Fast-track development of a lactase production process with Kluyveromyces lactis by a progressive parameter-control workflow

The time-to-market challenge is key to success for consumer goods affiliated industries. In recent years, the dairy industry faces a fast and constantly growing demand for enzymatically produced lactose-free milk products, mainly driven by emerging markets in South America and Asia. In order to take advantage of this opportunity, we developed a fermentation process for lactase (beta-galactosidase) from Kluyveromyces lactis within short time. Here, we describe the process of stepwise increasing the level of control over relevant process parameters during scale-up that established a highly efficient and stable production system. Process development started with evolutionary engineering to generate catabolite-derepressed variants of the K. lactis wild-type strain. A high-throughput screening mimicking fed-batch cultivation identified a constitutive lactase overproducer with 260-fold improved activity of 4.4U per milligram dry cell weight when cultivated in glucose minimal medium. During scale-up, process control was progressively increased up to the level of conventional, fully controlled fed-batch cultivations by simulating glucose feed, applying pH- and dissolved oxygen tension (DOT)-sensor technology to small scale, and by the use of a milliliter stirred tank bioreactor. Additionally, process development was assisted by design-of-experiments optimization of the growth medium employing the response surface methodology.