Aureobasidium melanogenum 11–1 was found to be able to produce over 281.7 ± 7.1 U/mL of β-fructofuranosidase activity. The protein deduced from the cloned β-fructofuranosidase1 gene had the conserved ...motif A (IGDP), motif D (RDP) and motif E (ET) and 11 N-glycosylation sites, indicating it was a β-fructofuranosidase with the high-level fructooligosaccharide (FOS) biosynthesis. Overexpression of the β-fructofuranosidase1 gene in the yeast strain 11–1 made a tranformant 33 produce 557.7 U/mL of β-fructofuranosidase activity. The molecular weight of the β-fructofuranosidase1 in which all the carbohydrates were removed by the Endo-H was 82.4 kDa. Within 7 h of the transfructosylation reaction, the yield of FOS was 0.66 g of FOS/g of sucrose and percentages of GF2, GF3 and GF4 were 79.5%, 18.9% and 1.6%. This demonstrated that the β-fructofuranosidase1 and the transformant 33 had highly potential applications in biotechnology for FOS production.
•The gene encoding β-fructofuranosidase1 with the high-level fructooligosaccharide (FOS) biosynthesis was overexpressed.•557.7 U/mL of β-fructofuranosidase was produced within 60 h.•The yield of FOS was 0.66 g of FOS/g of sucrose and most of the FOSs were GF2 (79.5%) and GF3 (18.9%).
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.
•The NSDD genes from different strains of A. melanogenum were characterized.•NsdD is a positive regulator for pullulan and polymarate biosynthesis.•However, NsdD is a negative regulator for melanin biosynthesis.•The negative regulation mode by NsdD was proposed.
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,
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.
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, MgSO
4
·7H
2
O, 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 H
2
O
2
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.
It has been thought that when different strains of Aureobasidium spp. were grown in sucrose, the produced fructooligosaccharides (FOSs) by β-d-fructofuranosidase were beneficial for their cell growth ...and pullulan biosynthesis. However, it is still unknown about how β-d-fructofuranosidases activity and synthesized FOSs influence on pullulan biosynthesis. It was found that the genomic DNA of Aureobasidium melanogenum P16, a high pullulan producing yeast, contained three genes encoding β-d-fructofuranosidase1, β-d-fructofuranosidase2 and β-d-fructofuranosidase3. The FTR1 factor, a transcriptional activator, activated expression of the three β-d-fructofuranosidase genes and invertase gene. Disruption of the FTR1 gene rendered a disruptant DF3 to produce less FOSs (12.1 ± 0.4 g/L), less β-d-fructofuranosidase activity (1.1 ± 0.2 U/mL), lower Mw (3.8 × 105) of the pullulan and more pullulan titer (77.0 ± 2.6 g/L) than the yeast strain P16. Similarly, removal of both the two genes encoding β-d-fructofuranosidase1 and β-d-fructofuranosidase3 resulted in a double mutant DF4–7 producing 77.5 ± 3.1 g/L pullulan with Mw of 3.4 × 105, 0.2 ± 0.0 U/mL of β-d-fructofuranosidase activity and the trace amount of FOSs while its wild type strain P16 yielded 65.7 ± 3.5 g/L pullulan with Mw of 4.4 × 105, 6.8 ± 0.0 U/mL of β-d-fructofuranosidase activity and 6.2 ± 0.5 g/L of FOSs. These confirmed that high β-d-fructofuranosidase activity, the presence of high level of FOSs negatively influenced pullulan biosynthesis, but positively increased Mw of the produced pullulan. However, the β-d-fructofuranosidase2 had no such function. Furthermore, complementation of the FTR1 gene, β-d-fructofuranosidase1 gene and β-d-fructofuranosidase3 gene enabled the corresponding transformants to restore β-d-fructofuranosidase activity, FOSs and pullulan biosynthesis and Mw of the pullulan.
•High β-d-fructofuranosidase activity and the presence of high-level FOSs negatively influenced pullulan biosynthesis•The presence of high-level FOSs positively increased Mw (4.4 × 105) of the produced pullulan•β-d-fructofuranosidase and invertase were induced by a transcriptional activator.
