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If you’re interested in fermentation-based production of enzymes and proteins, chances are you have heard of the filamentous fungus, Aspergillus niger. It is an efficient factory for making everything from citric acid to a variety of industrially relevant enzymes and proteins. But if you know A. niger, you’re likely aware of the challenges in engineering and fermentation scale-up that limit its potential applications for commercial production. Addressing these challenges requires hefty investment into developing suitable host strains. This is where Ginkgo can help. Our solution is a scalable, low viscosity best-in-class production host of A. niger designed to help you achieve your commercial production goals.
A. niger has long been valued for commercial-scale enzyme and protein production for a variety of factors:
High native production of industrially relevant enzymes and proteins
“Generally recognized as safe” (GRAS) by the FDA making an easier path towards commercialization
High-level protein secretion systems that reduce recombinant protein degradation by intracellular proteases and make downstream processing easier and cheaper
High Viscosity Fermentation
A major challenge of working with A. niger is its high viscosity fermentation, caused by long filamentous structures known as hyphae, which make production and purification difficult. Thick cell culture presents challenges in oxygenation, reactor mixing and nutrient feeding in scaling, which reduces enzyme and protein expression as well as making product recovery almost impossible. These factors may lead to increased time, cost, and resources required for commercial production.
Low-Throughput Strain Engineering
Beyond these challenges in manufacturing, strain improvement in A. niger is complicated by low transformation efficiency, difficulty in isolating correct edits from multinucleated cells, difficulty in automating HTP workflows because of filamentous growth, and secretion of many native proteases which can degrade the product. This can lead to longer, more expensive, and less predictable R&D programs to develop new products using A. niger as an expression host.
Our Solution: A Low Viscosity A. niger Expression Platform
We are pioneering new innovation in filamentous fungi-based manufacturing. We have applied our platform to create a new proprietary low viscosity A. niger production chassis, which we believe is a major advance against all of these challenges, and shows great promise for the development of other host strain expression platforms. By decreasing viscosity of our host strain, we unlock the ability to increase feed rate, oxygen transfer, and ultimately achieve higher expression rates for enzymes and proteins. The result? Increased titers at significantly reduced time and cost compared to conventional high viscosity strains.
Video (7 seconds): Fermentation process in a 10L reactor demonstrating Ginkgo’s low viscosity Aspergillus technology. The opaque broth contains a high cell density critical to industrial fermentation, yet low viscosity of the Ginkgo technology enables smooth continuous mixing with standard, scalable CapEx.
Value of Low Viscosity Fermentation
Reduced Energy Consumption: Lower viscosity requires less energy for stirring at the same biomass levels therefore contributing to lower COGS.
Enhanced Biomass Yield: With a low viscosity phenotype maintained at a constant stirring rate, higher biomass levels can be achieved as we can unlock increased feed rate and oxygen transfer. This increased biomass can be attributed to increased production, improving protein titers and decreasing cycle time per batch, therefore lowering manufactured COGS.
Streamlined Downstream Processing: The lower viscosity phenotype facilitates potentially easier downstream processing and purification, simplifying the overall production workflow.
Generating a scalable low-viscosity A. niger manufacturing platform means an easier path to commercialization. We achieved this phenotype by integrating base strain screening with rigorous fermentation process optimization. Our novel base strain shows reduced filamentation in culture, and our optimized process – achieved through strategic changes to media composition, pH and feed rates – promotes enhanced growth of our strain without needing improved aeration strategies. We can promote high efficiency sporulation on agar plates for starting cultures and aiding recombinant gene transfer, and also prevent sporulation in liquid media that causes batch-to-batch variations in bioreactors and other processing issues. Through these methods, we have generated a base production strain optimized for low viscosity growth in scale-up fermentation.
The improved process created by our low viscosity strain and the strain’s genetic make-up which included multiple copies of the target enzyme, allowed us to improve production of a native secreted enzyme to 120 g/L, five times higher than our initial titers. With further strain engineering, there is potential to achieve even higher performance for this protein. These successes present opportunities for manufacturing other native products of A. niger to achieve similar titers. With the development of a fully validated fermentation process from 250 mL to large-scale industrial bioreactors we have helped reduce risk and associated R&D costs for future programs.
Chassis Strain Development
We can offer solutions for customers interested in developing their own chassis strain. We have identified several genomic mutations that confer the low-viscosity phenotype, and have confirmed that the equivalent edits replicate the phenotype in other fungal species. We see potential to engineer other filamentous fungi, including existing production strains, with similar phenotypes to generate multiple low viscosity expression platforms.
Development of a Clean Background
Using multiple gene editing approaches – a combination of classical split marker homologous recombination and CRISPR – we made significant improvements to our strain background. We performed targeted knockouts of multi-copy genes for secreted proteins that would compete with heterologous products, and for major protease genes and those related to protease regulation that can result in degrading your targets. Through these modifications of the host strain, we have generated a clean background strain with diverse project applications.
Strain Engineering Standardization
In parallel to developing a scalable fermentation process, we made fungal strain engineering more efficient and predictable. While filamentous fungi are notoriously difficult to engineer, we have developed efficient transformation protocols. We made the strain compatible with our platform by creating high-throughput transformation protocols that can leverage our in-house liquid handling robots and other clonal separation technologies, streamlining the process further. These improvements can give you confidence in our ability to express a variety of heterologous enzymes and proteins.
Expanding the scope of products that can be manufactured in filamentous fungi like A. niger offers immense industry value. Our breakthrough low viscosity expression platform stands as a key innovation, enhancing oxygenation and feed to maximize titer output of industrial enzymes and proteins. By combining our low viscosity strain with extensive R&D efforts, we’ve not only pushed titer limits but also simplified scale-up fermentation and optimized strain engineering processes. We’ve taken on the heavy lifting for you, streamlining production and reducing fixed manufacturing costs. With our proprietary high-productivity, low viscosity strain of A. niger, we’ve eliminated the complexities of strain development. This means you can feasibly achieve faster, more cost-effective commercial-scale production with reduced risk.
We’re equipped to engineer your strains and any enzyme or protein of interest in our production host. Stay engaged with us as we continue to push boundaries and uncover the full potential of A. niger.
Our ready-to-use proprietary Aspergillus niger strain delivers:
Low viscosity – also in your own preferred production strain
High production of native enzymes and proteins
High-level enzyme secretion system
Clean background – Minimal native secreted protein / protease activity
Partner with us to unlock the potential of Aspergillus niger today!
Contact our technical consultants to learn more: Peter Punt & Kenneth Bruno
