Applicability of non-invasive and live-cell holotomographic imaging on fungi.

The ability to acquire three-dimensional (3D) information of cellular structures without the need for fluorescent tags or staining makes holotomographic imaging a powerful tool in cellular biology. It provides valuable insights by measuring the refractive index (RI), an optical parameter describing the phase delay of light that passes through the living cell. Here, we demonstrate holotomographic imaging on industrial relevant ascomycete fungi and study their development and morphogenesis. This includes conidial germination, subcellular dynamics, and cytoplasmic flow during hyphal growth in Aspergillus niger. In addition, growth and budding of Aureobasidium pullulans cells are captured using holotomographic microscopy. Coupled to fluorescence imaging, lipid droplets, vacuoles, the mitochondrial network, and nuclei are targeted and analyzed in the 3D RI reconstructed images. While lipid droplets and vacuoles can be assigned to a specific RI pattern, mitochondria and nuclei were not pronounced. We show, that the lower sensitivity of RI measurements derives from the fungal cell wall that acts as an additional barrier for the illumination light of the microscope. After cell wall digest of hyphae and protoplast formation of A. niger expressing GFP-tagged histone H2A, location of nuclei could be determined by non-invasive RI measurements. Furthermore, we used coupled fluorescence microscopy to observe migration of nuclei in unperturbed hyphal segments and duplication during growth on a single-cell level. Detailed micromorphological studies in Saccharomyces cerevisiae and Trichoderma reesei are challenging due to cell size restrictions. Overall, holotomography opens up new avenues for exploring dynamic cellular processes in real time and enables the visualization of fungi from a new perspective.

Handheld methanol detector for beverage analysis: interlaboratory validation.

Methanol is a toxic alcohol contained in alcoholic beverages as a natural byproduct of fermentation or added intentionally to counterfeits to increase profit. To ensure consumer safety, many countries and the EU have established strict legislation limits for methanol content. Methanol concentration is mostly detected by laboratory instrumentation since mobile devices for routine on-site testing of beverages in distilleries, at border stations or even at home are not available. Here, we validated a handheld methanol detector for beverage analysis in an ISO 5725 interlaboratory trial: a total of 119 measurements were performed by 17 independent participants (distilleries, universities, authorities, and competence centers) from six countries on samples with relevant methanol concentrations (0.1, 1.5 vol%). The detector was based on a microporous separation filter and a nanostructured gas sensor allowing on-site measurement of methanol down to 0.01 vol% (in the liquid) within only 2 min by laymen. The detector showed excellent repeatability (<5.4%), reproducibility (<9.5%) and small bias (<0.012 vol%). Additional measurements on various methanol-spiked alcoholic beverages (whisky, rum, gin, vodka, tequila, port, sherry, liqueur) indicated that the detector is not interfered by environmental temperature and spirit composition, featuring excellent linearity (R2 > 0.99) down to methanol concentrations of 0.01 vol%. This device has been recently commercialized (Alivion Spark M-20) with comparable accuracy to the gold-standard gas chromatography and can be readily applied for final product inspection, intake control of raw materials or to identify toxic counterfeit products.

Manganese(II) ions suppress the transcription of the citrate exporter encoding gene cexA in Aspergillus niger.

Aspergillus niger is an important filamentous fungus used for the industrial production of citric acid. One of the most important factors that affect citric acid production is the concentration of manganese(II) ions present in the culture broth. Under manganese(II)-limiting conditions, the fungus develops a pellet-like morphology that is crucial for high citric acid accumulation. The impact of manganese(II) ions on the transcription of the major citrate exporter encoding gene cexA was studied under manganese(II)-deficient and -sufficient conditions. Furthermore, citric acid production was analyzed in overexpression mutant strains of cexA in the presence and absence of manganese(II) ions, and the influence of CexA on fungal morphology was investigated by microscopy. Transcriptional upregulation of cexA in the absence of manganese(II) ions was observed and, by decoupling cexA expression from the native promoter system, it was possible to secrete more citric acid even in the presence of manganese. This effect was shown for both an inducible and a constitutive overexpression of cexA. Furthermore, it was found that the presence of CexA influences fungal morphology and promotes a more branched phenotype. According to this study, manganese(II) ions suppress transcription of the citrate exporter cexA in Aspergillus niger, causing citric acid secretion to decrease.

Customizing amino acid metabolism of Pichia pastoris for recombinant protein production.

