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Flexible metabolic pathway construction using modular and divisible selection gene regulators.

Peter Rugbjerg1, Nils Myling-Petersen1, Morten O A Sommer1

  • 1Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, DK-2970 Hørsholm, Denmark.

Metabolic Engineering
|August 26, 2015
PubMed
Summary

Divisible selection in yeast enables simultaneous introduction of multiple DNA fragments using split transcription factors. This biological engineering advancement triples selection gene utility for complex strain construction.

Keywords:
Chromosomal integrationPathway constructionSelection systemSplit transcription factorSynthetic biology

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Genetic Engineering

Background:

  • Genetic selection is crucial for biological engineering, but current methods limit DNA fragment introduction.
  • Complex strain engineering requires rapid, combinatorial gene introduction, which is hindered by limited selectable traits.

Purpose of the Study:

  • To develop a divisible selection system in Saccharomyces cerevisiae to triple the utility of existing selection genes.
  • To enable simultaneous introduction and selection of multiple independent DNA fragments in yeast.

Main Methods:

  • Developed a divisible selection system using split hybrid transcription factors (Escherichia coli LexA and Herpes simplex VP16).
  • Co-expressed split transcription factors to regulate a single selectable phenotype, ensuring transformants contain all desired DNA fragments.
  • Tested the system for ARS/CEN plasmid transformation and chromosome integration.

Main Results:

  • Achieved 94% of colonies containing all three DNA modules when using ARS/CEN plasmids.
  • Attained 95% of transformants containing all three modules during chromosome integration.
  • Successfully introduced an 11 kb fungal polyketide pathway using a single selection trait and one transformation step.

Conclusions:

  • The divisible selection system expands selection gene utility from one to three without genomic pre-modifications.
  • This method enhances flexibility and freedom in yeast strain engineering.
  • Divisible selection facilitates complex pathway and strain construction through combinatorial gene introduction.