Summary
Currently, piperazic acid is chemically synthesized using ecologically unfriendly processes. Microbial synthesis from glucose is an attractive alternative to chemical synthesis. In this ...study, we report the production of L‐piperazic acid via microbial fermentation with the first engineered fungal strain of Aureobasidium melanogenum; this strain was constructed by chassis development, genetic element reconstitution and optimization, synthetic rewiring and constitutive genetic circuit reconstitution, to build a robust L‐piperazic acid synthetic cascade. These genetic modifications enable A. melanogenum to directly convert glucose to L‐piperazic acid without relying on the use of either chemically synthesized precursors or harsh conditions. This bio‐based process overcomes the shortcomings of the conventional synthesis routes. The ultimately engineered strain is a very high‐efficient cell factory that can excrete 1.12 ± 0.05 g l‐1 of L‐piperazic acid after a 120‐h 10.0‐l fed‐batch fermentation; this is the highest titre of L‐piperazic acid reported using a microbial cell factory.
L‐piperazic acid via microbial fermentation with the first engineered fungal strain of Aureobasidium melanogenum was achieved. This strain was constructed by chassis development, genetic element reconstitution and optimization, synthetic rewiring, and constitutive genetic circuit reconstitution to build a robust L‐piperazic acid synthetic cascade. The ultimately engineered strain has the highest titer of L‐piperazic acid of 1.12 ± 0.05 g l‐1 ever reported.
•Three fermentation strategies for PMA production from malt syrup were studied.•High PMA titer 124.07 g/L was obtained by fed-batch fermentation.•PMA yield 1.10 g/g and productivity 0.70 g/L·h in ...repeated-batch fermentation.•PMA production cost is $ 1.716/kg by fed-batch fermentation at 2000 MT plant.•This provides an economical strategy for large-scale PMA production.
Malt syrup, as a low-cost substrate without any pretreatment, was proved to be able to replace maltose for ploymalic acid (PMA) production by Aureobasidium melanogenum GXZ-6. The PMA titer of 55.53 ± 1.72 g/L was obtained by batch fermentation in a 10-L fermentor with addition of malate, citrate and sodium malonate. Then, a higher PMA titer of 124.07 ± 2.28 g/L was obtained in fed-batch fermentation, which increased by 123.43% than that from batch fermentation. Moreover, repeated-batch fermentation with three batches gave a PMA titer of 64.06 g/L on average with a higher yield of 0.81 g/g and productivity of 0.56 g/L·h. Fermentation process and economics analysis were performed by SuperPro Designer for a 2000 metric tons plant. Results showed that PMA production cost was as low as $ 1.716/kg by fed-batch fermentation, which provides an economical strategy for large-scale PMA production.
Tannins biodegradation by a microorganism is one of the most efficient ways to produce bioproducts of high value. However, the mechanism of tannins biodegradation by yeast has been little explored. ...In this study,
T9 isolated from red wine starter showed the ability for tannins degradation and had its highest biomass when the initial tannic acid concentration was 20 g/L. Furthermore, the genes involved in the tannin degradation process were analyzed. Genes
,
and
encoding three different tannases respectively were identified in the
T9. Among these genes,
and
can be induced by tannin acid simultaneously at both gene transcription and protein expression levels. Our assay result showed that the deletion of
and
resulted in tannase activity decline with 51.3 ± 4.1 and 64.1 ± 1.9 U/mL, respectively, which is much lower than that of
T9 with 91.3 ± 5.8 U/mL. In addition, another gene coding gallic acid decarboxylase (
) was knocked out to better clarify its function. Mutant
completely lost gallic acid decarboxylase activity and no pyrogallic acid was seen during the entire cultivation process, confirming that there was a sole gene encoding decarboxylase in the
T9. These results demonstrated that
,
and
were crucial for tannin degradation and provided new insights for the mechanism of tannins biodegradation by yeast. This finding showed that
has potential in the production of tannase and metabolites, such as gall acid and pyrogallol.
