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Salt-enhanced cultivation as a morphology engineering tool for filamentous actinomycetes: Increased production of labyrinthopeptin A1 in Actinomadura namibiensis.

ABSTRACT: Salt-enhanced cultivation as a morphology engineering tool for the filamentous actinomycete Actinomadura namibiensis was evaluated in 500-mL shaking flasks (working volume 100 mL) with the aim of increasing the concentration of the pharmaceutically interesting peptide labyrinthopeptin A1. Among the inorganic salts added to a complex production medium, the addition of (NH4)2SO4 led to the highest amount of labyrinthopeptin A1 production. By using 50 mM (NH4)2SO4, the labyrinthopeptin A1 concentration increased up to sevenfold compared to the non-supplemented control, resulting in 325 mg L-1 labyrinthopeptin A1 after 10 days of cultivation. The performance of other ammonium- and sulfate-containing salts (e.g., NH4Cl, K2SO4) was much lower than the performance of (NH4)2SO4. A positive correlation between the uptake of glycerol as one of the main carbon sources and nongrowth-associated labyrinthopeptin productivity was found. The change in the cell morphology of A. namibiensis in conjunction with increased osmolality by the addition of 50 mM (NH4)2SO4, was quantified by image analysis. A. namibiensis always developed a heterogeneous morphology with pellets and loose mycelia present simultaneously. In contrast to the non-supplemented control, the morphology of (NH4)2SO4-supplemented cultures was characterized by smaller and circular pellets that were more stable against disintegration in the stationary production phase.

SUBMITTER: Tesche S

PROVIDER: S-EPMC6999293 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Salt-enhanced cultivation as a morphology engineering tool for filamentous actinomycetes: Increased production of labyrinthopeptin A1 in Actinomadura namibiensis.

Tesche Sebastian S Rösemeier-Scheumann René R Lohr Jonas J Hanke René R Büchs Jochen J Krull Rainer R

Engineering in life sciences 20190908 11

Salt-enhanced cultivation as a morphology engineering tool for the filamentous actinomycete Actinomadura namibiensis was evaluated in 500-mL shaking flasks (working volume 100 mL) with the aim of increasing the concentration of the pharmaceutically interesting peptide labyrinthopeptin A1. Among the inorganic salts added to a complex production medium, the addition of (NH4)2SO4 led to the highest amount of labyrinthopeptin A1 production. By using 50 mM (NH< ...[more]

PMID: 32624971

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Project description:BackgroundFungal fermentation is used to produce a diverse repertoire of enzymes, chemicals, and drugs for various industries. During submerged cultivation, filamentous fungi form a range of macromorphologies, including dispersed mycelia, clumped aggregates, or pellets, which have critical implications for rheological aspects during fermentation, gas/nutrient transfer, and, thus, product titres. An important component of strain engineering efforts is the ability to quantitatively assess fungal growth phenotypes, which will drive novel leads for morphologically optimized production strains.ResultsIn this study, we developed an automated image analysis pipeline to quantify the morphology of pelleted and dispersed growth (MPD) which rapidly and reproducibly measures dispersed and pelleted macromorphologies from any submerged fungal culture. It (i) enables capture and analysis of several hundred images per user/day, (ii) is designed to quantitatively assess heterogeneous cultures consisting of dispersed and pelleted forms, (iii) gives a quantitative measurement of culture heterogeneity, (iv) automatically generates key Euclidian parameters for individual fungal structures including particle diameter, aspect ratio, area, and solidity, which are also assembled into a previously described dimensionless morphology number MN, (v) has an in-built quality control check which enables end-users to easily confirm the accuracy of the automated calls, and (vi) is easily adaptable to user-specified magnifications and macromorphological definitions. To concomitantly provide proof of principle for the utility of this image analysis pipeline, and provide new leads for morphologically optimized fungal strains, we generated a morphological mutant in the cell factory Aspergillus niger based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for A. niger to identify a putative gamma-adaptin encoding gene (aplD) that was predicted to play a role in endosome cargo trafficking. Gene editing was used to generate a conditional aplD expression mutant under control of the titratable Tet-on system. Reduced aplD expression caused a hyperbranched growth phenotype and diverse defects in pellet formation with a putative increase in protein secretion. This possible protein hypersecretion phenotype could be correlated with increased dispersed mycelia, and both decreased pellet diameter and MN.ConclusionThe MPD image analysis pipeline is a simple, rapid, and flexible approach to quantify diverse fungal morphologies. As an exemplar, we have demonstrated that the putative endosomal transport gene aplD plays a crucial role in A. niger filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.

Dataset Information

Salt-enhanced cultivation as a morphology engineering tool for filamentous actinomycetes: Increased production of labyrinthopeptin A1 in Actinomadura namibiensis.

Publications

Salt-enhanced cultivation as a morphology engineering tool for filamentous actinomycetes: Increased production of labyrinthopeptin A1 in <i>Actinomadura namibiensis</i>.

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