A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE159524 | GEO | 2020/11/09
REPOSITORIES: GEO
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Project description:The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes synthetic neochromosomes as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular neochromosomes of 50 and 100 kb were fully assembled de novo from up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. Neochromosomes made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding neochromosomes were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.
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Project description:A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae
| PRJNA669988 | ENA
Project description:BackgroundDue to their modular repeat structure, Pumilio/fem-3 mRNA binding factor (PUF) proteins are promising candidates for designer RNA-binding protein (RBP) engineering. To further facilitate the application of the PUF domain for the sequence-specific RBP engineering, a rapid cloning approach is desirable that would allow efficient introduction of multiple key amino acid mutations in the protein. Here, we report the implementation of the Golden Gate cloning method for an efficient one-step assembly of a designer PUF domain for RNA specificity engineering.ResultsWe created a repeat module library that is potentially capable of generating a PUF domain with any desired specificity. PUF domains with multiple repeat modifications for the recognition of altered RNA targets were obtained in a one-step assembly reaction, which was found to be highly efficient. The new PUF variants exhibited high in vitro binding efficiencies to cognate RNA sequences, corroborating the applicability of the modular approach for PUF engineering. To demonstrate the application of the PUF domain assembly method for RBP engineering, we fused the PUF domain to a post-transcriptional regulator and observed a sequence-specific reporter and endogenous gene repression in human cell lines.ConclusionsThe Golden Gate based cloning approach thus should allow greater flexibility and speed in implementing the PUF protein scaffold for engineering designer RBPs, and facilitate its use as a tool in basic and applied biology and medicine.
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Project description:DNA is typically found as a double helix, however it must be separated into single strands during all phases of DNA metabolism; including transcription, replication, recombination and repair. Although recent breakthroughs have enabled the design of modular RNA- and double-stranded DNA-binding proteins, there are currently no tools available to manipulate single-stranded DNA (ssDNA). Here we show that artificial pentatricopeptide repeat (PPR) proteins can be programmed for sequence-specific ssDNA binding. Interactions occur using the same code and specificity as for RNA binding. We solve the structures of DNA-bound and apo proteins revealing the basis for ssDNA binding and how hydrogen bond rearrangements enable the PPR structure to envelope its ssDNA target. Finally, we show that engineered PPRs can be designed to bind telomeric ssDNA and can block telomerase activity. The modular mode of ssDNA binding by PPR proteins provides tools to target ssDNA and to understand its importance in cells.
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2021-09-09 | PXD023127 | Pride
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2024-05-17 | GSE267438 | GEO
Project description:Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the target sequence. We present here a strategy for engineering of TALE proteins with novel DNA binding specificities based on the 17.5 repeat-containing AvrBs3 TALE as a scaffold. For each of the 17 full repeats, four module types were generated, each with a distinct base preference. Using this set of 68 repeat modules, recognition domains for any 17 nucleotide DNA target sequence of choice can be constructed by assembling selected modules in a defined linear order. Assembly is performed in two successive one-pot cloning steps using the Golden Gate cloning method that allows seamless fusion of multiple DNA fragments. Applying this strategy, we assembled designer TALEs with new target specificities and tested their function in vivo.
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Project description:Easy operation, readily accessible starting materials, and short syntheses of the privileged scaffold indeno[1,2-c]isoquinolinone were achieved by an multicomponent reaction (MCR)-based protocol via an ammonia-Ugi-four component reaction (4CR)/copper-catalyzed annulation sequence. The optimization and scope and limitations of this short and general sequence are described. The methodology allows an efficient construction of a wide variety of indenoisoquinolinones in just two steps.
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