Practical guidance for the implementation of the CRISPR genome editing tool in filamentous fungi

Background: Within the last years, numerous reports described successful application of the CRISPR nucleases Cas9 and Cpf1 for genome editing in filamentous fungi. However, still a lot of efforts are invested to develop and improve protocols for the fungus and genes of interest with respect to applicability, scalability and targeting efficiencies. These efforts are often hampered by the fact that—although many different protocols are available— one have systematically analysed and compared different CRISPR nucleases and different application procedures thereof for the efficiency of single- and multiplex-targeting approaches in the same fungus. Results: We present here data for successful genome editing in the cell factory Thermothelomyces thermophilus, formerly known as Myceliophthora thermophila, using the three different nucleases SpCas9, FnCpf1, AsCpf1 guided to four different gene targets of our interest. These included a polyketide synthase (pks4.2), an alkaline protease (alp1), a SNARE protein (snc1) and a potential transcription factor (ptf1). For all four genes, guide RNAs were developed which enabled successful single-targeting and multiplex-targeting. CRISPR nucleases were either delivered to T. thermophilus on plasmids or preassembled with in vitro transcribed gRNA to form ribonucleoproteins (RNPs). We also evaluated the efficiency of single oligonucleotides for site-directed mutagenesis. Finally, we were able to scale down the transformation protocol to microtiter plate format which generated high numbers of positive transformants and will thus pave the way for future high-throughput investigations. Conclusion: We provide here the first comprehensive analysis and evaluation of different CRISPR approaches for a filamentous fungus. All approaches followed enabled successful genome editing in T. thermophilus; however, with different success rates. In addition, we show that the success rate depends on the respective nuclease and on the targeted gene locus. We finally present a practical guidance for experimental considerations aiming to guide the reader for successful implementation of CRISPR technology for other fungi.TU Berlin, Open-Access-Mittel - 201

Evolution of temperature and mobilization of terrigenous organic matter in the subarctic Northwest Pacific and adjacent Beringia since the Last Glacial Maximum

In the subarctic Northwest Pacific and adjacent Siberia mean climate changes between the Last Glacial Maximum and the Holocene are poorly understood since climate records spanning the full LGM-Holocene transition are sparse. This thesis shall contribute to a better understanding of climate and environmental change since the LGM and the controlling mechanisms in the region by investigating the development of temperature, glaciation and export of terrigenous organic matter into the North Pacific (N Pacific). Biomarkers in sediment cores from the Western Bering Sea and the NW Pacific are applied as palaeoclimate archives. In the first part of the thesis LGM-to-Holocene sea surface temperature (SST) records for the marginal Northwest Pacific and the Western Bering Sea are established using the TEXL86 (Tetraether IndeX)-SST proxy. It is found that SSTs in both settings are determined by rapid atmospheric teleconnections with abrupt climate changes in the North Atlantic (N Atlantic) since 15 ka BP. Before 15 ka BP, only the Bering Sea was connected to N-Atlantic climate change. The NW Pacific remained disconnected from the N-Atlantic until 15 ka BP due to an oceanic linkage with the NE Pacific through the Alaskan Stream. The second part investigates the LGM-to-Holocene evolution of mean air temperature (MAT) of the Kamchatka Peninsula where climate archives do not reach beyond 12 ka BP. Using the CBT/MBT-palaeothermometry (Cyclisation of Branched Tetreathers and the Methylation of Branched Tetraethers indices) a continuous record in summer MAT is provided for the past 20 ka. It is found that glacial summers were as warm as at present. Likely, strong southerly winds, associated with a pronounced North Pacific High pressure system (NPH) over the subarctic NW Pacific, accounted for the warm conditions on Kamchatka. The deglacial temperature development was characterized by abrupt millennial-scale temperature oscillations during the past 15 ka BP. Considering that NE-Siberian glaciation is supposed to have been more extensive than at present but restricted to mountain ranges during the LGM, the warm glacial-summers of Siberia suggest that summer temperature may have been an important limiting factor for ice sheet growth in the region. In the third part of the thesis, mass balance calculations for the LGM-glaciers on Kamchatka and the Kankaren Range (NE Siberia) are performed by degreeday-modelling in order to estimate the precipitation needed to sustain the glaciers under warm summer conditions. It is found that precipitation at least must have equaled or even exceeded the modern average, confirming the hypothesis that summer temperature limited ice-sheet expansion in NE Russia during the LGM. The fourth part of the thesis contributes to an ongoing debate about the sources of old, (14Cdepleted) carbon dioxide (CO2) which increased atmospheric CO2-levels (CO2atm) and concurrently decreased the atmospheric radiocarbon signature (14Catm) during the deglaciation. Permafrost-decomposition in the Northern Hemisphere (NH) triggered by deglacial warming and sea-level rise is considered as one possible source of 14C-depleted CO2, particularly at the onset of the B/A-interstadial (14.6 ka BP). However, the timing of carbon mobilization in permafrost areas of the NH is underconstrained. In order to investigate the potential role of permafrost decomposition in the subarctic N Pacific realm in the atmospheric, changes mass accumulation rates and the radiocarbon signature (14C) of leafwax-lipids are analyzed in order to identify intervals of intensified export of 14C-depleted terrigenous OM into the Western Bering Sea and the NW Pacific. Enhanced burial of nearly 14C-free carbon commenced during the HS1 and was likely triggered by increased runoff in the Yukon River due to retreating American ice-sheets. Since the B/A mobilization of 14Cdepleted seems to have been dominantly controlled by sea-level rise and thus by erosion of permafrost-covered shelves. Enhanced OM-export associated with permafrost-thaw on Kamchatka likely initiated during the second half of the B/A-interstadial and peaked during the YD-stadial. Lagging the rapid CO2atm/14Catm changes at 14.6 ka BP, the permafrost degradation in the Kamchatka region was probably irrelevant for the atmosphere. Instead, enhanced OM-export in the region coincided with abrupt CO2atm/14Catm changes during the YD suggesting that permafrost may have contributed to the atmospheric carbon-pool at that time

