How do filamentous fungi grow

Do these models indeed form the conceptual basis for wet-lab data interpretation? These macromorphological effects were likely caused by decreased hyphal growth rate, reduced ability to maintain hyphal tip polarity, and a defective actin ring position at the hyphal tip due to altered arfA expression [ 50 ].

The actin ring has been shown to be the site of endocytosis in A. Geometric models of the spatial distribution of the actin ring in A. In an arfA conditional expression strain of A. Modification of the actin ring location might thus represent a generic strategy for titrating morphology and enhancing protein secretion in industrial fungi [ 50 ]. Taken together, these studies demonstrate how increasingly sophisticated modelling of growth and morphology at macro and subcellular levels can lead to mechanistic explanations of fungal strain engineering in industrial settings.

Despite these advances in the fermentation control of fungal morphology and cognate modelling approaches, it is not currently possible to precisely predict the optimal morphology for a desired product and, consequently, it is necessary to invest significant efforts in process design.

However, as the fundamental understanding and associated models of the filamentous lifecycle advances, it may be possible to use the underlying molecular, cellular, and developmental biology of fungi to predict improved growth and macromorphology for certain classes of product i. Consequently, protein secretion is generally highest during rapid hyphal extension and periods of active growth Fig.

A growing body of evidence suggests that modifying fungal macromorphology for a maximum tip: biomass ratio is a useful approach for improving protein secretion in many fungal systems [ 55 , 56 , 57 ].

Interestingly, however, in some cases elevated hyphal tip number is not correlated with increased protein titre, which may suggest routes other than the tip are important in some instances.

One recent explanation for the discrepancies between elevated hyphal tip numbers and titres of extracellular proteins is that unconventional protein secretion UPS pathways may also play important roles during industrial fermentation [ 58 ]. Generally, in UPS, proteins do not pass through the classical Golgi-vesicle-apex-dependent route, but are transported to the cell membrane via as yet undefined alternative mechanisms.

Indeed, numerous extracellular proteins do not contain N-terminal signal peptides necessary for processing through the Golgi and packaging into extracellular vesicles, and consequently are predicted to be secreted via UPS [ 58 ].

A second possible complication in the relationship between hyphal tip number and extracellular protein titres is recent work suggesting that protein secretion may also occur at the hyphal septum.

For example, in A. Secretion at hyphal septa plays a fundamental role in branch initiation and thickening of the cell wall at sub-apical locations and, in A. Fluorescent monitoring of the major secreted and industrially fermented glucoamylase protein in A. These data support the hypothesis that septal secretion could be of industrial relevance, and it is interesting to speculate that optimising morphology to maximise septal junctions through strain engineering efforts may be a future avenue to enhance product titres.

Taken together, while several studies support the hypothesis that optimising fungal morphology by increasing hyphal tip numbers is a promising strategy to enhance protein production, both UPS and intercalary secretion pathways represent promising, yet underexplored, avenues for strain engineering efforts.

For acid production, specifically citric acid in A. An exciting and important piece of the puzzle that has recently been revealed is the identification of the CexA major facilitator superfamily transporter that is required for export of citrate from A. However, this protein has yet to be localised to precise positions in the hyphal plasma membrane e. Despite these limitations to our fundamental knowledge, however, it is clear that production of citrate occurs at specific stages of active hyphal growth.

For example, recent dynamic modelling approaches have demonstrated that both oxalic and citric acid syntheses in A. With regard to biosynthesis of secondary metabolites, a small pelleted morphology has been demonstrated to increase product titres in some instances, for example lovastatin fermentation by A. What is certain, however, is that the formation of fungal secondary metabolites mostly occurs during periods of extremely low, or zero growth Fig.

Thus, an optimal morphology for secondary metabolite biosynthesis, in contrast to protein production, must somehow be integrated with nutrient limitation, thus causing ultralow fungal growth. A possible avenue for this is to generate pelleted morphologies with densely compact core, which may limit nutrient and oxygen diffusion and thus growth at the colony centre, in turn activating secondary metabolism [ 29 ].

