Characterization of Nerolidol Biotransformation Based on Indirect On-Line Estimation of Biomass Concentration and Physiological State in Batch Cultures of Aspergillus niger

Hrdlicka, Patrick J.; Sørensen, Anders B.; Poulsen, Bjarne Rask; Ruijter, George J. G.; Visser, Jaap; Iversen, Jens

doi:10.1021/bp034137f

Cited by 14 publications

(6 citation statements)

References 34 publications

(41 reference statements)

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“…The defined medium had a pH of 3 and was balanced such, that carbon (maltose) was the growth limiting nutrient. During exponential growth ( μ max = 0.24h −1 ), pH 3 was maintained by alkaline addition (Figure 1B), which linearly correlated with the biomass accumulation and was previously shown to reflect ammonium uptake during balanced growth on minimal medium [24]. The end of the exponential growth phase was detected by an increase of the dissolved oxygen signal (Figure 1B) and depletion of the carbon source was confirmed by measurements of maltose and glucose concentrations (not shown).…”

Section: Resultssupporting

confidence: 53%

The carbon starvation response of Aspergillus niger during submerged cultivation: Insights from the transcriptome and secretome

et al. 2012

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BackgroundFilamentous fungi are confronted with changes and limitations of their carbon source during growth in their natural habitats and during industrial applications. To survive life-threatening starvation conditions, carbon from endogenous resources becomes mobilized to fuel maintenance and self-propagation. Key to understand the underlying cellular processes is the system-wide analysis of fungal starvation responses in a temporal and spatial resolution. The knowledge deduced is important for the development of optimized industrial production processes.ResultsThis study describes the physiological, morphological and genome-wide transcriptional changes caused by prolonged carbon starvation during submerged batch cultivation of the filamentous fungus Aspergillus niger. Bioreactor cultivation supported highly reproducible growth conditions and monitoring of physiological parameters. Changes in hyphal growth and morphology were analyzed at distinct cultivation phases using automated image analysis. The Affymetrix GeneChip platform was used to establish genome-wide transcriptional profiles for three selected time points during prolonged carbon starvation. Compared to the exponential growth transcriptome, about 50% (7,292) of all genes displayed differential gene expression during at least one of the starvation time points. Enrichment analysis of Gene Ontology, Pfam domain and KEGG pathway annotations uncovered autophagy and asexual reproduction as major global transcriptional trends. Induced transcription of genes encoding hydrolytic enzymes was accompanied by increased secretion of hydrolases including chitinases, glucanases, proteases and phospholipases as identified by mass spectrometry.ConclusionsThis study is the first system-wide analysis of the carbon starvation response in a filamentous fungus. Morphological, transcriptomic and secretomic analyses identified key events important for fungal survival and their chronology. The dataset obtained forms a comprehensive framework for further elucidation of the interrelation and interplay of the individual cellular events involved.

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Section: Resultssupporting

confidence: 53%

The carbon starvation response of Aspergillus niger during submerged cultivation: Insights from the transcriptome and secretome

et al. 2012

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“…In two independent runs, the maximal specific growth rate achieved was 0.24 h −1 and the biomass concentration peaked at 4.2 g · kg −1 culture broth after about 26 h post inoculation (Figure 7 A). During exponential growth, pH 3 was maintained by the addition of 1 M NaOH, which has been shown to linearly correlate with the biomass accumulation and reflecting ammonium uptake during balanced growth [ 9 ],[ 43 ]. The end of the exponential growth phase was detected by an increase of the dissolved oxygen signal (data not shown), after which the cell mass decreased by nearly 50% (Figure 7 A).…”

Section: Resultsmentioning

confidence: 99%

Engineering of Aspergillus niger for the production of secondary metabolites

Richter¹,

Wanka²,

Boecker³

et al. 2014

Fungal Biol Biotechnol

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BackgroundFilamentous fungi can each produce dozens of secondary metabolites which are attractive as therapeutics, drugs, antimicrobials, flavour compounds and other high-value chemicals. Furthermore, they can be used as an expression system for eukaryotic proteins. Application of most fungal secondary metabolites is, however, so far hampered by the lack of suitable fermentation protocols for the producing strain and/or by low product titers. To overcome these limitations, we report here the engineering of the industrial fungus Aspergillus niger to produce high titers (up to 4,500 mg • l−1) of secondary metabolites belonging to the class of nonribosomal peptides.ResultsFor a proof-of-concept study, we heterologously expressed the 351 kDa nonribosomal peptide synthetase ESYN from Fusarium oxysporum in A. niger. ESYN catalyzes the formation of cyclic depsipeptides of the enniatin family, which exhibit antimicrobial, antiviral and anticancer activities. The encoding gene esyn1 was put under control of a tunable bacterial-fungal hybrid promoter (Tet-on) which was switched on during early-exponential growth phase of A. niger cultures. The enniatins were isolated and purified by means of reverse phase chromatography and their identity and purity proven by tandem MS, NMR spectroscopy and X-ray crystallography. The initial yields of 1 mg • l−1 of enniatin were increased about 950 fold by optimizing feeding conditions and the morphology of A. niger in liquid shake flask cultures. Further yield optimization (about 4.5 fold) was accomplished by cultivating A. niger in 5 l fed batch fermentations. Finally, an autonomous A. niger expression host was established, which was independent from feeding with the enniatin precursor d-2-hydroxyvaleric acid d-Hiv. This was achieved by constitutively expressing a fungal d-Hiv dehydrogenase in the esyn1-expressing A. niger strain, which used the intracellular α-ketovaleric acid pool to generate d-Hiv.ConclusionsThis is the first report demonstrating that A. niger is a potent and promising expression host for nonribosomal peptides with titers high enough to become industrially attractive. Application of the Tet-on system in A. niger allows precise control on the timing of product formation, thereby ensuring high yields and purity of the peptides produced.Electronic supplementary materialThe online version of this article (doi:10.1186/s40694-014-0004-9) contains supplementary material, which is available to authorized users.

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“…When using nitrogen assimilation activity as a proxy for growth [62], this indicated fungal growth on IL pretreated substrates from 3 to 24 h, reflecting high digestibility of the IL pretreated substrates (Additional file 1: Fig. S1).…”

Section: Pretreatment and Feedstock Effects Dominate Gene Expression mentioning

confidence: 99%

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose

Munster

Daly

Blythe

et al. 2020

Biotechnol Biofuels

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Background: Understanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production. Results: We analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZymeencoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds. Conclusion: In this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism.

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Characterization of Nerolidol Biotransformation Based on Indirect On-Line Estimation of Biomass Concentration and Physiological State in Batch Cultures of Aspergillus niger

Cited by 14 publications

References 34 publications

The carbon starvation response of Aspergillus niger during submerged cultivation: Insights from the transcriptome and secretome

The carbon starvation response of Aspergillus niger during submerged cultivation: Insights from the transcriptome and secretome

Engineering of Aspergillus niger for the production of secondary metabolites

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose

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