Investigation of poly ( β-L-malic acid) production by strains of Aureobasidium pullulans

 Eight strains of the genus Aureobasidium obtained from culture collections were tested for their capability to produce poly(β-L-malic acid) (PMA). Four of the tested strains showed positive results. The most productive strain, A. pullulans CBS 591.75, was used to study the production of PMA in stirred-tank reactors. It was found that PMA was mainly produced in the late exponential phase, and the production related positively to glucose consumption. At the beginning of the fermentation the pH increased from 4.0 to about 7.0; subsequently the pH decreased and remained stable at around 3.0–3.5 for several days. Temperatures higher than 25^°C were detrimental to PMA production and cell growth. PMA production and cell growth at 20^°C and 25^°C exhibited no significant differences. PMA production and cell growth were studied under pH-controlled fermentation (at pH 2.0, 4.0, 5.5). The highest PMA production occurred at pH 4.0. PMA production was reduced at pH 2.0 although quite reasonable cell growth occurred at this pH value. Under optimized conditions 9.8 g PMA/l was produced during 9 days of fermentation in the stirred-tank reactors with an overall yield of 0.11 g PMA/g glucose. A procedure for the isolation of PMA and its separation from the other components of the fermentation broth was developed. The isolated PMA was characterized by ¹H and ¹³C-NMR spectroscopy as well as by infrared absorption spectroscopy. Gel-permeation chromatography revealed a relative molecular mass of approximately 3000–5000 by comparison with polyethylene glycol standards. Received: 13 February 1996/Received revision: 25 April 1996/Accepted: 1 May 1996     

Poly(β-L-malic acid) production by diverse phylogenetic clades of Aureobasidium pullulans

Poly(β-L-malic acid) (PMA) is a natural biopolyester that has pharmaceutical applications and other potential uses. In this study, we examined PMA production by 56 strains of the fungus Aureobasidium pullulans representing genetically diverse phylogenetic clades. Thirty-six strains were isolated from various locations in Iceland and Thailand. All strains from Iceland belonged to a newly recognized clade 13, while strains from Thailand were distributed among 8 other clades, including a novel clade 14. Thirty of these isolates, along with 26 previously described strains, were examined for PMA production in medium containing 5% glucose. Most strains produced at least 4 g PMA/L, and several strains in clades 9, 11, and 13 made 9–11 g PMA/L. Strains also produced both pullulan and heavy oil, but PMA isolated by differential precipitation in ethanol exhibited up to 72% purity with no more than 12% contamination by pullulan. The molecular weight of PMA from A. pullulans ranged from 5.1 to 7.9 kDa. Results indicate that certain genetic groups of A. pullulans are promising for the production of PMA.     

Production of poly(β-l-malic acid) (PMA) from agricultural biomass substrates by Aureobasidium pullulans

For the first time the production of poly(β-l -malic acid) (PMA) has been achieved using agricultural biomass substrates by the yeast-like fungus Aureobasidium pullulans. Strains NRRL Y-2311-1, NRRL 50382, NRRL 50383, and NRRL 50384, representing diverse isolation sources and phylogenetic clades, produced PMA from alkaline H₂O₂-pretreated corn fiber and wheat straw as sole carbon sources. Pretreated wheat straw was better than pretreated corn fiber, and strain NRRL 50383 gave the highest overall yields of PMA. The addition of CaCO₃ plus supplementary hydrolytic enzymes enhanced PMA production. Four basal media were compared for PMA production, and the best was found to be a N-limited pullulan production medium (PM). In this medium, PMA production took place during growth limitation. Under optimal conditions, strain NRRL 50383 produced more than 20 g PMA/l from 5 % (w/v) pretreated wheat straw in PM with 3 % (w/v) CaCO₃ and supplementary enzymes.     

