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

Flux Design: In silico design of cell factories based on correlation of pathway fluxes to desired properties

<p>Abstract</p> <p>Background</p> <p>The identification of genetic target genes is a key step for rational engineering of production strains towards bio-based chemicals, fuels or therapeutics. This is often a difficult task, because superior production performance typically requires a combination of multiple targets, whereby the complex metabolic networks complicate straightforward identification. Recent attempts towards target prediction mainly focus on the prediction of gene deletion targets and therefore can cover only a part of genetic modifications proven valuable in metabolic engineering. Efficient in silico methods for simultaneous genome-scale identification of targets to be amplified or deleted are still lacking.</p> <p>Results</p> <p>Here we propose the identification of targets via flux correlation to a chosen objective flux as approach towards improved biotechnological production strains with optimally designed fluxes. The approach, we name Flux Design, computes elementary modes and, by search through the modes, identifies targets to be amplified (positive correlation) or down-regulated (negative correlation). Supported by statistical evaluation, a target potential is attributed to the identified reactions in a quantitative manner. Based on systems-wide models of the industrial microorganisms <it>Corynebacterium glutamicum </it>and <it>Aspergillus niger</it>, up to more than 20,000 modes were obtained for each case, differing strongly in production performance and intracellular fluxes. For lysine production in <it>C. glutamicum </it>the identified targets nicely matched with reported successful metabolic engineering strategies. In addition, simulations revealed insights, e.g. into the flexibility of energy metabolism. For enzyme production in <it>A.niger </it>flux correlation analysis suggested a number of targets, including non-obvious ones. Hereby, the relevance of most targets depended on the metabolic state of the cell and also on the carbon source.</p> <p>Conclusions</p> <p>Objective flux correlation analysis provided a detailed insight into the metabolic networks of industrially relevant prokaryotic and eukaryotic microorganisms. It was shown that capacity, pathway usage, and relevant genetic targets for optimal production partly depend on the network structure and the metabolic state of the cell which should be considered in future metabolic engineering strategies. The presented strategy can be generally used to identify priority sorted amplification and deletion targets for metabolic engineering purposes under various conditions and thus displays a useful strategy to be incorporated into efficient strain and bioprocess optimization.</p

Aspergillus nidulans como modelo para manipulação de genes envolvidos no processo de unfolded protein response

Orientadores: André Ricardo de Lima Damásio, Fábio Márcio SquinaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaAgência de fomento: CAPESResumo: Em eucariotos, o unfolded protein response (UPR) regula positivamente genes responsáveis por restaurar a homeostase no retículo endoplasmático (RE) durante o acúmulo de proteínas enoveladas incorretamente. A homeostase é restaurada devido à ativação de genes relacionados à via secretória, como aqueles que codificam chaperonas e foldases, o que aumenta, por sua vez, a capacidade de enovelamento de proteínas pelo RE. Alguns sistemas de produção de proteínas heterólogas têm sido desenvolvidos com a super-expressão individual de chaperonas e foldases nas células. Entretanto, a taxa de sucesso com a aplicação dessa estratégia é baixa. Estudos têm mostrado que a manipulação de genes que respondem ao UPR em linhagens fúngicas podem levar ao aumento na produção de proteínas de interesse. Neste trabalho, inicialmente estudamos e identificamos o perfil de proteínas que são recrutadas para expressar e produzir proteínas heterólogas em A. nidulans por espectrometria de massas. Posteriormente, identificamos nas cepas de A. nidulans os genes que respondem ao tratamento com ditiotreitol e tunicamicina, drogas que induzem o UPR. Finalmente, selecionamos 12 genes associados à via de secreção em A. nidulans, os quais foram deletados em cepas recombinantes de A. nidulans, uma que secreta xilanase homóloga (xlnE, 5B3 strain) e outra xilanase heteróloga (tpet_0854, 854 strain). A deleção de uma ciclofilina e de uma chaperona molecular Hsp40 resultou no aumento de 1,25 e 1,70 vezes na secreção de xlnE, respectivamente. Da mesma forma, a deleção de uma tiorredoxina e uma manosiltransferase também aumentou, ainda que em níveis mais baixos, a secreção de xlnE. Os resultados ainda mostraram que a secreção de proteínas totais diminuiu nessas cepas delatadas. Considerando os resultados, essa abordagem demonstrou aumento expressivo na produção de enzimas-alvo, sugerindo que a manosiltransferase, a chaperona Hsp40, a ciclofilina e a tiorredoxina codificadas pelos genes deletados desempenham um papel importante na regulação da produção de proteína em A. nidulans. Entretanto, ainda não entendemos o mecanismo envolvido no aumento da secreção de xlnE após as deleções. Sugerimos que a maior produção de enzimas nas cepas deletadas possa estar relacionada à ativação do UPR e também a um "afrouxamento" no rigor do enovelamento de proteínas pela célula, resultando em um controle de qualidade mais brando e maior secreção de proteínas para o meio extracelularAbstract: In eukaryotes, the unfolded protein response (UPR) positively regulates genes responsible for restoring homeostasis in the endoplasmic reticulum (ER) during accumulation of misfolded proteins. The homeostasis is restored due to the activation of genes related to proteins secretion such as those encoding for chaperones and foldases, which, in turn, increase protein folding capacity by RE. In fact, some systems for heterologous protein production have been developed by the individual overexpression of chaperones and foldases in the hosting cells. However, the success rate of this strategy usually is quite low. Studies on the manipulation of genes that respond to UPR in fungal strains are interesting aiming a higher production of proteins. In this work, we initially identified the profile of proteins that are recruited to express and produce heterologous proteins in A. nidulans by mass spectrometry. Subsequently, we proceeded the identification of genes that respond to UPR-activating chemicals such as dithiothreitol and tunicamycin. Finally, we selected 12 genes with a predicted function in the A. nidulans secretion pathway. These 12 genes were deleted in an A. nidulans recombinant strain producing an homologous xylanase (xlnE, 5B3 strain) and another recombinant strain producing an heterologous xylanase (tpet_0854, 854 strain). The deletion of cyclophilin and a molecular chaperone Hsp40 resulted in an increase around 1.25 and 1.70-fold in the xlnE activity, respectively. Similarly, the deletion of thioredoxin and glycosyl phosphatidyl inositol-mannosyltransferase also increased the xlnE secretion even at lower levels. The results also showed a decreased production of total proteins production in these deleted strains. Thus, our results suggest that proteins such as glycosyl phosphatidyl inositol-mannosyltransferase, chaperone Hsp40, cyclophilin and thioredoxin play an important role in the regulation of proteins production by A. nidulans. However, we still do not understand the mechanism involved in increased secretion of 5B3 after the deletions. We suggest that the increased production of enzymes in the deleted strains is related to the activation of the UPR and a "less stringent" protein folding by the cell, resulting in a mild quality control and higher secretion of proteins into the extracellular mediumDoutoradoBioquímicaDoutora em Biologia Funcional e Molecular2014/15403-6FAPES

