Abstract
Industrial fungi need a strong environmental stress tolerance to ensure acceptable efficiency and yields. Previous studies shed light on the important role that Aspergillus nidulans gfdB, putatively encoding a NAD+-dependent glycerol-3-phosphate dehydrogenase, plays in the oxidative and cell wall integrity stress tolerance of this filamentous fungus model organism. The insertion of A. nidulans gfdB into the genome of Aspergillus glaucus strengthened the environmental stress tolerance of this xerophilic/osmophilic fungus, which may facilitate the involvement of this fungus in various industrial and environmental biotechnological processes. On the other hand, the transfer of A. nidulans gfdB to Aspergillus wentii, another promising industrial xerophilic/osmophilic fungus, resulted only in minor and sporadic improvement in environmental stress tolerance and meanwhile partially reversed osmophily. Because A. glaucus and A. wentii are phylogenetically closely related species and both fungi lack a gfdB ortholog, these results warn us that any disturbance of the stress response system of the aspergilli may elicit rather complex and even unforeseeable, species-specific physiological changes. This should be taken into consideration in any future targeted industrial strain development projects aiming at the fortification of the general stress tolerance of these fungi.
Key points
• A. wentii c’ gfdB strains showed minor and sporadic stress tolerance phenotypes.
• The osmophily of A. wentii significantly decreased in the c’ gfdB strains.
• Insertion of gfdB caused species-specific phenotypes in A. wentii and A. glaucus.
Introduction
Many industrial microbial biotechnological processes take advantage of the remarkable diversity, productivity, technological applicability, and high endurance of the ascomycete fungi belonging to the genus Aspergillus (de Vries et al. 2017; Park et al. 2017; Cairns et al. 2021; Huang et al. 2021; Jin et al. 2022). For example, the xerophilic/osmophilic Aspergillus wentii (Wheeler and Hocking 1993; de Lima Alves et al. 2015; de Vries et al. 2017) is a good enzyme producer (Sinha and Chakrabarty 1978; Chander et al. 1980; Gross et al. 1984; Lago et al. 2021) and can be a workhorse in future biodiesel transesterification processes as well (Shoaib et al. 2018).
Based on previous hyperosmotic stress studies, osmophily is widespread in the aspergilli and the growth stimulatory effect of either 2.0 M sorbitol or 1.0 M NaCl was the most significant for A. wentii and Aspergillus glaucus (de Vries et al. 2017). In these fungi, osmophily was hypothesized to be connected to the lack of the gfdB gene, encoding a putative NAD+-dependent glycerol-3-phosphate dehydrogenase enzyme in many Aspergillus spp., including the filamentous fungus model organism Aspergillus nidulans (Miskei et al. 2009; Balázs et al. 2010; de Vries et al. 2017). Importantly, the xerophilic/osmophilic A. glaucus may also find its industrial fermentation applications in enzyme (Tao et al., 2010, 2011; Abrashev et al. 2016; Li et al. 2018; Takenaka et al. 2019) and aspergiolide A (an anticancer-polyketide) (Cai et al. 2009, 2014; Sun et al. 2009; Wu et al. 2017) production, and A. nidulans is also a well-known enzyme producer fungus and represents a potential platform for heterologous protein expression (MacCabe et al. 2002; Kumar 2020; Lopes et al. 2021; Jin et al. 2022). It is noteworthy that A. glaucus, which has a remarkably high abiotic stress resistance, is also considered a suitable tool in saline-alkaline remediation technologies (Wei and Zhang 2018; Zhou et al. 2021), in the biomineralization of the insecticide fipronil (Gajendiran and Abraham 2017), and in the hydrolysis of sugar cane bagasse (Tao et al. 2010). A. glaucus stress tolerance genes transferred into other organisms can enhance the osmotic stress tolerance of recipient fungi and plants (Liu et al. 2014, 2015).
Unexpectedly, the deletion of gfdB resulted in a decreased oxidative and cell wall integrity stress tolerance in A. nidulans (Király et al. 2020a) although its expression was only responsive to 0.6 M NaCl exposure and not to oxidative stress (Balázs et al. 2010). Nevertheless, the hyphal-morphology of the A. nidulans ΔgfdA mutant strain was altered, and its growth was also attenuated at various carbon sources except glycerol (Fillinger et al. 2001). This defective growth was recoverable on all carbon sources in the presence of 1 M NaCl, which functioned as an osmotic stabilizer for the mutant strain. Furthermore, the mutant strain was also sensitive to cell wall integrity stress (Fillinger et al. 2001).
In A. glaucus, the implementation and expression of A. nidulans gfdB with its own promoter increased considerably the oxidative, cell wall integrity, and heavy metal (Cd2+) stress tolerance of the fungus cultured on 2 M sorbitol without affecting its osmophily (Király et al. 2020b). Because the adequate stress tolerance of industrial yeasts and filamentous fungi is of paramount importance in fermentation processes (Bai et al. 2003; Li et al. 2011; Teixeira et al. 2011; Hagiwara et al. 2016; Deparis et al. 2017; Steensels et al. 2019; Brandt et al. 2021; Yaakoub et al. 2022), we wanted to test if the genetic transfer of A. nidulans gfdB to A. wentii with its own promoter would also enhace the general stress tolerance of this fungus.
The outcomes of this study supported the original hypothesis of Miskei et al. (2009) on the possible involvement of GfdB in the control of xerophilic/osmophilic phenotypes, and not in the environmental stress tolerance of the aspergilli. The significance of the appearant species-specific physiological functions of GfdB has also been discussed here together with the implications for the future development of stress-tolerant industrial fungal strains.
Materials and methods
Fungal strains and culture conditions
The list of strains used in this study is presented in Table 1. A. wentii and A. glaucus conidia were produced on Malt Extract Agar (MEA) (1.5 % agar, 25 °C in the dark, 6 days), and all sporulation media were supplemented with 1.0 M NaCl in the case of A. glaucus (de Vries et al. 2017; Emri et al. 2018; Orosz et al. 2018). A. nidulans strains were sporulated on Barratt's nitrate minimal medium (NMM) under standard conditions (1.5 % agar, 25 °C in the dark, 6 days; (Barratt et al. 1965). Conidiospores were scraped in sterile water containing 9 g L-1 NaCl and 100 μL L-1 TWEEN-80, passed through two layers of Miracloth (Merck-Millipore, Burlington, MA, USA) and then quantified using a hemocytometer. All strains were grown on NMM nutrient agar plates under the culture conditions indicated and were stored in conidiospore suspension stocks prepared in 50 % glycerol and were kept at -75 oC (Szabó et al. 2020a,b).
