Draft Genome Sequence of the Aspergillus terreus High-Itaconic-Acid-Productivity Strain IFO6365.

Itaconic acid is an important organic acid used in the chemical industry. Aspergillus terreus strain IFO6365 is one of the highest-yielding itaconic acid-producing wild-type strains. Here, we report the draft genome sequence of IFO6365, enhancing the understanding of the role and biosynthesis of itaconic acid in this fungus.

Draft Genome Sequence of Aspergillus terreus High-Itaconic-Acid-Productivity Mutant TN-484.

Itaconic acid is an important organic acid used in the chemical industry. Aspergillus terreus strain TN-484 is a high-itaconic-acid-productivity mutant derived from strain IFO6365. Here, we report the draft genome sequence of strain TN-484, advancing the understanding of the biosynthesis of itaconic acid in filamentous fungi.

Aureobasidium pullulans produced ß-glucan is effective to enhance Kurosengoku soybean extract induced Thrombospondin-1 expression.

Black yeast, Aureobasidium pullulans is extracellularly produced ß-(1,3), (1,6)-D-glucan (ß-glucan) under certain conditions. In this study, using Glycine max cv. Kurosengoku (Kurosengoku soybeans), the production of ß-glucan through fermentation of A. pullulans was evaluated, and the effects of A. pullulans cultured fluid (AP-CF) containing ß-glucan made with Kurosengoku soybeans (kAP-CF) on a human monocyte derived cell line, Mono Mac 6 cells were investigated. Concentration of ß-glucan in kAP-CF reached the same level as normal AP-CF. An anti-angiogenic protein, Thrombospondin-1 (THBS1) was effectively induced after the stimulation with kAP-CF for comparison with AP-CF. The THBS1 is also induced after stimulation with hot water extract of Kurosengoku soybeans (KS-E), while the combined stimulation of ß-glucan with KS-E more effectively induced THBS1 than that with KS-E alone. These results suggest effects of A. pullulans-produced ß-glucan on the enhancement of Kurosengoku soybean-induced THBS1 expression.

Oral administration of the Aureobasidium pullulans-derived ß-glucan effectively prevents the development of high fat diet-induced fatty liver in mice.

Aureobasidium pullulans-derived ß-glucan (AP-PG) consisting of a ß-(1,3)-linked glucose main chain and ß-(1,6)-linked glucose branches is taken as a supplement to improve health. This study demonstrates that oral administration of AP-PG is effective to prevent the development of high-fat diet (HFD)-induced fatty liver in mice. Here, C57BL/6N mice were fed with a normal diet or HFD, and AP-PG diluted in drinking water was administered orally. After 16 weeks, the serological analysis showed that HFD-induced high blood cholesterol and triglyceride levels were reduced by the oral administration of AP-PG. Further, HFD induced-fatty liver was significantly reduced by the oral administration of AP-PG. The triglyceride accumulation in the liver was also significantly reduced in mice administered AP-PG. Liver injury as indicated by an increase in serum alanine aminotransferase (ALT) in the HFD-fed mice was significantly reduced in the mice administered AP-PG orally, and the gene expression of cholesterol 7 alpha-hydroxylase (CYP7A1) which is known to be involved in cholesterol degradation in the liver was significantly increased in the AP-PG administered mice. These results suggest the possibility that the oral administration of AP-PG is effective to prevent the development of non-alcoholic fatty liver disease (NAFLD).

Stimulation of macrophages with the ß-glucan produced by aureobasidium pullulans promotes the secretion of tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

A ß-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a ß-(1,3)-linked main chain with ß-(1,6)-linked glucose side residues. Various ß-glucans consisting of ß-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial ß-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.

Stimulation with the Aureobasidium pullulans-produced ß-glucan effectively induces interferon stimulated genes in macrophage-like cell lines.

A ß-(1,3),(1,6)-D-glucan produced by A. pullulans (AP-PG) is known to be an immune stimulating agent. In this study, we demonstrate that the stimulation with AP-PG effectively induces the interferon (IFN) stimulated genes (ISGs) in macrophage-like cell lines. The ISGs, Mx1, ISG15, and viperin mRNAs were significantly increased in RAW264.7 cells after stimulation with AP-PG. The stimulation with AP-PG transiently induced IFN-ß mRNA. However, the expression of viperin mRNA was also increased after stimulation with AP-PG even when new protein synthesis was completely blocked by treatment with cycloheximide. Further, in IFN-α receptor knockdown RAW264.7 cells, AP-PG stimulation more effectively induced viperin mRNA compared with that of IFN-α stimulation. The phosphorylation of Ser 727 in STAT1 involved in the enhancement of STAT1 activation was immediately increased after stimulation with AP-PG. In addition, viperin mRNA expression induced after stimulation with IFN-α was significantly increased by combined stimulation with AP-PG. These results suggest that stimulation with AP-PG effectively induces the ISGs through the induction of IFN and the enhancement of STAT1-mediated transcriptional activation.