Effects of culture conditions on β-poly(l-malate) production by Physarum polycephalum

Culture conditions for the fermentative production of β-poly(l-malate) (PMLA) by microplasmodia of Physarum polycephalum were investigated and optimized. Optimal production was achieved in the presence of CaCO₃. For 1.5% (w/v) d-glucose, 1% bactotryptone and 1% CaCO₃, a maximum of 1.7 g PMLA/l was secreted in 3 days. For 4.5% glucose and otherwise the same conditions, 2.7 g PMLA/l was produced in 6 days. The contribution of carbonate was inhibited by avidin. PMLA and biomass production were not strictly coupled: PMLA was also synthesized at the beginning of the stationary phase. At pH 5.5 PMLA production was twice that at pH 4.0, while biomass was not changed. Optimal temperatures were 24–28 °C. Received: 12 November 1998 / Received revision: 10 February 1999 / Accepted: 12 February 1999     

Stimulation of Macrophages with the β-Glucan Produced by Aureobasidium pullulans Promotes the Secretion of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL)

A β-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a β-(1,3)-linked main chain with β-(1,6)-linked glucose side residues. Various β-glucans consisting of β-(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 β-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.     

Which morphological forms of the fungus Aureobasidium pullulans are responsible for pullulan production?

Attempts were made to clarify the precise location and possible site of production of the alpha-glucan pullulan in different morphological forms of the fungus Aureobasidium pullulans. Gold-conjugated pullulanase was used as the specific probe for this purpose. No cell wall pullulan-like material was detected by transmission electron microscopy (TEM) in any morphological form of this fungus, although intracellular electron transparent material bound this probe. When silver enhancement of this gold-conjugated pullulanase probe was used, the data strongly suggested that only swollen cells and chlamydospores, and neither hyphae nor unicellular blastospores, often held responsible for pullulan formation, appeared to produce pullulan-like material.     

Does the agitation rate and/or oxygen saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture?

  When Aureobasidium pullulans was grown at a number of agitation rates under batch conditions, exopolysaccharide yields were dramatically reduced at high rates i.e. at least 750 rpm. Investigations with gas blending, which allowed pO₂ manipulation and control independently of the agitation rate, showed that this yield reduction was due solely to the high pO₂ levels that occurred at these agitation rates. Thus, polysaccharide production at 1000 rpm could be elevated by maintaining the pO₂ at a low level during the initial phase of the fermentation. However, both the timing of the pO₂ decrease and the level at which it was maintained were crucial for obtaining yields at 1000 rpm, similar to those observed at low agitation rates. Received: 29 February 1996 / Received revision: 11 July 1996 / Accepted: 15 July 1996     

The effects of gene disruption of Kre6-like proteins on the phenotype of β-glucan-producing Aureobasidium pullulans

Applied Microbiology and Biotechnology - Killer toxin resistant 6 (Kre6) and its paralog, suppressor of Kre null 1 (Skn1), are thought to be involved in the biosynthesis of cell wall...     

Nutritional status of Aureobasidium sp. ATCC 20524 for the production of β-fructofuranosidase

Sucrose at 10 to 20% (w/v) was the best carbon source for the production of -fructofuranosidase by Aureobasidium sp. ATCC 20524. At higher concentrations, it arrested growth. Glucose and fructose were also good carbon sources for the enzyme production. Yeast extract at 1.5 to 2% (w/v) was the best nitrogen source for the enzyme production and for cell growth. Addition of NaNO₃ (1 to 2%, w/v) and MgSO₄·7H₂O (0.5 to 1.5%, w/v) to the cultivation medium increased the intracellular enzymatic activity. The total enzymatic activity and cell growth reached 1.2×10⁴ U/flask and 2.5 g dry cell/flask, respectively after 48 h.Sachio Hayashi, Yoshihiko Shimokawa, Masaharu Nonoguchi, Yoshiyuki Takasaki and Kiyohisa Imada are with the Department of Industrial Chemistry, Faculty of Engineering, Miyazaki University, 1-1 Nishi, Gakuen Kibanadai, Miyazaki, 889-21 Japan. Hideo Ueno is with the Nippon Oligo Corporation, 588 Izumisawa, Jyohana-chyo, Tonami-gun, Toyama, 939-18, Japan.     