Generation of the A. nidulans gfdB complemented strains of A. wentii
The plasmid pAN7.1 (Punt et al. 1987; full sequence map is available at https://www.addgene.org/168129/) containing the hygromycin B resistance gene was used to transform A. wentii protoplasts with the A. nidulans gfdB (locus ID: AN6792) gene with its native promoter and terminator sequences (for primer pair see Supplemental Table S1), which was cloned to the XbaI-HindIII site. Protoplasts were generated from exponential growth phase (13-14 h old) submerged cultures of A. wentii grown on complex medium (NMM containing 2% glucose and supplemented with 0.5% yeast extract and 1% peptone) using the lysing enzymes from Trichoderma harzianum (Sigma, St Louis, MO, USA) with the polyethylene glycol (PEG)-mediated transformation method as previously described in the protocol of Szewczyk et al. (2006). We used 106–107 protoplasts in 100 μl suspension and 5–8 μg pAN7.1 plasmid in 10 μl aliquot per transformation. Transformants were regrown from a single conidium on NMM containing 100 μg mL−1 hygromycin at 25 °C after 3–5 days incubation. For the genomic DNA isolation, transformants were incubated in a rotary shaker overnight at 25 °C, 220 rpm in NMM containing 100 μg mL−1 hygromycin. Genomic DNA was isolated from mycelial mat collected by centrifugation (Szabó et al. 2020a). To prove the successful incorporation of the gfdB gene, after genomic DNA isolation, Emerald PCR reactions (EmeraldAmp MAX PCR Master Mix, Takara Bio, San Jose, CA, USA) were carried out with the AN6792 XbaI FW and AN6792 HindIII REV primers (Supplemental Table S1).
Copy number analysis of the gfdB gene using the quantitative polymerase chain reaction (qPCR) method
For the qPCR reaction, we used the Fast SYBR® Green master mix (Applied Biosystems by Life Technologies, Waltham, MA, USA) kit. To determine the copy number of the gfdB gene, a dilution series (320 ng, 160 ng, 80 ng, 40 ng, 20 ng, 10 ng DNA per 7 μl volume) was prepared from the genomic DNA of A. wentii transgenic strains of known concentration. The single copy Aspwe1_39921 gene, encoding the A. nidulans γ-glutamylcysteine synthetase (locus ID: AN3150) ortholog in A. wentii, was used as a copy reference gene. qPCR reactions were carried out in 96 well plates, and the reaction mixtures contained 7 μl of a given concentration of genomic DNA, 10 μl of Fast SYBR® Green master mix, 0.4 μl of forward primer, 0.4 μl of reverse primer, and 2.2 μl of nuclease-free water. Three parallel measurements were performed with each primer pair at each genomic DNA concentration (for the complete list of primer pairs see Supplemental Table S1) on a LightCycler®480 equipment (Roche, Basel, Switzerland). PCR cycles were performed according to the following protocol: 1. 95 °C 2 min; 40× cycles: 95 °C 5 s, 51 °C 10 s, 65 °C 20 s; 95 °C 15 s, 51 °C 15 s, 95 °C continuous, 37 °C 1 s (Szabó et al. 2020a).
The copy number of the gfdB gene incorporated in the A. wentii genome was quantified as previously described {(Király et al. 2020a, b; Szabó et al. 2020a, b); using the equation of (Herrera et al. 2009)}.
Determination of gfdB gene expression by quantitative real-time reverse transcription PCR (qRT-PCR) in A. wentii 'c gfdB complemented strains
For the RNA isolation, mycelial samples were collected after 3-day incubation in a rotary shaker (NMM, 220 rpm, 25 °C). Total RNA was isolated from lyophilized mycelia using TRIzol reagent (Chomczynski 1993), and qRT-PCR experiments were performed as described earlier (Emri et al. 2015) using a Xceed SG 1-step 2× Mix Lo-ROX qPCR Kit (Institute of Applied Biotechnologies, Prague, Czech Republic). In qRT-PCR measurements, 500 ng of total RNA per reaction was added and the reactions were halted after 40 cycles according to the manufacturer’s recommendations. The applied primer pairs are summarized in Supplemental Table S1. Relative transcription levels were quantified with the ΔΔCP method, where ΔCT = CT reference gene − CT gfdB, and CT stands for the qRT-PCR cycle numbers corresponding to the crossing points. Relative transcript levels were also examined using the following reference gene: Aspwe1_38228 (A. fumigatus tef1 ortholog), and these reactions gave similar results (Szabó et al. 2020a).
Stress tolerance studies
Large-scale stress agar plate assays were performed (Balázs et al. 2010) to study and compare the stress sensitivities of the tested reference and mutant strains (Table 1). Following standard stress agar plate protocols routinely used in our laboratory (de Vries et al. 2017, Orosz et al. 2018), 1×105 freshly harvested spores were point-inoculated on Barratt’s NMM agar and were incubated at 25 °C for 5 and 10 days. NMM agar was supplemented with various stress-generating agents as required. For the osmophilic A. wentii and A. glaucus strains, a similar set of stress sensitivity experiments was repeated where NMM agar was also supplemented with 2 M sorbitol in addition to the stressors. The following stress-eliciting agents were employed at the concentrations indicated in parentheses: cell wall integrity stress: Congo Red (54, 81, and 108 μM); oxidative stress: tert-butyl hydroperoxide (tBOOH; 0.4, 1.6, and 2.4 mM), hydrogen peroxide (9 and 18 mM), menadione sodium bisulphite (MSB; 0.096, 0.19, 0.38, 0.62 mM), diamide (1.5 mM); heavy metal stress: CdCl2 (0.1, 0.15, 0.2, and 0.5 mM); hyperosmotic stress (when this tearm is applicable)): sorbitol (2 M), NaCl (0.5, 1, and 1.5 mM). Following stress treatments, the stress sensitivity of the strains was characterized by the diameters of the colonies (Balázs et al. 2010; de Vries et al. 2017; Orosz et al. 2018; Király et al. 2020a,b).
Cluster analysis and multidimensional scaling of stress tolerance
To perform cluter analysis on the general stress sensitivity observed in the genus of the aspergilli, growths of the A. wentii (CBS141173), A. wentii (CBS141173) 'c gfdB1, A. glaucus (CBS516.65), A. glaucus (CBS516.65) 'c gfdB1, A. nidulans THS30.3, and A. nidulans ΔgfdB strains recorded in current and pervious studies (Király et al. 2020a,b) were compared to growth data gained with other Aspergillus spp. and deposited in the Fungal Stress Database (FSD; https://www.fung-stress.org/; de Vries et al. 2017; Orosz et al. 2018; Emri et al. 2018). Other Aspergillus species (15 spp. in total) whose stress sensitivity data are available in the Fungal Stress Database include the following species: A. aculeatus (CBS 172.66), A. brasiliensis (CBS 101740), A. carbonarius (CBS 141172 = DTO 115-B6), A. clavatus (CBS 513.65 = NRRL1), A. fischeri (CBS 544.65), A. flavus (CBS 128202 = NRRL 3357), A. fumigatus (CBS 126847 = Af293), A. luchuensis (CBS 106.47), A. niger (represented by two strains: CBS 113.46 and N402), A. oryzae (Rib40), A. sydowii (CBS 593.65), A. terreus (NIH2624), A. tubingensis (CBS 134.48), and A. versicolor (CBS 795.97).
