Combinatorial assembly of large biochemical pathways into yeast chromosomes for improved production of value-added compounds.
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 δ-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 δ-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.
Department of Chemical and Biomolecular Engineering, National University of Singapore , 4 Engineering Drive 4, Singapore 117585, Singapore.
Jifeng
J
Yuan
Chi Bun
CB
Ching
D000440:Butanols / Q000378:metabolism; D015825:Chromosomes, Fungal / Q000235:genetics*; D015723:Gene Library; D005813:Genes, Synthetic; D005818:Genetic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008798:Mevalonic Acid / Q000378:metabolism; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D058615:Synthetic Biology
D000440:Butanols; C040507:isobutyl alcohol; D008798:Mevalonic Acid
Saccharomyces cerevisiae; antibiotic selection; isobutanol pathway; mevalonate pathway; pathway assembly; δ-integration