Attachment of SUMO to proteins regulates protein-protein interactions through noncovalent binding of the SUMO moiety to specialized SUMO interaction motifs (SIMs). A core of hydrophobic amino acids has been described as the major determinant of SIM function. Using the transcriptional coregulator and SUMO ligase PIAS1 as a model, we define an extended phospho-regulated SIM module. We show that serine residues adjacent to the hydrophobic core are phosphorylated by CK2 and demonstrate that this dictates binding of free SUMO and SUMO conjugates to PIAS1 in vivo. We provide evidence that the phosphorylated residues contact lysine 39 and 35 in SUMO1 and SUMO2, respectively. Phospho-dependent SUMO binding does not impair the ligase activity but affects the transcriptional coregulatory potential of PIAS1 and other PIAS family members. CK2-regulated phosphoSIM modules were also dissected in the tumor suppressor PML and the exosome component PMSCL1, indicating that these modules serve as general platforms that integrate CK2- and SUMO-regulated signaling networks.

Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches. Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule. In the present study, we describe the fermentative production of the cyclic dipeptide D-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases. E. coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes. D-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis. Coexpression of the corresponding genes in E. coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment. Furthermore, the cyclic dipeptide is stable and not toxic to E. coli and is secreted into the culture medium without the need for any additional factors. Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature. By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of D-Phe-Pro-diketopiperazine/liter. As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals.

Highlights d The N-terminal region of SENP6 binds to SUMO chains d SENP6 controls SUMOylation of cohesin and regulates sister chromatid cohesion d SENP6 regulates the ATR-Chk1 DNA damage checkpoint d SENP6 orchestrates chromatin dynamics by balancing chromatin residency of proteins

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