Peroxicretion: a novel secretion pathway in the eukaryotic cell

Background: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles. Results: Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extracellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies. Conclusion: Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared t

Metabolic peculiarities of Aspergillus niger disclosed by comparative metabolic genomics

A genome-scale metabolic network and an in-depth genomic comparison of Aspergillus niger with seven other fungi is presented, revealing more than 1,100 enzyme-coding genes that are unique to A. niger

Organic acid production in Aspergillus niger and other filamentous fungi

The aim of the thesis was to increase the understanding of organic acid production in Aspergillus niger and other filamentous fungi, with the ultimate purpose to improve A. niger as biotechnological production host. In Chapter 1, the use of microbial cell-factories for the production of various compounds of interest, with a focus on organic acid production in A. niger, is introduced. To convert A. niger into a cell-factory for the production of fumarate, an organic acid that this fungus does not naturally accumulate extracellularly, we need to know the key components that lead to high extracellular fumarate accumulation. This can be achieved by studying a natural fumarate producer, in our case the filamentous fungus Rhizopus delemar. To increase both the understanding of R. delemar fumarate production, and identify a possible candidate fumarate exporter protein for heterologuous expression in A. niger, we studied differences in the transcriptional and proteomic responses of R. delemar under high and low fumarate producing conditions, described in Chapter 2. Based on our analyses, we propose that a substantial part of the fumarate accumulated in R. delemar during nitrogen starvation results from the urea cycle due to amino acid catabolism. Thus, although we failed to identify the correct fumarte exporter (discussed in Chapter 8), the results of these analyses lead to a broader understanding of the mechanism underlying fumarate accumulation in R. delemar. In order to make A. niger a suitable production host for other organic acids, we also delved deeper into the understanding of why A. niger has an innate ability to secrete various organic acids, especially citrate, described in Chapter 3. We show that an increase in citrate secretion under iron limited conditions is a physiological response consistent with a role of citrate as A. niger iron siderophore. We found that A. niger citrate secretion increases with decreasing amounts of iron added to the culture medium and, in contrast to previous findings, this response is independent of the nitrogen source. Differential transcriptomics analyses of the two A. niger mutants NW305 (gluconate non-producer) and NW186 (gluconate and oxalate non-producer) revealed up-regulation of the citrate biosynthesis gene citA under iron limited conditions compared to iron replete conditions. In addition, we show that A. niger can utilise Fe(III) citrate as iron source. Finally, we discuss our findings in the general context of the pH-dependency of A. niger organic acid production, offering an explanation, besides competition, for why A. niger organic acid production is a sequential process influenced by the external pH of the culture medium. In Chapter 4, we further unravel the various different mechanisms underlying extracellular A. niger citrate accumulation. We show that the phenotype of increased extracellular citrate accumulation can have fundamentally different underlying mechanisms, depending on how this response was triggered. We found that varying the amount and supplement of an arginine auxotrophic A. niger strain induces increased citrate productivity. Transcriptomics analysis shows down-regulation of citrate metabolising enzymes in the conditions in which more citrate is accumulated extracellularly. This contrasts with the transcriptional adaptations triggered by iron limited conditions, described in Chapter 3. By combining data obtained from both experimental setups described in Chapters 3 and 4, we compiled a list of likely citrate transporter candidates. Two promising citrate exporter candidates were tested in the yeast Saccharomyces cerevisiae, of which one was successfully identified as citrate exporter. Our findings provide the first steps in untangling the complex interplay of different mechanisms underlying A. niger citrate accumulation, and we pinpoint, for the first time, a promising A. niger citrate exporter candidate, offering a valuable tool for improvement of A. niger as biotechnological cell-factory for citrate production. For the identification of different A. niger substrate importers, we combined in silico and in vivo approaches, and established a reliable pipeline to identify and test candidate transport proteins. The in silico approach, in which likely glucose transporter candidates are inferred from good matches with a glucose transporter specific Hidden Markov model (HMMgluT), and the in