A Novel Protein AoHPS1 Is Involved In Oxidative Stress and Kojic Acid Synthesis in Aspergillus Oryzae

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A Novel Protein AoHPS1 Is Involved In Oxidative Stress and Kojic Acid Synthesis in Aspergillus Oryzae

https://doi.org/10.21203/rs.3.rs-491523/v1

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Aspergillus oryzae usually suffers from oxidative stress during the process of aerobic fermentation. However, there is little information on what genes involve in oxidative stress of A. oryzae. Here, we found that the expression of a novel gene Aohps1 was induced during the growth and development of A. oryzae with and without oxidative stress. Sequence analysis revealed that Aohps1 has four transmembrane regions and is conserved in Aspergillus species. Overexpression and deletion of Aohpi1 caused the growth defects with and without oxidative stress, including mycelium growth, conidia formation and biomass. Meanwhile, the Aohpi1 overexpression strain exhibited more sensitivity to oxidative stress than the Aohpi1 disrupted mutant. Furthermore, overexpression and disruption of Aohps1 resulted in the inhibition of kojic acid production with and without oxidative stress, consistent with the reduced expression of kojA that directly contributed to the synthesis of kojic acid. Additionally, the yield of kojic acid is less in the Aohps1 overexpression strain than in the Aohps1 deletion mutant under oxidative stress. Collectively, the discovery of Aohps1 provides new insights into oxidative stress and kojic acid synthesis in A. oryzae.

Physiology

Biotechnology and Bioengineering

Applied & Industrial Microbiology

Kojic acid

Aspergillus oryzae

Oxidative stress

CRISPR

Aspergillus oryzae as an important filamentous fungus is used in the fermentation industry for more than a thousand years (Kobayashi et al. 2007; Machida et al. 2008). During the course of fermentation, on the one hand, it has to face and deal with a wide variety of environmental challenges, including oxidative stress. For example, during oxidative stress the high levels of reactive oxygen species (ROS) to which A. oryzae must rise are produced by neutrophil cells, evoking cell damage. In response to oxidative stress, A. oryzae has to rapidly reprogram gene expression and gene products to buffer the lethal elevation in ROS. On the other hand, there is abundant evidence to show that secondary metabolism is closely related to oxidative stress in filamentous fungi (Hong et al. 2013). A. oryzae has been widely used to produce several industrially important secondary metabolites, such as kojic acid (Marui et al. 2010; Terabayashi et al. 2010). However, whether and how oxidative stress affects secondary metabolism in A. oryzae is not clear yet.

For defence against ROS, cells have developed various antioxidant strategies, including antioxidant enzymatic strategies and non-enzyme antioxidant strategies (Bai et al. 2003; Breitenbach et al. 2015; Finkel 2003; Li et al. 2009). The enzymatic systems involved in ROS detoxification mainly include superoxide dismutases (SODs), catalases (CATs) and peroxidases (PODs), while non-enzyme antioxidants consist of GSH, ascorbate or vitamin E (Herrero et al. 2008; Li et al. 2009). A key feature of the response to oxidative stress is to sense the stress signaling rapidly, in turn leading to a reprogramming of expression of genes and their products to protect cells from oxidative stress. Although fungi can utilize multiple different means to control the expression of genes responsible for oxidative challenge, a common feature is the invocation of a transcriptional response to oxidative stress (Hong et al. 2013; Montibus et al. 2015). In yeast, Yap1 and Skn7 are thought as the main two oxidative stress-related transcription factors. Yap1 encodes a leucine-zipper (bZip) AP-1 transcriptional factor and mediates the expression of genes encoding most yeast antioxidants in response to oxidants (Kuge and Jones 1994; Schnell et al. 1992). Disruption of Yap1 leads to be hypersensitive to H₂O₂ (Schnell et al. 1992). Likely, Skn7 is identified as a response regulator protein involved in H₂O₂ hypersensitivity and found to regulate the expression of anti-oxidant genes, including CTT1 (cytosolic catalase), SOD1 (superoxide dismutases) in response to oxidative stress (Krems et al. 1996; Morgan et al. 1997). Additionally, the stress response element binding protein Msn2p is required for activating the expression of CTT1 in response to oxidative stress (Schmitt and McEntee 1996).