As described before (Emri et al. 2018), MIC50 values, which were defined as the lowest concentrations of the tested stress initiating agents, causing 50% decreases in colony growth, were calculated for stress agar cultures exposed to H2O2, MSB, and CdCl2 at 25 °C for 5 and 10 days. In the case of NaCl, Congo Red and sorbitol, relative growth values (% of those recorded in unstressed control cultures) measured at 1.0 M, 108 μM, and 2.0 M concentrations, respectively, were taken into consideration on the cluster analyses, which were performed with the R version 4.2.0 software. The values were standardized for comparability, and the data are available in Supplemental Table S2. The “dist,” “hclust,” and “cmdscale” functions of the R Project (www.R-project.org/) were used to calculate Euclidian distances between strains and to generate the cladogram and MDS plot, respectively.
Similarities and differences between the stress tolerance of the A. wentii CBS141173, A. wentii CBS141173 'c gfdB1, 'c gfdB2 and 'c gfdB3, A. glaucus CBS516.65, A. glaucus CBS516.65 'c gfdB1 and 'c gfdB2 as well as A. nidulans THS30.3 and A. nidulans ΔgfdB strains based on the mean colony diameter values recorded under different stress conditions were also visualized with MDS. In this case, colony diameters, measured in the presence of the following stressors: 54 μM Congo Red, 2 M sorbitol, 0.096 mM MSB, 0.4 mM tBOOH, 9 mM H2O2, 1 M NaCl, 0.5 mM CdCl2, were taken into consideration to compare the strains. In the case of the highly osmophilic A. glaucus, all NMM stress agar plates were supplemented with 2 M sorbitol.
Statistical analysis
The effects of stress treatments and gene manipulations on the growth of the A. wentii CBS141173, A. wentii CBS141173 'c gfdB1, 'c gfdB2 and 'c gfdB3, as well as the A. glaucus CBS516.65 and A. glaucus CBS516.65 'c gfdB1 and 'c gfdB2 strains were analyzed by two-way ANOVA followed by Tukey’s post hoc test. The difference between the mean colony diameter values were considered significant if the adjusted p-value was less than 0.05 (Király et al. 2020a,b).
Results
Supplementation of A. wentii with A. nidulans gfdB gene and phenotypic characterization of the A. wentii wild-type and ’c gfdB strains
To construct A. wentii ’c gfdB mutant strains, the pAN7.1 plasmid, containing the A. nidulans gfdB gene with its native promoter and terminator sequences, was introduced in A. wentii. The presence of the gfdB gene was verified with the AN6792 XbaI FW and AN6792 HindIII REV primers (Supplemental Table S1). After the verification of the expected genotypes, the copy number of the incorporated A. nidulans gfdB gene (Table 2) was determined, and the expression of gfdB in A. wentii was demonstrated by qRT-PCR method (Supplemental Fig. S1).
The osmophily and the stress sensitivity phenotypes of the A. wentii 'c gfdB1, 'c gfdB2, and 'c gfdB3 strains (independent transformants) were compared to those of the A. wentii wild-type strain. Remarkably, the supplementation of A. wentii with A. nidulans gfdB partially complemented the osmophily of wild-type A. wentii exposed to 2 M sorbitol or to 0.5 M or 1 M NaCl (Figs. 1 and 2), meanwhile exposure to a higher (1.5 M) NaCl concentration did not result in any osmophily in the wild-type strain and was even inhibitory for the ’c gfdB strains (Fig. 3). A slow growth phenotype was observed for the gfdB supplemented A. wentii mutants in the absence of sorbitol with appr. 16–30% decreases in the colony diameters (Figs. 1, 2, and 3). In the presence of 2M sorbitol, unstressed colony diameters were appr. 28–35% smaller for the A. wentii 'c gfdB1, 'c gfdB2, and 'c gfdB3 mutant strains than those recorded in the case of the A. wentii wild type strain (Fig. 4). Some sporadic minor stress sensitivity phenotypes were observed in oxidative (H2O2, tBOOH, MSB, diamide), cell wall integrity (Congo Red), and heavy metal (Cd2+) exposed A. wentii cultures which were typically enhanced in the ’c gfdB strains with the exception of high (0.2 mM) concentration CdCl2 treatments when the supplementation of A. nidulans gfdB mildly mitigated the observed heavy metal stress sensitivity (Figs. 1 and 2).
Stress sensitivity phenotypes of the A. wentii wild-type and the gfdB-complemented 'c gfdB1, 'c gfdB2, and 'c gfdB3 strains under various stress conditions, after 10 d incubation at 25 °C on NMM stress agar plates. In each experiment, letters “a” and “b” indicate significant differences between the growths of stress treated and untreated cultures and significant interactions between the effects of genetic manipulations and stress exposures, respectively
Varying stress tolerance of the A. wentii wild-type and the gfdB complemented 'c gfdB1, 'c gfdB2, and 'c gfdB3 strains (10 d incubation, 25 °C, NMM stress agar plates). Letters “a” and “b” stand for significant differences between the growths of stress treated and untreated cultures and significant interactions between the effects of genetic manipulations and stress exposures, respectively
Stress phenotypes of the A. wentii gfdB complemented 'c gfdB1, 'c gfdB2, and 'c gfdB3 strains (10 d incubation, 25 °C, NMM stress agar plates). “a” significant differences between the growths of stress treated and untreated cultures. “b” significant interactions between the effects of genetic manipulations and stress exposures
Versatile stress sensitivity phenotypes recorded in A. wentii gfdB complemented 'c gfdB1, 'c gfdB2, and 'c gfdB3 strains, grown on NMM stress agar plates (10 d, 25 °C, in the presence of 2 M sorbitol). “a” significant differences between the growths of stress treated and untreated cultures. “b” significant interactions between the effects of genetic manipulations and stress exposures
Combined osmolyte treatments (2 M sorbitol with 1 M or 1.5 M NaCl) were disadvantageous for A. wentii and the inhibitory power of the osmolyte mixtures was further enhanced in the ’c gfdB strains (Fig. 4). In some cases, the addition of 2 M sorbitol to culture media increased the severity of environmental stress, e.g., the ’c gfdB strains did not even grow out in the presence 1.5 mM diamide (Fig. 4), which stress treatment gave us only a minor phenotype on NMM stress agar without any sorbitol supplementation (Fig. 1). In contrast, 2 M sorbitol helped the ’c gfdB strains to grow out when exposed to 0.38 mM MSB (Fig. 4), while no outgrowth of the gfdB supplemented A. wentii strains was recorded in the absence of the osmolyte (Fig. 3). Interestingly, the addition of 2 M sorbitol to NMM agar also slightly increased the oxidative stress tolerance of the A. wentii ’c gfdB strains in the presence of 9 mM H2O2 (Fig. 4), which was not observed in NMM stress agar experiments in the absence of sorbitol (Fig. 1).
