Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillusoryzae

Abstract

Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucoseinduced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although ␣-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on ␣-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB. The ␣-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the ␣-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae. IMPORTANCEIn eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucoseinduced endocytosis of transporters is mediated by their ubiquitination, and arrestinlike proteins act as adaptors of transporters and ubiquitin ligases. In this study, we showed that CreD, an arrestin-like protein, is involved in glucose-induced endocytosis of maltose permease and carbon catabolite derepression of amylase gene expression in Aspergillus oryzae. Dephosphorylation of CreD was required for CCR relief triggered by the disruption of creB, which encodes a deubiquitinating enzyme; a combination of the phosphorylation-defective mutation of CreD and creB disruption dramatically improved ␣-amylase production. This study shows the dual function of an arrestin-like protein and provides a novel approach for improving the production of amylolytic enzymes in A. oryzae.

Nuclear export‐dependent degradation of the carbon catabolite repressor CreA is regulated by a region located near the C‐terminus in Aspergillus oryzae

“…Because the A. nidulans stain used to express CreA‐GFP contained the creB allele ( creB15 ), which had a mutation comprising a single amino acid substitution, we hypothesize that this mutation is not sufficient to abrogate the reduction in the level of CreA protein. The reduced CreA abundance in the creB disruption mutant was consistent with our recent study that showed the transcriptional relief of α‐amylase gene expression from CCR by A. oryzae creB disruption (Tanaka et al, ). However, a recent report suggests that CreA is not a direct target for CreB, although interaction between CreA and CreB was observed when they were overexpressed (Alam et al, ).…”

“…It is thought that CreA is degraded via ubiquitination by HulA and CreD, and stabilized through deubiquitination by the CreB/CreC complex (Lockington and Kelly, ; Boase and Kelly, ). Previously, we reported that A. oryzae CreD is required for carbon catabolite de‐repression (Tanaka et al, ). When FLAG‐CreA was expressed from its own promoter in the Δ creD strain, the abundance of FLAG‐CreA was comparable with that in the wild‐type strain, even after incubation in 1% maltose medium (Fig.…”

“…The A. oryzae Δ ligD :: ptrA strain ( niaD − , sC − ; Mizutani et al, ), derived from the wild‐type strain A. oryzae RIB40 (National Research Institute of Brewing Stock Culture), and the Δ snfA strain (Δ ligD :: loxP , Δ snfA :: pyrG , niaD − , sC − ; Tanaka et al, ) were used as the recipient strains of creA deletion for the expression of FLAG‐ or GFP‐fused CreA proteins. The Δ creA pyrG − strain (Δ ligD :: loxP , Δ creA :: sC , niaD − , pyrG − ; Ichinose et al, ) was used as the recipient strain for disruption of creC and creD genes.…”

“…Plasmids pNthiA and pNPcreA contain a terminator of the α‐glucosidase gene ( agdA ) downstream of a promoter‐attached multi‐cloning site, and the niaD gene encoding nitrate reductase as a fungal selectable marker. The 3 × FLAG‐encoding sequence was amplified by PCR using the N‐3FLAGsenNotI and N‐3FLAGantiPmaCI primers, and the plasmid pUSC‐CreD3FLAG (Tanaka et al, ) as a template, and inserted into Not I/ Pma CI‐digested pNPcreA and pNthiA, yielding pNPcreA‐3FLAG and pNT‐3FLAG. Finally, a DNA fragment containing the creA coding region was amplified by PCR using the primers creAsenPmaCI and creAantiSphI, and inserted into Pma CI/ Sph I‐digested pNPcreA‐3FLAG and pNT‐3FLAG.…”

“…However, three additional factors (CreB, CreC and CreD) have been identified as CCR regulatory factors in A. nidulans using a genetic approach (Hynes and Kelly, ; Kelly and Hynes, ). Interestingly, CreB and CreC form a deubiquitinating enzyme complex (Lockington and Kelly, ), and CreD is an arrestin‐like protein that serves as an adaptor of ubiquitin ligase and its target (Boase and Kelly, ; Tanaka et al, ). The function of these factors has led us to the hypothesis that proteolytic degradation of the CreA protein is involved in CCR regulation.…”