Kojic acid is a secondary metabolite naturally produced by Aspergillus oryzae, which is used widely in the food, pharmaceutical and cosmetic area (Mohamad 2010; Saeedi et al. 2019). The related genes for kojic acid have been identified in recent studies (Arakawa et al. 2019; Chang et al. 2011; Marui et al. 2011; Terabayashi et al. 2010). Firstly, a kojic acid cluster in A.oryzae is found, including kojR, kojA and kojT (Terabayashi et al. 2010). KojR encoding a zinc finger protein, serves as a positive regulator to promote kojic acid synthesis through activating its target genes, kojA and kojT (Marui et al. 2011). KojA is identified as a FAD-dependent oxidoreductase and catalyzes the conversion of glucose to kojic acid, while kojT is proposed to be a kojic acid transporter helping to transport kojic acid to the extracellular (Terabayashi et al. 2010). LaeA, a global transcriptional regulator of secondary metabolism, works as the upstream of kojR to positively regulate the synthesis of kojic acid (Oda et al. 2014). Recently, another regulatory protein KepA is also found to be involved in kojic acid production through mediating kojR and kojA expression (Arakawa et al. 2019). Additionally, MsnA, homologous to Msn2, is proved to be related to kojic acid biosynthesis in A. parasiticus and A. flavus (Chang et al. 2011). Moreover, disruption of MsnA leads to not only elevated amounts of kojic acid but also higher levels of ROS, making cells under increased oxidative stress (Chang et al. 2011). However, the information on what genes involve in oxidative stress and kojic acid production are unknown in A. oryzae.

In this study, an uncharacterized gene (Ao090009000298) was induced during the process of growth and development of A. oryzae, and it was overexpressed in A. oryzae, resulting in the sensitivity of A. oryzae to hydrogen peroxide. Therefore, Ao090009000298 was named Aohps1 (hydrogen peroxide sensitivity). Aohps1 was overexpressed by the A. oryzae amyB promoter and Aohps1 knock-out strain was constructed by CRIPSR/Cas9 system. The growth and kojic acid production of Aohps1 overexpression and deletion strains with and without oxidative stress were investigated. The study helps us understand kojic acid synthesis and oxidative stress in A. oryzae.

Microorganisms and cultivations

In this study, the A. oryzae 3.042 strain (CICC 40092) served as wild type (WT). The strain Δ3.042 (ΔpyrG) was derived from 3.042 strain by the CRISPR/Cas9 system (Fan et al. 2020). Czapek-Dox (CD) medium (2% soluble starch, 0.2% NaNO₃, 0.1% KH₂PO₄, 0.05% MgSO₄, 0.05% KCl, 0.05% NaCl, 0.002% FeSO₄ pH 5.5) was used to grow the A. oryzae strains. CD medium with 0.02% H₂O₂ (v/v) was used for oxidative stress. The modified CD medium (2% soluble starch, 0.2% NaNO₃, 0.1% KH₂PO₄, 0.05% MgSO₄, 0.05% KCl, 0.05% NaCl, 0.002% FeSO₄, 500 µM FeCl₃, 200 µM ZnSO₄ pH 5.5) was used to observe kojic acid. All fungal strains were cultured at 30°C.

Sequence analysis of Aohps1

The “Secondary structure consensus prediction” tool was used to predict α-helices, random coil, and other protein features. The protein sequence of Aohps1 was submitted to TMHMM Server v. 2.0 to predict transmembrane helices. For multiple alignment, amino acid sequences of Aohps1 and its orthologous genes from different species were retrieved from the Ensembl Genome Databases and aligned with ClustalW program.