Interactions between stress exposures and A. nidulans gfdB supplementations were also investigated by two-way ANOVA followed by Tukey’s post hoc test. As shown in Figs. 1, 2, 3, and 4, stress exposure - gfdB interactions were rather sporadic but some clear-cut interactions (marked by letter “b” in the upper parts of the figures) were recorded. Interestingly, no interaction between 2.4 mM tBOOH treatment and gfdB supplementation was observed in A. wentii (Fig. 2) although A. nidulans gfdB fully restored the growth of A. glaucus exposed to 0.4 mM tBOOH on stress agar plates supplemented with 2 M sorbitol (Király et al. 2020b). The addition of 2 M sorbitol did not influence these interactions considerably in A. wentii but the 54 μM Congo Red treatment – gfdB supplementation interaction (Fig. 1) was lost in the presence of 2 M sorbitol (Fig. 4).
Stress tolerance-based positioning of A. glaucus and A. wentii among aspergilli
The evolutionary distances of 17 selected Aspergillus species (A. niger was represented by two strains) based on stress sensitivity patterns publicly available in FSD (Orosz et al. 2018) were visualized by the generation of multidimensional scale plots using the MIC50 values (H2O2, MSB, CdCl2) and colony diameters measuered at selected concentrations (sorbitol, NaCl, Congo Red) as a result of current and previous stress tolerance studies (Emri et al. 2018). In addition, cluster analysis was also performed to construct dendrograms (Fig. 5; 5 and 10 d incubations, 25 °C; Emri et al. 2018).
Comparison of the phylogenetic positions and the stress tolerance of the tested Aspergillus strains. Part A, maximum likelihood phylogeny of the Aspergillus spp. as reproduced from the previous publication of de Vries et al. (2017) with modifications. Note that A. niger ATCC 1015 is identical to CBS 113.46. Part B, cluster analysis dendrogram constructed on the stress tolerance data gained for A. glaucus, A. nidulans, and A. wentti strains in current and previous (Király et al. 2020a,b) studies or reposited in the Fungal Stress Database (all other strains; Orosz et al. 2018; http://www.fung-stress.org/). Part C, multidimensional scale plot presentation of the stress tolerance variability of the Aspergillus species tested (de Vries et al. 2017; Emri et al. 2018)
In this study, cluster analysis and multidimensional scaling were performed on an extended stress database to track changes in the stress tolerance-based positioning of the two osmophilic species A. glaucus and A. wentii, which were elicited by osmolytes and gfdB supplementation. To reach this aim, stress sensitivity data gained in A. glaucus and A. wentii NMM stress agar cultures supplemented with 0.5 and 1 M NaCl (A. glaucus), with 2 M sorbitol (A. glaucus and A. wentii wild-type strains, A. glaucus and A. wentii ’c gfdB strains) (de Vries et al. 2017; Emri et al. 2018; Orosz et al. 2018; Király et al. 2020b), as well as those recorded with the A. nidulans ΔgfdB gene deletion mutant (Király et al. 2020a) were also taken into consideration in the construction of both the dendogram (Fig. 5B) and the multidimensional scale plot (Fig. 5C).
Neither the construction of a dendrogram via cluster analysis (Fig. 5B) nor multidimensional scale plot presentation of the distances between the environmental stress tolerance of the tested Aspergillus spp. (Fig. 5C) separated remarkably well the osmolyte exposed and gfdB supplemented A. glaucus and A. wentii strains from the wild-type A. wentii, wild-type A. nidulans, and the A. nidulans ΔgfdB strains. Nevertheless, A. wentii and A. nidulans ΔgfdB both lacking gfdB genes were relatively closer to each other than to wild-type A. nidulans (Fig. 5). No similar tendencies were observed for A. glaucus, which is a strictly xerophilic/osmophilic fungus that can hardly grow without the supplementation of any osmolyte (Supplemental Fig. S2; de Vries et al. 2017; Orosz et al. 2018; Király et al. 2020b).
As shown in Fig. 5B and C, all A. wentii wild-type and ’c gfdB strains grown on either NMM or 2 M sorbitol supplemented NMM agars remained in the proximity of A. terreus (Fig. 5B; cluster analysis dendrogram) or close to A. aculeatus (Fig. 5C; multidimensional scale plot presentation). Similarly, all osmolyte (NaCl, sorbitol) supplemented A. glaucus strains remained close to A. carbonarius and A. clavatus (Fig. 5B; cluster analysis dendrogram) or in the proximity of A. carbonarius and A. terreus (Fig. 5C; multidimensional scale plot presentation).
Stress tolerance-based positioning of the tested A. glaucus, A. wentii, and A. nidulans strains
The stress tolerances of A. glaucus and A. wentii wild-type and ’c gfdB mutant strains were also compared to those of the A. nidulans wild-type and ΔgfdB mutant strains via the generation of multidimensional scale plots. In this set of experiments, colony diameters measured on NMM agar plates supplemented with selected stress initiating agents (54 μM Congo Red, 2 M sorbitol, 0.096 mM MSB, 0.4 mM tBOOH, 9 mM H2O2, 1 M NaCl, and 0.5 mM CdCl2) were taken into consideration (Fig. 6). Unlike the 17 Aspergillus species-based cluster analysis dendrogram and multidimensional scale plot presented in Fig. 5, this approach clearly indicated that the supplementation of A. wentii and A. glaucus with the A. nidulans gfdB gene increased the distance between these species (Fig. 6). This result is in line with the observation that the insertion of gfdB massively increased the oxidative stress tolerance of A. glaucus without influencing its osmophily (Király et al. 2020b), while the insertion of the same gene into the A. wentii genome reduced osmophily without giving a clear, unidirectional change in its oxidative stress tolerance (Figs. 1, 2, 3, and 4). Interestingly, the same method did not show profound differences in the stress tolerance of the A. nidulans wild-type and ΔgfdB strains, and both strains remained separated, far apart from the A. glaucus and A. wentii strains tested (Fig. 6).
Multidimensional scale plot presentation of the stress tolerance variability of the Aspergillus wild-type (A. nidulans, A. glaucus, A. wentii) and mutant (A. nidulans ΔgfdB; A. glaucus 'c gfdB1 and 'c gfdB2; A. wentii 'c gfdB1, 'c gfdB2, and 'c gfdB3) strains tested. Stress sensitivity assays were carried out on NMM stress agar plates (10 d incubation, 25 °C) and culture media prepared for the A. glaucus strains always contained 2 M sorbitol
Discussion
Fungi are exposed to various environmental stresses, such as heat shock, oxidative, and osmotic stress, and glycerol has an important role in overcoming various stress conditions and microenvironments. Glycerol 3-phosphate dehydrogenase (G3PDH) can catalyze the reversible redox conversion of dihydroxyacetone phosphate to glycerol 3-phosphate, which is then dephosphorylated into glycerol. However, current knowledge on the functions of G3PDH genes in aspergilli is limited (Zhang et al. 2018). Nevertheless, the ancient G3PDH-encoding gfd gene was duplicated before the diversification of the ascomycetous fungal species belonging to the genus Aspergillus (Miskei et al. 2009; Balázs et al. 2010; de Vries et al. 2017), and the potentially harmful disturbance in gfd gene dosage seems to be successfully resolved by various subfunctionalization and neofunctionalization events (Wapinski et al. 2007; Ames et al. 2010; Levasseur and Pontarotti 2011; Emri et al. 2018).