Amino acids are the building blocks of proteins. In this respect, a reciprocal effect of recombinant protein production on amino acid biosynthesis as well as the impact of the availability of free amino acids on protein production can be anticipated. In this study, the impact of engineering the amino acid metabolism on the production of recombinant proteins was investigated in the yeast Pichia pastoris (syn Komagataella phaffii). Based on comprehensive systems-level analyses of the metabolomes and transcriptomes of different P. pastoris strains secreting antibody fragments, cell engineering targets were selected. Our working hypothesis that increasing intracellular amino acid levels could help unburden cellular metabolism and improve recombinant protein production was examined by constitutive overexpression of genes related to amino acid metabolism. In addition to 12 genes involved in specific amino acid biosynthetic pathways, the transcription factor GCN4 responsible for regulation of amino acid biosynthetic genes was overexpressed. The production of the used model protein, a secreted carboxylesterase (CES) from Sphingopyxis macrogoltabida, was increased by overexpression of pathway genes for alanine and for aromatic amino acids, and most pronounced, when overexpressing the regulator GCN4. The analysis of intracellular amino acid levels of selected clones indicated a direct linkage of improved recombinant protein production to the increased availability of intracellular amino acids. Finally, fed batch cultures showed that overexpression of GCN4 increased CES titers 2.6-fold, while the positive effect of other amino acid synthesis genes could not be transferred from screening to bioreactor cultures.

Nitrogen recovery from low-value biogenic feedstocks via steam gasification to methylotrophic yeast biomass.

Carbon and nitrogen are crucial elements for life and must be efficiently regenerated in a circular economy. Biomass streams at the end of their useful life, such as sewage sludge, are difficult to recycle even though they contain organic carbon and nitrogen components. Gasification is an emerging technology to utilize such challenging waste streams and produce syngas that can be further processed into, e.g., Fischer-Tropsch fuels, methane, or methanol. Here, the objective is to investigate if nitrogen can be recovered from product gas cleaning in a dual fluidized bed (DFB) after gasification of softwood pellets to form yeast biomass. Yeast biomass is a protein-rich product, which can be used for food and feed applications. An aqueous solution containing ammonium at a concentration of 66 mM was obtained and by adding other nutrients it enables the growth of the methylotrophic yeast Komagataella phaffii to form 6.2 g.L-1 dry yeast biomass in 3 days. To further integrate the process, it is discussed how methanol can be obtained from syngas by chemical catalysis, which is used as a carbon source for the yeast culture. Furthermore, different gas compositions derived from the gasification of biogenic feedstocks including sewage sludge, bark, and chicken manure are evaluated for their ability to yield methanol and yeast biomass. The different feedstocks are compared based on their potential to yield methanol and ammonia, which are required for the generation of yeast biomass. It was found that the gasification of bark and chicken manure yields a balanced carbon and nitrogen source for the formation of yeast biomass. Overall, a novel integrated process concept based on renewable, biogenic feedstocks is proposed connecting gasification with methanol synthesis to enable the formation of protein-rich yeast biomass.

The importance of complete and high-quality genome sequences in Aspergillus niger research.

The possibility to sequence the entire genome of an organism revolutionized the fields of biology and biotechnology. The first genome sequence of the important filamentous fungus Aspergillus niger was obtained in 2007, 11 years after the release of the first eukaryotic genome sequence. From that moment, genomics of A. niger has seen major progresses, facilitated by the advances in the sequencing technologies and in the methodologies for gene function prediction. However, there are still challenges to face when trying to obtain complete genomes, equipped with all the repetitive sequences that they contain and without omitting the mitochondrial sequences. The aim of this perspective article is to discuss the current status of A. niger genomics and draw attention to the open challenges that the fungal community should address to move research of this important fungus forward.

Genome sequencing of the neotype strain CBS 554.65 reveals the MAT1-2 locus of Aspergillus niger.