Kinetics of β-N-methylamino-L-alanine (BMAA) and 2, 4-diaminobutyric acid (DAB) production by diatoms: the effect of nitrogen

The neurotoxins β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) are produced by cyanobacteria, diatoms and dinoflagellates and have been detected in seafood worldwide. Our present knowledge of their metabolism or biosynthesis is limited. In this study, the production of BMAA and DAB as a function of time was monitored in five strains representing four species of diatoms, i.e. Phaeodactylum tricornutum, Thalassiosira weissflogii, Thalassiosira pseudonana and Navicula pelliculosa, previously identified as BMAA and DAB producers. Subsequently, three strains were selected and exposed to three nitrogen treatments – starvation, control (the standard concentration in f/2 medium) and enrichment, because BMAA metabolism has been suggested to be closely associated with cellular nitrogen metabolism in both cyanobacteria and diatoms. Chlorophyll a and total protein concentrations were also determined. Our results indicate that BMAA and DAB production in diatoms is species- and strain-specific. However, production might also be affected by stress, particularly as related to nitrogen starvation and cell density. Furthermore, this study shows a significant correlation between the production of the two neurotoxins which might further suggest common steps in the metabolic pathways.     

β-Glucan Derived from Aureobasidium pullulans Is Effective for the Prevention of Influenza in Mice

β-(1→3)-D-glucans with β-(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 β-(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 β-(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.     

Involvement of Branched Units at Position 6 in the Reactivity of a Unique Variety of β-D-Glucan from Aureobasidium pullulans to Antibodies in Human Sera

We recently determined the structure of a unique type of 1,3-β-D-glucan obtained from Aureobasidium pullulans (AP-FBG) and found that it reacted with the antibodies in human sera. The reactivity of AP-FBG to the antibodies was stronger than that of 1,3-β-D-glucan obtained Grifola frondosa (GRN) but weaker than that of 1,3-β-D-glucan from Candida albicans (CSBG). Here, we demonstrated that AP-FBG reacted to IgG antibodies, especially those of the subclasses IgG2, IgG1, and IgG3, in human sera. Moreover, the results of competitive enzyme-linked immunosorbent assays (ELISAs) using various glucan competitors showed that these IgGs recognized branched chains at position 6. This is the first study to report that the branched chains at position 6 of β-D-glucan strongly contribute to its recognition by antibodies in human sera. This high reactivity of AP-FBG to human IgG could be advantageous for the use of this glucan in medicine, e.g., as an immunostimulatory agent.     

Nox4 modulates collagen production stimulated by transforming growth factor β1 in vivo and in vitro

The synthesis of extracellular matrix including collagen during wound healing responses involves signaling via reactive oxygen species (ROS). We hypothesized that NADPH oxidase isoform Nox4 facilitates the stimulatory effects of the profibrotic cytokine transforming growth factor (TGF) β₁ on collagen production in vitro and in vivo. TGFβ₁ stimulated collagen synthesis and hydrogen peroxide generation in mouse cardiac fibroblasts, and both responses were attenuated by a scavenger of superoxide and hydrogen peroxide (EUK-134). Furthermore, by expressing a dominant negative form of Nox4 (Adv-Nox4^ΔNADPH) in fibroblasts, TGFβ₁-induced hydrogen peroxide production and collagen production were abrogated, suggesting that Nox4-dependent ROS are important for TGFβ₁ signaling in collagen production. This was confirmed by the inhibitory effect of an adenovirus carrying siRNA targeting Nox4 (Adv-Nox4i) on TGFβ₁-induced collagen synthesis and expression of activated myofibroblasts marker smooth muscle alpha actin. Finally we used a mouse model of subcutaneous sponge implant to examine the role of Nox4 in the local stimulatory effects of TGFβ₁ on collagen accumulation in vivo. TGFβ₁-induced collagen accumulation was significantly reduced when the sponges were instilled with Adv-Nox4^ΔNADPH. In conclusion, Nox4 acts as an intermediary in the signaling of TGFβ₁ to facilitate collagen synthesis.     