Previous studies clearly indicated that A. nidulans gfdA and gfdB found their important physiological functions in the maintenance of cellular growth and cell wall integrity (gfdA; Fillinger et al. 2001) and in oxidative stress defence (gfdB; against H2O2, tBOOH, and diamide; Király et al. 2020a). It is important to note that the physiological functions of A. nidulans gfdA and gfdB did not separate hermetically because the ΔgfdB strain also showed minor reduction in growth and cell wall integrity (Congo Red) phenotypes (Király et al. 2020a).
The loss of one of the gfd paralogs is a relatively rare event in aspergilli but two xerophilic/osmophilic species, A. glaucus and A. wentii, evolutionarily lost their gfdB orthologous gene (de Vries et al. 2017). Therefore, it was reasonable to assume a causal connection between this gene loss event and the appearance of osmophily (de Vries et al. 2017; Orosz et al. 2018). This hypothesis was further strengthened by the upregulation of gfdB (but not gfdA) under 0.6 M NaCl exposure in A. nidulans (Balázs et al. 2010).
In a previous study by Király et al. (2020b), we managed to supplement A. glaucus with A. nidulans gfdB resulting in ’c gfdB strains with considerably increased tBOOH and, to a lesser extent, increased H2O2, MSB, Congo Red, and CdCl2 tolerance. Since A. glaucus is a promising enzyme (Tao et al. 2010, 2011; Abrashev et al. 2016; Li et al. 2018; Takenaka et al. 2019; Chen et al. 2020) and polyketide (Cai et al. 2009, 2014; Sun et al. 2009; Wu et al. 2017) producer and bioremediation (Gajendiran and Abraham 2017; Wei and Zhang 2018; Zhou et al. 2021) fungus, and a satisfactory stress tolerance is highly recquired for any industrial fungal strains (Bai et al. 2003; Li et al. 2011; Teixeira et al. 2011; Hagiwara et al. 2016; Deparis et al. 2017; Steensels et al. 2019; Brandt et al. 2021; Yaakoub et al. 2022), these observations raised the question if the contribution of A. nidulans gfdB to oxidative stress tolerance could be exploited in other Aspergillus spp. as well.
After supplementation of A. wentii with A. nidulans gfdB gene, only minor changes in the stress tolerance were observed in the A. wentii ’c gfdB strains in comparison to the A. wentii wild-type strain, including slightly increased CdCl2 (Figs. 1 and 2) as well as oxidative (MSB, H2O2; only in the presence of 2 M sorbitol; Figs. 1, 2, 3, and 4) stress tolerance. Therefore, we reached the conclusion that these sporadic and hardly significant improvements in environmental stress tolerance would be difficult to take advantage of under industrial conditions (Sinha and Chakrabarty 1978; Chander et al. 1980; Gross et al. 1984; Shoaib et al. 2018; Lago et al. 2021).
Stress tolerance-based positioning of A. glaucus and A. wentii among the aspergilli was achieved using cluster analysis dendrogram and multidimensional scale plot presentation approaches as shown in Fig. 5B and C, respectively. It is noteworthy that the tested A. wentii and all osmolyte supplemented A. glaucus wild-type and ’c gfdB strains took their positions near some industrially and/or agriculturally important Aspergillus spp. including A. aculeatus (a promising hydrolase producing fungus; Mhuantong et al. 2020; Wang et al. 2021), A. carbonarius (a major ochratoxin A producer in grapes; Mondani et al. 2020), A. clavatus (a rich source of secondary metabolites; Zutz et al. 2013), or in the proximity of A terreus (producing lovastatin, itaconic acid and hydrolyses; Ryngajłło et al. 2021). Therefore, further studies should aim at shedding light onto any possible link between the environmental stress tolerance of these fungi and the presence of a gfdB ortholog in their genomes (de Vries et al. 2017; Emri et al. 2018; Orosz et al. 2018).
When stress tolerance-based positioning of the tested A. glaucus, A. wentii, and A. nidulans strains was carried out using data obtained after a careful selection of stress initiating agents, these species sperated well and the supplementation of A. wentti and A. glaucus with A. nidulans gfdB increased the distance between these Aspergillus species, meanwhile their positions relative to the A. nidulans wild-type and ΔgfdB strains remained essentially unaltered (Fig. 6).
These observations on the stress tolerance-based positions of the tested Aspergillus spp. and their mutants warn us that any alterations in the stress response systems of fungi may trigger rather complex, often unpredictable physiological changes. Furthermore, we can determine the size and direction of the phenotypic changes only based on the results of a large number of carefully executed experiments, after carrying out properly chosen mathematical analyses.
Industrial fungi are confronted with a wide spectrum of environmental stress factors including oxidative stress (Bai et al. 2003; Li et al. 2011; Teixeira et al. 2011; Hagiwara et al. 2016; Deparis et al. 2017; Steensels et al. 2019; Brandt et al. 2021; Yaakoub et al. 2022). Moreover, some aspergilli (A. glaucus, A. wentii, A. versicolor, A. sydowii, and A. oryzae) show osmophily under various culture conditions (de Vries et al. 2017; Orosz et al. 2018). A deeper understanding of the molecular background of osmophily may pave the way for the development of new, highly stress tolerant industrial strains (Király et al. 2020a,b). Future screening studies in submerged Aspergillus cultures on stress-elicited changes in the concentrations of intracellular compatible solutes, e.g., glycerol, mannitol, erythritol, and arabitol (Sánchez-Fresneda et al. 2013; de Lima Alves et al. 2015; Király et al. 2020a) will likely provide ways to increase sugar alcohol yields in industrial fermentation processes — an important field mostly dedicated to osmotolerant/osmophilic yeasts thus far (Moon et al. 2010; Yang et al. 2021; Erian and Sauer 2022; Yaakoub et al. 2022).