A novel yeast strain Aureobasidium melanogenum 13-2 isolated from the Taklimakan desert was found to be able to produce a high level of extracellular polysaccharide (EPS). Under the optimal ...conditions, the yeast strain could yield 73.25 ± 2.3 g/L of EPS within 5 days at a flask level. During a 10-liter fermentation, the yeast strain could produce 78.05 ± 3.5 g/L of EPS within 120 h. The FT-IR spectra of the standard pullulan from Sigma and the purified EPS produced by A. melanogenum 13-2 were almost identical and the purified EPS could be actively hydrolyzed by a pullulanase, demonstrating that the purified EPS was pullulan. The molecular weight (Mw) of the purified pullulan was estimated to be 7.703 × 105 g/moL. Disruption of a pullulan synthase gene (PUL1) made a mutant DAG27 lose the ability to synthesize any pullulan. The mutant DAG27 was more sensitive to radiation of UV light, high NaCl concentration, heat treatment, strong oxidation of H2O2 and desiccation than its wild type strain 13-2, suggesting that the produced pullulan could play an important role in resistance of the yeast cells to various stresses. This was the first time to show that the yeast strain obtained from the desert could produce such high level pullulan and the produced pullulan had an obviously protective effect on its producer.
•A novel yeast strain 13-2 produced 78.05 ± 3.5 g/L of pullulan.•Disruption of a pullulan synthase gene (PUL1) made a mutant DAG27 synthesize no pullulan.•The produced pullulan played an important role in resistance of the yeast cells to various stresses.
Melanin plays an important role in the stress adaptation of
Aureobasidium melanogenum
XJ5-1 isolated from the Taklimakan desert. A trehalose-6-phosphate synthase gene (
TPS1
gene) was cloned from K5, ...characterized, and then deleted to determine the role of trehalose in the stress adaptation of the albino mutant K5. No stress response element and heat shock element were found in the promoter of the
TPS1
gene. Deletion of the
TPS1
gene in the albino mutant rendered a strain DT43 unable to synthesize any trehalose, but DT43 still could grow in glucose, suggesting that its hexokinase was insensitive to inhibition by trehalose-6-phosphate. Overexpression of the
TPS1
gene enhanced trehalose biosynthesis in strain ET6. DT43 could not grow at 33 °C, whereas K5, ET6, and XJ5-1 could grow well at this temperature. Compared with K5 and ET6, DT43 was highly sensitive to heat shock treatment, high oxidation, and high desiccation, but all the three strains demonstrated the same sensitivity to UV light and high NaCl concentration. Therefore, trehalose played an important role in the adaptation of K5 to heat shock treatment, high oxidation, and high desiccation.
Summary
Aureobasidium pullulans and A. melanogenum are black‐yeast‐like surface colonisers and are commonly encountered as contaminants in the hospital. The species are able to produce melanin which ...play a role in protection against environmental stress and irradiation. Aureobasidium melanogenum shows higher frequency in opportunistic infections compared to A. pullulans. Comparative pathogenicity of opportunistic black yeasts between Aureobasidium pullulans and A. melanogenum to explain the observed differences in frequency in infection. Degrees of melanisation and thermotolerance were measured, and virulence of strains from different sources was examined in Galleria mellonela and murine infection models. Aureobasidium melanogenum responds with increased melanisation to temperature stress and generally survives at 37°C, A. pullulans on average scored less on these parameters. In the murine model, differences between species were not significant, but the melanised A. melanogenum group showed the highest virulence. This result was not reproducible in Galleria mellonella larvae at 25°C. The A. melanogenum black group showed higher pathogenicity in murine model, indicating that the combination of melanisation and thermotolerance rather than species affiliation is instrumental. Galleria larvae did not survive very well at 37°C, and hence, this model is judged insufficient to detect the small virulence differences observed in Aureobasidium.
The widespread use of shared bicycles has increased the demand and sanitary requirements for shared bicycles. Previous studies have identified potentially pathogenic bacteria on the surfaces of ...shared bicycles, but fungal communities have not been investigated.
We sampled shared-bicycle handles and saddles from five selected locations in a metropolis (Chengdu, China, n = 98) and used surrounding air deposition samples as controls (n = 12). Full-length ITS sequencing and multiple bioinformatic analyses were utilized to reveal fungal community structures and differences.
Aspergillus was dominant on both the handles and saddles of shared bicycles, and Alternaria and Cladosporium were the most abundant families in the air samples. Significant differences in fungal community structures were found among the three groups. The handle samples contained higher abundances of Aureobasidium melanogenum and Filobasidium magnum than the saddle and air samples. The saddle samples had a higher abundance of Cladosporium tenuissimum than the other two sample types (P < 0·05). A higher abundance of fungal animal pathogens on shared-bicycle surfaces than in air by FUNGuild (P < 0·05). Moreover, the co-occurrence network of fungi on handles was more stable than that on saddles.
There were more potential pathogens, including Aspergillus pseudoglaucus, Aureobasidium melanogenum, Kazachstania pintolopesii, Filobasidium magnum, Candida tropicalis, and Malassezia globose were found on shared bicycles than in air, suggesting that hands should not contact mucous membrane after cycling, especially in susceptible individuals, and hygiene management of shared bicycles should be given more attention by relevant organizations worldwide.