Export of fungal secondary metabolites is also an extremely complex puzzle. Fungal natural products are biosynthesised by physically linked contiguous gene clusters, many of which contain genes encoding putative transporters that are predicted to be involved in extracellular secretion of the respective natural product [ 3 ]. Intriguingly, functional analyses of transporter genes in mycotoxin encoding clusters demonstrate that some of these transporters are functionally redundant, as deletion causes no reduction in secondary metabolite secretion [ 70 ].

Interestingly, in the model organism A. Critically, determining the exact distribution of these transporters throughout the fungal cell or colony could enable rational design of morphology for maximum secretion of these molecules. In summary, a complex relationship between fungal growth, morphology and protein, acid, and secondary metabolite titres emerges. Strain engineering efforts, increasingly informed by omics datasets, promise to deliver both the lead genes and platform strains for optimisation of filamentous morphology during diverse industrial applications.

Initial strain engineering efforts for optimal morphologies began in the s, whereby industrial fungal isolates were mutagenized for improved biotechnological applications [ 72 ].

For a diverse range of fungi, strains displaying modified morphology following mutagenesis screens have generated elevated product titres and improved hydrodynamic performance in bioreactors. For example, UV and nitrous acid mutagenesis resulted in several hyperbranched A. Elsewhere, diethyl sulfite mutagenesis of T. The genomes of these production strain lineages are currently being sequenced in community-wide efforts to identify candidate genes for strain improvement from comparative genomic approaches to identify desirable properties with respect to morphology and hyperproductivity [ 15 ].

At present, however, studies which attempt to identify single nucleotide polymorphisms SNPs responsible for advantageous growth or production phenotypes in production strain lineages are limited.

One such example used comparative genomics between the high protein producing industrial A. The hypersecretion phenotype of isolate SH2 is thought to be at least partially attributed to the highly branched hyphal fragments produced by this strain in submerged culture [ 74 ].

Comparative genomics between this strain and CBS Confirmation of these hypotheses, however, would require gene functional characterisation, and given that SNPs in several genes may synergistically contribute to the SH2 morphology, such wet-lab verification would require highly labour-intensive generation of combinations of A.

Thus, while the genes identified from this comparative genomic study remain high priority candidates for engineering filamentous fungi for optimal industrial growth [ 74 ], their exact application in biotechnology remains to be determined.

Elsewhere, interrogation of UV mutagenized penicillin platform isolates of P. LaeA is a component of the heterotrimeric velvet complex in filamentous fungi [ 76 ] that was originally discovered in A.

The velvet complex consists of VeA, which is predominantly expressed in the dark and physically interacts with the protein VelB, which is expressed during hyphal growth and development [ 76 ].

VeA bridges VelB to LaeA, which in turn is hypothesised to reverse the formation of transcriptionally silent heterochromatin by DNA or H3K9 methylation activity [ 79 ], thus activating secondary metabolite gene loci during hyphal growth. The velvet complex is, therefore, a molecular nexus connecting light responses, hyphal growth, and secondary metabolism. LaeA mutants have been generated in numerous fungal cell factories, which have been used to concomitantly activate natural product formation and modify morphology in many [ 75 , 80 , 81 ] but not all species [ 82 ].

It is likely that other such key regulators of development e. Clearly, comparative genomics is a powerful approach to unlock lead genes from mutagenized isolates for strain improvement programs.

A recent experimental technique developed in A. This approach requires a sexual or parasexual cycle in the fungus of interest, as the mutagenized isolate is firstly crossed with a wild-type strain. Importantly, the SNP absent in the progenitor strain, and concomitantly present in all segregants, is responsible for the mutant phenotype.

In a proof of principle experiment, the developers of this technique analysed a non-acidifying phenotype of a UV-mutated A. Thus, bulk segregant analysis is a powerful approach which could in future be applied to conclusively reverse engineer the SNPs, and encoding genes, that result in biotechnologically advantageous growth and morphology from libraries of mutagenized fungal isolates.

In addition to genomics approaches, RNA seq and microarray gene expression profiling during experimental models of enzyme, acid, and natural product fermentation have revealed potential gene candidates for optimising fungal morphology across diverse industrial processes.