BACKGROUND: Aspergillus niger is a ubiquitous filamentous fungus widely employed as a cell factory thanks to its abilities to produce a wide range of organic acids and enzymes. Its genome was one of the first Aspergillus genomes to be sequenced in 2007, due to its economic importance and its role as model organism to study fungal fermentation. Nowadays, the genome sequences of more than 20 A. niger strains are available. These, however, do not include the neotype strain CBS 554.65. RESULTS: The genome of CBS 554.65 was sequenced with PacBio. A high-quality nuclear genome sequence consisting of 17 contigs with a N50 value of 4.07 Mbp was obtained. The assembly covered all the 8 centromeric regions of the chromosomes. In addition, a complete circular mitochondrial DNA assembly was obtained. Bioinformatic analyses revealed the presence of a MAT1-2-1 gene in this genome, contrary to the most commonly used A. niger strains, such as ATCC 1015 and CBS 513.88, which contain a MAT1-1-1 gene. A nucleotide alignment showed a different orientation of the MAT1-1 locus of ATCC 1015 compared to the MAT1-2 locus of CBS 554.65, relative to conserved genes flanking the MAT locus. Within 24 newly sequenced isolates of A. niger half of them had a MAT1-1 locus and the other half a MAT1-2 locus. The genomic organization of the MAT1-2 locus in CBS 554.65 is similar to other Aspergillus species. In contrast, the region comprising the MAT1-1 locus is flipped in all sequenced strains of A. niger. CONCLUSIONS: This study, besides providing a high-quality genome sequence of an important A. niger strain, suggests the occurrence of genetic flipping or switching events at the MAT1-1 locus of A. niger. These results provide new insights in the mating system of A. niger and could contribute to the investigation and potential discovery of sexuality in this species long thought to be asexual.

Sclerotia formed by citric acid producing strains of Aspergillus niger: Induction and morphological analysis.

Some strains of Aspergillus niger have been previously reported to produce sclerotia under certain conditions. Sclerotia are aggregations of hyphae which can act either as survival or as sexual structures in species related to A. niger. In this study, we were able to induce the formation of sclerotia in the progenitor of the industrial citric acid producing strains of A. niger, ATCC 1015, and in pyrG mutants derived from it. Sclerotia can be stably formed by ATCC 1015 on malt extract agar medium supplemented with raisins, showing a spatial differentiation of the fungus dependent on the addition and on the position of the fruits into the medium. On other media, including malt extract agar, pyrG auxotrophs also form abundant sclerotia, while the complementation of this gene reverses this phenotype. Additionally, a macro- and microscopical analysis of the sclerotia is reported. Our results show that the sclerotia formed by A. niger are similar to those formed by other fungi, not only in their morphology but also in their ability to germinate and regenerate the organism.

Flow Cytometry for Filamentous Fungi.

Flow cytometry is a powerful high-throughput method, which enables a fast and multi-parameter analysis of single cells and particles. A plethora of different dyes for flow cytometry are available to label different parts of a cell in addition to in vivo markers like fluorescent proteins. Trichoderma species as well as other filamentous fungi show hyphal growth, which  makes analysis in a flow cytometer difficult. Nevertheless, conidia can be readily analyzed in conventional flow cytometers. Many different applications can be envisaged. This protocol describes how conidia  can be prepared for flow cytometry and the occurrence of genetic markers such as GFP can be measured. Furthermore, a guideline how to fix and stain cells is given.

Microscale Perfusion-Based Cultivation for Pichia pastoris Clone Screening Enables Accelerated and Optimized Recombinant Protein Production Processes.

Pichia pastoris has emerged in the past years as a promising host for recombinant protein and biopharmaceutical production. In the establishment of high cell density fed-batch biomanufacturing, screening phase and early bioprocess development (based on microplates and shake flasks) still represent a bottleneck due to high-cost and time-consuming procedures as well as low experiment complexity. In the present work, a screening protocol developed for P. pastoris clone selection is implemented in a multiplexed microfluidic device with 15 µL cultivation chambers able to operate in perfusion mode and monitor dissolved oxygen content in the culture in a non-invasive way. The setup allowed us to establish carbon-limited conditions and evaluate strain responses to different input variables. Results from micro-scale perfusion cultures are then compared with 1L fed-batch fermentation. The best producer in terms of titer and productivity is rapidly identified after 12 h from inoculation and the results confirmed by lab-scale fermentation. Moreover, the physiological analyses of the strains under different conditions suggested how more complex experimental conditions are achievable despite the relatively easy, straight-forward, and cost-effective experimental setup. Implementation and standardization of these micro-scale protocols could reduce the demand for lab-scale bioreactor cultivations thus accelerating the development of protein production processes.

The fungal sexual revolution continues: discovery of sexual development in members of the genus Aspergillus and its consequences.

Asexuality was considered to be a common feature of a large part of fungi, including those of the genus Aspergillus. However, recent advances and the available genomic and genetic engineering technologies allowed to gather more and more indications of a hidden sexuality in fungi previously considered asexual. In parallel, the acquired knowledge of the most suitable conditions for crossings was shown to be crucial to effectively promote sexual reproduction in the laboratory. These discoveries not only have consequences on our knowledge of the biological processes ongoing in nature, questioning if truly asexual fungal species exist, but they also have important implications on other research areas. For instance, the presence of sexuality in certain fungi can have effects on their pathogenicity or on shaping the ecosystem that they normally colonize. For these reasons, further investigations of the sexual potential of Aspergillus species, such as the industrially important A. niger, will be carried on.