Effect of culture conditions on mycelial growth and production of γ-linolenic acid by the fungus Mortierella ramanniana

Summary Effect of culture conditions on cell growth, lipid accumulation and -linolenic acid production is reported for four Mortierella species. The highest concentration as well as the highest productivity of -linolenic acid in lipid was determined in strains of M. ramanniana. M. ramanniana CBS 112.08 was used in the studies of the influence of medium composition, concentration of carbon- and nitrogen sources and growth temperature. Several carbon sources provided good growth and a high lipid content in biomass. The highest dry weights (11–12g/l) and lipid contents (24%, w/w), were observed if glucose or fructose was used as carbon source, whereas the highest amount of -linolenic acid (26%) was determined in starch-grown cells. The fatty acid composition in the lipid was influenced by the cultivation time, growth temperature and, to a minor extent, by the carbon source used. In fermentor cultures, both strains of Mortierella ramanniana showed relatively poor growth and incomplete consumption of glucose. M. vinacea, on the other hand, grew well in tower reactors. M. vinacea, which has a different morphology than M. ramanniana strains, also showed higher yields of biomass and lipid and higher yield coefficients than the latter.     

Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants

We have generated transgenic maize seed containing -glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant -glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant -glucuronidase (GUS) was compared to native -glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a K_m of 0.20 mM and a V_max of 0.29 mM MUG per hour, and the native enzyme had a K_m and V_max of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of -glucuronidase, the K_i of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor.     

Effects of cyclodextrins on the hydrolysis of ganglioside GM1 by acid β-galactosidases

The hydrolysis of ganglioside G_M1 by acid -galactosidases was greatly enhanced by the inclusion of heptakis(2,6-di-O-methyl)--cyclodextrin or -cyclodextrin in the assay mixture. The other cyclodextrins tested were not effective. The extent of stimulation by these cyclodextrins was relatively smaller than those by taurodeoxycholate and taurochenodeoxycholate. However, it is suggested that stimulation by bile salts may be partly a reflection of the detergent effects of bile salts on G_M1 and partly a reflection of the interaction between bile salts and the enzyme itself. On the other hand, the stimulation by the cyclodextrins seems to correlate to the formation of an inclusion complex between G_M1 and cyclodextrin without enzyme protein interaction.     

Joint production of IL-22 participates in the initial phase of antigen-induced arthritis through IL-1β production

IntroductionRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by neutrophil articular infiltration, joint pain and the progressive destruction of cartilage and bone. IL-22 is a key effector molecule that plays a critical role in autoimmune diseases. However, the function of IL-22 in the pathogenesis of RA remains controversial. In this study, we investigated the role of IL-22 in the early phase of antigen-induced arthritis (AIA) in mice.MethodsAIA was induced in C57BL/6, IL-22^−/−, ASC^−/− and IL-1R1^−/− immunized mice challenged intra-articularly with methylated bovine serum albumin (mBSA). Expression of IL-22 in synovial membranes was determined by RT-PCR. Articular hypernociception was evaluated using an electronic von Frey. Neutrophil recruitment and histopathological analyses were assessed in inflamed knee joint. Joint levels of inflammatory mediators and mBSA-specific IgG concentration in the serum were measured by ELISA.ResultsThe IL-22 mRNA expression and protein levels in synovial tissue were increased during the onset of AIA. In addition, pharmacological inhibition (anti-IL-22 antibody) and genetic deficiency (IL-22^−/− mice) reduced articular pain and neutrophil migration in arthritic mice. Consistent with these findings, recombinant IL-22 joint administration promoted articular inflammation per se in WT mice, restoring joint nociception and neutrophil infiltration in IL-22^−/− mice. Moreover, IL-22-deficient mice showed reduced synovitis (inflammatory cell influx) and lower joint IL-1β levels, whereas the production of IL-17, MCP-1/CCL2, and KC/CXCL1 and the humoral immune response were similar, compared with WT mice. Corroborating these results, the exogenous administration of IL-22 into the joints induced IL-1β production in WT mice and reestablished IL-1β production in IL-22^−/− mice challenged with mBSA. Additionally, IL-1R1^−/− mice showed attenuated inflammatory features induced by mBSA or IL-22 challenge. Articular nociception and neutrophil migration induced by IL-22 were also reduced in ASC^−/− mice.ConclusionsThese results suggest that IL-22 plays a pro-inflammatory/pathogenic role in the onset of AIA through an ASC-dependent stimulation of IL-1β production.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0759-2) contains supplementary material, which is available to authorized users.     