Various experimental designs have been utilised, for example, time-series analysis throughout A. It is clear that genes belonging to common morphology and growth-associated processes are transcriptionally deployed, including classical and non-classical secretory pathways, cytoskeleton components, endocytosis, exocytosis, cell wall and cell membrane biosynthesis Fig.

Including the various signalling pathways driving and controlling these subcellular processes, it has been estimated that as many as genes encode proteins that at a certain level may participate in filamentous fungal growth and development [ 63 , 84 , 85 , 88 ]. Cellular processes that are essential for morphogenesis in filamentous fungi as deduced from transcriptomic studies.

Note, for each fungal species, it is common for several hundred differentially expressed genes to belong to each cohort. TORC2 signalling likely plays a crucial role in maintaining polarity by directly controlling actin polarisation but also by inhibiting calcineurin signalling. Inositolphosphate IP is also proposed to control actin polarisation.

For details, see [ 24 ]. As just one example, the A. This hypothesis has been validated by RNAi knockdown of chitin synthase encoding genes in A. More generally, numerous transcriptional studies support the hypothesis that diverse cell signalling networks orchestrate growth, morphology, and development in multiple filamentous cell factories [ 24 , 63 , 84 , 85 , 86 , 87 , 88 ]. Signalling cascades are interconnected networks that transduce extracellular environmental signals into cellular responses, including, for example, nutrient availability, cell wall integrity in response to sheer stress, and osmotic perturbation see next section for detail [ 91 ].

Based on transcriptomics signatures, a signalling network controlling morphogenesis was reconstructed for A. It has been hypothesized that phospholipid signalling, sphingolipid signalling, target of rapamycin kinase TORC2 signalling, calcium signalling and cell wall integrity CWI signalling pathways concertedly act to control polar growth in A.

The reconstructed transcriptomic network model obtained implies that these pathways become integrated to control sterol, ion transport, amino acid metabolism and protein trafficking to ensure cell membrane and cell wall expansion during hyphal growth. Most importantly, this transcriptomic network predicted that the transcription factors RlmA, CrzA and at least a third, so far unknown, transcription factor are output genes of the CWI signalling pathway.

This was subsequently experimentally confirmed by identification of the transcription factor MsnA which—at least in A. A final example for the successful deduction of lead genes from transcriptomic data for improved morphology and productivity is the Rho GTPase RacA, which was hypothesized to control filamentous growth via actin polymerisation and depolymerisation at the hyphal apex in A.

Transcriptional profiling of a racA deletion and dominant activation allele suggested that this protein plays a critical role in morphology and protein secretion [ 87 ] and that deletion of racA in A. Given that racA is highly conserved in filamentous fungi [ 17 ], it is possible that racA mutant isolates could be widely applied to enhance protein secretion in other systems, including Trichoderma spp. Genome wide metabolic models GWMM of various fungal cell factories have recently been developed and offer novel avenues to accurately predict gene knockout phenotypes or maximum product yields under different nutritional sources.

The ultimate aim of GWMM is to predict most of the metabolite content of an organism and link these with cognate reactions and catalytic enzymes. Arguably, the best such model in the fungal kingdom is for the budding yeast Saccharomyces cerevisiae , which contains over metabolites, biochemical reactions, and genes encoding the catalysing enzymes [ 95 ].

These models have enabled sophisticated predictions of protein function related to fungal growth, for example regulation of acetyl-COA biosynthesis by the Oaf1 transcription factor encoding gene in yeast [ 96 ]. GWMMs for numerous filamentous cell factories have been developed over the last decade [ 97 , 98 , 99 ] and have been used to model conditions for maximum production of fermentation products, for example secreted proteins in A.

More recently, strain-specific models have been updated, for example in A. Integration of these GWMM into publicly available data repositories including FungiDB [ 17 ], MycoCosm [ 15 ] and Ensembl [ ] promises to facilitate numerous avenues towards improved growth, nutrient utilisation, activation of secondary metabolism, and other diverse applications in subsequent strain engineering experimentation [ 1 ].

While currently linking metabolism and filamentous morphology is challenging, these public models will likely be critical for future hypothesis generation. In summary, comparative genomics, transcriptomics, and metabolic models have identified hundreds, or even thousands of genes that are promising candidates for engineering morphology in industrial fungi.