Aureobasidium melanogenum P16, the high pullulan producer, had only one GATA type transcriptional activator AreA and one GATA type transcriptional repressor AreB. It was found that 2.4 g/L of ...(NH4)2SO4 had obvious nitrogen repression on pullulan biosynthesis by A. melanogenum P16. Removal of the AreB gene could make the disruptant DA6 produce 34.8 g/L pullulan while the P16 strain only produced 28.8 g/L pullulan at the efficient nitrogen condition. Further both removal of the native AreA gene and overexpression of the mutated AreAS628-S678 gene with non-phosphorylatable residues could render the transformant DEA12 to produce 39.8 g/L pullulan. The transcriptional levels of most of the genes related to pullulan biosynthesis in the transformant DEA12 were greatly enhanced. The mutated AreAS628-S678 was localized in the nuclei of the transformant DEA12 while the native AreA was distributed in the cytoplasm in A. melanogenum P16. This meant that nitrogen repression on pullulan biosynthesis in the transformant DEA12 was indeed significantly relieved. This was the first time to report that the GATA type transcriptional factors of nitrogen catabolite repression system could regulate pullulan biosynthesis in Aureobasidium spp.
•The GATA type transcriptional activator AreA and the transcriptional repressor AreB were removed.•The mutated AreA gene with nonphosphorylatable residues was overexpressed.•The obtained derepressed mutant could produce 39.8 g/L pullulan.•The mutated AreA was only localized in the nuclei of the derepressed mutant.
Poly (β-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries. In this study, electrolytic stimulation assisted PMLA production was ...developed. Firstly, it was found that the pentavalent nitrogen source (i.e., NO3−) was more suitable for PMLA production. Secondly, a usual single-chamber bioelectric-fermentation system (BES) cannot improve PMLA production, which can only promote cell growth. Then, a new single-chamber BES with an external circulation was developed, where the PMLA metabolism was further intensified. Finally, the integration of NO3− addition and electrolytic stimulation mode (c) showed a positive synergy on the PMLA production. Compared to the case without NO3− addition and electrolytic stimulation, the PMLA production was increased by 22.9 % using the integrated process. Moreover, compared to the case without the electrolytic stimulation mode (c), it was revealed that the different genes involved in 12 metabolic subsystems using the integrated process, where 31 and 177 genes were up-regulated and down-regulated, respectively. The up-regulated genes were mainly participated in melanin metabolic process, catalase activity, and oxidoreductase activity. Hence, the integration of electrolytic stimulation represents a novel approach to improve PMLA production.
•A novel single-chamber BES with an external circulation was developed.•An oxidation state inorganic nitrogen source was more suitable for the PMLA production.•Electrolytic stimulation promotes PMLA production from A. melanogenum ipe-1.•The integration of NO3− addition and electrolytic stimulation showed positive synergy.
Biosynthesis enhancement of liamocins and intracellular lipids by overexpressing PYC1 gene.
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•A transformant M39 over-expressing the PYC1 gene could produce 43.04 ± 0.4 g/l of ...liamocin, and 23. 8 ± 0.2% (w/w) of intracellular lipids.•The alkaline hydrolysis products of the liamocin contained mainly 5-hydroxy-2-decenoic acid lactone;.•The intracellular lipids contained mainly C16:0, C16:1, C18:0, C18:1 and C18:2 fatty acids.
