High-performance production strains for enzymes and proteins

BRAIN Biotech's project teams are on a mission to revolutionize the production of enzymes and proteins through microbial fermentation. They're embracing an interdisciplinary approach, bringing together experts from microbiology, molecular biology, and bioprocess development to work in parallel on strain and bioprocess optimization. BRAIN Biotech's Aspergillus niger strain has shown itself to be an amazingly efficient production strain for enzymes and alternative proteins, with yields in the "double-digit" gram-per-liter range!

Aspergillus niger as a production strain

Here is one case study: A customer is on the lookout for a microbial strain that can produce a specific molecule in the highest possible quantity. How do we select such an amazing production strain? Christian Naumer, Director and Unit Head of Bioprocess Development at BRAIN Biotech, is eager to explain: "If the customer does not specify a strain, the team decides which microorganism to use after a scientific assessment of the target molecule. Usually, this is a strain that is already recognized as a production strain for the respective application and is classified as safe."

Production strains include Escherichia coli, Pichia pastoris (Komagataella phaffii), Bacillus subtilis, and the fungus Aspergillus niger. The latter is particularly popular in biotechnology because it naturally secretes proteins in high titers and this property can be exploited.

The Aspergillus niger used at BRAIN Biotech belongs to biosafety level 1 (BSL1) and has already shown its potential in Zwingenberg several times. In Aspergillus niger, production successes with yields in the “double-digit” gram per liter range, which is relevant for industry, have long been demonstrated by the project teams for the secretion of homologous proteins. These yields are now also being achieved with heterologous proteins from eukaryotic hosts.

Alexander Pelzer, Head of Research and Development at BRAIN Biotech's R&D site in Zwingenberg, explains: "Aspergillus niger has played an important role in the production of enzymes for many years. Now, there are also many requests from the field of alternative proteins: in the future, animal proteins will be replaced by sequence-identical but microbially produced proteins.”

Hand in hand: strain and bioprocess development

The food industry needs powerful microorganisms to produce animal proteins. Researchers often modify the metabolism of a microorganism to enable it to produce high levels of a target protein. Molecular biologists at BRAIN Biotech achieve this with the help of genome editing and a very special enzyme: the nuclease BMC, an in-house development of BRAIN Biotech's Akribion Genomics team. Genome editing involves the targeted integration of multiple optimized gene expression cassettes encoding the target protein into the genome of the microorganism.

In addition, the production organism is modified so that it ultimately produces only minimal amounts of unwanted proteins without reducing the secretion capacity for the target protein. The targeted deletion of the host's genes plays a crucial role in this process. The fewer "secondary proteins" the microorganism produces and secretes, the less complex the subsequent downstream process (DSP). Alexander Pelzer emphasizes: "Such a result is extremely important for our customers because it means a faster and more cost-effective production process - and ultimately a cheaper product.”

At BRAIN Biotech, strain optimization and bioprocess development are considered from the outset as an interdisciplinary task from the perspective of microbiology, molecular biology and bioprocess development. Alexander Pelzer formulates the idea as follows: "I need to improve the strain at the molecular level, but I also need to understand the strain in the bioreactor."

The strain and the bioprocess are optimized in parallel during the development of the production strain. Christian Naumer can also confirm that the close teamwork at BRAIN Biotech has paid off: "We would not have recognized the potential of some microorganisms if we had not developed the respective process in parallel. This is especially true for bioprocesses with the complex organism Aspergillus niger.”

Keywords: Bioprocess development, alternative proteins, precision fermentation, production strains, genome editing, Aspergillus niger, DSP