This work, combined with numerous gene functional characterisation experiments in industrial and model fungi, has identified what is arguably one of the most promising strain engineering strategies for controlling growth and morphology: genetic targeting of fungal signalling cascades. The next section introduces some key aspects of fungal signal transduction and highlights how these are currently being rationally manipulated for optimised industrial applications.

Given the crucial role that cell signalling plays in regulating morphology, numerous strain engineering efforts have targeted components of these cascades to optimise growth for improved biotechnological applications. Selected examples will be discussed in the following section.

Simplified schematic depiction of the major signalling cascades in filamentous fungal cell factories. MAPK cascades are initiated at the plasma membrane by two main processes. Secondly, in the two-component signal transduction system, a transmembrane histidine kinase HK is activated by extracellular ligands and a response regulator REG activates a histidine-containing phospho-transmitter HP that subsequently activates MAPK signalling.

Rho1 and protein kinase C PkcA. Increases in concentration of the second messenger cAMP activates protein kinase A PKA , which phosphorylates various target proteins, including transcription factors.

These enter the nucleus and regulate diverse responses. Once activated, calcineurin dephosphorylates the transcription factor CrzA, which causes elevated expression of genes necessary for growth and diverse stress responses.

All pathways have critical control of filamentous growth, fungal morphology, and development. Gene names are taken from A. Note that extensive cross talk occurs between pathways, and that in this schematic not all possible membrane receptors, signalling proteins, or transcription factors are depicted. Interested readers are guided to excellent reviews which cover fungal signalling cascades in greater depth [ 91 , ]. In several instances, MAPK phosphorylation of downstream transcription factors that control filamentous growth and development have been identified, mainly in the model organism A.

For instance, the MpkB controls the regulator SteA, which concomitantly induces sexual development and inhibits the activation of transcription factor MedA, which is also involved in conidiophore and sexual development reviewed in [ ]. Also in A. Consequently, deletion, overexpression or RNAi-based knock down of various levels of MAPK signalling cascades can cause diverse changes in morphology in filamentous fungi that may be biotechnologically advantageous, including hyperbranching e.

Despite the pleiotropic consequences of genetic targeting of MAPK signalling cascades, recent work has demonstrated that they can be used in rational strain engineering efforts.

In a proof of principle experiment, deletion of the gene predicted to encode an MkpB orthologue in T. It remains to be determined how strain engineering of other components of MAPK signalling can be applied in other species.

The activated PKA phosphorylates various target proteins, including transcription factors, resulting in their entry to the nucleus and modification of gene expression Fig. In addition to deletion, overexpression of PKA signalling can be used as a strategy to modify fungal macromorphology. For instance, in A. For example, in the model organism A. Once activated, calcineurin dephosphorylates the transcription factor CrzA, which causes elevated expression of genes necessary for growth and diverse stress responses [ ].

The calcineurin signalling pathway is an important regulator of asexual growth, for example in Aspergillus spp. Moreover, CrzA is necessary for responses to withstand cell wall stress encountered during high bioreactor stir speeds, and this pathway is required for elevated chitin, glucan and cell wall protein levels in A. In addition to these main signalling mechanisms, there are numerous other signal transduction pathways in filamentous fungi that regulate morphology, growth and development, including responses to pH via membrane receptor PalH and transcription factor PacC , light via the velvet complex, see above , additional nutrient sensing pathways via the target of rapamycin protein kinase TORC2 , response to reactive oxygen species via transmembrane NADPH oxidases , and RAS signalling [ 91 , ].

Given that all of these pathways transduce extracellular signals to regulate interconnected and diverse aspects of morphology and development, they are also promising targets for strain engineering.

It remains to be seen if the pleiotropic consequences of genetic manipulation of these pathways are advantageous, or a limitation for strain engineering of industrial fungi.

One example of the limitations to this strategy involves the heterotrimeric velvet complex Fig. These studies highlight potential pitfalls of manipulating signalling cascades and proteins that are components of the complex and dynamic architecture for fungal environmental sensing and adaptation.

