Growth and product formation in chemostat and recycling cultures by Aspergillus niger N402 and a glucoamylase overproducing transformant, provided with multiple copies of the glaA gene

ABSTRACTExposure to an aerial environment or severe nutrient limitation induces asexual differentiation in filamentous fungi. Submerged cultivation ofAspergillus nigerin carbon- and energy-limited retentostat cultures both induces and fuels conidiation. Physiological and transcriptomic analyses have revealed that this differentiation strongly affects product formation. Since conidiation is inherent in the aerial environment, we hypothesized that product formation near zero growth can be influenced by affecting differentiation or development of aerial hyphae in general. To investigate this idea, three developmental mutants (ΔfwnA,scl-1, andscl-2mutants) that have no apparent vegetative growth defects were cultured in maltose-limited retentostat cultures. The secondary-metabolite profile of the wild-type strain defined flavasperone, aurasperone B, tensidol B, and two so far uncharacterized compounds as associated with conidium formation, while fumonisins B2, B4, and B6were characteristic of early response to nutrient limitation by the vegetative mycelium. The developmental mutants responded differently to the severe substrate limitation, which resulted in distinct profiles of growth and product formation.fwnAencodes the polyketide synthase responsible for melanin biosynthesis during aerial differentiation, and we show that conidial melanin synthesis in submerged retentostat cultures and aurasperone B production arefwnAdependent. Thescl-1andscl-2strains are two UV mutants generated in the ΔfwnAbackground that displayed reduced asexual conidiation and formed sclerotium-like structures on agar plates. The reduced conidiation phenotypes of thescl-1andscl-2strains are reflected in the retentostat cultivation and are accompanied by elimination or severely reduced accumulation of secondary metabolites and distinctly enhanced accumulation of extracellular protein. This investigation shows that submerged conidiation and product formation of a mitosporic fungus cultured at low specific growth rates can be fundamentally affected by interfering with the genetic program for differentiation of aerial hyphae, opening new perspectives for tailoring industrial performance.

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Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger

10.21203/rs.3.rs-42423/v1 ◽

2020 ◽

Author(s):

Yu-fei Sui ◽

Tabea Schütze ◽

Li-Ming Ouyang ◽

Hong-zhong Lu ◽

Peng Liu ◽

...

Keyword(s):

Metabolic Engineering ◽

Aspergillus Niger ◽

Gene Copy ◽

Cell Factory ◽

Flux Analysis ◽

Cofactor Engineering ◽

Nadph Regeneration ◽

Gene Copies ◽

Glaa Gene ◽

Glucoamylase Production

Abstract Background: Nicotinamide 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. Results: We 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 regeneration enzymes under the control of 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 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. Conclusions: This 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 reverse TCA cycle ( maeA gene) followed by engineering the flux through the pentose phosphate pathway ( gndA 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.

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Studi Kinetika Konsumsi Glukosa oleh Aspergillus Niger dalam Produksi Bioethanol dari Lignoselulosa

INTEK Jurnal Penelitian ◽

10.31963/intek.v4i1.98 ◽

2017 ◽

Vol 4 (1) ◽

pp. 14

Author(s):

Setyo Erna Widiyanti

Keyword(s):

Growth Rate ◽

Citric Acid ◽

Aspergillus Niger ◽

Kinetic Parameters ◽

Glucose Concentration ◽

Raw Materials ◽

Glucose Consumption ◽

Product Formation ◽

Initial Glucose Concentration ◽

Kinetics Of

Global warming resulted from CO2 level increase in the atmosphere has caused elevation of earth temperature and uncertain climate changes. To prevent the rise of CO2 in the atmosphere can be done by using biomass fuel such as bioethanol. The raw materials of bioethanol can be derived from oil palm empty fruit bunch. Enzymatic hydrolysis utilizes cellulase-producing fungus and in this research, Aspergillus niger was chosen. The glucose produced is consumed by A niger as carbon source and this is undesirable, therefore it should be minimized as low as possible. Knowing the rate of glucose consumption is important to have a model of the hydrolysis reaction rate which will be helpful in the design process on an industrial scale hydrolysis reactor. This study aimed to determine the equations that can be used to approximate the growth rate of A. niger, glucose consumption, the formation of citric acid, and the kinetic parameters used to modeling the kinetics of glucose consumption by A. niger. Kinetics of glucose consumption by A. niger was studied in batch system with variation of initial glucose concentration of 30, 50, 70 g/l. The growth rate of A. niger, glucose consumption, and the formation of citric acid were modeled using 3 equations; i.e. Monod with non-competitive product inhibition, Luedeking-Piret, and Luedeking-Piret growth associated product formation, respectively. The values of kinetic parameters such as μmax, Ks, Kp, were 0.65 hour-1, 157.5 g/l, 0.3 g/l, for initial glucose concentration of 30, 50, 70 g/l, respectively. The values of α (kinetic parameter for growth associated product formation and α would be equal to Yp/x) and Yx/s were 0.4903, 0.8531, 0.9863; 0.5124, 0.2704, 0.2381, for initial glucose concentration of 30, 50, 70 g/l, respectively. Higher initial glucose concentration would increase α but it lowered Yx/s.

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Transcriptomic Insights into the Physiology of Aspergillus niger Approaching a Specific Growth Rate of Zero

Applied and Environmental Microbiology ◽

10.1128/aem.00450-10 ◽

2010 ◽

Vol 76 (16) ◽

pp. 5344-5355 ◽

Cited By ~ 45

Author(s):

Thomas R. Jørgensen ◽

Benjamin M. Nitsche ◽

Gerda E. Lamers ◽

Mark Arentshorst ◽

Cees A. van den Hondel ◽

...

Keyword(s):

Growth Rate ◽

Aspergillus Niger ◽

Specific Growth Rate ◽

Secondary Metabolism ◽

Filamentous Fungi ◽

Growth Rates ◽

Specific Growth ◽

Product Formation ◽

Transcriptomic Analysis ◽

Genes Encoding

ABSTRACT The physiology of filamentous fungi at growth rates approaching zero has been subject to limited study and exploitation. With the aim of uncoupling product formation from growth, we have revisited and improved the retentostat cultivation method for Aspergillus niger. A new retention device was designed allowing reliable and nearly complete cell retention even at high flow rates. Transcriptomic analysis was used to explore the potential for product formation at very low specific growth rates. The carbon- and energy-limited retentostat cultures were highly reproducible. While the specific growth rate approached zero (<0.005 h−1), the growth yield stabilized at a minimum (0.20 g of dry weight per g of maltose). The severe limitation led to asexual differentiation, and the supplied substrate was used for spore formation and secondary metabolism. Three physiologically distinct phases of the retentostat cultures were subjected to genome-wide transcriptomic analysis. The severe substrate limitation and sporulation were clearly reflected in the transcriptome. The transition from vegetative to reproductive growth was characterized by downregulation of genes encoding secreted substrate hydrolases and cell cycle genes and upregulation of many genes encoding secreted small cysteine-rich proteins and secondary metabolism genes. Transcription of known secretory pathway genes suggests that A. niger becomes adapted to secretion of small cysteine-rich proteins. The perspective is that A. niger cultures as they approach a zero growth rate can be used as a cell factory for production of secondary metabolites and cysteine-rich proteins. We propose that the improved retentostat method can be used in fundamental studies of differentiation and is applicable to filamentous fungi in general.

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