vivo approach, in which a sub-cellular proteomics approach is applied to isolate plasmalemmal glucose transporters, is described in Chapter 5. In the presented research work, a hidden Markov model (HMM), that shows a good performance in the identification and segmentation of functionally validated glucose transporters, was constructed. The model (HMMgluT) was used to analyse the A. niger membrane-associated proteome response to high and low glucose concentrations at a low pH. By combining the abundance patterns of the proteins found in the A. niger plasmalemma proteome with their HMMgluT scores, two new putative high affinity glucose transporters, denoted MstG and MstH, were identified. MstG and MstH were functionally validated and biochemically characterised by heterologous expression in a S. cerevisiae glucose transport null mutant. They were shown to be a high affinity glucose transporter (Km = 0.6 ± 0.1 mM) and a very high affinity glucose transporter (Km = 0.06 ± 0.005 mM) respectively. The concepts developed in Chapter 5 were applied in Chapter 6 to identify further substrate importer proteins in both A. niger and another filamentous fungus, Trichoderma reesei. Again a hidden Markov model, this time for the identification of xylose transporters, was constructed and used to analyse the A. niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterised through their expression in a S. cerevisiae hexose transport null mutant, engineered to be able to metabolise xylose, but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose, but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs associated to xylose transporters. Transcriptional analysis of A. niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, implying a biological role for these transporters in xylose utilisation. Thus, our findings show that our approach using HMMs is a robust pipeline to identify different substrate importer candidates. In Chapter 7, comparative plasmalemma proteomic analysis was used to identify candidate L-rhamnose transporters in A. niger. Further analysis was focused on protein ID 1119135 (RhtA) (JGI A. niger ATCC 1015 genome database). RhtA was classified as a Family 7 Fucose:H+ Symporter (FHS) within the Major Facilitator Superfamily. Family 7 currently includes exclusively bacterial transporters able to use different sugars. Strong indications for its role in L-rhamnose transport were obtained by functional complementation of the Saccharomyces cerevisiae EBY.VW.4000 strain in growth studies with a range of potential substrates. Biochemical analysis using L-[3H(G)]-rhamnose confirmed that RhtA is a L-rhamnose transporter. The RhtA gene is located in tandem with a hypothetical alpha-L-rhamnosidase gene (rhaB). Transcriptional analysis of rhtA and rhaB confirmed that both genes have a coordinated expression, being strongly and specifically induced by L-rhamnose, and controlled by RhaR, a transcriptional regulator involved in the release and catabolism of the methyl-pentose. RhtA is the first eukaryotic L-rhamnose transporter identified and functionally validated to date. In Chapter 8, the findings presented in this thesis with regards to our attempts at improving A. niger as biotechnological production host are summarised, and further implications for metabolic engineering approaches based on the conclusions drawn are discussed.</p

Abstracts from the 11th European Conference on Fungal Genetics

Programs and Abstracts from the 11th European Conference on Fungal Genetic

Predicting Protein Producibility: Binary classification of recombinant proteins produced in filamentous fungi

Recombinant protein synthesis aims to produce specific protein products of interest in living cells. However, protein production is subject to failure, and thus the successful development of a computational tool to predict protein sequence success prior to laboratory experimentation would save time and resources. We demonstrate the ability of an SVM trained on protein amino acid composition to predict successful protein production in a dataset of sequences tested in the host species Trichoderma reesei. We found that predictive models generalize well between two species of filamentous fungi, and furthermore that 50 training sequences are sufficient to train a model that yields an AUC of over .7. We introduced novel predictive features using protein domains detected with the InterProScan tool, which were modestly successful in the predictive task but whose addition did not improve over the use of amino acid composition alone. Experiments applying semi-supervised SVM formulations to the predictive task did not yield significant improvement, most likely because the spatial distribution of data points under the chosen numeric representations did not conform to the assumptions of the semi-supervised models. We explored the species of origin and enzyme function of sequences from the UniProt SwissProt database predicted to be successful by the trained SVM models, and showed that models trained with an RBF kernel were the most conservative in terms of the number of predicted successes