Journal
ACS SYNTHETIC BIOLOGY
Volume 4, Issue 1, Pages 23-31Publisher
AMER CHEMICAL SOC
DOI: 10.1021/sb500079f
Keywords
mevalonate pathway; isobutanol pathway; pathway assembly; delta-integration; antibiotic selection; Saccharomyces cerevisiae
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Funding
- National University of Singapore [R279 000 364 133]
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Saccharomyces cerevisiae as a eukaryotic organism is particularly suitable as microbial cell factory because it has interesting features such as membrane environments for supporting membrane-associated enzymes and its capability for post-translational modifications of enzymes from plants. However, S. cerevisiae does not readily express polycistronic transcriptional units, which represents a significant challenge for constructing large biochemical pathways in budding yeast. In the present study, we developed a novel approach for rapid construction of large biochemical pathways into yeast chromosomes. Our approach takes advantage of antibiotic selection for combinatorial assembly of large pathways into the delta-sites of retrotransposon elements of yeast chromosomes. As proof-of-principle, a five-gene isobutanol pathway and an eight-gene mevalonate pathway were successfully assembled into yeast chromosomes in one-step fashion. To our knowledge, this is the first report to exploit delta-integration coupled with antibiotic selection for rapid assembly of large biochemical pathways in budding yeast. We envision our new approach could serve as a generalized technique for large pathway construction in yeast-a method that would be of significant interest to the synthetic biology community.
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