An engineered Sox17 induces somatic to neural stem cell fate transitions independently from pluripotency reprogramming.

Advanced strategies to interconvert cell types provide promising avenues to model cellular pathologies and to develop therapies for neurological disorders. Yet, methods to directly transdifferentiate somatic cells into multipotent induced neural stem cells (iNSCs) are slow and inefficient, and it is unclear whether cells pass through a pluripotent state with full epigenetic reset. We report iNSC reprogramming from embryonic and aged mouse fibroblasts as well as from human blood using an engineered Sox17 (eSox17FNV). eSox17FNV efficiently drives iNSC reprogramming while Sox2 or Sox17 fail. eSox17FNV acquires the capacity to bind different protein partners on regulatory DNA to scan the genome more efficiently and has a more potent transactivation domain than Sox2. Lineage tracing and time-resolved transcriptomics show that emerging iNSCs do not transit through a pluripotent state. Our work distinguishes lineage from pluripotency reprogramming with the potential to generate more authentic cell models for aging-associated neurodegenerative diseases.

Colorimetric High-Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) are important biocatalysts for the enzymatic synthesis of chiral sulfoxides, including chiral sulfoxide-type proton pump inhibitors for the treatment of gastrointestinal diseases. However, native BVMOs are not yet suitable for practical application due to their unsatisfactory activity and thermostability. Although protein engineering approaches can help address these issues, few feasible high-throughput methods are available for the engineering of such enzymes. Herein, a colorimetric detection method to distinguish sulfoxides from sulfides and sulfones was developed for prazole sulfide monooxygenases. Directed evolution enabled by this method has identified a prazole sulfide monooxygenase CbBVMO variant with improved activity and thermostability that catalyzes the asymmetric oxidation of lansoprazole sulfide. A 71.3 % increase in conversion and 6 °C enhancement in the melting point were achieved compared with the wild-type enzyme. This new method is feasible for high-throughput screening of prazole sulfide monooxygenase variants with improved activity, thermostability, and/or substrate specificity.

Identification two key residues at the intersection of domains of a thioether monooxygenase for improving its sulfoxidation performance.

AcCHMO, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus, is a typical Type I Baeyer-Villiger monooxygenase (BVMO). We previously obtained the AcCHMOM6 mutant, which oxidizes omeprazole sulfide (OPS) to the chiral sulfoxide drug esomeprazole. To further improve the catalytic efficiency of the AcCHMOM6 mutant, a focused mutagenesis strategy was adopted at the intersections of the FAD-binding domain, NADPH-binding domain, and α-helical domain based on structural characteristics of AcCHMO. By using focused mutagenesis and subsequent global evolution two key residues (L55 and P497) at the intersections of the domains were identified. Mutant of L55Y improved catalytic efficiency significantly, whereas the P497S mutant alleviated substrate inhibition remarkably. AcCHMOM7 (L55Y/P497S) was obtained by combining the two mutations, which increased the specific activity from 18.5 (M6) to 108 U/g, and an increase in the Ki of the substrate OPS from 34 to 265 µM. The results indicate that catalytic performance can be elevated by modification of the sensitive sites at the intersection of the domains of AcCHMO. The results also provided some insights for the engineering of other Type I BVMOs or other multidomain proteins.