Impact of overexpressing NADH kinase on glucoamylase production in Aspergillus niger

Glucoamylase has a wide range of applications in the production of glucose, antibiotics, amino acids, and other fermentation industries. Fungal glucoamylase, in particular, has attracted much attention because of its wide application in different industries, among which Aspergillus niger is the most popular strain producing glucoamylase. The low availability of NADPH was found to be one of the limiting factors for the overproduction of glucoamylase. In this study, 3 NADH kinases (AN03, AN14, and AN17) and malic enzyme (maeA) were overexpressed in aconidial A. niger by CRISPR/Cas9 technology, significantly increasing the size of the NADPH pool, resulting in the activity of glucoamylase was improved by about 70%, 50%, 90%, and 70%, respectively; the total secreted protein was increased by about 25%, 22%, 52%, and 26%, respectively. Furthermore, the combination of the mitochondrial NADH kinase (AN17) and the malic enzyme (maeA) increased glucoamylase activity by a further 19%. This study provided an effective strategy for enhancing glucoamylase production of A. niger.     

Physicochemical Properties Analysis and Secretome of Aspergillus niger in Fermented Rapeseed Meal

The nutrient digestibility and feeding value of rapeseed meal (RSM) for non-ruminant animals is poor due to the presence of anti-nutritional substances such as glucosinolate, phytic acid, crude fiber etc. In the present study, a solid state fermentation (SSF) using Aspergillus niger was carried out with the purpose of improving the nutritional quality of RSM. The chemical composition and physicochemical properties of RSM before and after fermentation were compared. To further understand possible mechanism of solid state fermentation, the composition of extracellular enzymes secreted by Aspergillus niger during fermentation was analysed using two-dimentional difference gel electrophoresis (2D-DIGE) combined with matrix assisted laser desorption ionization—time of flight—mass spectrometer (MALDI-TOF-MS). Results of the present study indicated that SSF had significant effects on chemical composition of RSM. The fermented rapeseed meal (FRSM) contained more crude protein (CP) and amino acid (AA) (except His) than unfermented RSM. Notably, the small peptide in FRSM was 2.26 time larger than that in unfermented RSM. Concentrations of anti-nutritional substrates in FRSM including neutral detergent fiber (NDF), glucosinolates, isothiocyanate, oxazolidithione, and phytic acid declined (P < 0.05) by 13.47, 43.07, 55.64, 44.68 and 86.09%, respectively, compared with unfermented RSM. A. niger fermentation disrupted the surface structure, changed macromolecular organic compounds, and reduced the protein molecular weights of RSM substrate. Total proteins of raw RSM and FRSM were separated and 51 protein spots were selected for mass spectrometry according to 2D-DIGE map. In identified proteins, there were 15 extracellular hydrolases secreted by A. niger including glucoamylase, acid protease, beta-glucanase, arabinofuranosidase, xylanase, and phytase. Some antioxidant related enzymes also were identified. These findings suggested that A. niger is able to secrete many extracellular degradation enzymes (especially lignocellulosic hydrolyzing enzymes, acid proteases and phytase) during fermentation of RSM, thus altering chemical composition and physicochemical properties of RSM.     

产糖化酶黑曲霉固定化方法比较的研究

采用海藻酸钙凝胶电埋法、以沸石、多孔聚酯等材料为固定化载体的吸附法固定黑曲霉（Aspergillus niger AS3.4309)菌丝细胞,以游离菌丝体作为对照,进行发酵产糖化酶的比较,结果表明：以聚酯泡沫作为固定化载体吸附固定化菌丝细胞产糖化酶活力最高。在产糖化酶的发酵过程中,与游离菌丝体细胞相比,固定化黑曲霉持续产酶时间有一定程度的延长。     

Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae

The DNA (glaB) and a cDNA-encoding glucoamylase produced in solid-state culture of Aspergillus oryzae were cloned using oligodeoxyribonucleotide probes derived from internal amino acid sequences of the enzyme. Comparison of the nucleotide sequences of a genomic DNA fragment with its cDNA showed the glaB gene carried three exons interrupted by two introns and had an open reading frame encoding 493 aa residues. The 5′-flanking region had a TATA box at nt −87 from the start codon and two putative CAAT sequences at nt −276 and −288. The glaB gene shared 57% homology at the aa level with the glaA gene which was cloned previously from A. oryzae. Interestingly, the glucoamylase encoded by the glaB gene had no C-terminal domain such as that proposed to have starch binding activity in Aspergillus glucoamylases. Introduction of cDNA of the glaB gene to Saccharomyces cerevisiae caused the secretion of active glucoamylase to culture medium and introduction of the glaB gene to A. oryzae increased glucoamylase productivity in solid-state culture. Northern blot analysis showed the glaB gene was expressed in solid-state culture, but not in submerged culture.     

