Search

Currently, there is no consensus regarding the mechanism underlyingAspergillus nigercitrate biosynthesis and secretion, although it is amongst the most studied biotechnological production processes. Carbon excess relative to various other medium constituents is key, but the complex interplay between the limiting factors required for extracellular citrate accumulation remains elusive. It is thought that one of the industrial bottlenecks for citrate production is citrate export, however, noA. nigercitrate exporter has yet been identified. Here, we show that the phenotype of increased extracellular citrate accumulation can have fundamentally different underlying mechanisms, depending on how this response is triggered, and that combining gene expression analyses of the different conditions can lead to the compilation of a shortlist of the most promising citrate exporter candidates. Specifically, we found that varying the amount and type of supplement of an arginine auxotrophicA. nigerstrain shows down-regulation of citrate metabolising enzymes in the condition in which more citrate is accumulated extracellularly. This contrasts with the transcriptional adaptations triggered by iron limitation, which also induces increasedA. nigercitrate production. By combining data obtained from these two manners of inducing comparatively high extracellular citrate accumulation, we were able to compile a shortlist of the most likely citrate transporter candidates. Two of the most promising candidates were tested in the yeastSaccharomyces cerevisiae, one of which showed the ability to secrete citrate. Deletion of the endogenousA. nigergene encoding the corresponding transporter abolished the ability of this fungus to secrete citrate. Instead, under conditions that usually favourA. nigercitrate production, we found increased accumulation of extracellular oxalate. Our findings provide steps in untangling the complex interplay of different mechanisms underlyingA. nigercitrate accumulation, and we identify, for the first time, a fungal citrate exporter, offering a valuable tool for improvement ofA. nigeras biotechnological cell-factory for organic acid production.Author SummaryCitrate is widely applied as acidifier, flavouring and chelating agent. Industrial citrate production currently relies on the filamentous fungusAspergillus niger. Although the industrial production process usingA. niger has vastly improved since initiated almost 100 years ago, citrate export remains a bottleneck. Here, we studied the gene expression pattern ofA. nigerunder various citrate producing conditions. Using these expression patterns and different computational approaches, we compiled a shortlist of putative citrate exporter candidates. In this way, we were able to identify a gene encoding a transporter protein capable of citrate export. We show that the yeastSaccharomyces cerevisiae, normally a citrate non-producer, secretes detectable amounts of citrate when harbouring this gene. In addition, we verify the biological function of this gene inA. nigeritself, as removing this gene resulted in a citrate non-producing phenotype, which is atypical for this fungus. This finding is particularly exciting, as it is the first identification of a eukaryotic citrate exporter. With this, we not only provide a tool for improvement of industrial citrate production, but knowledge of this gene should help develop new methods for improvement ofA. nigeras biotechnological cell-factory for the production of other organic acids.

Aspergillus niger has an innate ability to secrete various organic acids, including citrate. The conditions required for A. niger citrate overproduction are well described, but the physiological reasons underlying extracellular citrate accumulation are not yet fully understood. One of the less understood culture conditions is the requirement of growth-limiting iron concentrations. While this has been attributed to iron-dependent citrate metabolizing enzymes, this straightforward relationship does not always hold true. Here, 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 utilize 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.

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.

The study of plant biomass utilization by fungi is a research field of great interest due to its many implications in ecology, agriculture and biotechnology. Most of the efforts done to increase the understanding of the use of plant cell walls by fungi have been focused on the degradation of cellulose and hemicellulose, and transport and metabolism of their constituent monosaccharides. Pectin is another important constituent of plant cell walls, but has received less attention. In relation to the uptake of pectic building blocks, fungal transporters for the uptake of galacturonic acid recently have been reported in Aspergillus niger and Neurospora crassa. However, not a single L-rhamnose (6-deoxy-L-mannose) transporter has been identified yet in fungi or in other eukaryotic organisms. L-rhamnose is a deoxy-sugar present in plant cell wall pectic polysaccharides (mainly rhamnogalacturonan I and rhamnogalacturonan II), but is also found in diverse plant secondary metabolites (e.g. anthocyanins, flavonoids and triterpenoids), in the green seaweed sulfated polysaccharide ulvan, and in glycan structures from viruses and bacteria. Here, a 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., Author Summary The growth of filamentous fungi on plant biomass, which occurs through the utilization of its components (e.g. D-glucose, D-xylose, L-arabinose, L-rhamnose) as carbon sources, is a highly regulated event. L-rhamnose (6-deoxy-L-mannose) is a deoxy-sugar present in plant cell wall pectic polysaccharides (mainly rhamnogalacturonan I and rhamnogalacturonan II), but also in diverse plant secondary metabolites, ulvan from green seaweeds and glycan structures from virus and bacteria. The utilization, transformation or detoxification of this monosaccharide by fungi involves a first step of chemical hydrolysis, performed by alpha-L-rhamnosidases, and a second step of transport into the cell, prior to its metabolization. While many rhamnosidases have been identified, not a single eukaryotic plasma membrane L-rhamnose transporter is known to date. In this study we identified and characterized, for the first time, a fungal L-rhamnose transporter (RhtA), from the industrial workhorse Aspergillus niger. We also found that RhtA putative orthologs are conserved throughout different fungal orders, opening the possibility of identifying new transporters of its kind.

Background The development of biological processes that replace the existing petrochemical-based industry is one of the biggest challenges in biotechnology. Aspergillus niger is one of the main industrial producers of lignocellulolytic enzymes, which are used in the conversion of lignocellulosic feedstocks into fermentable sugars. Both the hydrolytic enzymes responsible for lignocellulose depolymerisation and the molecular mechanisms controlling their expression have been well described, but little is known about the transport systems for sugar uptake in A. niger. Understanding the transportome of A. niger is essential to achieve further improvements at strain and process design level. Therefore, this study aims to identify and classify A. niger sugar transporters, using newly developed tools for in silico and in vivo analysis of its membrane-associated proteome. Results In the present 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 = 0.5 ± 0.04 mM) and a very high-affinity glucose transporter (Km = 0.06 ± 0.005 mM), respectively. Conclusions This study, focusing for the first time on the membrane-associated proteome of the industrially relevant organism A. niger, shows the global response of the transportome to the availability of different glucose concentrations. Analysis of the A. niger transportome with the newly developed HMMgluT showed to be an efficient approach for the identification and classification of new glucose transporters. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0317-9) contains supplementary material, which is available to authorized users.

Aspergillus niger is a filamentous fungus that is extensively used in industrial fermentations for protein expression and the production of organic acids. Inherent biosynthetic capabilities, such as the capacity to secrete these biomolecules in high amounts, make A. niger an attractive production host. Although A. niger is renowned for this ability, the knowledge of the molecular components that underlie its production capacity, intercellular trafficking processes and secretion mechanisms is far from complete. Here, we introduce a standardized set of tools, consisting of an N-terminal GFP-actin fusion and codon optimized eforRed chromoprotein. Expression of the GFP-actin construct facilitates visualization of the actin filaments of the cytoskeleton, whereas expression of the chromoprotein construct results in a clearly distinguishable red phenotype. These experimentally validated constructs constitute the first set of standardized A. niger biomarkers, which can be used to study morphology, intercellular trafficking, and secretion phenomena.