PROTAC-Based Protein Degradation as a Promising Strategy for Targeted Therapy in Sarcomas.

Sarcomas are heterogeneous bone and soft tissue cancers representing the second most common tumor type in children and adolescents. Histology and genetic profiling discovered more than 100 subtypes, which are characterized by peculiar molecular vulnerabilities. However, limited therapeutic options exist beyond standard therapy and clinical benefits from targeted therapies were observed only in a minority of patients with sarcomas. The rarity of these tumors, paucity of actionable mutations, and limitations in the chemical composition of current targeted therapies hindered the use of these approaches in sarcomas. Targeted protein degradation (TPD) is an innovative pharmacological modality to directly alter protein abundance with promising clinical potential in cancer, even for undruggable proteins. TPD is based on the use of small molecules called degraders or proteolysis-targeting chimeras (PROTACs), which trigger ubiquitin-dependent degradation of protein of interest. In this review, we will discuss major features of PROTAC and PROTAC-derived genetic systems for target validation and cancer treatment and focus on the potential of these approaches to overcome major issues connected to targeted therapies in sarcomas, including drug resistance, target specificity, and undruggable targets. A deeper understanding of these strategies might provide new fuel to drive molecular and personalized medicine to sarcomas.

Charting a New Path Towards Degrading Every Protein.

Strategies to directly alter protein abundance such as small-molecule-induced targeted protein degradation (TPD) are innovative pharmacological modalities with promising clinical potential. Herein, I describe my experience with the development of the degradation tag (dTAG) system, which is a chemical biology strategy to induce rapid and precise degradation of any target protein. Open-source collaborative discovery has been critical for advancing the versatility and accessibility of the dTAG system and will be necessary to understand the benefits and limits of TPD-based strategies in the clinic.

Synthetic Gene Regulation in Cyanobacteria.

Cyanobacteria are appealing hosts for green chemical synthesis due to their use of light and carbon dioxide. To optimize product yields and titers, specific and tunable regulation of the metabolic pathways is needed. Synthetic biology has increased and diversified the genetic tools available for biological process control. While early tool development focused on commonly used heterotrophs, there has been a recent expansion of tools for cyanobacteria. CRISPR-Cas9 has been used to edit the genome of cyanobacterial strains, while transcriptional regulation has been accomplished with CRISPR interference and RNA riboswitches. Promoter development has produced a significant number of transcriptional regulators, including those that respond to chemicals, environmental signals, and metabolic states. Trans-acting RNAs have been utilized for posttranscriptional and translational control. The regulation of translation initiation is beginning to be explored with ribosome binding sites and riboswitches, while protein degradation tags have been used to control expression levels. Devices built from multiple parts have also been developed to create more complex behaviors. These advances in development of synthetic cyanobacterial regulatory parts provide the groundwork for creation of new, even more sophisticated bioprocess control devices, bolstering the viability of cyanobacteria as sustainable biotechnology platforms.

Highly Efficient Biosynthesis of Protocatechuic Acid via Recombinant Pseudomonas putida KT2440.

Owing to their physiological activities, plant-derived phenolic acids, such as protocatechuic acid (PCA), have extensive applications and market prospects. However, traditional production processes present numerous challenges and cannot meet increasing market demands. Hence, we aimed to biosynthesize PCA by constructing an efficient microbial factory via metabolic engineering of Pseudomonas putida KT2440. Glucose metabolism was engineered by deleting the genes for gluconate 2-dehydrogenase to enhance PCA biosynthesis. To increase the biosynthetic metabolic flux, one extra copy of the genes aroGopt, aroQ, and aroB was inserted into the genome. The resultant strain, KGVA04, produced 7.2 g/L PCA. By inserting the degradation tags GSD and DAS to decrease the amount of shikimate dehydrogenase, PCA biosynthesis was increased to 13.2 g/L in shake-flask fermentation and 38.8 g/L in fed-batch fermentation. To the best of our knowledge, this was the first use of degradation tags to adjust the amount of a key enzyme at the protein level in P. putida KT2440, evidencing the remarkable potential of this method for naturally producing phenolic acids.

Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression.