Aspergillus niger Spores Are Highly Resistant to Space Radiation

The filamentous fungus Aspergillus niger is one of the main contaminants of the International Space Station (ISS). It forms highly pigmented, airborne spores that have thick cell walls and low metabolic activity, enabling them to withstand harsh conditions and colonize spacecraft surfaces. Whether A. niger spores are resistant to space radiation, and to what extent, is not yet known. In this study, spore suspensions of a wild-type and three mutant strains (with defects in pigmentation, DNA repair, and polar growth control) were exposed to X-rays, cosmic radiation (helium- and iron-ions) and UV-C (254 nm). To assess the level of resistance and survival limits of fungal spores in a long-term interplanetary mission scenario, we tested radiation doses up to 1000 Gy and 4000 J/m2. For comparison, a 360-day round-trip to Mars yields a dose of 0.66 ± 0.12 Gy. Overall, wild-type spores of A. niger were able to withstand high doses of X-ray (LD90 = 360 Gy) and cosmic radiation (helium-ion LD90 = 500 Gy; and iron-ion LD90 = 100 Gy). Drying the spores before irradiation made them more susceptible toward X-ray radiation. Notably, A. niger spores are highly resistant to UV-C radiation (LD90 = 1038 J/m2), which is significantly higher than that of other radiation-resistant microorganisms (e.g., Deinococcus radiodurans). In all strains, UV-C treated spores (1000 J/m2) were shown to have decreased biofilm formation (81% reduction in wild-type spores). This study suggests that A. niger spores might not be easily inactivated by exposure to space radiation alone and that current planetary protection guidelines should be revisited, considering the high resistance of fungal spores

How a fungus shapes biotechnology: 100 years of Aspergillus niger research

In 1917, a food chemist named James Currie made a promising discovery: any strain of the filamentous mould Aspergillus niger would produce high concentrations of citric acid when grown in sugar medium. This tricarboxylic acid, which we now know is an intermediate of the Krebs cycle, had previously been extracted from citrus fruits for applications in food and beverage production. Two years after Currie’s discovery, industrial-level production using A. niger began, the biochemical fermentation industry started to flourish, and industrial biotechnology was born. A century later, citric acid production using this mould is a multi-billion dollar industry, with A. niger additionally producing a diverse range of proteins, enzymes and secondary metabolites. In this review, we assess main developments in the field of A. niger biology over the last 100 years and highlight scientific breakthroughs and discoveries which were influential for both basic and applied fungal research in and outside the A. niger community. We give special focus to two developments of the last decade: systems biology and genome editing. We also summarize the current international A. niger research community, and end by speculating on the future of fundamental research on this fascinating fungus and its exploitation in industrial biotechnology.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität Berli

Vita activa in biotechnology: what we do with fungi and what fungi do with us

Filamentous fungi are fascinating microorganisms. One of the reasons why it is so worthwhile to take a closer look at them is their capacity to produce secondary metabolites. Some of these substances have the potential to be of great use for mankind, such as it was the case with penicillin and its discovery in 1928. Almost a century later, the situation in healthcare could possibly turn back to the state before the development of the first antibiotics. Due to an overuse of antibiotics we are facing a surge of multiresistant bacteria that are not inhibited by any of the currently known drugs. That was part of the background why a European research project was launched in October 2013, titled "Quantitative Biology for Fungal Secondary Metabolite Producers", or "QuantFung". Fifteen young scientists embarked on a new phase in their career, moving to new work environments within Europe and dedicating their work lives intensively to the quest for useful secondary metabolites. After 4 years, the QuantFung project concluded in October this year. In this commentary, we aim to convey what it means to work in this field of fungal biotechnology and how important it is to improve the efficiency of the research therein. We introduce five out of the fifteen fellows at length and let them have their say about the adventure of science, euphoric moments, prospects and doubts. We also raise questions about the current state of research in academia, something the QuantFung fellows experienced first-hand. Being a scientist often goes beyond earning money to make one's living. This is why we also reflect on aspects of the meaning of work in our western society, where production for profit's sake is a main driver. For that we refer to one of the most distinguished thinkers of the twentieth century, to Hannah Arendt

