Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity

Abstract

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

Fig 1

Verification of gene replacement. (A) Schematic showing primer binding sites. Primers (see Table 2 for details): 1, Ao_creB_US_F2; 2, pPTR_for_creB_US2p; 3, Ao_DS_creBKOtest_F; 4, Ao_DS_creBKOtest_R. (B) Agarose gel showing PCR products. Lane 1, molecular size markers; lane 2, blank; lanes 3 to 6, primers and DNA templates for strains, as indicated. +, creB⁺ strain; Δ, creBΔ strain.

Fig 2

Phenotypic effects of deleting creB in A. oryzae. The indicated strains were grown for 4 days at 30°C on the indicated media. Sucrose was added at 1%; proline, acetamide, glucuronate, and quinate were added at 50 mM, as carbon sources; urea, proline, and acetamide were added at 10 mM, as nitrogen sources; and bovine serum albumin (BSA) was added at 0.01%. PDA, potato dextrose agar.

Fig 3

Deletion of creB increases secretion of cellulases and xylanases under inducing conditions. Triplicate spore-inoculated 50-ml shake flask cultures of the three strains were grown under noninducing (1% sorbitol), inducing (1% xylan), and repressing (1% xylan plus 2% sucrose) conditions. After 48 h, biomass and supernatants were harvested, and total secreted enzyme activities were measured using EnzChek cellulase substrate or EnzChek xylanase substrate and are expressed as activities per unit of dry weight biomass, normalized such that activity in the wild-type strain under inducing conditions was 100 units. (A) Growth in liquid medium. (B) Total secreted cellulase activity. (C) Total secreted xylanase activity.

Fig 4

Inhibition of germination of A. oryzae by glucose. Strain RIB40, the creBΔ strain, and the creBΔ::creB⁺ strain (left to right) were grown from 500 spores for 2 days at 30°C on 1% fructose (top) or 1% glucose (bottom).

Fig 5

Deletion of creB increases secretion of amylases under various conditions via an increase in gene transcription. (A) Triplicate spore-inoculated 50-ml shake flask cultures of the three strains were grown under noninducing (1% sorbitol), inducing (1% sorbitol plus 1% starch), and repressing (1% sorbitol plus 1% starch plus 2% sucrose) conditions. After 48 h, biomass and supernatants were harvested, and total secreted amylase activities were measured using EnzChek amylase substrate and are expressed as activities per unit of dry weight biomass, normalized such that activity in the wild-type strain under inducing conditions was 100 units. (B) qRT-PCR analysis of total α-amylase transcript levels in A. oryzae RIB40 and the creBΔ strain. (C) qRT-PCR analysis of glucoamylase A transcript levels in A. oryzae RIB40 and the creBΔ strain. Strains were induced using starch (1%) or repressed using sucrose (2%) and were grown at 30°C for 24 h. Transcript levels were standardized against β-tubulin levels. (D) qRT-PCR analysis of alcA transcript levels in A. nidulans wild-type and creB1936 strains. Strains were induced using EMK (50 mM) or repressed using glucose (1%) and were grown at 37°C for 16 to 18 h. Transcript levels were standardized against β-tubulin levels. Results shown are fold changes compared to the wild type induced with ethanol.

Fig 6

Analysis of DAR3699 creB locus. The schematic shows a representation of the region at the 5′ end of the creB gene in A. nidulans, A. oryzae RIB40, and A. oryzae DAR3699 (not to scale). The top line represents the 5′ region of A. nidulans creB, showing the mapped start points of transcription (6) and an upstream open reading frame of 12 codons that is conserved in A. oryzae. The middle line represents the 5′ region of A. oryzae RIB40 creB, showing a putative 11-codon upstream open reading frame spanning the site that is the major start point of transcription in A. nidulans. The bottom line represents the 5′ region of A. oryzae DAR3699 creB, showing the effect of the insertion of one base pair into the upstream open reading frame sequence, lengthening it to 46 codons.