Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries

• Published in: Nature communications Vol. 14; no. 1; p. 4871
• Main Authors: Englund, Elias, Schmidt, Matthias, Nava, Alberto A., Klass, Sarah, Keiser, Leah, Dan, Qingyun, Katz, Leonard, Yuzawa, Satoshi, Keasling, Jay D.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 12.08.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 38/22; 38/23; 38/47; 38/77; 49/31; 49/40; 631/61/318; 631/61/32; 631/61/338/469; 631/61/338/552; 631/92/60; 96/10; Acyltransferase; assay systems; Assembly lines; BASIC BIOLOGICAL SCIENCES; Biosensors; biosynthesis; Boundaries; Exchanging; Humanities and Social Sciences; Hybrids; metabolic engineering; Modular engineering; multidisciplinary; Optimization; protein design; Protein folding; Science; Science (multidisciplinary); Solubility; Structural integrity; synthetic biology
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-40464-x

Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules. Engineering polyketide synthases can be challenging due to the absence of efficient high-throughput methods. Here, the authors used a solubility biosensor to identify stable variants from libraries of modified polyketide synthases.