Causes of the production of multiple forms of β-galactosidase by Bacillus circulans

The presence of multiple types of β-galactosidases in a commercial enzyme preparation from Bacillus circulans ATCC 31382 and differences in their transgalactosylation activity were investigated. Four β-galactosidases, β-Gal-A, β-Gal-B, β-Gal-C, and β-Gal-D, which were immunologically homologous, were isolated and characterized. The N-terminal amino acid sequences of all of the enzymes were identical and biochemical characteristics were similar, except for galactooligosaccharide production. β-Gal-B, β-Gal-C, and β-Gal-D produced mainly tri- and tetra saccharides at maximum yields of 20-30 and 9-12%, while β-Gal-A produced trisaccharide with 7% with 5% lactose as substrate. The Lineweaver-Burk plots for all of the enzymes, except for β-Gal-A, showed biphasic behavior. β-Gal-A was truncated to yield multiple β-galactosidases by treatment with protease isolated from the culture broth of B. circulans. Treatment of β-Gal-A with trypsin yielded an active 91-kDa protein composed of 21-kDa and 70-kDa proteins with characteristics similar to those for β-Gal-D.     

Chaperone-aided expression of LipA and LplA followed by the increase in α-lipoic acid production

α-Lipoic acid (LA), a naturally occurring cofactor reported to be present in a diverse group of microorganisms, plants, and animal tissues, has been widely and successfully used as a therapy for a variety of diseases, including diabetes and heart disease. However, to date, recombinant DNA technology has not been applied for higher LA production due mainly to difficulties in the functional expression of key enzymes involved in LA production. Here, we report a study for higher LA production with the aid of chaperone plasmids, DnaKJE and trigger factor (Tf). The lipA and lplA genes encoding lipoate synthase and lipoate protein ligase in Pseudomonas fluorescens, respectively, were cloned and transformed into Escherichia coli K12. When they were overexpressed in E. coli, both LipA and LplA were expressed as inclusion bodies leading to no increase in LA production. However, when chaperone plasmids DnaKJE and Tf were coexpressed with lipA and lplA, the resulting recombinant E. coli strains showed higher LA production than the wild-type E. coli by 32–111%, respectively.     

Roles of N-acetylglucosaminyltransferase III in epithelial-to-mesenchymal transition induced by transforming growth factor β1 (TGF-β1) in epithelial cell lines

The epithelial-to-mesenchymal transition (EMT) plays crucial roles in embryonic development, wound healing, tissue repair, and cancer progression. Results of this study show how transforming growth factor β1 (TGF-β1) down-regulates expression of N-acetylglucosaminyltransferase III (GnT-III) during EMT-like changes. Treatment with TGF-β1 resulted in a decrease in E-cadherin expression and GnT-III expression, as well as its product, the bisected N-glycans, which was confirmed by erythro-agglutinating phytohemagglutinin lectin blot and HPLC analysis in human MCF-10A and mouse GE11 cells. In contrast with GnT-III, the expression of N-acetylglucosaminyltransferase V was slightly enhanced by TGF-β1 treatment. Changes in the N-glycan patterns on α3β1 integrin, one of the target proteins for GnT-III, were also confirmed by lectin blot analysis. To understand the roles of GnT-III expression in EMT-like changes, the MCF-10A cell was stably transfected with GnT-III. It is of particular interest that overexpression of GnT-III influenced EMT-like changes induced by TGF-β1, which was confirmed by cell morphological changes of phase contrast, immunochemical staining patterns of E-cadherin, and actin. In addition, GnT-III modified E-cadherin, which served to prolong E-cadherin turnover on the cell surface examined by biotinylation and pulse-chase experiments. GnT-III expression consistently inhibited β-catenin translocation from cell-cell contact into the cytoplasm and nucleus. Furthermore, the transwell assay showed that GnT-III expression suppressed TGF-β1-induced cell motility. Taken together, these observations are the first to clearly demonstrate that GnT-III affects cell properties, which in turn influence EMT-like changes, and to explain a molecular mechanism for the inhibitory effects of GnT-III on cancer metastasis.