Cloning Whole Bacterial Genomes in Yeast

Benders, Gwynedd A.

doi:10.1007/978-1-61779-564-0_13

Cited by 31 publications

(39 citation statements)

References 39 publications

(30 reference statements)

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“…The central technique for building large heterologous DNA constructs in yeast is transformationassociated recombination (TAR), which leverages yeast's unique capability to efficiently combine many DNA fragments by homologous recombination in a single step (Larionov et al, 1996). Recently, researchers have demonstrated the ability to assemble and clone whole bacterial genomes in yeast (Gibson et al, 2008) using a combination of enzymatic in vitro assembly (Gibson, 2011) and TAR-based assembly (Benders et al, 2010) methods. This foundational advance opens up the possibility of rapidly assembling large biosynthetic pathways using a combination of oligonucleotide synthesis, in vitro assembly, and yeast-mediated TAR assembly, providing a scalable alternative to traditional recombinant DNA cloning methods.…”

Section: Rapid Assembly Of Biosynthetic Pathwaysmentioning

confidence: 99%

Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools

et al. 2012

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Secondary metabolites are an important source of high-value chemicals, many of which exhibit important pharmacological properties. These valuable natural products are often difficult to synthesize chemically and are commonly isolated through inefficient extractions from natural biological sources. As such, they are increasingly targeted for production by biosynthesis from engineered microorganisms. The budding yeast species Saccharomyces cerevisiae has proven to be a powerful microorganism for heterologous expression of biosynthetic pathways. S. cerevisiae's usefulness as a host organism is owed in large part to the wealth of knowledge accumulated over more than a century of intense scientific study. Yet many challenges are currently faced in engineering yeast strains for the biosynthesis of complex secondary metabolite production. However, synthetic biology is advancing the development of new tools for constructing, controlling, and optimizing complex metabolic pathways in yeast. Here, we review how the coupling between yeast biology and synthetic biology is advancing the use of S. cerevisiae as a microbial host for the construction of secondary metabolic pathways.

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Section: Rapid Assembly Of Biosynthetic Pathwaysmentioning

confidence: 99%

Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools

et al. 2012

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“…While intensive efforts have been made to turn E. coli into a universal molecular biology tool, the power of S. cerevisiae as DNA assembly platform has so far been largely untapped. Despite the pivotal role played by S. cerevisiae in the last decade in the assembly of genomes (14,(43)(44)(45)(46) there is so far relatively little effort invested in turning this yeast into a universal and powerful DNA assembly platform (47,48), a situation that we expect to see change in the future.…”

Section: Discussionmentioning

confidence: 99%

A supernumerary designer chromosome for modularin vivopathway assembly inSaccharomyces cerevisiae

Postma

Dashko

Breemen

et al. 2020

Preprint

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The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes Synthetic Chromosomes (SynChs) as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular SynChs of 50 and 100 Kb were fully assembled de novo from up to 44 transcriptionalunit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. SynChs made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding SynChs 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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“…Yeast and Mycoplasma strains used in this study and culture conditions Saccharomyces cerevisiae strain W303a containing the marked Mmc genome YCpMmyc1.1 (Lartigue et al, 2009;Benders et al, 2010) and its derivative YCpMmyc1.1-Δglf were grown on synthetic minimal medium containing glucose (SD) either lacking histidine, or histidine and uracil, or lacking histidine and supplemented with 1 mg ml −1 5-fluoroorotic acid (5-FOA, Carl Roth, Germany) (Kouprina and Larionov, 2008;Noskov et al, 2010).…”

Section: Methodsmentioning

confidence: 99%

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance

Schieck

Lartigue

Frey

et al. 2015

Molecular Microbiology

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SummaryMycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-Δglf strain did not produce the galactofuranosecontaining glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also 'leaking' as revealed by a β-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.

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Cloning Whole Bacterial Genomes in Yeast

Cited by 31 publications

References 39 publications

Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools

Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools

A supernumerary designer chromosome for modularin vivopathway assembly inSaccharomyces cerevisiae

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance

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