Efficient and Direct Fermentation of Starch to Ethanol by Sake Yeast Strains Displaying Fungal Glucoamylases

Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.     

Glucoamylase production by Aspergillus niger on rice bran is improved by adding nitrogen sources

Corn steep liquor, peptone or NH _inf4 ^sup+ salts increased the yield of glucoamylase from Aspergillus niger growing in a solid-state fermentation on rice bran up to 360 IU/g dry substrate over 96 h at 30°C.The authors are with the Biotechnology Unit, Regional Research Laboratory, CSIR, Trivandrum-695 019, India;     

The Effect of organic nitrogen sources on recombinant glucoamylase production by Aspergillus niger in chemostat culture

Aspergillus niger B1, a recombinant strain carrying 20 extra copies of the native glucoamylase gene, was grown in glucose-limited chemostat cultures supplemented with various organic nitrogen sources (dilution rate 0.12 +/- 0.01 h(-1), pH 5.4). In cultures supplemented with l-alanine, l-methionine, casamino acids, or peptone, specific glucoamylase (GAM) production rapidly decreased to less than 20% of the initial level. Reducing the pH of the culture to 4.0 resulted in stable GAM production for up to 400 h. Morphological mutants (a light brown and a dark brown mutant) appeared in each fermentation and generally displaced B1. Light brown mutants had higher selection coefficients relative to B1 than dark brown mutants and became the dominant strain in all fermentations except those maintained at pH 4.0. Several mutants isolated from these cultures had reduced ability to produce GAM in batch culture, although few had lost copies of the glaA gene. Some mutants had methylated DNA.     

Screening for strains of Aspergillus oryzae that degrade carbamide for use in sake brewing

We screened 65 strains of Aspergillus oryzae for ones that degraded carbamide, Two strains, IFO 5238 and IFO 30113, were selected and used to prepare rice koji. Sake made with the rice koji prepared with these strains had less than half (less than 15 ppm) of the carbamide in sake made with rice koji prepared with commercial spores of A. oryzae.     

Influence of water activity on growth and activity of Aspergillus niger for glycoamylase production in solid-state fermentation

Growth of Aspergillus niger and glucoamylase production correlated well with the water activity of the substrate (wheat bran plus corn flour) in a solid-state fermentation. Both were maximal at an initial water activity of 0.936. Glycoamylase reached 550 units/g dry substrate after 96 h.The authors are with the Biotechnology Unit, Regional Research Laboratory, CSIR, Trivandrum-695 019, India     

Isolation and Sequencing of a New Glucoamylase Gene from an Aspergillus niger Aggregate Strain (DSM 823) Molecularly Classified as Aspergillus tubingensis

Based on morphological characteristics the taxa included in the Aspergillus aggregate can hardly be differentiated. For that reason the phylogeny of this genus was revised several times as different criteria, from morphological to later molecular, were used. We found, comparing nucleotide sequences of the ITS-region, that the strain Aspergillus niger (DSM 823) which is claimed to be identical to the strains ATCC 10577, IMI 027809, NCTC 7193 and NRRL 2322 can be molecularly classified as Aspergillus tubingensis, exhibiting 100% identity with the A. tubingensis CBS strains 643.92 and 127.49. We amplified, cloned and sequenced a new glucoamylase gene (glaA) from this strain of A. tubingensis (A. niger DSM 823) using primers derived from A. niger glucoamylase G1. The amplified cDNA fragment of 2013 bp contained an open reading frame encoding 648 amino acid residues. The calculated molecular mass of the glucoamylase, deduced from the amino acid sequence, was 68 kDa. The nucleotide sequence of glaA showed 99% similarity with glucoamylases from Aspergillus kawachii and Aspergillus shirousami, whereas the similarity with the glucoamylase G1 from A. niger was 92% An erratum to this article is available at .     

13C n.m.r. studies of the carbohydrate portion of glucoamylase from Aspergillus oryzae

Proton-decoupled, natural abundance ¹³C n.m.r. spectroscopy was used to investigate the carbohydrate structure and content of glucoamylase from Aspergillus oryzae. We found α-d-mannopyranose was the dominant sugar present (⋍91 residues). The Elson-Morgan assay showed that hexosamine was also present as a minor component (2.6% of the total carbohydrate). The intermannose linkages appear to be random. Integration data suggest that 41 α-d-mannopyranose residues are O-2 and O-3 glycosylated and 17 α-d-mannopyranose residues are involved in O-4 glycosylation. Treatment of glucoamylase with α-mannosidase appeared to remove all the carbohydrate residues present.     

