Understanding and controlling filamentous growth of fungal cell factories: novel tools and opportunities for targeted morphology engineering

Abstract

Filamentous fungal cell factories are efficient producers of platform chemicals, proteins, enzymes and natural products. Stirred-tank bioreactors up to a scale of several hundred m³ are commonly used for their cultivation. Fungal hyphae self-assemble into various cellular macromorphologies ranging from dispersed mycelia, loose clumps, to compact pellets. Development of these macromorphologies is so far unpredictable but strongly impacts productivities of fungal bioprocesses. Depending on the strain and the desired product, the morphological forms vary, but no strain- or product-related correlations currently exist to improve process understanding of fungal production systems. However, novel genomic, genetic, metabolic, imaging and modelling tools have recently been established that will provide fundamental new insights into filamentous fungal growth and how it is balanced with product formation. In this primer, these tools will be highlighted and their revolutionary impact on rational morphology engineering and bioprocess control will be discussed.

A Library of Aspergillus niger Chassis Strains for Morphology Engineering Connects Strain Fitness and Filamentous Growth With Submerged Macromorphology

“…Filamentous fungal growth in submerged culture produces a range of macromorphologies, each of which have advantages and disadvantages from the perspective of product titres and process engineering ( Cairns et al, 2019c ). It is not currently possible to predict an optimal macromorphology for a specific product a priori ( Meyer et al, 2021 ). Consequently, testing product titres in strains with distinct macromorphologies constitutes a major limitation to developing the next generation of ultra-efficient fungal cell factories ( Cairns et al, 2021 ).…”

“…Filamentous fungi are used to produce a diverse portfolio of molecules worth several billion dollars per year, including platform chemicals, proteins, enzymes, secondary metabolites, and organic acids ( Meyer et al, 2016 , 2020 ). The majority of industrially produced fungal metabolites are generated in stirred tank bioreactors which are often several hundred litres in volume ( Meyer et al, 2021 ). Maximizing product titres from minimum substrate and energy inputs promises to both reduce environmental impacts of fermentation and limit costs, which may ultimately enable fungal biotechnology to strengthen bioeconomy supposed to replace the fossil fuel economy in the near future ( Cairns et al, 2018 ; Meyer et al, 2020 ).…”

“…During submerged growth, filamentous fungi produce a range of different macromorphologies, including dispersed mycelial networks, loose clumps, spherical pellets several millimetres in diameter, or heterogenous mixtures of these structures ( Cairns et al, 2019a ; Meyer et al, 2021 ). The formation of respective macromorphologies is a complex process that is not comprehensively understood.…”

“…The formation of respective macromorphologies is a complex process that is not comprehensively understood. In general, clumped and pelleted growth is thought to involve coagulation of spores or hyphal fragments, whereas dispersed mycelial growth occurs following low levels of cell coagulation, in addition to fragmentation of large aggregates during bioreactor stirring ( Cairns et al, 2019a , 2021 ; Meyer et al, 2021 ). It is clear that the precise macromorphologies formed in submerged culture has critical implications for titres of proteins, enzymes, secondary metabolites, and acids ( Zhang and Zhang, 2016 ; Veiter et al, 2018 ; Cairns et al, 2019a ).…”

“…Additionally, each growth morphology has advantages and limitations from a process engineering perspective. For example, the rheological consequences of dispersed mycelial growth are elevated medium viscosity, and consequently temperature/substrate concentration gradients ( Žnidaršič and Pavko, 2001 ; Papagianni, 2004 ; Meyer et al, 2021 ). In contrast, clumped/pelleted macromorphologies improve transfer of oxygen and increase efficiency for removing biomass from growth media during downstream processing.…”