Aureobasidium melanogenum
P16 is a high pullulan-producing yeast. However, glucose repression on its pullulan biosynthesis must be relieved. After the gene encoding a glucose repressor was cloned, ...characterized and analyzed, it was found that the repressor belonged to one member of the CreA in filamentous fungi, not to one member of the Mig1 in yeasts. After the
CREA
gene was fully removed from the yeast strain P16, the glucose repression in the disruptant DG41 was relieved. At the same time, the pullulan production by the disruptant DG41 was enhanced compared to that by its wild-type strain P16, and the transcriptional levels of the gene encoding a glucosyltransferase, three genes encoding glucose transporters, the gene encoding a 6-P-glucose kinase and the genes encoding α-amylase, glucoamylase and pullulanase in the disruptant DG41 were also promoted. However, the transcriptional levels of the genes encoding the CreA and another two glucose transporters were greatly reduced. During the 10-liter fermentation, the disruptant DG41 produced 64.93 ± 1.33 g/l pullulan from 120 g/l of glucose, while its wild-type strain P16 produced only 52.0 ± 1.95 g/l pullulan within 132 h. After the
CREA
gene was complemented in the disruptant D373, the pullulan production by the transformant BC4 was greatly reduced compared to that by its wild-type strain P16, and the transcriptional levels of the many genes in the transformant BC4 were also decreased. All the results confirmed that the CreA played an important role in the regulation of pullulan biosynthesis in
A. melanogenum
P16, and that glucose derepression on pullulan biosynthesis could improve pullulan production from glucose. This study opened the possibility for improving the industrial production of this exopolysaccharide by genetic engineering.
The melanin produced by
Aureobasidium melanogenum
XJ5-1 obtained from the Taklimakan Desert can play an important role in adaptation of the yeast strain to various stress treatments. It is very ...important to know how the desert-derived yeast sense, respond and adapt to the harsh environments. However, it is still unclear how melanin is genetically controlled by signaling pathways and transcriptional factors. In this study, it was found that the mitogen-activated protein kinase (MAPK) Slt2 in the cell wall integrity (CWI) signal pathway could regulate activity of the transcriptional activator Swi4; in turn, the Swi4 could control the expression of the
CMR1
gene. The melanin-specific transcriptional activator Cmr1 encoded by the
CMR1
gene was specifically bound to the promoter with the sequence TTCTCTCCA of the
PKS1
gene and strongly stimulated expression of the
PKS1
gene and any other genes responsible for melanin biosynthesis, so that a large amount of melanin could be produced by
A. melanogenum
XJ5-1. Therefore, melanin biosynthesis in the desert-derived
A. melanogenum
XJ5-1 was controlled mainly by the CWI signal pathway among the cell wall-related signal pathways via a transcriptional activator Cmr and regulation of the melanin biosynthesis in
A. melanogenum
XJ5-1 was completely different from that of the melanin biosynthesis in any other fungi. This is the first time to show that melanin biosynthesis in the desert-derived
A. melanogenum
XJ5-1 is controlled mainly by the CWI signal pathway via a transcriptional activator Cmr1. This would provide the fundamentals for further research on the desert-derived yeast to sense, respond and adapt to the harsh environments.
The black yeast-like fungus Aureobasidium melanogenum is an opportunistic human pathogen frequently found indoors. Its traits, potentially linked to pathogenesis, have never been systematically ...studied. Here, we examine 49 A. melanogenum strains for growth at 37 °C, siderophore production, hemolytic activity, and assimilation of hydrocarbons and human neurotransmitters and report within-species variability. All but one strain grew at 37 °C. All strains produced siderophores and showed some hemolytic activity. The largest differences between strains were observed in the assimilation of hydrocarbons and human neurotransmitters. We show for the first time that fungi from the order Dothideales can assimilate aromatic hydrocarbons. To explain the background, we sequenced the genomes of all 49 strains and identified genes putatively involved in siderophore production and hemolysis. Genomic analysis revealed a fairly structured population of A.melanogenum, raising the possibility that some phylogenetic lineages have higher virulence potential than others. Population genomics indicated that the species is strictly clonal, although more than half of the genomes were diploid. The existence of relatively heterozygous diploids in an otherwise clonal species is described for only the second time in fungi. The genomic and phenotypic data from this study should help to resolve the non-trivial taxonomy of the genus Aureobasidium and reduce the medical hazards of exploiting the biotechnological potential of other, non-pathogenic species of this genus.
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