Fine-Tuning of Transcription in Pichia pastoris Using dCas9 and RNA Scaffolds.

For metabolic engineering approaches, fast and reliable tools are required to precisely manipulate the expression of target genes. dCas9 can be fused via RNA scaffolds to trans-activator domains and thus regulate the gene expression when targeted to the promoter region of a gene. In this work we show that this strategy can be successfully implemented for the methylotrophic yeast Pichia pastoris. It is shown that the thiamine repressible promoter of THI11 can be activated under repression conditions using a scgRNA/dCas9 construct. Furthermore, the RIB1 gene required for riboflavin production was activated, leading to increased riboflavin production exceeding the riboflavin titers of a conventional RIB1 overexpression with a pGAP promoter.

Matrix-Augmented Bone Marrow Stimulation With a Polyglycolic Acid Membrane With Hyaluronan vs Microfracture in Local Cartilage Defects of the Femoral Condyles: A Multicenter Randomized Controlled Trial.

BACKGROUND: Microfracture (MF) is an established operative treatment for small, localized chondral defects of the knee joint. There is evidence from animal studies that matrix augmentation of bone marrow stimulation (m-BMS) can improve the quality of the repair tissue formation. PURPOSE: To evaluate the therapeutic outcome of a matrix made of polyglycolic acid and hyaluronan as compared with a conventional MF technique. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: Patients between the ages of 18 and 68 years who had an articular femoral cartilage defect of 0.5 to 3 cm2 in the weightbearing area of the femoral condyles with indication for MF were included in this study. Patients were randomized and treated with either MF or m-BMS with Chondrotissue. Defect filling, as assessed on magnetic resonance imaging (MRI), at postoperative 12 weeks was defined as the primary outcome measure, with follow-up MRI at weeks 54 and 108. Follow-up data were also collected at 12, 54, and 108 weeks after surgery and included patient-reported clinical scores: visual analog scale for pain, Knee injury and Osteoarthritis Outcome Score (KOOS), International Knee Documentation Committee score, and 36-Item Short Form Health Survey. RESULTS: MRI scans confirmed cartilage repair tissue formation in both groups 12 weeks after treatment. There was no significant difference between the m-BMS and MF groups in the percentage of defect filling at 12, 54, and 108 weeks postoperatively. No significant difference was found in terms of patient-reported clinical scores. Both groups showed significant improvement in 4 KOOS subscales-Pain, Activities of Daily Living, Sport and Recreation, and Quality of Life-at 54 and 108 weeks after treatment. CONCLUSION: This is the first randomized controlled trial comparing m-BMS with a polyglycolic acid matrix with hyaluronan with MF. The use of the Chondrotissue implant in m-BMS has been proven to be a safe procedure. No difference was found between m-BMS and MF in terms of patient-reported outcome scores and MRI assessment until postoperative 2 years. Long-term follow-up studies including histological assessment are desirable for further investigation. REGISTRATION: EUCTR2011-003594-28-DE (EU Clinical Trials Register).

Downscaling screening cultures in a multifunctional bioreactor array-on-a-chip for speeding up optimization of yeast-based lactic acid bioproduction.

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. We present a multifunctional microfluidic device containing 12 individual bioreactors (Vt = 15 µl) in a 26 mm × 76 mm area with in-line biosensing of dissolved oxygen and biomass concentration. Following initial device characterization, the bioreactor lab-on-a-chip was used in a proof-of-principle study to identify the most productive cell line for lactic acid production out of two engineered yeast strains, evaluating whether it could reduce the time needed for collecting meaningful data compared with shake flasks cultures. Results of the study showed significant difference in the strains' productivity within 3 hr of operation exhibiting a 4- to 6-fold higher lactic acid production, thus pointing at the potential of microfluidic technology as effective screening tool for fast and parallelizable industrial bioprocess development.

The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2.

