Fungal Pellet Morphology Control for Submerged Culture Viscosity
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Solution Overview
Problem
Current methods for genetically manipulating filamentous fungi are laborious, inefficient, and result in random DNA integration, leading to unwanted genetic modifications and contamination issues, particularly in submerged cultures where hyphal growth increases viscosity, affecting oxygen and nutrient distribution and productivity.
Innovation Solution
The development of a variant filamentous fungal strain with a non-mycelium, pellet-forming phenotype achieved through genetic alterations in the osmotic response pathway, using automated co-transformation and screening to control morphology and reduce hyphal growth, allowing for improved growth in submerged cultures and product yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If filamentous fungi are grown in submerged cultures for large-scale production, then productivity and product yield are improved, but the mycelium formed increases broth viscosity, adversely affecting oxygen and nutrient dissolution and distribution
Solution Approach 1:
The patent applies parameter changes by modifying the genetic composition of the fungal strain through heterologous gene expression. Specifically, overexpressing genes involved in cell wall synthesis, chitin metabolism, and morphological regulation alters the physical parameters of hyphal growth, resulting in reduced viscosity and improved suspension characteristics without sacrificing productivity.
Solution Approach 2:
The patent replaces mechanical agitation requirements with biological control mechanisms. Instead of relying on mechanical mixing to overcome high viscosity, the invention uses genetically engineered morphological changes to inherently reduce viscosity, thereby decreasing the need for intensive mechanical agitation and energy input.
2Ease of manufacture
If typical transfection systems for filamentous fungi are used, then genetic manipulation can be achieved, but the process is laborious and yields are low due to protoplast formation requirements and difficult separation of heterokaryotic from homokaryotic protoplasts
Solution Approach 1:
The patent extracts and eliminates the problematic protoplast formation step from the transfection process. By using alternative transformation methods that work directly with intact fungal cells or spores, the invention removes the time-consuming steps of protoplast preparation, viscosity handling, and differential centrifugation required to separate heterokaryotic from homokaryotic cells.
Solution Approach 2:
The patent segments the complex transfection process into simpler, more manageable steps. Instead of a single laborious protoplast transformation procedure, the invention uses a multi-step approach involving spore preparation, direct transformation, and selective screening, which collectively reduce overall processing time and improve efficiency.
3Ease of manufacture
If DNA is introduced into fungus using current methods, then genetic transformation can occur, but DNA integrates randomly within the genome, resulting in multiple tandem repeats and unwanted modification of the host genome
Solution Approach 1:
The patent introduces a mediator mechanism in the form of targeted integration systems. Instead of random integration, the invention uses homologous recombination with specific genomic loci as intermediaries to guide precise DNA insertion. This ensures controlled integration at defined sites, preventing random tandem repeats and unwanted genomic modifications while maintaining transformation efficiency.
4Productivity
If filamentous fungi are grown as hyphae, then natural growth and sporulation occur, but the dense mycelium networks formed interfere with oxygen and nutrient dissolution, adversely affecting growth and productivity
Solution Approach 1:
The patent applies parameter changes by genetically modifying key regulatory genes that control hyphal growth dynamics. By altering the expression levels of genes involved in cell wall synthesis, actin cytoskeleton organization, and tip growth regulation, the invention changes the physical parameters of mycelium structure, resulting in more open, less dense networks that improve mass transfer while maintaining growth rate.
Data Source
AI summary
The present disclosure provides a microbial genomic engineering method and system for transforming, screening, and selecting filamentous fungal cells that have altered morphology and/or growth under specific growth conditions. The method and system utilize high-throughput (HTP) methods to produce filamentous fungal production strains with a desired morphological phenotype.