The evolution of the stress response system of fungi is remarkably fast especially at the level of transcriptional regulation (Nikolaou et al. 2009; Zhang et al. 2016; Emri et al. 2018). We can also assume that the A. nidulans gfdB inserted with its own promoter into the genomes of A. glaucus and A. wentii (Fig. 5A) interacts with different transcription factors with altered stress responsiveness and modified promoter preferences (Wohlbach et al. 2009), contributing to the observed phenotypic differences of the A. glaucus and A. wentii c’ gfdB strains (Figs. 1, 2, 3, and 4; Király et al. 2020b). Future studies should therefore focus on the development of suitable tools for the constitutive expression of A. nidulans gfdB gene using high-expressed promoter and terminator sequences of host fungi, like A. wentii.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Abrashev R, Feller G, Kostadinova N, Krumova E, Alexieva Z, Gerginova M, Spasova B, Miteva-Staleva J, Vassilev S, Angelova M (2016) Production, purification, and characterization of a novel cold-active superoxide dismutase from the Antarctic strain Aspergillus glaucus 363. Fungal Biol 120:679–689. https://doi.org/10.1016/j.funbio.2016.03.002
Ames RM, Rash BM, Hentges KE, Robertson DL, Delneri D, Lovell SC (2010) Gene duplication and environmental adaptation within yeast populations. Genome Biol Evol 2:591–601. https://doi.org/10.1093/gbe/evq043
Bai Z, Harvey LM, McNeil B (2003) Oxidative stress in submerged cultures of fungi. Crit Rev Biotechnol 23:267–302. https://doi.org/10.1080/07388550390449294
Balázs A, Pócsi I, Hamari Z, Leiter E, Emri T, Miskei M, Oláh J, Tóth V, Hegedus N, Prade RA, Molnár M, Pócsi I (2010) AtfA bZIP-type transcription factor regulates oxidative and osmotic stress responses in Aspergillus nidulans. Mol Genet Genomics 283:289–303. https://doi.org/10.1007/s00438-010-0513-z
Barratt RW, Johnson GB, Ogata WN (1965) Wild-type and mutant stocks of Aspergillus nidulans. Genetics 52:233–246. https://doi.org/10.1093/genetics/52.1.233
Brandt BA, Jansen T, Volschenk H, Görgens JF, Van Zyl WH, Den Haan R (2021) Stress modulation as a means to improve yeasts for lignocellulose bioconversion. Appl Microbiol Biotechnol 105:4899–4918. https://doi.org/10.1007/s00253-021-11383-y
Cai M, Zhang Y, Hu W, Shen W, Yu Z, Zhou W, Jiang T, Zhou X, Zhang Y (2014) Genetically shaping morphology of the filamentous fungus Aspergillus glaucus for production of antitumor polyketide aspergiolide A. Microb Cell Fact 13:73. https://doi.org/10.1186/1475-2859-13-73
Cai MH, Zhou XS, Sun XQ, Tao KJ, Zhang YX (2009) Statistical optimization of medium composition for aspergiolide A production by marine-derived fungus Aspergillus glaucus. J Ind Microbiol Biotechnol 36:381–389. https://doi.org/10.1007/s10295-008-0507-6
Cairns TC, Barthel L, Meyer V (2021) Something old, something new: challenges and developments in Aspergillus niger biotechnology. Essays Biochem 65:213–224. https://doi.org/10.1042/ebc20200139
Chander H, Batish VK, Sannabhadti SS, Srinivasan RA (1980) Factors affecting lipase production in Aspergillus wentii. J Food Sci 45:598–600. https://doi.org/10.1111/j.1365-2621.1980.tb04109.x
Chen L, Wei Y, Shi M, Li Z, Zhang SH (2020) Statistical optimization of a cellulase from Aspergillus glaucus CCHA for hydrolyzing corn and rice straw by RSM to enhance yield of reducing sugar. Biotechnol Lett 42(4):583-595. https://doi.org/10.1007/s10529-020-02804-5
Chomczynski P (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15(532-534):536–537
de Lima AF, Stevenson A, Baxter E, Gillion JL, Hejazi F, Hayes S, Morrison IE, Prior BA, McGenity TJ, Rangel DE, Magan N, Timmis KN, Hallsworth JE (2015) Concomitant osmotic and chaotropicity-induced stresses in Aspergillus wentii: compatible solutes determine the biotic window. Curr Genet 61:457–477. https://doi.org/10.1007/s00294-015-0496-8
de Vries RP, Riley R, Wiebenga A, Aguilar-Osorio G, Amillis S, Uchima CA, Anderluh G, Asadollahi M, Askin M, Barry K, Battaglia E, Bayram Ö, Benocci T, Braus-Stromeyer SA, Caldana C, Cánovas D, Cerqueira GC, Chen F, Chen W et al (2017) Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus. Genome Biol 18:28. https://doi.org/10.1186/s13059-017-1151-0
Deparis Q, Claes A, Foulquié-Moreno MR, Thevelein JM (2017) Engineering tolerance to industrially relevant stress factors in yeast cell factories. FEMS Yeast Res 17:fox036. https://doi.org/10.1093/femsyr/fox036
Emri T, Antal K, Riley R, Karányi Z, Miskei M, Orosz E, Baker SE, Wiebenga A, de Vries RP, Pócsi I (2018) Duplications and losses of genes encoding known elements of the stress defence system of the Aspergilli contribute to the evolution of these filamentous fungi but do not directly influence their environmental stress tolerance. Stud Mycol 91:23–36. https://doi.org/10.1016/j.simyco.2018.10.003
Emri T, Szarvas V, Orosz E, Antal K, Park H, Han KH, Yu JH, Pócsi I (2015) Core oxidative stress response in Aspergillus nidulans. BMC Genomics 16:478. https://doi.org/10.1186/s12864-015-1705-z
Erian AM, Sauer M (2022) Utilizing yeasts for the conversion of renewable feedstocks to sugar alcohols - a review. Bioresour Technol 346:126296. https://doi.org/10.1016/j.biortech.2021.126296 Epub 2021 Nov 16
Fillinger S, Ruijter G, Tamás MJ, Visser J, Thevelein JM, d'Enfert C (2001) Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans. Mol Microbiol 39:145–157. https://doi.org/10.1046/j.1365-2958.2001.02223.x
Gajendiran A, Abraham J (2017) Biomineralisation of fipronil and its major metabolite, fipronil sulfone, by Aspergillus glaucus strain AJAG1 with enzymes studies and bioformulation. 3 Biotech 7:212. https://doi.org/10.1007/s13205-017-0820-8
Gross M, Levy R, Toepke H (1984) Occurrence and analysis of the mycotoxin emodin. Nahrung 28:31–44. https://doi.org/10.1002/food.19840280116
Hagiwara D, Sakamoto K, Abe K, Gomi K (2016) Signaling pathways for stress responses and adaptation in Aspergillus species: stress biology in the post-genomic era. Biosci Biotechnol Biochem 80:1667–1680. https://doi.org/10.1080/09168451.2016.1162085
Herrera ML, Vallor AC, Gelfond JA, Patterson TF, Wickes BL (2009) Strain-dependent variation in 18S ribosomal DNA copy numbers in Aspergillus fumigatus. J Clin Microbiol 47:1325–1332. https://doi.org/10.1128/jcm.02073-08