Aureobasidium pullulans culture supernatant significantly stimulates R-848-activated phagocytosis of PMA-induced THP-1 macrophages.

Toll-like receptors (TLRs), which recognize a wide range of microbial pathogens and pathogen-related products, play important roles in innate immunology. Macrophages have a variety of TLRs, and pathogen binding to TLR resulted in the activation of macrophages. R-848, an immune response modifier, is an analog of imidazoquinoline derivative and binds to an endosome-localized TLR to exert an anti-viral response on leukocytes. In the present study, we verified that co-treatment of R-848 with other TLR agonists would enhance immune response. The culture supernatant of Aureobasidium pullulans (A. pullulans, which contains predominantly soluble ß-glucan), which binds to cell membrane-localized TLR, and to C-type lectin receptor Dectin-1, was treated together with R-848 to THP-1 macrophages. Compared to R-848 treatment alone, co-treatment of R-848 with A. pullulans culture supernatant significantly augmented TNF-α and IL-12p40 cytokine expression. Next, we investigated whether or not apoptotic cell uptake would be increased by co-treatment of R-848 with A. pullulans culture supernatant. To detect engulfed apoptotic cells, we induced apoptosis in human lymphoma Jurkat cells by 5-fluorouracil and stained them with fluorescent dye 5(6)-carboxytetramethylrhodamine (TAMRA), whereas THP-1 macrophage was labeled with fluorescein isothiocyanate-anti-CD14 and determined the percentage increase in TAMRA-positive THP-1 macrophages by flow cytometric assay. Since R-848 or A. pullulans treatment alone stimulated THP-1 macrophages to induce phagocytosis, co-treatment of R-848 with A. pullulans culture supernatant significantly augmented phagocytosis of apoptotic Jurkat cells. These results suggest that the activation of several different innate immune receptor pathways may enhance the immune response of R-848 significantly.

ß-Glucan derived from Aureobasidium pullulans is effective for the prevention of influenza in mice.

ß-(1→3)-D-glucans with ß-(1→6)-glycosidic linked branches produced by mushrooms, yeast and fungi are known to be an immune activation agent, and are used in anti-cancer drugs or health-promoting foods. In this report, we demonstrate that oral administration of Aureobasidium pullulans-cultured fluid (AP-CF) enriched with the ß-(1→3),(1→6)-D-glucan exhibits efficacy to protect mice infected with a lethal titer of the A/Puerto Rico/8/34 (PR8; H1N1) strain of influenza virus. The survival rate of the mice significantly increased by AP-CF administration after sublethal infection of PR8 virus. The virus titer in the mouse lung homogenates was significantly decreased by AP-CF administration. No significant difference in the mRNA expression of inflammatory cytokines, and in the population of lymphocytes was observed in the lungs of mice administered with AP-CF. Interestingly, expression level for the mRNA of virus sensors, RIG-I (retinoic acid-inducible gene-I) and MDA5 (melanoma differentiation-associated protein 5) strongly increased at 5 hours after the stimulation of A. pullulans-produced purified ß-(1→3),(1→6)-D-glucan (AP-BG) in murine macrophage-derived RAW264.7 cells. Furthermore, the replication of PR8 virus was significantly repressed by pre-treatment of AP-BG. These findings suggest the increased expression of virus sensors is effective for the prevention of influenza by the inhibition of viral replication with the administration of AP-CF.

A small scale study on the effects of oral administration of the ß-glucan produced by Aureobasidium pullulans on milk quality and cytokine expressions of Holstein cows, and on bacterial flora in the intestines of Japanese black calves.

BACKGROUND: The ß-(1 → 3),(1 → 6)-D-glucan extracellularly produced by Aureobasidium pullulans exhibits immunomodulatory activity, and is used for health supplements. To examine the effects of oral administration of the ß-(1 → 3),(1 → 6)-D-glucan to domestic animals, a small scale study was conducted using Holstein cows and newborn Japanese Black calves. FINDINGS: Holstein cows of which somatic cell count was less than 3 x 105/ml were orally administered with or without the ß-(1 → 3),(1 → 6)-D-glucan-enriched A. pullulans cultured fluid (AP-CF) for 3 months, and the properties of milk and serum cytokine expression were monitored. Somatic cell counts were not significantly changed by oral administration of AP-CF, whereas the concentration of solid non fat in the milk tended to increase in the AP-CF administered cows. The results of cytokine expression analysis in the serum using ELISA indicate that the expressions of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in all cows which were orally administered with AP-CF became slightly lower than that of control cows after the two-month treatment. On the other hand, IL-8 expression tended to indicate a moderately higher level in all treated cows after the three-month administration of AP-CF in comparison with that of the control cows. Peripartum Japanese Black beef cows and their newborn calves were orally administered with AP-CF, and bacterial flora in the intestines of the calves were analyzed by T-RFLP (terminal restriction fragment length polymorphism). The results suggest that bacterial flora are tendentiously changed by oral administration of AP-CF. CONCLUSIONS: Our data indicated the possibility that oral administration of the ß-(1 → 3),(1 → 6)-D- glucan produced by A. pullulans affects cytokine expressions in the serum of Holstein cows, and influences bacterial flora in the intestines of Japanese Black calves. The findings may be helpful for further study on the efficacies of oral administration of ß-(1 → 3),(1 → 6)-D-glucans on domestic animals.

