Crosstalk between chromatin and Shavenbaby defines transcriptional output along the Drosophila intestinal stem cell lineage.

The transcription factor Shavenbaby (Svb), the only member of the OvoL family in Drosophila, controls the fate of various epithelial embryonic cells and adult stem cells. Post-translational modification of Svb produces two protein isoforms, Svb-ACT and Svb-REP, which promote adult intestinal stem cell renewal or differentiation, respectively. To define Svb mode of action, we used engineered cell lines and develop an unbiased method to identify Svb target genes across different contexts. Within a given cell type, Svb-ACT and Svb-REP antagonistically regulate the expression of a set of target genes, binding specific enhancers whose accessibility is constrained by chromatin landscape. Reciprocally, Svb-REP can influence local chromatin marks of active enhancers to help repressing target genes. Along the intestinal lineage, the set of Svb target genes progressively changes, together with chromatin accessibility. We propose that Svb-ACT-to-REP transition promotes enterocyte differentiation of intestinal stem cells through direct gene regulation and chromatin remodeling.

Assessment of Four Engineered PET Degrading Enzymes Considering Large-Scale Industrial Applications.

In recent years, enzymatic recycling of the widely used polyester polyethylene terephthalate (PET) has become a complementary solution to current thermomechanical recycling for colored, opaque, and mixed PET. A large set of promising hydrolases that depolymerize PET have been found and enhanced by worldwide initiatives using various methods of protein engineering. Despite the achievements made in these works, it remains difficult to compare enzymes' performance and their applicability to large-scale reactions due to a lack of homogeneity between the experimental protocols used. Here, we pave the way for a standardized enzymatic PET hydrolysis protocol using reaction conditions relevant for larger scale hydrolysis and apply these parameters to four recently reported PET hydrolases (LCCICCG, FAST-PETase, HotPETase, and PES-H1L92F/Q94Y). We show that FAST-PETase and HotPETase have intrinsic limitations that may not permit their application on larger reaction scales, mainly due to their relatively low depolymerization rates. With 80% PET depolymerization, PES-H1L92F/Q94Y may be a suitable candidate for industrial reaction scales upon further rounds of enzyme evolution. LCCICCG outperforms the other enzymes, converting 98% of PET into the monomeric products terephthalic acid (TPA) and ethylene glycol (EG) in 24 h. In addition, we optimized the reaction conditions of LCCICCG toward economic viability, reducing the required amount of enzyme by a factor of 3 and the temperature of the reaction from 72 to 68 °C. We anticipate our findings to advance enzymatic PET hydrolysis toward a coherent assessment of the enzymes and materialize feasibility at larger reaction scales.