Dataset Information

Manipulation of an α-glucosidase in the industrial glucoamylase-producing Aspergillus niger strain O1 to decrease non-fermentable sugars production and increase glucoamylase activity.

ABSTRACT: Dextrose equivalent of glucose from starch hydrolysis is a critical index for starch-hydrolysis industry. Improving glucose yield and decreasing the non]-fermentable sugars which caused by transglycosylation activity of the enzymes during the starch saccharification is an important direction. In this study, we identified two key α-glucosidases responsible for producing non-fermentable sugars in an industrial glucoamylase-producing strain Aspergillus niger O1. The results showed the transglycosylation product panose was decreased by more than 88.0% in agdA/agdB double knock-out strains than strain O1. Additionally, the B-P1 domain of agdB was found accountable as starch hydrolysis activity only, and B-P1 overexpression in ΔAΔB-21 significantly increased glucoamylase activity whereas keeping the glucoamylase cocktail low transglycosylation activity. The total amounts of the transglycosylation products isomaltose and panose were significantly decreased in final strain B-P1-3 by 40.7% and 44.5%, respectively. The application of engineered strains will decrease the cost and add the value of product for starch biorefinery.

SUBMITTER: Guo W

PROVIDER: S-EPMC9633098 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Manipulation of an α-glucosidase in the industrial glucoamylase-producing <i>Aspergillus niger</i> strain O1 to decrease non-fermentable sugars production and increase glucoamylase activity.

Guo Wenzhu W Liu Dandan D Li Jingen J Sun Wenliang W Sun Tao T Wang Xingji X Wang Kefen K Liu Qian Q Tian Chaoguang C

Frontiers in microbiology 20221020

Dextrose equivalent of glucose from starch hydrolysis is a critical index for starch-hydrolysis industry. Improving glucose yield and decreasing the non]-fermentable sugars which caused by transglycosylation activity of the enzymes during the starch saccharification is an important direction. In this study, we identified two key α-glucosidases responsible for producing non-fermentable sugars in an industrial glucoamylase-producing strain <i>Aspergillus niger</i> O1. The results showed the transg ...[more]

PMID: 36338048

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Project description:BACKGROUND: Filamentous fungi such as Aspergillus niger are well known for their exceptionally high capacity for secretion of proteins, organic acids, and secondary metabolites and they are therefore used in biotechnology as versatile microbial production platforms. However, system-wide insights into their metabolic and secretory capacities are sparse and rational strain improvement approaches are therefore limited. In order to gain a genome-wide view on the transcriptional regulation of the protein secretory pathway of A. niger, we investigated the transcriptome of A. niger when it was forced to overexpression the glaA gene (encoding glucoamylase, GlaA) and secrete GlaA to high level. RESULTS: An A. niger wild-type strain and a GlaA over-expressing strain, containing multiple copies of the glaA gene, were cultivated under maltose-limited chemostat conditions (specific growth rate 0.1 h-1). Elevated glaA mRNA and extracellular GlaA levels in the over-expressing strain were accompanied by elevated transcript levels from 772 genes and lowered transcript levels from 815 genes when compared to the wild-type strain. Using GO term enrichment analysis, four higher-order categories were identified in the up-regulated gene set: i) endoplasmic reticulum (ER) membrane translocation, ii) protein glycosylation, iii) vesicle transport, and iv) ion homeostasis. Among these, about 130 genes had predicted functions for the passage of proteins through the ER and those genes included target genes of the HacA transcription factor that mediates the unfolded protein response (UPR), e.g. bipA, clxA, prpA, tigA and pdiA. In order to identify those genes that are important for high-level secretion of proteins by A. niger, we compared the transcriptome of the GlaA overexpression strain of A. niger with six other relevant transcriptomes of A. niger. Overall, 40 genes were found to have either elevated (from 36 genes) or lowered (from 4 genes) transcript levels under all conditions that were examined, thus defining the core set of genes important for ensuring high protein traffic through the secretory pathway. CONCLUSION: We have defined the A. niger genes that respond to elevated secretion of GlaA and, furthermore, we have defined a core set of genes that appear to be involved more generally in the intensified traffic of proteins through the secretory pathway of A. niger. The consistent up-regulation of a gene encoding the acetyl-coenzyme A transporter suggests a possible role for transient acetylation to ensure correct folding of secreted proteins.

Project description:BACKGROUND:The lifestyle of filamentous fungi depends on the secretion of hydrolytic enzymes into the surrounding medium, which degrade polymeric substances into monomers that are then taken up to sustain metabolism. This feature has been exploited in biotechnology to establish platform strains with high secretory capacity including Aspergillus niger. The accepted paradigm is that proteins become mainly secreted at the tips of fungal hyphae. However, it is still a matter of debate if the amount of growing hyphal tips in filamentous fungi correlates with an increase in secretion, with previous studies showing either a positive or no correlation. RESULTS:Here, we followed a systematic approach to study protein secretion in A. niger. First, we put the glaA gene encoding for glucoamylase (GlaA), the most abundant secreted protein of A. niger, under control of the tunable Tet-on system. Regulation of glaA gene expression by omitting or adding the inducer doxycycline to cultivation media allowed us to study the effect of glaA under- or overexpression in the same isolate. By inducing glaA expression in a fluorescently tagged v-SNARE reporter strain expressing GFP-SncA, we could demonstrate that the amount of post-Golgi carriers indeed depends on and correlates with glaA gene expression. By deleting the racA gene, encoding the Rho-GTPase RacA in this isolate, we generated a strain which is identical to the parental strain with respect to biomass formation but produces about 20% more hyphal tips. This hyperbranching phenotype caused a more compact macromorphology in shake flask cultivations. When ensuring continuous high-level expression of glaA by repeated addition of doxycycline, this hyperbranching strain secreted up to four times more GlaA into the culture medium compared to its parental strain. CONCLUSION:The data obtained in this study strongly indicate that A. niger responds to forced transcription of secretory enzymes with increased formation of post-Golgi carriers to efficiently accommodate the incoming cargo load. This physiological adaptation can be rationally exploited to generate hypersecretion platforms based on a hyperbranching phenotype. We propose that a racA deletion background serves as an excellent chassis for such hypersecretion strains.

Project description:BackgroundNicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction.ResultsWe thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production.ConclusionsThis data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway (gndA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.

Project description:BackgroundOur recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction.ResultsBased on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction.ConclusionThis study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory.

Dataset Information

Manipulation of an α-glucosidase in the industrial glucoamylase-producing Aspergillus niger strain O1 to decrease non-fermentable sugars production and increase glucoamylase activity.

Publications

Manipulation of an α-glucosidase in the industrial glucoamylase-producing <i>Aspergillus niger</i> strain O1 to decrease non-fermentable sugars production and increase glucoamylase activity.

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