Construction of Aohps1 disruption and overexpression strains

To obtain Aohps1 disruption strain, a 20 bp protospacer sequence targeted to Aohps1 was obtained using CRISPRdirect (https://crispr.dbcls.jp/). The U6 promoter attached with the protospacer sequence for Aohps1 was amplified from our previous vector pPTRII-Cas9-AoGld3 (Fan et al. 2020) using the forward primer PU6-F and reverse primer PU6-Aohps1-R with the protospacer sequence for Aohps1, generating the DNA fragment PU6-Aohps1. The U6 terminator and sgRNA sequence were obtained by PCR using the vector pPTRII-Cas9-AoGld3 as a temple with the forward primer containing the protospacer sequence for Aohps1 and reverse primer Aohps1-TU6-R, producing the fragment Aohps1-TU6. The two fragments with the protospacer sequence for Aohps1 were fused by overlapping PCR and inserted into the SmaI-digested pPTRII-Cas9 vector, generating the plasmid pPTRII-Cas9-Aohps1. The resulting plasmid was introduced into the strain 3.042 using the PEG–protoplast method as described previously (Maruyama and Kitamoto 2011), and then screened through the pyrithiamine resistance marker ptrA and DNA sequencing.

To construct Aohps1 overexpression strain, the open read frame (ORF) of Aohps1 was amplified using the primers listed in Table S2, and cloned into the AflII/BamHI-digested pEX2B vector under the control of amyB promoter. The sequenced plasmid was transformed into the strain Δ3.042 through a standard protoplast method. The positive transformants were examined by PCR.

Expression analysis

Total RNA extraction from grounded mycelia was performed using a Fungal Total RNA Extraction Kit (Omega) according to the manufacturer’s instructions. The genomic DNA contamination in the RNA samples was eliminated using gDNA Eraser (TaKaRa). The total RNA (1 µg) was taken as a template for a first-strand cDNA synthesis using a PrimeScript™ RT reagent Kit following the instruction manual. The CFX96 Real-Time PCR Detection System (Bio-Rad) was used to detect gene expression levels using a SYBR Green PCR Kit (TaKaRa) with the primers listed in Table S2. Histone H1 (AO090012000496) serves as a reference gene. Each gene expression was normalized with the Histone H1. Three independent biological replicates were carried out. Relative expression levels were calculated with the previously described method (Livak and Schmittgen 2001).

Morphological analysis and determination of kojic acid

The fungal strains were point inoculated on CD agar plates and cultured at 30°C. The fungal colonies were photographed and their diameters were measured after fungal strains were cultured for three days. To measure dry cell weight, spore stock suspension (10⁸ spores per mL) was spread on CD agar medium covered with cellophane and incubated at 30°C for three days, then the mycelia on the cellophanes were harvested and dried at 65°C overnight. To quantify the number of conidia, the conidia cultured on CD agar medium for three days were collected by washing solution (0.025% Tween 80, 0.8% NaCl) and counted using a hemocytometer. To detect the kojic acid on the modified CD agar plates, the fungal strains were harvested from three 3 cm cores together with the medium, and extracted with 3 mL absolute ethanol, then the supernatant was collected to determine the content of kojic acid by the colorimetric method with modification after centrifugation at 10,000 rpm for 5 min.

Expression analysis of Aohps1 in A. oryzae

To explore the expression pattern of Aohps1, samples from three different stages of A. oryzae were taken, which included that stage I mycelia expansion spans about 24 h; stage II early sporulation spans about 48 h and stage III mature sporulation spans about 72 h. RT-qPCR analysis showed that the expression of Aohps1 was induced in three development stages of A. oryzae (Fig. 1). After three days of growth under H₂O₂ treatment, the transcription level of Aohps1 had no significant changes compared to that without H₂O₂ treatment (Fig. 1).

Characterization of Aohps1

The Aohps1 gene encodes an uncharacterized protein of 306 amino acid residuals according to Aspergillus Genome Database (Fig. 2A). In silico analysis of Aohps1 protein showed that AoHPS1 had no given domains and contained 54% random coil, 29% α-helix and 10% extended strand (Fig. 2A). The prediction of transmembrane region of AoHPS1 sequence had four distinct hydrophobic regions (Fig. 2B). Multiple alignments showed that AoHPS1 is conserved in Aspergillus species (Fig. 2C).

Generation of Aohps1 overexpression and deletion mutants

To reveal the function of Aohps1, Aohps1 overexpression and deletion mutants were constructed using A. oryzae amyB gene promoter and the CRISPR/Cas9 system, respectively (Fig. 3). In the Aohps1-overexpressing strain (OE-Aohps1), qPCR analysis showed that the transcription level of Aohps1 increased by 5-fold relative to the control WT strain on the starch-containing medium (Fig. 3C). For the Aohps1-disrupted strain (ΔAohpi1), one base-pair deletion in the second exon of Aohps1 led to the termination of translation (Fig. 3A). But disruption of Aohps1 didn’t result in the reduced expression of Aohps1 (Fig. 3C).

