Felix Hamburger ¹ Niels Schlichting ¹ Michael Eichenlaub ² Paul Igor Costea ² Christopher Sauer ² Stefan Jenewein ^2* Johannes Kabisch ^1,3*

• ¹ Darmstadt University of Technology, Darmstadt, Hesse, Germany
• ² BASF (Germany), Ludwigshafen am Rhein, Germany
• ³ Norwegian University of Science and Technology, Trondheim, Norway

Proteins face an obstacle race on their way to successful folding. Chaperones facilitate the proper folding of proteins by ensuring they remain on the correct path towards their final tertiary structure. In Bacilli, the PrsA chaperone is essential for the correct folding and stabilisation of proteins within the cell wall. Overexpression of PrsA chaperone has been shown to improve the successful folding and secretion of many biotechnology relevant, secreted enzymes. This results in a double benefit: Firstly, it promotes the efficient release of properly folded enzymes from the cell wall and secondly, it reduces the folding stress for the cell, thereby enhancing the overall fitness of the production organism. This paper presents a workflow in which different wild-type PrsA molecules in Bacillus subtilis are co-expressed with different amylases having different signal peptides and promoters. To achieve this, six genome reduced strains and nine PrsA proteins were systematically selected in respect to their cultivation performance as well as production of two reference amylases. After strain selection and deletion of major extracellular proteases, several hundred individual strains were created and screened using a step wise and modular automation approach combined with amplicon sequencing. Besides sharing the key learnings from the workflow, it could be shown that no single PrsA molecule consistently improved amylase production, but genetic constructs combining different elements showed up to a 10-fold variation in yield. Among the screened constructs the signal peptides YdjM and YvcE demonstrated the best performance.