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

Timothy C Cairns,Vera Meyer,Xiaomei Zheng,Ping Zheng,Jibin Sun,Claudia Feurstein

doi:10.3389/fbioe.2021.820088

Abstract

Submerged fermentation using filamentous fungal cell factories is used to produce a diverse portfolio of useful molecules, including food, medicines, enzymes, and platform chemicals. Depending on strain background and abiotic culture conditions, different macromorphologies are formed during fermentation, ranging from dispersed hyphal fragments to approximately spherical pellets several millimetres in diameter. These macromorphologies are known to have a critical impact on product titres and rheological performance of the bioreactor. Pilot productivity screens in different macromorphological contexts is technically challenging, time consuming, and thus a significant limitation to achieving maximum product titres. To address this bottleneck, we developed a library of conditional expression mutants in the organic, protein, and secondary metabolite cell factory Aspergillus niger. Thirteen morphology-associated genes transcribed during fermentation were placed via CRISPR-Cas9 under control of a synthetic Tet-on gene switch. Quantitative analysis of submerged growth reveals that these strains have distinct and titratable macromorphologies for use as chassis during strain engineering programs. We also used this library as a tool to quantify how pellet formation is connected with strain fitness and filamentous growth. Using multiple linear regression modelling, we predict that pellet formation is dependent largely on strain fitness, whereas pellet Euclidian parameters depend on fitness and hyphal branching. Finally, we have shown that conditional expression of the putative kinase encoding gene pkh2 can decouple fitness, dry weight, pellet macromorphology, and culture heterogeneity. We hypothesize that further analysis of this gene product and the cell wall integrity pathway in which it is embedded will enable more precise engineering of A. niger macromorphology in future.

Highlights

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)
We have shown that conditional expression of the putative kinase encoding gene pkh2 can decouple fitness, dry weight, pellet macromorphology, and culture heterogeneity
We hypothesize that further analysis of this gene product and the cell wall integrity pathway in which it is embedded will enable more precise engineering of A. niger macromorphology in future

Summary

Introduction

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). 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). 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). Clumped or pelleted fungal macromorphologies may result in hypoxia at internal pellet areas, limiting growth, metabolism, and possibly production of a desired molecule (Driouch et al, 2010)

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