Research paperFunctional and transcriptomic analysis of the key unfolded protein response transcription factor HacA in Aspergillus oryzae
Introduction
In eukaryotic cells, the endoplasmic reticulum (ER) is crucial for the production of membrane and secreted proteins, and its production ability is limited by the level of ER-resident chaperones, foldases, and other modifying enzymes that assist in protein folding. When protein folding requirements exceed the ER's folding capabilities, misfolded proteins can accumulate and elicit stress to the ER, which will activate the unfolded protein response (UPR) mechanism to decrease ER stress (Feng et al., 2011, Montenegro-Montero et al., 2015, Tanaka et al., 2015). In Saccharomyces cerevisiae and filamentous fungi, the UPR mainly depends on an evolutionarily conserved signaling cascade that is mediated by the ER-resident transmembrane kinase/endoribonuclease IRE1 and the basic leucine zipper (bZIP) transcription factor Hac1p/HacA (HacA in filamentous fungi) (Carvalho et al., 2012, Heimel, 2015, Montenegro-Montero et al., 2015). In the UPR process, the hac1/hacA mRNAs of yeast and filamentous fungi undergo similar, unconventional splicing reactions to produce functional Hac1p/HacA proteins, which are then shuttled into the nucleus where they induce the expression of ER chaperone genes and ER-associated degradation (ERAD) genes to promote the refolding or degradation of unfolded proteins (Moon et al., 2015, Tanaka et al., 2015).
A transcriptome analysis under UPR-inducing conditions in fungi suggested that the target genes of the UPR are predominantly enriched in functional categories that are associated with the secretory pathway, including ER-resident chaperones, phospholipid metabolism, fatty acid synthesis, translocation, protein glycosylation, cell wall biosynthesis, vesicular transport, vacuolar protein targeting, and protein degradation (Travers et al., 2000, Sims et al., 2005, Arvas et al., 2006, Guillemette et al., 2007, Wang et al., 2010). However, the majority of these studies induced the UPR signaling pathway through the use of harsh chemicals (dithiothreitol (DTT) or tunicamycin) (Guillemette et al., 2007, Wang et al., 2010), homologous or heterologous protein expression (Guillemette et al., 2007, Kwon et al., 2012, Liu et al., 2014), or growth conditions that induced secretory hydrolytic enzyme production (Jorgensen et al., 2009, Benz et al., 2014). Additionally, these studies were performed on wild-type (WT) strains; thus, the specific contributions of HacA could not be evaluated. To address this, genome-wide expression profiles have been generated for a hacA disruption mutant and a strain that constitutively expresses hacA (Feng et al., 2011, Carvalho et al., 2012, Fan et al., 2015). In filamentous fungi, hacA deletion mutants of Aspergillus fumigatus (Feng et al., 2011), Neurospora crassa (Fan et al., 2015), and Aspergillus niger (Carvalho et al., 2010) have been constructed successfully, and the hacA disruptions resulted in drastic growth defects in A. niger and N. crassa, and nearly normal growth in A. fumigatus. However, a homokaryotic hacA disruption mutant has not been successfully generated in Aspergillus oryzae (Tanaka et al., 2015), and further understanding the contribution of A. oryzae hacA to ER stress adaptation is still needed.
A. oryzae, an organism that is generally recognized as safe, is a suitable host for homologous and heterologous protein production (Nevalainen et al., 2005, Ward et al., 2006). To comprehensively evaluate the role of HacA as the master regulator of the UPR in the A. oryzae protein secretory pathway, we successfully generated a homokaryotic hacA disruption mutant (HacA-DE) and a strain that constitutively expressed an activated form of hacA (HacA-CA). Our results demonstrated that hyphal growth and sporulation were impaired in the HacA-DE and HacA-CA strains when they were cultured in complete medium, and minimal media containing glucose, maltose, dextrin, and starch as carbon sources. Additionally, the growth impairment of the HacA-CA strain was more pronounced than that of the HacA-DE strain. Here, we performed a transcriptome analysis of the HacA-DE and HacA-CA strains to more thoroughly characterize the effect of hacA on the secretory pathway and whole cell metabolism.
Section snippets
Strains and culture conditions
A. oryzae strains used in this study (Table 1) were cultivated in minimal medium (Czapek–Dox (CD) medium) (Zhou et al., 2015) containing 1 or 2% (w/v) of glucose as a carbon source (or other carbon sources as indicated), 0.3% (w/v) NaNO2, 0.1% (w/v) K2HPO4, 0.2% KCl, 0.05% (w/v) MgSO4·7H2O, and 0.001% (w/v) FeSO4·7H2O, pH 5.5; or in complete medium (dextrose-peptone-yeast extract (DPY) medium) containing 2% (w/v) glucose, 1% (w/v) peptone, 0.5% (w/v) yeast extract, 0.1% (w/v) K2HPO4, and 0.05%
Construction and analysis of the hacA deletion strain and a complemented strain expressing the activated form of hacA
To obtain an A. oryzae hacA deletion strain (HacA-DE, niaD−; ΔhacA::pyrG; Δku70::ptrA), the hacA ORF was replaced by the pyrG cassette. Correct integration of the pyrG cassette was verified by PCR amplification of the chromosomal sequences (Supplementary Fig. S3). Furthermore, to obtain a complemented strain that expresses a constitutively activated form of HacA, we integrated the spliced form of hacA, which lacks the 20-nucleotide intron, into the hacA locus in the HacA-DE-RE (niaD−; ΔhacA; Δ
Discussion
HacA is the master regulator of the UPR, which is a key cellular response for homeostatic adaptation to ER stress. Here, we examined the role of the hacA gene and the UPR control mechanisms of A. oryzae by constructing a hacA knockout strain (HacA-DE) and a strain that expresses constitutively active HacA (HacA-CA). In an earlier study, Tanaka et al. (2015) obtained an A. oryzae hacA disruption mutant only as a heterokaryon. However, in this study, we successfully generated a homokaryotic hacA
Conclusions
Here, we successfully generated a homokaryotic hacA disruption mutant (HacA-DE) and a strain that constitutively expresses an activated form of HacA (HacA-CA). Growth and phenotypic profiles demonstrated that hyphal growth and sporulation were impaired in the HacA-DE and HacA-CA strains, and the growth rate impairment was more pronounced for the HacA-CA strain. The combination of a genetically defined, constitutively activated HacA mutant and a hacA disruption mutant have provided a solid basis
Acknowledgements
We wish to acknowledge the financial support by the State 863 Project (grant no 2014AA021304), the Science and Technology Planning Project of Guangdong Province (grant nos. 2013B010404007, 2013B090800003, and 2016A050503016), the Fundamental Research Funds for the Central Universities (grant nos. 2015ZP032 and 2015ZZ040), and the Science and Technology Planning Project of Guangzhou City (grant no. 201510010191).
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