Species-Specific Differences in the Susceptibility of Fungi to the Antifungal Protein AFP Depend on C-3 Saturation of Glycosylceramides

AFP is an antimicrobial peptide (AMP) produced by the filamentous fungus Aspergillus giganteus and is a very potent inhibitor of fungal growth that does not affect the viability of bacteria, plant, or mammalian cells. It targets chitin synthesis and causes plasma membrane permeabilization in many human- and plant-pathogenic fungi, but its exact mode of action is not known. After adoption of the “damage-response framework of microbial pathogenesis” regarding the analysis of interactions between AMPs and microorganisms, we have recently proposed that the cytotoxic capacity of a given AMP depends not only on the presence/absence of its target(s) in the host and the AMP concentration applied but also on other variables, such as microbial survival strategies. We show here using the examples of three filamentous fungi (Aspergillus niger, Aspergillus fumigatus, and Fusarium graminearum) and two yeasts (Saccharomyces cerevisiae and Pichia pastoris) that the important parameters defining the AFP susceptibilities of these fungi are (i) the presence/absence of glycosylceramides, (ii) the presence/absence of Δ3(E) desaturation of the fatty acid chain therein, and (iii) the (dis)ability of these fungi to respond to AFP inhibitory effects with the fortification of their cell walls via increased chitin and β-(1,3)-glucan synthesis. These observations support the idea of the adoption of the damage-response framework to holistically understand the outcome of AFP inhibitory effects.TU Berlin, Open-Access-Mittel - 201

Thunderstorm Tracking and Monitoring on the Basis of Three Dimensional Lightning Data and Conventional and Polarimetric Radar Data

The aim of this work is to assess the benefit of total-lightning information as independent data source for thunderstorm tracking and short-term prediction (nowcasting) of storm evolution. Special focus has been laid on the three-dimensional lightning information and the in-cloud and cloud-to-ground discrimination provided by the lightning detection network LINET. The reliability of the lightning information and its usability for nowcasting purposes have been tested both separately and in combination with other data sources which are commonly used for thunderstorm nowcasting. The new thunderstorm tracker ec-TRAM (tracking and monitoring of electrically charged cells; Meyer et al. (2009)) has been developed to identify, track, and monitor thunderstorms in high temporal and spatial resolution by combining the information of independently tracked convective ground-precipitation cells and lightning-cells to new cell objects. The algorithm builds on the autonomously operating routines rad-TRAM (tracking and monitoring of radar cells; Kober and Tafferner (2009)) and li-TRAM (tracking and monitoring of lightning cells). The latter has also been developed within this work. The new tracking algorithm has been tested based on a thunderstorm data set of more than 500 storm tracks which were recorded by ec-TRAM in southern Germany during summer 2008. It is found that the newly composed cell objects comprehensively describe simple as well as complex thunderstorm structures and the cell tracking method of ec-TRAM proves to be more coherent and stable in comparison with the tracking performances of rad-TRAM and li-TRAM. For two selected thunderstorms the time series of cell parameters monitored by ec-TRAM have been complemented with three-dimensional polarimetric radar data and satellite data to assess how the temporal evolution and parameter correlation of total lightning strokes, hydrometeor formation, ground precipitation patterns, and cloud top temperature can be used to estimate the storm state and predict its development. The parameter evolutions are found to be consistent with the current state of knowledge. A principal life-cycle scheme can be identified for the cell parameters on large time scales. The stronger fluctuating short-term parameter evolutions are found to refl ect the momentary storm dynamic. Based on the lifetime diagrams several warning parameters for subsequent storm events can be suggested. Significant cell parameter correlations, which can be parameterized, are also found in statistical analyses over the complete data set. Strong positive correlations are found between cell extension, discharge frequency, and in-cloud discharge height. Two cell regimes, sharply separated at a specific cell characteristic, can clearly be identified in all correlation diagrams. Interpreted on the basis of previous studies and in terms of the current state of knowledge, it seems most likely that the two cell-regimes refl ect the storm characteristics of different storm organization forms. The parameterized correlation curves could then be used as cell parameterizations in operational nowcasting tools to predict the dynamic evolution, duration, and danger potential of a storm, provided that the storm system can be classified. Finally, it can be concluded that this study demonstrates the usability and the promising potential of total-lightning data as reliable and independent data source for future nowcasting tools