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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 (β-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 4.4 U per mg 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 DOT-sensor technology to small scale, and by the use of a milliliter stirred tank bioreactor. Additionally, process development was assisted by DOE-optimization of the growth medium employing the response surface methodology. This article is protected by copyright. All rights reserved