed an organic solvent and CaSO4 -free process for the industrial-scale production of PMLA. Firstly, calcium ion was removed at pH 9.2 by pH adjustment with Na2CO3, and the generated CaCO3 was reused in the fermentation process. Then, the D296 resin was selected to isolate the PMLA from the Ca2+-free broth, where the adsorption data were both primely described by the Freundlich and Langmuir equation, while Freundlich model better fit the process than Langmuir equation, indicating that it was non-monolayer adsorption of PMLA on the resin. Meanwhile, a three-step gradient elution with phosphate buffer (i.e., 0.2 mol/L, pH 7.0) containing 0.1, 0.2 and 1 mol/L NaCl was developed to recover PMLA. Finally, a PES15 membrane was selected to recover the PMLA from the elution solution, which could be reused in the next cycle. As a result, the PMLA with a purity of 98.89 % was obtained with the developed green process. In the developed process, it removed the pollution of organic solvent and calcium waste for the biosynthesis of PMLA on an industrial scale, which also offers a sustainable and green route for the biosynthesis of other carboxylic acids.

Fractionation and characterization of poly(ß-L-malic acid) produced by Aureobasidium melanogenum ipe-1.

Poly (ß-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries, whose functions primarily depend upon its molecular size and chemical structure. Up to now, the fractionation and characterization of PMLA produced by Aureobasidium spp. were still unclear. In this study, the product from A. melanogenum ipe-1 was effectively fractionated using 300 and 50 kDa membranes. During the filtration, the mechanisms of membrane fouling were illegible since the PMLA can both reject and permeate the membrane, while the main fouling mechanism varied between standard blocking and complete blocking during the diafiltration. After fractionation, 14.0, 8.4 and 77.6 % of the PMLAs with Mws of 75,134, 21,344 and 10,056 Da were distributed in the 300 kDa retentate after diafiltrating, 50 kDa retentate after diafiltrating, and the 50 kDa permeate, respectively. The Mw/Mns of the PMLAs were 4.12, 1.92, and 1.12 in the three fractions. Based on characteristic spectra of NMR, HPLC and FTIR, the product was not usual L-malic acid monomers, but glucose-terminated PMLA. The glucose was located at the terminal hydroxyl of PMLA. These results would serve as a valuable guide for process design and practical operation in subsequent industrial application.

Assembly and comparative genome analysis of a Patagonian Aureobasidium pullulans isolate reveals unexpected intraspecific variation.

Aureobasidium pullulans is a yeast-like fungus with remarkable phenotypic plasticity widely studied for its importance for the pharmaceutical and food industries. So far, genomic studies with strains from all over the world suggest they constitute a genetically unstructured population, with no association by habitat. However, the mechanisms by which this genome supports so many phenotypic permutations are still poorly understood. Recent works have shown the importance of sequencing yeast genomes from extreme environments to increase the repertoire of phenotypic diversity of unconventional yeasts. In this study, we present the genomic draft of A. pullulans strain from a Patagonian yeast diversity hotspot, re-evaluate its taxonomic classification based on taxogenomic approaches, and annotate its genome with high-depth transcriptomic data. Our analysis suggests this isolate could be considered a novel variant at an early stage of the speciation process. The discovery of divergent strains in a genomically homogeneous group, such as A. pullulans, can be valuable in understanding the evolution of the species. The identification and characterization of new variants will not only allow finding unique traits of biotechnological importance, but also optimize the choice of strains whose phenotypes will be characterized, providing new elements to explore questions about plasticity and adaptation.

First case of neonatal fungemia caused by Aureobasidium melanogenum.