The methylotrophic yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals. Like most biotechnological production hosts, P. pastoris is heterotrophic and grows on organic feedstocks that have competing uses in the production of food and animal feed. In a step toward more sustainable industrial processes, we describe the conversion of P. pastoris into an autotroph that grows on CO2. By addition of eight heterologous genes and deletion of three native genes, we engineer the peroxisomal methanol-assimilation pathway of P. pastoris into a CO2-fixation pathway resembling the Calvin-Benson-Bassham cycle, the predominant natural CO2-fixation pathway. The resulting strain can grow continuously with CO2 as a sole carbon source at a µmax of 0.008 h-1. The specific growth rate was further improved to 0.018 h-1 by adaptive laboratory evolution. This engineered P. pastoris strain may promote sustainability by sequestering the greenhouse gas CO2, and by avoiding consumption of an organic feedstock with alternative uses in food production.

Metabolic specialization in itaconic acid production: a tale of two fungi.

Some of the oldest and most established industrial biotechnology processes involve the fungal production of organic acids. In these fungi, the transport of metabolites between cellular compartments, and their secretion, is a major factor. In this review we exemplify the importance of both mitochondrial and plasma membrane transporters in the case of itaconic acid production in two very different fungal systems, Aspergillus and Ustilago. Homologous and heterologous overexpression of both types of transporters, and biochemical analysis of mitochondrial transporter function, show that these two fungi produce the same compound through very different pathways. The way these fungi respond to itaconate stress, especially at low pH, also differs, although this is still an open field which clearly needs additional research.

Microbial production of high value molecules using rayon waste material as carbon-source.

Rayon filaments composed of regenerated cellulose are used as reinforcement materials in tires and to a lower extent in the clothing industry as personal protective equipment e.g. flame retardant cellulosic based materials. After use, these materials are currently transferred to landfills while chemical degradation does not allow the recovery of the cellulose (as glucose) nor the separation of the high valuable flame-retardant pigment. In this study, rayon fibers were enzymatically hydrolyzed to allow recovery of glucose and valuable additives. The glucose was successfully used as carbon source for the production of high value compounds such as itaconic acid, lactic acid and chitosan. 14.2 g/L of itaconic acid, 36.5 g/L of lactic acid and 39.2 g/L of chitosan containing biomass were produced from Escherichia coli, Lactobacillus paracasei and Aspergillus niger, respectively, comparable to yields obtained when using commercial glucose as carbon source.

Engineering of the citrate exporter protein enables high citric acid production in Aspergillus niger.

Aspergillus niger was engineered using a gene responsible for citric acid transport, which has a significant impact on citric acid secretion when overexpressed. The transport gene was identified by a homology search using an itaconic acid transporter from Ustilago maydis as template. The encoding homologous protein CexA belongs to the major facilitator superfamily subclass DHA1 and members of this family work as drug-H+ antiporter. The disruption of this gene completely abolishes citric acid secretion, which indicates that this protein is the main citric acid transporter in A. niger. In the disruption strain, the metabolism is re-routed mainly to oxalic acid, which is a known by-product during citric acid production. The gene can be heterologously expressed in Saccharomyces cerevisiae, which leads to the secretion of citric acid during the growth on glucose. These results confirm the functionality of CexA as the main transporter for citric acid of A. niger. Overexpression of cexA leads to a significant increase in secreted citric acid. Thereby, striking differences between a strong constitutive expression system using pmbfA as a promoter and an inducible expression system using ptet-on can be observed. The inducible system significantly outcompetes the constitutive expression system yielding up to 109 g/L citric acid, which is 5 times higher compared to the parental wild-type strain and 3 times higher compared to the constitutive expression system. These results demonstrate the importance of the cellular transport system for an efficient production of metabolites. By overexpressing a single gene, it is possible to significantly improve the citric acid secretion capability of a moderately producing parental strain.

A single Gal4-like transcription factor activates the Crabtree effect in Komagataella phaffii.

The Crabtree phenotype defines whether a yeast can perform simultaneous respiration and fermentation under aerobic conditions at high growth rates. It provides Crabtree positive yeasts an evolutionary advantage of consuming glucose faster and producing ethanol to outcompete other microorganisms in sugar rich environments. While a number of genetic events are associated with the emergence of the Crabtree effect, its evolution remains unresolved. Here we show that overexpression of a single Gal4-like transcription factor is sufficient to convert Crabtree-negative Komagataella phaffii (Pichia pastoris) into a Crabtree positive yeast. Upregulation of the glycolytic genes and a significant increase in glucose uptake rate due to the overexpression of the Gal4-like transcription factor leads to an overflow metabolism, triggering both short-term and long-term Crabtree phenotypes. This indicates that a single genetic perturbation leading to overexpression of one gene may have been sufficient as the first molecular event towards respiro-fermentative metabolism in the course of yeast evolution.