A long-term goal for maximum control of fungal morphology during industrial applications may thus be to develop strains with reduced genome complexity. We thus discuss several recent technological developments in the field of fungal synthetic biology below. As stated above, thousands of genes may contribute to the complex phenotype of fungal morphology. This complexity results in emergent properties that cannot currently be predicted or explained based on understanding of the constituent components [ ].

In this regard, the revolutions in the field of synthetic biology promise to deliver the next generation of filamentous cell factories by delivering chassis cells that contain either designer chromosomes, or minimal genomes, with drastically reduced complexity and thus improved engineering capabilities. Progress towards a minimalized fungal genome has moved at a rapid pace in the unicellular yeast S. Remarkably, the 16 S. Although much less advanced than in S.

In a proof of principle experiment, a cluster necessary for the biosynthesis of the mycotoxin fumonisin was removed. While the gene content of filamentous fungi is considerably higher than that of yeast e.

Thus, it is conceivable that minimal genomes exclusively containing the necessary genes required for a user-defined growth phenotype or morphology could be developed in the future. Other than CRISPR—Cas, what other synthetic tools and techniques promise to revolutionise fungal cell factories, both from morphological perspectives and for increasing the associated product portfolio? Several filamentous fungi have been engineered to heterologously express key natural product biosynthetic genes, such as those encoding nonribosomal peptide synthetases, or polyketide synthases, including A.

Excitingly, new-to-nature compounds can also be generated, either by swapping of enzyme domains, subunits, or modules [ , ], or by feeding various amino acid precursors in growth media, which are incorporated into nonribosomal peptide molecules [ 6 ]. Thus, in future, fungal cell factories can not only be optimised for improved morphology, but also to heterologously express high-value products including new-to-nature compounds. Further synthetic biological advances are complimentary to the above natural product discovery pipelines.

This includes, for example, the development of polycistronic gene expression approaches in filamentous fungi [ , , ]. Given that transcriptomic analyses reveal highly coordinated and stage-specific transcriptional deployment of gene cohorts throughout growth in industrial applications [ 63 , 85 , ], the capability of concomitantly controlling the expression of multiple morphological regulatory genes using a single promoter may offer an attractive solution for improved morphological engineering studies.

A further important conceptual point with regard to engineering morphology, revealed from the use of the synthetic Tet-on gene switch in A. For example, transcriptional profiling during carbon-dependent enhancement of protein secretion in A. Subsequent functional analysis of this gene by replacement of the native promoter with the tunable Tet-on gene switch revealed that it is essential, and, moreover, that distinct morphologies and protein production phenotypes were revealed from titratable control of arfA expression [ 50 ].

Consequently, conditional and tuneable synthetic gene switches which are functional in filamentous fungi and have gone through multiple rounds of engineering and optimisation [ 94 , , ] represent an attractive tool that offers more precise interrogation of the relationship between gene function and strain morphology when compared to classical deletion or constitutive over-expression approaches. These molecular advances have occurred concomitantly with developments in fungal imaging.

Excitingly, this technology opens up new avenues for accurately quantifying hyphal distributions in the pellet core, including hyphal density, hyphal branch rates, and tip numbers. Thus, future studies on pellet morphology can now begin to access how different pellet phenotypes impact product titres. In summary, these technological advances highlight how many synthetic biological tools are already optimised for filamentous fungi.

We predict that these will enable the development of new cell factories with optimised morphologies, minimalised genomes, and improved product formation based on precise gene transcriptional control. Advances in fundamental science and modelling approaches are beginning to reveal the molecular and cellular basis of product formation and secretion in filamentous fungi under industrial, i.

A wealth of omics data is currently available and comparative analyses have already shown on how to unlock these data. Hence, targeted genetic manipulation of candidate genes controlling or indirectly impacting morphology can increasingly be used to generate and test novel strains for optimal growth. In parallel with these trends, fundamental progress in synthetic biology promises to reduce genome complexity of filamentous fungi, which ultimately may deliver chassis cells that have highly controlled and predictable growth and development for maximum product titres and enhanced performance in bioreactor cultivations.

Hence, the technological tools are thus in place for data-driven strain improvement programs. Still, the insights generated so far also touch on some fundamental questions, which need to be addressed to fully exploit the potential of filamentous fungi for a sustainable bio-economy: from an evolutionary point of view, are multicellularity and polar growth a prerequisite for high protein secretion?

