Enhanced Production of Bovine β-Lactoglobulin in an Industrial <i>Aspergillus oryzae</i> Host: A Step Forward in Alternative Protein Biomanufacturing

Precision fermentation of animal food proteins is an area of intense research as the requirements for more sustainable production increase. Despite significant strain engineering advances, the optimization of cell factories, including existing industrial fungal hosts, to achieve cost-effective protein yields is a major challenge. It is envisaged that the cost point of precision fermentation proteins needs to reach 10 USD/kg by 2025 and 1 USD/kg by 2035. This is a gigantic challenge to obtain high protein titers through strain and process development. A major challenge is the cost of carbon source, and improving the yield is therefore crucial for the economic feasibility. In this study, the influence of pH, temperature, and carbon-input rate was examined in a design of experiment approach to identify optimal fermentation parameters for the production of bovine beta-lactoglobulin by an industrial Aspergillus oryzae production strain. Carbon-input is a measure of carbon dosing relative to a benchmark feeding profile. The carbon-input was defined as the feed concentration multiplied by the feed-rate. This approach was used to show the relevance of the study and without compromising the confidentiality of the specific feed data. The optimal production yield, representing a protein production increase of almost 80% and a carbon yield above 70%, was achieved at pH 6.9, 36°C, and a carbon-input of 1.23×. The carbon-input correlated with biomass formation and significantly affected carbon yield. These results represent a significant step toward cost reduction and implementation of feasible precision fermentation for sustainable production of alternative food protein for a growing population. The production strain used in this study has been used to produce registration batches for a current Generally Regarded As Safe notice submission to the Food and Drug Administration.