Biotechnological production of itaconic acid and its biosynthesis in Aspergillus terreus.

More than 80,000 tons of itaconic acid (IA) is produced worldwide each year and is sold at a price of around US$ 2/kg. The IA production yield from sugar is higher than 80 g/l. The widespread use of IA in synthetic resins, synthetic fibers, plastics, rubbers, surfactants, and oil additives has resulted in an increased demand for this product. However, at present, the IA production capacity exceeds the demand because this product has a restricted range of applications. Studies have been actively conducted in different biomedical fields--dental, ophthalmic, and drug delivery--to extend the range of applications of IA. Recently, many researchers have attempted to replace the carbon source used for microbial production of IA with cheaper alternative substrates. However, there is still a need for new biotechnology innovations that would help to reduce the production costs, such as innovative process development and strain improvement to allow the use of a low-quality carbon source. In this short review, we discuss the following aspects of IA production: strain improvement, process development, identification of the key enzyme cis-aconitic acid decarboxylase (CAD) in the IA metabolic pathway, metabolic importance of CAD, and new applications of IA.

Cloning and functional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus.

A filamentous fungus Aspergillus terreus produces itaconic acid, which is predicted to be derived from cis-aconitic acid via catalysis by cis-aconitic acid decarboxylase (CAD) in the carbon metabolism of the fungus. To clarify the enzyme's function and a pathway for itaconic acid biosynthesis, we cloned a novel gene encoding the enzyme. The open reading frame of this gene (CAD1) consists of 1,529 bp encoding 490 amino acids and is interrupted by a single intron. Among the identified proteins in the database, the primary structure of the protein encoded by CAD1 shared high identity with the MmgE/PrpD family of proteins, including a number of 2-methylcitrate dehydratases of bacteria. The cloned gene excluding an intron was introduced into the expression plasmid pAUR-CAD1 controlled by the ADH1 promoter. The CAD activity in Saccharomyces cerevisiae was confirmed by directly detecting itaconic acid as a product from cis-aconitic acid as a substrate. This result reveals for the first time that this gene encodes CAD, which is essential for itaconic acid production in A. terreus.

Itaconic acid production using sago starch hydrolysate by Aspergillus terreus TN484-M1.

Sago starch was hydrolyzed using either chemical agents, or enzymes at various pH and concentrations. Hydrolysis using 5000 AUN/ml (0.5%, w/v) glucoamylase exhibited the highest itaconic acid yield up to 0.36 g/g sago starch, whereas hydrolysis using nitric acid at pH 2.0 yielded 0.35 g/g sago starch. The medium was optimized and the composition was (g/l) 140 sago starch, 1.8 corn steep liquor, 1.2 MgSO(4).7H(2)O and 2.9 NH(4)NO(3). When the optimal conditions of hydrolysis and medium composition were applied to itaconic acid production in a 3-l jar fermentor, the itaconic acid production was 48.2 g/l with a yield of 0.34 g/g sago starch. This was filtered from the cultured broth and 37.1g of itaconic acid was recovered with a purity of 97.2%. This result showed that sago starch could be converted to a value-added product with only a simple pretreatment.

Scale-up of L-lactic acid production by mutant strain Rhizopus sp. MK-96-1196 from 0.003 m3 to 5 m3 in airlift bioreactors.

In order to study the feasibility of commercial-scale L-lactic acid production by Rhizopus sp. MK-96-1196 using large scale airlift bioreactors (ALBs), a scale-up study from 0.003 m3 to 5 m3 ALB was carried out using oxygen transfer rate (OTR) as the scale-up criterion. Enhanced L-lactic acid production was achieved at OTRs higher than 0.28 (g-O2/l/h) irrespective of the scale of the bioreactor in question: in the range of 0.003 m3 to 5 m3, more than 90 g/lL-lactic acid was produced with a yield of approximately 80%, based on the initial glucose concentration. In future research, we plan to design an ALB greater than 3000 m3 (working volume: 2000 m3) for further studies on the production of L-lactic acid in large quantities.

Enhanced production of L-lactic acid by ammonia-tolerant mutant strain Rhizopus sp. MK-96-1196.