Can the molecular basis of filamentous and multicellular growth be significantly reduced, or are too many of the components essential for high productivities? From a bioprocess engineering perspective, is it possible to develop a universal model of fungal growth, from dynamic changes in subcellular structures in young un branched hyphae to macroscopic units?

Are generic solutions to engineering morphology and growth in the diverse repertoire of industrial filamentous fungi possible, or do deviations in gene and protein function make this goal unrealistic?

As with the last decades, fundamental and applied sciences on filamentous fungi have to go hand in hand to mutually benefit from each other and to synergistically contribute to answering these questions. Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper.

Fungal Biol Biotechnol. Article Google Scholar. How a fungus shapes biotechnology: years of Aspergillus niger research. Fungal secondary metabolism: from biochemistry to genomics. Nat Rev Micro. Natural products as sources of new drugs over the 30 years from to J Nat Prod. Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides. III ed. Smith and D. Berry , Arnold, London, 51— Grove, S. Berry , Arnold, London, 28— Gull, K.

Berry , Arnold, London, 78— Hunsley, D. Microbiol , 62 , — Peberdy, J. Berry , Arnold, London, — Prosser, J. Microbiol , — Polacheck, I. Rosenberger, R. Ruiz-Herrera, J. Biol Chem. Stewart, P. Sypherd, P. State-of-the-art microscopy and molecular biological methods are enhancing our understanding of the mechanisms by which these intriguing and important organisms grow and differentiate.

A further project, led by Prof Meritxell Riquelme is shedding light on the precise nature of the transport of vesicles found at the growing hyphal tip.

Remarkably, it turns out that there are separate populations of differently-sized vesicles carrying different enzymes for building various components of the fungal cell wall.

From repair to reprogramming In addition to spontaneous growth, organisms also need to repair themselves from damage, and research into filamentous fungi is shedding light onto how this may be achieved. This molecular pathway promotes cell differentiation and regeneration in the face of damage and — using ROS as signals — could be shared with both plants and animals.

Therefore, understanding it could have important applications in medicine. As molecular biology advances, and the genomes of filamentous fungi are sequenced, our understanding of the precise molecular nature of these interactions will unfold. From fungi to further afield The diverse and enlightening findings of this high-profile programme have implications far beyond the fungal kingdom.

The methods used — particularly novel ways of imaging microscopic and rapidly-changing structures — have the potential to revolutionise studies of subcellular processes across the living world. Professor Fischer and his collaborators are finally bringing the filamentous fungi from the soil firmly into the limelight. How numerous, widespread and significant are the filamentous fungi? Fungi are found in nearly all ecosystems, where they fulfil important functions for nutrient recycling.

Some species are important plant pathogens, such as Magnaporthe oryzae or rust fungi. There are also animal pathogens. Many moulds contaminate food and feed and cause tremendous losses due to mycotoxin formation. Why is their growth and development such an interesting area to study? The fungal hypha is able to grow indefinitely at the tip.

It is one of the few examples of extreme polar growth of individual cells. Other examples are pollen tubes, root hairs and nerve cells. Thus the study of the mechanisms of polar fungal extension may help to improve our understanding of polarity in general.

Likewise, simple hyphae are able to differentiate rather complicated structures such as fruiting bodies. This requires massive changes in gene expression. It can be an example for other differentiation processes, e. What recent technological advances have helped further your research? The advent of molecular biological methods in the s, the use of GFP and other fluorescent proteins since in combination with steadily improved microscopy techniques and the recent development of super-resolution microscopy techniques have boosted fungal research.

How do the cells of filamentous fungi differ from those of other organisms? Fungi are in many aspects identical to human cells. One important difference, however, is the presence of a rigid cell wall consisting of different carbohydrate polymers, including chitin.

Because of this difference, the fungal cell wall or the biosynthesis machinery may be targets for drug development. What biological features are conserved between filamentous fungi and animals such as humans? Basic cell biological processes such as mitosis, meiosis, the functioning of organelles, or the principles of gene regulation, are highly conserved between humans and both filamentous and yeast-like fungi.