RNA-Seq Based Transcriptome Analysis of Aspergillus oryzae DSM 1863 Grown on Glucose, Acetate and an Aqueous Condensate from the Fast Pyrolysis of Wheat Straw.

Due to its acetate content, the pyrolytic aqueous condensate (PAC) formed during the fast pyrolysis of wheat straw could provide an inexpensive substrate for microbial fermentation. However, PAC also contains several inhibitors that make its detoxification inevitable. In our study, we examined the transcriptional response of Aspergillus oryzae to cultivation on 20% detoxified PAC, pure acetate and glucose using RNA-seq analysis. Functional enrichment analysis of 3463 significantly differentially expressed (log2FC >2 & FDR < 0.05) genes revealed similar metabolic tendencies for both acetate and PAC, as upregulated genes in these cultures were mainly associated with ribosomes and RNA processing, whereas transmembrane transport was downregulated. Unsurprisingly, metabolic pathway analysis revealed that glycolysis/gluconeogenesis and starch and sucrose metabolism were upregulated for glucose, whereas glyoxylate and the tricarboxylic acid (TCA) cycle were important carbon utilization pathways for acetate and PAC, respectively. Moreover, genes involved in the biosynthesis of various amino acids such as arginine, serine, cysteine and tryptophan showed higher expression in the acetate-containing cultures. Direct comparison of the transcriptome profiles of acetate and PAC revealed that pyruvate metabolism was the only significantly different metabolic pathway and was overexpressed in the PAC cultures. Upregulated genes included those for methylglyoxal degradation and alcohol dehydrogenases, which thus represent potential targets for the further improvement of fungal PAC tolerance.

Detoxification of a pyrolytic aqueous condensate from wheat straw for utilization as substrate in Aspergillus oryzae DSM 1863 cultivations.

BACKGROUND: The pyrolytic aqueous condensate (PAC) formed during the fast pyrolysis of wheat straw contains a variety of organic carbons and might therefore potentially serve as an inexpensive substrate for microbial growth. One of its main components is acetic acid, which was recently shown to be a suitable carbon source for the filamentous fungus Aspergillus oryzae. However, the condensate also contains numerous toxic compounds that inhibit fungal growth and result in a tolerance of only about 1%. Therefore, to enable the use of the PAC as sole substrate for A. oryzae cultivations, a pretreatment seems to be necessary. RESULTS: Various conditions for treatments with activated carbon, overliming, rotary evaporation and laccase were evaluated regarding fungal growth and the content of inhibitory model substances. Whereas the first three methods considerably increased the fungal tolerance to up to 1.625%, 12.5% and 30%, respectively, the enzymatic treatment did not result in any improvement. The optimum carbon load for the treatment with activated carbon was identified to be 10% (w/v) and overliming should ideally be performed at 100 °C and an initial pH of 12. The best detoxification results were achieved with rotary evaporation at 200 mbar as a complete removal of guaiacol and a strong reduction in the concentration of acetol, furfural, 2-cyclopenten-1-one and phenol by 84.9%, 95.4%, 97.7% and 86.2%, respectively, were observed. Subsequently, all possible combinations of the effective single methods were performed and rotary evaporation followed by overliming and activated carbon treatment proved to be most efficient as it enabled growth in 100% PAC shake-flask cultures and resulted in a maximum cell dry weight of 5.21 ± 0.46 g/L. CONCLUSION: This study provides a comprehensive insight into the detoxification efficiency of a variety of treatment methods at multiple conditions. It was revealed that with a suitable combination of these methods, PAC toxicity can be reduced to such an extent that growth on pure condensate is possible. This can be considered as a first important step towards a microbial valorization of the pyrolytic side-stream with A. oryzae.

Incubation time after pulsed electric field treatment of microalgae enhances the efficiency of extraction processes and enables the reduction of specific treatment energy.

Pulsed Electric Field (PEF) pre-treatment, applied on fresh microalgae Auxenochlorella protothecoides, induces spontaneous release of a substantial water fraction and enables subsequent lipid extraction using ethanol-hexane blends. In this study, fresh microalgae suspensions were treated with PEF and incubated under inert conditions. Incubation promotes the release of ions and carbohydrates and increases the yields of subsequent lipid extraction thus enabling a considerable reduction of PEF-treatment energy. With a 20 h incubation period at 25 °C, almost total lipid extraction is achieved with a specific PEF-treatment energy of only 0.25 MJ/kgDW. Incubation on ice remains beneficial but less efficient than at 25 °C. Additionally, incubating microalgae cells in suspension at 100gDW/L or in a dense paste, was almost equally efficient. Correlation between the different results suggests that spontaneous release of ions and carbohydrates facilitates more successful lipid extraction. A direct causality between the two phenomena remains to be demonstrated.

Conditional Expression of the Small GTPase ArfA Impacts Secretion, Morphology, Growth, and Actin Ring Position in Aspergillus niger.

In filamentous fungi, growth and protein secretion occurs predominantly at the tip of long, thread like cells termed hyphae. This requires coordinated regulation of multiple processes, including vesicle trafficking, exocytosis, and endocytosis, which are facilitated by a complex cytoskeletal apparatus. In this study, functional analyses of the small GTPase ArfA from Aspergillus niger demonstrate that this protein functionally complements the Saccharomyces cerevisiae ARF1/2, and that this protein is essential for A. niger. Loss-of-function and gain-of-function analyses demonstrate that titration of arfA expression impacts hyphal growth rate, hyphal tip morphology, and protein secretion. Moreover, localization of the endocytic machinery, visualized via fluorescent tagging of the actin ring, was found to be abnormal in ArfA under- and overexpressed conditions. Finally, we provide evidence that the major secreted protein GlaA localizes at septal junctions, indicating that secretion in A. niger may occur at these loci, and that this process is likely impacted by arfA expression levels. Taken together, our results demonstrate that ArfA fulfills multiple functions in the secretory pathway of A. niger.

Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion.

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.

HisB as novel selection marker for gene targeting approaches in Aspergillus niger.

BACKGROUND: For Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. niger. RESULTS: A histidine-auxotrophic strain was established via disruption of the A. niger hisB gene by using the counterselectable pyrG marker. After curing, a hisB - , pyrG - strain was obtained, which served as recipient strain for further studies. We show here that both hisB orthologs from A. nidulans and A. niger can be used to reestablish histidine prototrophy in this recipient strain. Whereas the hisB gene from A. nidulans was suitable for efficient gene targeting at different loci in A. niger, the hisB gene from A. niger allowed efficient integration of a Tet-on driven luciferase reporter construct at the endogenous non-functional hisB locus. Subsequent analysis of the luciferase activity revealed that the hisB locus is tight under non-inducing conditions and allows even higher luciferase expression levels compared to the pyrG integration locus. CONCLUSION: Taken together, we provide here an alternative selection marker for A. niger, hisB, which allows efficient homologous integration rates as well as high expression levels which compare favorably to the well-established pyrG selection marker.