Dataset Information

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

ABSTRACT: Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the ?-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L(-1) of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.

SUBMITTER: Maury J

PROVIDER: S-EPMC4775045 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

Maury Jérôme J Germann Susanne M SM Baallal Jacobsen Simo Abdessamad SA Jensen Niels B NB Kildegaard Kanchana R KR Herrgård Markus J MJ Schneider Konstantin K Koza Anna A Forster Jochen J Nielsen Jens J Borodina Irina I

PloS one 20160302 3

Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence ...[more]

PMID: 26934490

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Project description:BackgroundManipulations in Saccharomyces cerevisiae classically depend on use of auxotrophy selection markers. There are several disadvantages to this in a microbial cell factory setting: (1) auxotrophies must first be engineered in prototrophic strains, and many industrial strains are polyploid/aneuploid prototrophs (2) available strain auxotrophies must be paired with available repair plasmids (3) remaining auxotrophies must be repaired prior to development of industrial bioprocesses. Use of dominant antibiotic resistance markers can circumvent these problems. However, there are relatively few yeast antibiotic resistance marker vectors available; furthermore, available vectors contain only one expression cassette, and it is often desirable to introduce more than one gene at a time.ResultsTo overcome these problems, eight new shuttle vectors have been developed. The plasmids are maintained in yeast under a 2 μm ori and in E. coli by a pUC ori. They contain two yeast expression cassettes driven by either (1) the constitutive TEF1 and PGK1 promoters, or (2) the constitutive TEF1 promoter and the inducible GAL10 or HXT7 promoters. Expression strength of these promoters over a typical production time frame in glucose/galactose medium was examined, and identified the TEF1 and HXT7 promoters as preferred promoters over long term fermentations. Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. coli) or ble (conferring resistance to phleomycin in both yeast and E. coli). Selection conditions for these plasmids/antibiotics in defined media were examined, and selection considerations are reviewed. In particular, medium pH has a strong effect on both G418 and phleomycin selection.ConclusionsThese vectors allow manipulations in prototrophic yeast strains with expression of two gene cassettes per plasmid, and will be particularly useful for metabolic engineering applications. The vector set expands the (currently limited) selection of antibiotic marker plasmids available for use in yeast, and in addition makes available dual gene expression cassettes on individual plasmids using antibiotic selection. The resistance gene cassettes are flanked by loxP recognition sites to allow CreA-mediated marker removal and recycling, providing the potential for genomic integration of multiple genes. Guidelines for selection using G418 and phleomycin are provided.

Dataset Information

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

Publications

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

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