Aureobasidium melanogenum is a saprophytic, dematiaceous, yeast-like fungus rarely implicated in human infections. Here, we report the first case of A. melanogenum fungemia in a 30-week-old preterm, very low birth weight neonate born to a primigravida with history of gestational diabetes, pregnancy induced hypertension and oligohydramnios. The baby developed respiratory distress, hypotension, bradycardia, coagulopathy and septic shock shortly after birth, and eventually succumbed to multiple organ dysfunction syndrome on day 9 of life. Paired blood culture showed growth of a dematiaceous yeast-like fungus which was identified as A. melanogenum by rDNA internal transcribed spacer (ITS) sequencing. Antifungal susceptibility testing of the isolate showed high minimum inhibitory concentration of fluconazole (32 µg/mL), indicating resistance. Diagnosis of A. melanogenum fungemia is difficult as it is easily confused with Candida species in Gram stained smears and similar colony morphology during the initial stages of growth. Also, the conventional diagnostic methods, such as VITEK 2 and MALDI-TOF MS are unreliable for identification of this pathogen. Accurate identification using molecular techniques is crucial for making treatment decisions as A. melanogenum shows substantial antifungal resistance. Clinicians should be aware that yeast-like cells in blood culture are not only indicative of Candida species, but also rare pathogens like A. melanogenum and should exercise caution while starting fluconazole therapy. At present, there are no established susceptibility breakpoints for Aureobasidium spp. Further studies are needed to determine the optimal treatment for such infections.

Hyper-Production of Pullulan by a Novel Fungus of Aureobasidium melanogenum ZH27 through Batch Fermentation.

Pullulan, which is a microbial exopolysaccharide, has found widespread applications in foods, biomedicines, and cosmetics. Despite its versatility, most wild-type strains tend to yield low levels of pullulan production, and their mutants present genetic instability, achieving a limited increase in pullulan production. Therefore, mining new wild strains with robust pullulan-producing abilities remains an urgent concern. In this study, we found a novel strain, namely, Aureobasidium melanogenum ZH27, that had a remarkable pullulan-producing capacity and optimized its cultivation conditions using the one-factor-at-a-time method. To elucidate the reasons that drove the hyper-production of pullulan, we scrutinized changes in cell morphology and gene expressions. The results reveal that strain ZH27 achieved 115.4 ± 1.82 g/L pullulan with a productivity of 0.87 g/L/h during batch fermentation within 132 h under the optimized condition (OC). This pullulan titer increased by 105% compared with the initial condition (IC). Intriguingly, under the OC, swollen cells featuring 1-2 large vacuoles predominated during a rapid pullulan accumulation, while these swollen cells with one large vacuole and several smaller ones were prevalent under the IC. Moreover, the expressions of genes associated with pullulan accumulation and by-product synthesis were almost all upregulated. These findings suggest that swollen cells and large vacuoles may play pivotal roles in the high level of pullulan production, and the accumulation of by-products also potentially contributes to pullulan synthesis. This study provides a novel and promising candidate for industrial pullulan production.

Atypical pulmonary phaeohyphomycosis due to Aureobasidium spp. - case report and brief literature review.

We report on a case of probable invasive Auerobasidium spp. pulmonary infection in a patient with myelodysplastic syndrome. The patient was successfully treated with liposomal amphotericin B monotherapy, with transition to orally administered isavuconazole. This case shows an atypical initial radiological presentation with diffuse ground-glass opacities, as previously demonstrated in cases of Aureobasidium spp. hypersensitivity pneumonitis. Moreover this case further highlights the difficulties associated with the diagnosis and complexity in the management of Aureobasidium spp. infections.

Metabolic engineering of Aureobasidium melanogenum 9-1 for overproduction of liamocins by enhancing supply of acetyl-CoA and ATP.

In this study, it was found that reducing consumption of acetyl-CoA in mitochondria, peroxisome and lipid biosynthesis could not obviously enhance liamocin biosynthesis by engineered strains of Aureobasidium melanogenm 9-1, but decreased cell growth of the mutants. On the contrary, expression of heterologous PTA gene for phosphotransacetylase in PK pathway and native ALD gene for acetaldehyde dehydrogenase and ACS gene encoding acetyl-CoA synthetase in the PDH bypass pathway reduced liamocin biosynthesis. However, expression the PK gene for phosphoketolase, the PDC gene encoding pyruvate decarboxylase and VHb gene coding for Vitreoscilla hemoglobin (VHb) in the glucose derepression mutants could greatly enhance liamocin production. The resulting strain V33 could produce 55.38 g/L of liamocin and 25.10 g/L of cell dry weight from 117.27 g/L of glucose within 168 h of 10-liter fermentation, leading to the yield of 0.47 g/g of glucose, the productivity of 0.33 g/L/h and rate of glucose utilization of 0.70 ± 0.01 g/L/h. This was a new and efficient strategy for overproduction of liamocin by A. melanogenm.