Riboswitches are promising regulatory tools in synthetic biology. To date, 25 theophylline riboswitches have been developed for regulation of gene expression in bacteria. However, no one has systematically evaluated their regulatory effects. To promote efficient selection and application of theophylline riboswitches, we examined 25 theophylline riboswitches in Escherichia coli MG1655 and found that they varied widely in terms of activation/repression ratios and expression levels in the absence of theophylline. Of the 20 riboswitches that activate gene expression, only one exhibited a high activation ratio (63.6-fold) and low expression level without theophylline. Furthermore, none of the five riboswitches that repress gene expression were more than 2.0-fold efficient. To obtain an effective repression system, we rationally designed a novel theophylline riboswitch to control a downstream gene or genes by premature transcription termination. This riboswitch allowed theophylline-dependent downregulation of the TurboRFP reporter in a dose- and time-dependent manner. Its performance profile exceeded those of previously described repressive theophylline riboswitches. We then introduced as the second part a RepA tag (protein degradation tag) coding sequence fused at the 5'-terminal end of the turborfp gene, which further reduced protein level, while not reducing the repressive effect of the riboswitch. By combining two tandem theophylline riboswitches with a RepA tag, we constructed a regulatory cassette that represses the expression of the gene(s) of interest at both the transcriptional and posttranslational levels. This regulatory cassette can be used to repress the expression of any gene of interest and represents a crucial step toward harnessing theophylline riboswitches and expanding the synthetic biology toolbox. IMPORTANCE A variety of gene expression regulation tools with significant regulatory effects are essential for the construction of complex gene circuits in synthetic biology. Riboswitches have received wide attention due to their unique biochemical, structural, and genetic properties. Here, we have not only systematically and precisely characterized the regulatory properties of previously developed theophylline riboswitches but also engineered a novel repressive theophylline riboswitch acting at the transcriptional level. By introducing coding sequences of a tandem riboswitch and a RepA protein degradation tag at the 5' end of the reporter gene, we successfully constructed a simple and effective regulatory cassette for gene regulation. Our work provides useful biological components for the construction of synthetic biology gene circuits.

Development of a Highly Efficient Copper-Inducible GAL Regulation System (CuIGR) in Saccharomyces cerevisiae.

Dynamic regulation of gene expression to decouple growth and production has been proven to be effective for improving the biosynthetic efficiency of microbial cell factories. However, the number of efficient regulatory systems available for regulation of Saccharomyces cerevisiae is limited. In the present study, a novel copper-inducible gene expression system (CuIGR) composed of the copper-induced transcriptional activator Gal4 and the copper-inhibited repressor Gal80 was constructed in S. cerevisiae. When Gal80 was fused with a N-degron tag (K15), the resulting CuIGR4 system exhibited the most stringent regulation of gene expression driven by GAL1/2/7/10 promoters. As compared to the native Cu2+-inducible CUP1 promoter, the CuIGR4 system amplified the response to copper by as much as 2.7 folds, resulting in 72-fold induction of EGFP expression and a 33-fold change in lycopene production (3-100 mg/L) with addition of 20 µM copper. This newly developed copper-inducible system provides a powerful tool for gene expression control in S. cerevisiae, which is expected to be widely applicable in the regulation of yeast cell factories for enhanced biosynthesis of valuable products.

A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution.

BACKGROUND: Aldehyde decarbonylases (ADs), which convert acyl aldehydes into alkanes, supply promising solution for producing alkanes from renewable feedstock. However the instability of ADs impedes their further application. Therefore, the current study aimed to investigate the degradation mechanism of ADs and engineer it towards high stability. RESULTS: Here, we describe the discovery of a degradation tag (degron) in the AD from marine cyanobacterium Prochlorococcus marinus using error-prone PCR-based directed evolution system. Bioinformatic analysis revealed that this C-terminal degron is common in bacterial ADs and identified a conserved C-terminal motif, RMSAYGLAAA, representing the AD degron (ADcon). Furthermore, we demonstrated that the ATP-dependent proteases ClpAP and Lon are involved in the degradation of AD-tagged proteins in E. coli, thereby limiting alkane production. Deletion or modification of the degron motif increased alkane production in vivo. CONCLUSION: This work revealed the presence of a novel degron in bacterial ADs responsible for its instability. The in vivo experiments proved eliminating or modifying the degron could stabilize AD, thereby producing higher titers of alkanes.