The pyruvate carboxylase-encoding PYC1 gene from the yeast strain Aureobasidium sp. P6, which is a polymalate over-producer, was actively overexpressed in the marine-derived yeast Aureobasidium melanogenum 9-1, which is a heavy oil (liamocin) producer. The resulting PYC1-over-expressing transformant M39 produced 35.3 ± 1.2 g/l liamocins and 24.6 ± 0.5% (w/w) intracellular oil, and its cell dry weight was 17.0 ± 1.6 g/l. On the other hand, wild-type strain 9-1 only produced 27.4 ± 0.3 g/l liamocins and 22.6 ± 0.8% (w/w) intracellular oil and had a cell dry weight of 15.3 ± 2.0 g/l within 168 h. The results suggested that the over-expression of the PYC1 gene enhanced the biosynthesis of liamocins and intracellular lipids in transformant M39 due to the enhanced biosynthesis of citric acid, which could be the precursor for the biosynthesis of the intracellular lipids and extracellular liamocins. During fermentation, 43.04 ± 1.2 g/l of extracellular liamocins, 17.7 ± 0.5 g/l of cell dry weight and 23.8 ± 0.2 g/l of intracellular lipids were produced by transformant M39 within 156 h, leaving 2.1% reducing sugar in the fermented medium. Moreover, the strong alkaline hydrolysis products of the extracellular liamocins mainly contained 5-hydroxy-2-decenoic acid lactone (92.1 ± 0.3%), hexadecanoic acids (C16:0, 3.3 ± 0.04%), octadecadienoic acids (C18:2, 1.0 ± 0.2%) and octadecanoic acids (C18:0, 3.6 ± 0.01%), while the intracellular lipids mainly contained C16:0, C16:1, C18:0, C18:1 and C18:2 fatty acids.
Abstract
Background
The great diversity of lifestyles and survival strategies observed in fungi is reflected in the many ways in which they reproduce and recombine. Although a complete absence of ...recombination is rare, it has been reported for some species, among them 2 extremotolerant black yeasts from Dothideomycetes: Hortaea werneckii and Aureobasidium melanogenum. Therefore, the presence of diploid strains in these species cannot be explained as the product of conventional sexual reproduction.
Results
Genome sequencing revealed that the ratio of diploid to haploid strains in both H. werneckii and A. melanogenum is about 2:1. Linkage disequilibrium between pairs of polymorphic loci and a high degree of concordance between the phylogenies of different genomic regions confirmed that both species are clonal. Heterozygosity of diploid strains is high, with several hybridizing genome pairs reaching the intergenomic distances typically seen between different fungal species. The origin of diploid strains collected worldwide can be traced to a handful of hybridization events that produced diploids, which were stable over long periods of time and distributed over large geographic areas.
Conclusions
Our results, based on the genomes of over 100 strains of 2 black yeasts, show that although they are clonal, they occasionally form stable and highly heterozygous diploid intraspecific hybrids. The mechanism of these apparently rare hybridization events, which are not followed by meiosis or haploidization, remains unknown. Both extremotolerant yeasts, H. werneckii and even more so A. melanogenum, a close relative of the intensely recombining and biotechnologically relevant Aureobasidium pullulans, provide an attractive model for studying the role of clonality and ploidy in extremotolerant fungi.
Marine fungi represent an important and sustainable resource, from which the search for novel biological substances for application in the pharmacy or food industry offers great potential. In our ...research, novel polysaccharide (AUM-1) was obtained from marine
SCAU-266 were obtained and the molecular weight of AUM-1 was determined to be 8000 Da with 97.30% of glucose, 1.9% of mannose, and 0.08% galactose, owing to a potential backbone of α-D-Glcp-(1→2)-α-D-Manp-(1→4)-α-D-Glcp-(1→6)-(SO
)-4-α-D-Glcp-(1→6)-1-β-D-Glcp-1→2)-α-D-Glcp-(1→6)-β-D-Glcp-1→6)-α-D-Glcp-1→4)-α-D-Glcp-6→1)-α-D-Glcp-4
→1)-α-D-Glcp and two side chains that consisted of α-D-Glcp-1 and α-D-Glcp-(1→6)-α-D-Glcp residues. The immunomodulatory effect of AUM-1 was identified. Then, the potential molecular mechanism by which AUM-1 may be connected to ferroptosis was indicated by metabonomics, and the expression of COX2, SLC7A11, GPX4, ACSL4, FTH1, and ROS were further verified. Thus, we first speculated that AUM-1 has a potential effect on the ferroptosis-related immunomodulatory property in RAW 264.7 cells by adjusting the expression of GPX4, regulated glutathione (oxidative), directly causing lipid peroxidation owing to the higher ROS level through the glutamate metabolism and TCA cycle. Thus, the ferroptosis related immunomodulatory effect of AUM-1 was obtained.