Phenotypic characterization of Aohps1 overexpression and disruption strains

After cultivation for three days on CD agar medium, the growth of Aohps1 overexpression and disruption strains slightly decreased compared with the WT strain, while the Aohps1 overexpression strain exhibited a more severe growth defect than the Aohps1 deletion mutant and the WT strain under H₂O₂ treatment (Fig. 4A, B). Moreover, Aohps1 overexpression and disruption strains exhibited severe conidiation defects (Fig. 4C). Additionally, the biomass of Aohps1 overexpression and disruption strains markedly reduced relative to the WT strain with H₂O₂ stress, whereas the Aohps1 deletion mutant had no changes in the biomass compared with the WT strain without H₂O₂ treatment (Fig. 4D).

The effects of overexpression and disruption of Aohps1 on kojic acid synthesis

To study the effects of Aohps1 on secondary metabolism of A. oryzae, the control WT, OE-Aohps1 and ΔAohpi1 strains were cultivated on CD agar medium with ferric ions chelated by kojic acid, showing a red color. The control WT, OE-Aohps1 and ΔAohpi1 strains all displayed a visible red color on the agar medium after cultivation for three days (Fig. 5A). But the control WT strains showed a red color much more intense than OE-Aohps1 and ΔAohpi1 strains (Fig. 5A). Quantitative analysis displayed that the yield of kojic acid in the control WT was about 1.7 and 2.1 times higher than those in the OE-Aohps1 and ΔAohpi1 strains without H₂O₂ treatment, respectively (Fig. 5A). Under H₂O₂ treatment, the production of kojic acid in the WT, OE-Aohps1 and ΔAohpi1 strains was down-regulated and kojic acid production was significantly lower in the OE-Aohps1 and ΔAohpi1 strains than in the control strain (Fig. 5). Moreover, the yield of kojic acid in the OE-Aohps1 strain decreased more than that in ΔAohpi1 strains compared with WT (Fig. 5). To clarify whether the declined production of kojic acid is related to transcription levels, the expression levels of LaeA, kojA, kojR and kojT involved in kojic acid synthesis were analyzed. We found that kojA but not kojR and kojT was decreased significantly in the OE-Aohps1 and ΔAohpi1 strains without H₂O₂ treatment (Fig. 6B, C and D). However, the expression profiles of LaeA, kojR and kojT in the OE-Aohps1 and ΔAohpi1 strains were up-regulated markedly under H₂O₂ treatment relative to those without H₂O₂ treatment (Fig. 6A, B and C). Impressively, the transcription level of kojA in the OE-Aohps1 strain had no significant changes with and without H₂O₂ treatment whereas kojA expression in the ΔAohpi1 strain was increased significantly after H₂O₂ treatment (Fig. 6B), consistent with the fact that kojic acid production was significantly less in the OE-Aohps1 strain than in the ΔAohpi1 mutant under H₂O₂ treatment (Fig. 5B).

A. oryzae is widely used in the fermentation industry to produce fermented food, enzymes and secondary metabolites (Abe et al. 2006; Gomi 2019; Ichishima 2016). During the fermentation process, the aeration and agitation contribute to not only the growth of A. oryzae but also the development of oxidative stress (Li et al. 2009). However, there is little information on the involvement of A. oryzae in oxidative stress. In this study, an uncharacterized gene Aohps1 was found to be involved in oxidative stress. At the transcription level, Aohps1 expression didn’t be inhibited by H₂O₂ (Fig. 1). In terms of the growth and development of A. oryzae, the overexpressing Aohps1 strain became more susceptible to H₂O₂ than WT, and Aohps1 overexpression and deletion strains exhibited obvious growth inhibition in mycelium growth, conidia formation and biomass compared with the control WT under oxidative stress (Fig. 4). These results indicate that Aohps1 has a role in oxidative stress and the growth and development of A. oryzae.