Huang X, Men P, Tang S, Lu X (2021) Aspergillus terreus as an industrial filamentous fungus for pharmaceutical biotechnology. Curr Opin Biotechnol 69:273–280. https://doi.org/10.1016/j.copbio.2021.02.004
Jin FJ, Wang BT, Wang ZD, Jin L, Han P (2022) CRISPR/Cas9-based genome editing and its application in Aspergillus species. J Fungi (Basel) 8:467. https://doi.org/10.3390/jof8050467
Király A, Hámori C, Gyémánt G, Kövér KE, Pócsi I, Leiter É (2020a) Characterization of gfdB, putatively encoding a glycerol 3-phosphate dehydrogenase in Aspergillus nidulans. Fungal Biol 124:352–360. https://doi.org/10.1016/j.funbio.2019.09.011
Király A, Szabó IG, Emri T, Leiter É, Pócsi I (2020b) Supplementation of Aspergillus glaucus with gfdB gene encoding a glycerol 3-phosphate dehydrogenase in Aspergillus nidulans. J Basic Microbiol 60:691–698. https://doi.org/10.1002/jobm.202000067
Kumar A (2020) Aspergillus nidulans: a potential resource of the production of the native and heterologous enzymes for industrial applications. Int J Microbiol 2020:8894215. https://doi.org/10.1155/2020/8894215
Lago MC, Dos Santos FC, Bueno PSA, de Oliveira MAS, Barbosa-Tessmann IP (2021) The glucoamylase from Aspergillus wentii: purification and characterization. J Basic Microbiol 61:443–458. https://doi.org/10.1002/jobm.202000595
Levasseur A, Pontarotti P (2011) The role of duplications in the evolution of genomes highlights the need for evolutionary-based approaches in comparative genomics. Biol Direct 6:11. https://doi.org/10.1186/1745-6150-6-11
Li Z, Pei X, Zhang Z, Wei Y, Song Y, Chen L, Liu S, Zhang SH (2018) The unique GH5 cellulase member in the extreme halotolerant fungus Aspergillus glaucus CCHA is an endoglucanase with multiple tolerance to salt, alkali and heat: prospects for straw degradation applications. Extremophiles 22:675–685. https://doi.org/10.1007/s00792-018-1028-5
Li Q, Bai Z, O'Donnell A, Harvey LM, Hoskisson PA, McNeil B (2011) Oxidative stress in fungal fermentation processes: the roles of alternative respiration. Biotechnol Lett 33:457–467. https://doi.org/10.1007/s10529-010-0471-x
Liu XD, Wei Y, Zhou XY, Pei X, Zhang SH (2015) Aspergillus glaucus aquaglyceroporin gene glpF confers high osmosis tolerance in heterologous organisms. Appl Environ Microbiol 81:6926–6937. https://doi.org/10.1128/aem.02127-15
Liu XD, Xie L, Wei Y, Zhou X, Jia B, Liu J, Zhang S (2014) Abiotic stress resistance, a novel moonlighting function of ribosomal protein RPL44 in the halophilic fungus Aspergillus glaucus. Appl Environ Microbiol 80:4294–4300. https://doi.org/10.1128/aem.00292-14
Lopes AMM, Martins M, Goldbeck R (2021) Heterologous expression of lignocellulose-modifying enzymes in microorganisms: current status. Mol Biotechnol 63:184–199. https://doi.org/10.1007/s12033-020-00288-2
MacCabe AP, Orejas M, Tamayo EN, Villanueva A, Ramón D (2002) Improving extracellular production of food-use enzymes from Aspergillus nidulans. J Biotechnol 96:43–54. https://doi.org/10.1016/s0168-1656(02)00036-6
Mhuantong W, Charoensri S, Poonsrisawat A, Pootakham W, Tangphatsornruang S, Siamphan C, Suwannarangsee S, Eurwilaichitr L, Champreda V, Charoensawan V, Chantasingh D (2020) High quality Aspergillus aculeatus genomes and transcriptomes: a platform for cellulase activity optimization toward industrial applications. Front Bioeng Biotechnol 8:607176. https://doi.org/10.3389/fbioe.2020.607176
Miskei M, Karányi Z, Pócsi I (2009) Annotation of stress-response proteins in the aspergilli. Fungal Genet Biol 46(Suppl 1):S105–S120. https://doi.org/10.1016/j.fgb.2008.07.013
Mondani L, Palumbo R, Tsitsigiannis D, Perdikis D, Mazzoni E, Battilani P (2020) Pest management and ochratoxin A contamination in grapes: a review. Toxins (Basel) 12:303. https://doi.org/10.3390/toxins12050303
Moon HJ, Jeya M, Kim IW, Lee JK (2010) Biotechnological production of erythritol and its applications. Appl Microbiol Biotechnol 86(4):1017–1025. https://doi.org/10.1007/s00253-010-2496-4
Nikolaou E, Agrafioti I, Stumpf M, Quinn J, Stansfield I, Brown AJ (2009) Phylogenetic diversity of stress signalling pathways in fungi. BMC Evol Biol 9:44. https://doi.org/10.1186/1471-2148-9-44
Orosz E, van de Wiele N, Emri T, Zhou M, Robert V, de Vries RP, Pócsi I (2018) Fungal Stress Database (FSD)--a repository of fungal stress physiological data. Database (Oxford) 2018:bay009. https://doi.org/10.1093/database/bay009
Park HS, Jun SC, Han KH, Hong SB, Yu JH (2017) Diversity, application, and synthetic biology of industrially important Aspergillus fungi. Adv Appl Microbiol 100:161–202. https://doi.org/10.1016/bs.aambs.2017.03.001
Park HS, Man YY, Lee MK, Jae Maeng P, Chang Kim S, Yu JH (2015) Velvet-mediated repression of β-glucan synthesis in Aspergillus nidulans spores. Sci Rep 5:10199. https://doi.org/10.1038/srep10199
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56(1):117–124. https://doi.org/10.1016/0378-1119(87)90164-8
R Core Team (2022) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria https://www.R-project.org/. Accessed 04 Aug 2022
Ryngajłło M, Boruta T, Bizukojć M (2021) Complete genome sequence of lovastatin producer Aspergillus terreus ATCC 20542 and evaluation of genomic diversity among A. terreus strains. Appl Microbiol Biotechnol 105:1615–1627. https://doi.org/10.1007/s00253-021-11133-0
Sánchez-Fresneda R, Guirao-Abad JP, Argüelles A, González-Párraga P, Valentín E, Argüelles JC (2013) Specific stress-induced storage of trehalose, glycerol and D-arabitol in response to oxidative and osmotic stress in Candida albicans. Biochem Biophys Res Commun 430(4):1334–1339. https://doi.org/10.1016/j.bbrc.2012.10.118
Shoaib A, Bhran A, Rasmey AH, Mikky Y (2018) Optimization of cultural conditions for lipid accumulation by Aspergillus wentii Ras101 and its transesterification to biodiesel: application of response surface methodology. 3 Biotech 8:417. https://doi.org/10.1007/s13205-018-1434-5
Sinha S, Chakrabarty SL (1978) Production of amylase by a submerged culture of Aspergillus wentii. Folia Microbiol (Praha) 23:6–11. https://doi.org/10.1007/bf02876589
Steensels J, Gallone B, Voordeckers K, Verstrepen KJ (2019) Domestication of industrial microbes. Curr Biol 29(10):R381–R393. https://doi.org/10.1016/j.cub.2019.04.025
