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

A quantitative image analysis pipeline for the characterization of filamentous fungal morphologies as a tool to uncover targets for morphology engineering: a case study using aplD in Aspergillus niger

Background Fungal fermentation is used to produce a diverse repertoire of enzymes, chemicals, and drugs for various industries. During submerged cultivation, filamentous fungi form a range of macromorphologies, including dispersed mycelia, clumped aggregates, or pellets, which have critical implications for rheological aspects during fermentation, gas/nutrient transfer, and, thus, product titres. An important component of strain engineering efforts is the ability to quantitatively assess fungal growth phenotypes, which will drive novel leads for morphologically optimized production strains. Results In this study, we developed an automated image analysis pipeline to quantify the morphology of pelleted and dispersed growth (MPD) which rapidly and reproducibly measures dispersed and pelleted macromorphologies from any submerged fungal culture. It (i) enables capture and analysis of several hundred images per user/day, (ii) is designed to quantitatively assess heterogeneous cultures consisting of dispersed and pelleted forms, (iii) gives a quantitative measurement of culture heterogeneity, (iv) automatically generates key Euclidian parameters for individual fungal structures including particle diameter, aspect ratio, area, and solidity, which are also assembled into a previously described dimensionless morphology number MN, (v) has an in-built quality control check which enables end-users to easily confirm the accuracy of the automated calls, and (vi) is easily adaptable to user-specified magnifications and macromorphological definitions. To concomitantly provide proof of principle for the utility of this image analysis pipeline, and provide new leads for morphologically optimized fungal strains, we generated a morphological mutant in the cell factory Aspergillus niger based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for A. niger to identify a putative gamma-adaptin encoding gene (aplD) that was predicted to play a role in endosome cargo trafficking. Gene editing was used to generate a conditional aplD expression mutant under control of the titratable Tet-on system. Reduced aplD expression caused a hyperbranched growth phenotype and diverse defects in pellet formation with a putative increase in protein secretion. This possible protein hypersecretion phenotype could be correlated with increased dispersed mycelia, and both decreased pellet diameter and MN. Conclusion The MPD image analysis pipeline is a simple, rapid, and flexible approach to quantify diverse fungal morphologies. As an exemplar, we have demonstrated that the putative endosomal transport gene aplD plays a crucial role in A. niger filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.DFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

Anatomical location of Macrophage Migration Inhibitory Factor in urogenital tissues, peripheral ganglia and lumbosacral spinal cord of the rat

BACKGROUND: Previous work suggested that macrophage migration inhibitory factor (MIF) may be involved in bladder inflammation. Therefore, the location of MIF was determined immunohistochemically in the bladder, prostate, major pelvic ganglia, sympathetic chain, the L6-S1 dorsal root ganglia (DRG) and the lumbosacral spinal cord of the rat. RESULTS: In the pelvic organs, MIF immunostaining was prominent in the epithelia. MIF was widely present in neurons in the MPG and the sympathetic chain. Some of those neurons also co-localized tyrosine hydroxylase (TH). In the DRGs, some of the neurons that stained for MIF also stained for Substance P. In the lumbosacral spinal cord, MIF immunostaining was observed in the white mater, the dorsal horn, the intermediolateral region and in the area around the central canal. Many cells were intensely stained for MIF and glial fibrillary acidic protein (GFAP) suggesting they were glial cells. However, some cells in the lumbosacral dorsal horn were MIF positive, GFAP negative cells suggestive of neurons. CONCLUSIONS: Therefore, MIF, a pro-inflammatory cytokine, is localized to pelvic organs and also in neurons of the peripheral and central nervous tissues that innervate those organs. Changes in MIF's expression at the end organ and at peripheral and central nervous system sites suggest that MIF is involved in pelvic viscera inflammation and may act at several levels to promote inflammatory changes

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

Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture

Background: Filamentous fungal cell factories are used to produce numerous proteins, enzymes, and organic acids. Protein secretion and filamentous growth are tightly coupled at the hyphal tip. Additionally, both these processes require ATP and amino acid precursors derived from the citric acid cycle. Despite this interconnection of organic acid production and protein secretion/filamentous growth, few studies in fungi have identified genes which may concomitantly impact all three processes. Results: We applied a novel screen of a global co-expression network in the cell factory Aspergillus niger to identify candidate genes which may concomitantly impact macromorphology, and protein/organic acid fermentation. This identified genes predicted to encode the Golgi localized ArfA GTPase activating protein (GAP, AgeB), and ArfA guanine nucleotide exchange factors (GEFs SecG and GeaB) to be co-expressed with citric acid cycle genes. Consequently, we used CRISPR-based genome editing to place the titratable Tet-on expression system upstream of ageB, secG, and geaB in A. niger. Functional analysis revealed that ageB and geaB are essential whereas secG was dispensable for early filamentous growth. Next, gene expression was titrated during submerged cultivations under conditions for either protein or organic acid production. ArfA regulators played varied and culture-dependent roles on pellet formation. Notably, ageB or geaB expression levels had major impacts on protein secretion, whereas secG was dispensable. In contrast, reduced expression of each predicted ArfA regulator resulted in an absence of citric acid in growth media. Finally, titrated expression of either GEFs resulted in an increase in oxaloacetic acid concentrations in supernatants. Conclusion: Our data suggest that the Golgi may play an underappreciated role in modulating organic acid titres during industrial applications, and that this is SecG, GeaB and AgeB dependent in A. niger. These data may lead to novel avenues for strain optimization in filamentous fungi for improved protein and organic acid titres.TU Berlin, Open-Access-Mittel - 201

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