By a monospore isolation technique, Rhizopus sp. MK-96-1 was selected from colonies of Rhizopus sp. MK-96, which was isolated from the soil sample collected in Fujieda, Japan, and used as a parent strain. By the ammonia-concentration-gradient agar plate technique after mutation using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) method, a mutant strain designated Rhizopus sp. MK-96-1196 producing more than 90 g/l L-lactic acid under pH control using liquid ammonia in an airlift bioreactor was successfully isolated. Compared with the parent strain, this mutant strain produced about twofold the amount of L-lactic acid in half fermentation time under the same culture conditions. Ammonium L-lactate was recovered and purified as free L-lactic acid via n-butyl L-lactate. The ammonia used for pH control in the fermentation broth was recovered as liquid ammonia during the recovery and purification process and subsequently reused for the next fermentation. Thus, we have developed a new highly purified L-lactic acid production process without producing recalcitrant wastes, e.g., CaSO4 (gypsum).

Production of L-lactic acid from corncob.

The optimum temperature, initial pH, amount of added enzyme and substrate (corncob) for the hydrolysis of corncob by Acremonium cellulase were 35 degrees C, 4.5, 10 u/g-corncob and 100 g/l, respectively. Under the optimum conditions, more than 55 g/l of reducing sugars were hydrolyzed from 100 g/l of corncob to 34 g/l of glucose and 12 g/l of xylose based on dried corncob. More than 25 g/l of L-lactic acid was produced from this enzymatic hydrolyzate and less than 5 g/l of xylose remained in the 3-l airlift bioreactor. The production of L-lactic acid by simultaneous saccharification and fermentation (SSF) was also carried out in the 3-l airlift bioreactor using Acremonium thermophilus (cellulose-producer) and Rhizopus sp. MK-96-1196 (lactic acid-producer). More than 24 g/l of L-lactic acid was produced from 100 g/l of untreated raw corncob.

Optimization and scale-up of L-lactic acid fermentation by mutant strain Rhizopus sp. MK-96-1196 in airlift bioreactors.

We determined the optimum culture conditions such as inoculum size, initial starch concentration, pH during the fermentation and aeration rate for L-lactic acid production by Rhizopus sp. MK-96-1196 in a 3-l airlift bioreactor. More than 90 g/l of L-lactic acid was produced from only partially enzymatically hydrolyzed corn starch with a production rate of 2.6 g/l/h and a product yield of 87% based on the starch consumed under the optimum conditions in the 3-l airlift bioreactor. Scale-up from the 3-l to a 100-l airlift bioreactor for L-lactic acid fermentation was carried out using V(s)(cm/s) as a scale-up criterion. The production rates and yields of L-lactic acid in both bioreactors appeared to be fairly well correlated with k(L)a (1/h).

Production of epsilon-polylysine in an airlift bioreactor (ABR).

This paper deals with studies on epsilon-poly-L-lysine (epsilon-PL) production in an airlift bioreactor (ABR) using Streptomyces albulus S410 (S410) to minimize the production cost including the downstream processing of epsilon-PL. In a 5-l ABR, 30 g/l of epsilon-PL was produced with a power consumption of 0.3 kW/m3, the production level being similar to that in a 5-l jar fermentor with a power consumption of 8.0 kW/m3. Furthermore, the leakage of intracellular nucleic acid (INA)-related substances into the culture broth in the ABR was less than that in the jar fermentor. Due to the high-level power consumption (8.0 kW/m3) in the jar fermentor, the morphology of the cells changed from the pellet to filament form due to the extensive shear stress arising from continuous agitation, thereby increasing the leakage of the INA-related substances into the culture broth. This suggested that ABR would have an advantage in the low-cost production of epsilon-PL over stirred tank type reactors (STR).

Purification and characterization of cis-aconitic acid decarboxylase from Aspergillus terreus TN484-M1.

cis-Aconitic acid decarboxylase (CAD) was assumed to be a key enzyme in the production of itaconic acid by comparing the activity of CAD from Aspergillus terreus TN484-M1 with that of CAD from the low-itaconate yielding strain Aspergillus terreus CM85J. The constitutive CAD was purified to homogeneity from A. terreus TN484-M1 by ammonium sulfate fractionation, and column chromatography on DEAE-toyopearl, Butyl-toyopearl, and Sephacryl S200HR, and then characterized. A molecular mass of 55 kDa for the native enzyme was determined by SDS-PAGE. The enzymic activity was optimal at a pH of 6.2 and temperature of 45 degrees C. The K(m) value for cis-aconitic acid was determined as 2.45 mM (pH 6.2, 37 degrees C). The enzyme was completely inactivated by Hg+, Cu2+, Zn2+, p-chloromercuribenzoate, and 5,5'-dithio-bis(2-nitrobenzoate).