Aohps1 encodes a hypothetical protein according to the Aspergillus genome database. To explore its possible function, we attempted to search its homologous proteins to provide research clues, but there were no known proteins homologous with AoHPS1 to be found in other species. Here, sequence analysis showed AoHPS1 contains four transmembrane regions and the transmembrane domains are mainly located at the N terminus of AoHPS1 protein (Fig. 2B, C), suggesting that AoHPS1 might function in cell surface. In addition, multiple alignments revealed that AoHPS1 and its homologous proteins are conserved in Aspergillus species (Fig. 2C).

Kojic acid is widely used in food and cosmetic fields as anti-oxidant (El-Kady et al. 2014; Mohamad 2010; Saeedi et al. 2019). Previous study has been revealed that disruption of MsnA encoding a zinc finger protein, increased the production of kojic acid followed by higher levels of reactive oxygen species (ROS) in Aspergillus parasiticus and Aspergillus flavus (Chang et al. 2011), indicating kojic acid might be used to combat oxidative stress in cells. However, it remains unclear what genes involve in oxidative stress by affecting the synthesis of kojic acid in A. oryzae. In this present study, overexpression and deletion of Aohps1 inhibited the synthesis of kojic acid under no oxidative stress (Fig. 5). Furthermore, under oxidative stress, kojic acid production is significantly less in the Aohps1 overexpression strain than in the Aohps1 disrupted mutant, while the Aohps1-overexpressing strain is more sensitive to H₂O₂ than the Aohps1 deletion mutant (Fig. 4, 5), suggesting that Aohps1 is involved in protecting A. oryzae from oxidative stress through affecting the synthesis of kojic acid. How Aohps1 affects kojic acid synthesis in A. oryzae ? We found that oxidative stress contributed to the expression of LaeA, kojR and kojT in the Aohps1 overexpression and deletion strains (Fig. 6A, C and D), inconsistent with the reduced production of kojic acid in the Aohps1 overexpression and deletion (Fig. 5). This is probably because LaeA and kojR as the regulatory proteins are indirectly related to kojic acid and there are other genes as the targets of LaeA and kojR involved in the response to oxidative stress. It has been reported that kojA is proposed to directly contribute to kojic acid synthesis as an oxidoreductase (Terabayashi et al. 2010), which is also supported by the kojA expression and kojic acid production in the Aohps1 overexpression and deletion strains. Without oxidative stress, overexpression and disruption of Aohps1 inhibited the expression of kojA (Fig. 6B), resulting in the reduced production of kojic acid (Fig. 5). Under oxidative stress, the transcription level of kojA in the Aohps1 deletion mutant was up-regulated whereas that in the Aohps1 overexpression strain had no changes relative to the control WT without oxidative stress (Fig. 6B), in agreement with the yield of kojic acid is more in the Aohps1 deletion mutant than the Aohps1 overexpression strain (Fig. 5). These data indicate that Aohps1 might be involved in kojic acid synthesis by affecting the expression of kojA.

In summary, a novel gene Aohps1 was found to be involved in oxidative stress and kojic acid synthesis in A. oryzae. Aohps1 overexpression strain exhibited reduced oxidative stress resistance. Meanwhile, Overexpression and deletion of Aohpi1 led to growth defects, including mycelium growth, conidia formation and biomass with and without oxidative stress. Furthermore, overexpression and disruption of Aohps1 caused the inhibition of kojic acid production, consistent with the declined expression of kojA that was directly involved in the production of kojic acid. Additionally, Aohps1 overexpression strain is more susceptible to oxidative stress than Aohps1 disrupted mutant, consistent with the yield of kojic acid is less in the Aohps1 overexpression strain than in the Aohps1 deletion mutant. These data suggest that Aohps1 plays an important role in oxidative stress and kojic acid synthesis in A. oryzae.

Acknowledgements

This work was supported by Natural Science Foundation of China (32000049), Jiangxi Provincial Natural Science Foundation (20202BABL213042), Science and Technology Research Project of Jiangxi Provincial Department of Education (GJJ190611) and Doctoral Scientific Research Foundation of Jiangxi Science and Technology Normal University (2018BSQD030).

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The authors declare that they have no conflict of interest.

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This article does not contain any studies with human participants or animals experiment.

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A Novel Protein AoHPS1 Is Involved In Oxidative Stress and Kojic Acid Synthesis in Aspergillus Oryzae

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