Sun X, Zhou X, Cai M, Tao K, Zhang Y (2009) Identified biosynthetic pathway of aspergiolide A and a novel strategy to increase its production in a marine-derived fungus Aspergillus glaucus by feeding of biosynthetic precursors and inhibitors simultaneously. Bioresour Technol 100:4244–4251. https://doi.org/10.1016/j.biortech.2009.03.061
Szabó Z, Pákozdi K, Murvai K, Kecskeméti Á, Oláh V, Logrieco AF, Madar A, Dienes B, Csernoch L, Emri T, Hornok L, Pócsi I, Leiter É (2020a) FvmnSOD is involved in oxidative stress defence, mitochondrial stability and apoptosis prevention in Fusarium verticillioides. J Basic Microbiol 60:994–1003. https://doi.org/10.1002/jobm.202000560
Szabó Z, Pákozdi K, Murvai K, Pusztahelyi T, Kecskeméti Á, Gáspár A, Logrieco AF, Emri T, Ádám AL, Leiter É, Hornok L, Pócsi I (2020b) FvatfA regulates growth, stress tolerance as well as mycotoxin and pigment productions in Fusarium verticillioides. Appl Microbiol Biotechnol 104:7879–7899. https://doi.org/10.1007/s00253-020-10717-6
Szewczyk E, Nayak T, Oakley CE, Edgerton H, Xiong Y, Taheri-Talesh N, Osmani SA, Oakley BR (2006) Fusion PCR and gene targeting in Aspergillus nidulans. Nat Protoc 1:3111–3120. https://doi.org/10.1038/nprot.2006.405
Takenaka S, Lim L, Fukami T, Yokota S, Doi M (2019) Isolation and characterization of an aspartic protease able to hydrolyze and decolorize heme proteins from Aspergillus glaucus. J Sci Food Agric 99:2042–2047. https://doi.org/10.1002/jsfa.9339
Tao YM, Xu XQ, Ma SJ, Liang G, Wu XB, Long MN, Chen QX (2011) Cellulase hydrolysis of rice straw and inactivation of endoglucanase in urea solution. J Agric Food Chem 59:10971–10975. https://doi.org/10.1021/jf203712n
Tao YM, Zhu XZ, Huang JZ, Ma SJ, Wu XB, Long MN, Chen QX (2010) Purification and properties of endoglucanase from a sugar cane bagasse hydrolyzing strain, Aspergillus glaucus XC9. J Agric Food Chem 58:6126–6130. https://doi.org/10.1021/jf1003896
Teixeira MC, Mira NP, Sá-Correia I (2011) A genome-wide perspective on the response and tolerance to food-relevant stresses in Saccharomyces cerevisiae. Curr Opin Biotechnol 22:150–156. https://doi.org/10.1016/j.copbio.2010.10.011
Wang J, Gao Z, Qian Y, Hu X, Li G, Fu F, Guo J, Shan Y (2021) Citrus segment degradation potential, enzyme safety evaluation, and whole genome sequence of Aspergillus aculeatus strain ZC-1005. Front Microbiol 12:671200. https://doi.org/10.3389/fmicb.2021.671200
Wapinski I, Pfeffer A, Friedman N, Regev A (2007) Natural history and evolutionary principles of gene duplication in fungi. Nature 449:54–61. https://doi.org/10.1038/nature06107
Wei Y, Zhang SH (2018) Abiostress resistance and cellulose degradation abilities of haloalkaliphilic fungi: applications for saline-alkaline remediation. Extremophiles 22:155–164. https://doi.org/10.1007/s00792-017-0986-3
Wheeler KA, Hocking AD (1993) Interactions among xerophilic fungi associated with dried salted fish. J Appl Bacteriol 74:164–169. https://doi.org/10.1111/j.1365-2672.1993.tb03010.x
Wohlbach DJ, Thompson DA, Gasch AP, Regev A (2009) From elements to modules: regulatory evolution in Ascomycota fungi. Curr Opin Genet Dev 19:571–578. https://doi.org/10.1016/j.gde.2009.09.007
Wu Y, Ren Y, Zhou X, Cai M, Zhang Y (2017) Transcription factor Agseb1 affects development, osmotic stress response, and secondary metabolism in marine-derived Aspergillus glaucus. J Basic Microbiol 57:873–882. https://doi.org/10.1002/jobm.201700123
Yaakoub H, Sanchez NS, Ongay-Larios L, Courdavault V, Calenda A, Bouchara JP, Coria R, Papon N (2022) The high osmolarity glycerol (HOG) pathway in fungi. Crit Rev Microbiol 48:657–695. https://doi.org/10.1080/1040841X.2021.2011834
Yang L, Kong W, Yang W, Li D, Zhao S, Wu Y, Zheng S (2021) High D-arabitol production with osmotic pressure control fed-batch fermentation by Yarrowia lipolytica and proteomic analysis under nitrogen source perturbation. Enzyme Microb Technol 152:109936. https://doi.org/10.1016/j.enzmictec.2021.109936
Zhang C, Meng X, Gu H, Ma Z, Lu L (2018) Predicted glycerol 3-phosphate dehydrogenase homologs and the glycerol kinase GlcA coordinately adapt to various carbon sources and osmotic stress in Aspergillus fumigatus. G3 (Bethesda) 8:2291–2299. https://doi.org/10.1534/g3.118.200253
Zhang L, Zhou Z, Guo Q, Fokkens L, Miskei M, Pócsi I, Zhang W, Chen M, Wang L, Sun Y, Donzelli BG, Gibson DM, Nelson DR, Luo JG, Rep M, Liu H, Yang S, Wang J, Krasnoff SB et al (2016) Insights into adaptations to a near-obligate nematode endoparasitic lifestyle from the finished genome of Drechmeria coniospora. Sci Rep 6:23122. https://doi.org/10.1038/srep23122
Zhou Z, Wu X, Lin Z, Pang S, Mishra S, Chen S (2021) Biodegradation of fipronil: current state of mechanisms of biodegradation and future perspectives. Appl Microbiol Biotechnol 105:7695–7708. https://doi.org/10.1007/s00253-021-11605-3
Zutz C, Gacek A, Sulyok M, Wagner M, Strauss J, Rychli K (2013) Small chemical chromatin effectors alter secondary metabolite production in Aspergillus clavatus. Toxins (Basel) 5:1723–1741. https://doi.org/10.3390/toxins5101723
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Open access funding provided by University of Debrecen. This work was supported by the European Union and the European Social Fund through project EFOP-3.6.1-16-2016-00022, by the National Research, Development and Innovation Office (Hungary) projects NN125671 and K131767. Project no. TKP2021-EGA-20 (Biotechnology) has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the TKP2021-EGA funding scheme.
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IP and VB conceived and designed research. VB, AK, and EO conducted experiments. VB, MM, TE, ZK, ÉL, RPV, and IP analyzed data. IP, VB, ÉL, and RPV wrote the manuscript. All authors read and approved the manuscript.
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Bodnár, V., Király, A., Orosz, E. et al. Species-specific effects of the introduction of Aspergillus nidulans gfdB in osmophilic aspergilli. Appl Microbiol Biotechnol 107, 2423–2436 (2023). https://doi.org/10.1007/s00253-023-12384-9
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DOI: https://doi.org/10.1007/s00253-023-12384-9
Keywords
- Aspergillus nidulans
- Aspergillus wentii
- Aspergillus glaucus
- Osmophily
- Environmental stress
- Oxidative stress tolerance