Enhancement of operation and storage stability of glucoamylase from Aspergillus awamori by a protease inhibitor preparation

Glucoamylase was produced extracellularly by fermentation of strain Aspergillus awamori, which had been genetically modified to have high-level glucoamylase activity. Initial experiments showed that the enzyme deactivated quickly, with a half-life of less than 6 days even stored at 5°C. A possible reason for the rapid deactivation was the presence of proteases, attacking and degrading the glucoamylase. Therefore a liquid protease inhibitor cocktail (Sigma, USA) was selected and applied to enhance the stability of the enzyme. The activity of the enzyme (stored at 5°C) measured by the Schoorl-method with starch as substrate showed that the cocktail was effective with the enzyme maintaining 95% of its initial storage activity for almost one year. The enzyme preparation has been used for starch hydrolysis in a flat-sheet membrane bioreactor at 60°C to manufacture glucose solution and its operation stability extended by using the cocktail.     

The effect of combining signal sequences with the N28 fragment on GFP production in Aspergillus oryzae

Effective secretion of green fluorescent protein (GFP) was investigated by the screening signal sequences for GFP secretion in Aspergillus oryzae. GFP production in A. oryzae was evaluated using fusions with signal sequences from Taka-amylase A (TAA), glucoamylase A, glucoamylase B, and triacylglycerol lipase. The TAA signal sequence promoted the highest protein secretion of GFP. Fusing this signal sequence with an N-terminal 28-amino acid region (N28 fragment) from the Rhizopus oryzae lipase signal sequence increased protein secretion. In addition, using multiple copies of this signal sequence, instead of the N28 fragment, also induced protein secretion. These results show that using multiple signal sequences or combining a signal sequence with the N28 fragment can be used to improve heterogeneous protein secretion in A. oryzae.     

The effect of point amino acid substitutions in an internal α-helix on thermostability of Aspergillus awamori X100 glucoamylase

Conformational flexibility of α-helices in glucoamylase of the fungus Aspergillus awamori was studied by molecular dynamics methods. Several amino acid substitutions (G127A, P128A, I136L, G137A, and G139A) optimizing intrinsic interactions in one of the α-helices (D) within the hydrophobic core of this protein were constructed and studied. It was found that these point mutations had different effects on the glucoamylase thermal inactivation constant. Unlike amino acid substitution P128A and substitutions G137A and A246C, I136L and G139A displayed a pronounced additive thermostabilizing effect.     

Development of a method for efficient cost-effective screening of <Emphasis Type="Italic">Aspergillus niger</Emphasis> mutants having increased production of glucoamylase

Objectives

To develop an efficient cost-effective screening process to improve production of glucoamylase in Aspergillus niger.

Results

The cultivation of A. niger was achieved with well-dispersed morphology in 48-deep-well microtiter plates, which increased the throughput of the samples compared to traditional flask cultivation. There was a close negative correlation between glucoamylase and its pH of the fermentation broth. A novel high-throughput analysis method using Methyl Orange was developed. When compared to the conventional analysis method using 4-nitrophenyl α-D-glucopyranoside as substrate, a correlation coefficient of 0.96 by statistical analysis was obtained.

Conclusion

Using this novel screening method, we acquired a strain with an activity of 2.2 × 10³ U ml^?1, a 70% higher yield of glucoamylase than its parent strain.

     

携多拷贝glaA的重组黑曲霉过量合成糖化酶的研究

以工业生产菌株黑曲霉CICIMF0410基因组DNA为模板,扩增出糖化酶glaA基因,测序并进行表达研究。GlaA基因的核苷酸序列长为2167bp,包含4个内含子。氨基酸序列比对表明此黑曲霉糖化酶与其他曲霉属来源的糖化酶有很高的同源性。将glaA基因克隆到pBC-Hygro载体中,构建重组质粒pBC-Hygro-glaA并转化A.nigerF0410。携多拷贝glaA的转化子用150μg/mL潮霉素抗性筛选并通过荧光实时定量PCR鉴定。结果表明,在染色体整合2~3倍糖化酶基因对糖化酶的过量合成是适宜的,有助于提高糖化酶活力。对转化子进行摇瓶发酵研究,发酵终止时转化子GB0506的糖化酶活力比出发菌株F0410提高了17.5%。因此,增加黑曲霉染色体糖化酶基因的拷贝数可以显著提高糖化酶活力。