Enzymes are crucial to biotechnology and consumer goods. These enzymes are produced by micro-organisms, which are also referred to as cell-factories (in contrast to concrete buildings). One such specific cell-factory organism is the bacterium Bacillus subtilis. A wide range of industries uses B. subtilis to manufacture enzymes used in laundry detergents, dairy products, textiles, bakery products, or beverages. Due to the importance of biotechnology in achieving a modern society, additional enzyme production improvements are needed to develop societies further. However, these developments cannot be reached by merely increasing production quantity. More importantly, enzyme production must become more efficient concerning its environmental impact due to energy and material consumption.

Genomic modifications to enzyme-producing micro-organisms can increase enzyme production efficiency, however, modifications relating to the class of non-coding RNA genes are relatively unexplored.

This Ph.D. thesis was part of a larger academic-industrial collaboration that investigated what role RNA, particularly non-coding RNA (ncRNA), might have in the production of enzymes in B. subtilis.

To conduct such an investigation, knowledge of what the coding and non-coding RNA transcripts in B. subtilis are is needed; such knowledge comes from genome annotations. Due to the innovative recency of RNA technologies, the quality of ncRNA annotations lacks behind those of coding genes, despite tremendous research community efforts. To study ncRNA in their biotechnological context, the work in this thesis also includes efforts to provide such annotations.

The work presented in this thesis comprises multiple components. Firstly, a comprehensive genome annotation of both coding and non-coding RNA transcripts, including untranslated regions and RNA structures, was generated by integrating and unifying a wide variety of existing resources. Secondly, experimental RNA sequencing data from the industrial enzyme production process were investigated. The experimental data allowed inference of additional potential no vel non-coding transcripts. In combination with advanced expression analysis methods, RNA candidates were identified to increase enzyme production yields in a knockdown expression experiment. Thirdly, in combination with the alternative differential RNA sequencing protocol, the specific promoter activities during enzyme production were investigated. Moreover, the data gave rise to an unprecedented high-resolution annotation of transcription start sites. 

Therefore, the work presented in this thesis show how RNA-based technologies could also revolutionize enzyme-producing biotechnology. Although this thesis focused on enzyme production in B. Subtilis, the methodology developed in this theses can also be used to study of other micro-organisms, both in the production context and other biotechnological or academic studies. 

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