Journal article
Flexible metabolic pathway construction using modular and divisible selection gene regulators
Genetic selections are important to biological engineering. Although selectable traits are limited,currently each trait only permits simultaneous introduction of a single DNA fragment. Complex pathwayand strain construction however depends on rapid, combinatorial introduction of many genes thatencode putative pathway candidates and homologs.
To triple the utility of existing selection genes, wehave developed divisible selection in Saccharomyces cerevisiae. Here, independent DNA fragments can beintroduced and selected for simultaneously using a set of split hybrid transcription factors composed ofparts from Escherichia coli LexA and Herpes simplex VP16 to regulate one single selectable phenotype ofchoice.
Only when co-expressed, these split hybrid transcription factors promote transcription of aselection gene, causing tight selection of transformants containing all desired DNA fragments. Upontransformation, 94% of the selected colonies resulted strictly from transforming all three modules basedon ARS/CEN plasmids.
Similarly when used for chromosome integration, 95% of the transformantscontained all three modules. The divisible selection system acts dominantly and thus expands selectiongene utility from one to three without any genomic pre-modifications of the strain. We demonstrate theapproach by introducing the fungal rubrofusarin polyketide pathway at a gene load of 11 kb distributedon three different plasmids, using a single selection trait and one yeast transformation step.
By triplingthe utility of existing selection genes, the employment of divisible selection improves flexibility andfreedom in the strain engineering process.
Language: | English |
---|---|
Year: | 2015 |
Pages: | 189-197 |
ISSN: | 10967184 and 10967176 |
Types: | Journal article |
DOI: | 10.1016/j.ymben.2015.08.004 |
ORCIDs: | Rugbjerg, Peter and Sommer, Morten Otto Alexander |