Metabolic engineering has emerged as a promising strategy for the production of therapeutically important natural products and their analogs. However, the construction and manipulation of pathways of biosynthetic genes poses a key bottleneck to efforts to engineer cells for the production of complex natural products and their derivatives, and a new generation of transformative technologies will be needed to overcome this barrier. Towards this end, we have developed Reiterative Recombination as a strategy for the efficient in vivo assembly of multi-gene DNA constructs. Here we propose to develop our technology for metabolic engineering applications by challenging it in the context of optimizing terpenoid yields, specifically for taxol intermediates, in yeast. If successful, this research will make the construction of libraries of natural product biosynthetic pathways a routine, affordable, and highly efficient process that can be carried out by the non-expert without any specialized equipment.

Public Health Relevance

The objective of this proposal is to create a transformative technology for in vivo assembly of multi-gene DNA constructs. This technology will greatly facilitate the construction of engineered strains for metabolic engineering and other synthetic biology applications. By providing a robust, efficient, and accessible method for the assembly of large DNA constructs, this technology, if successful, will significantly further efforts to produce therapeutically important natural products in recombinant microorganisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM096064-03S1
Application #
8786758
Study Section
Special Emphasis Panel (ZRG1 (02))
Program Officer
Preusch, Peter
Project Start
2012-02-01
Project End
2016-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
3
Fiscal Year
2014
Total Cost
$53,677
Indirect Cost
$18,263
Name
Columbia University (N.Y.)
Department
Chemistry
Type
Other Domestic Higher Education
DUNS #
049179401
City
New York
State
NY
Country
United States
Zip Code
10027
Ostrov, Nili; Wingler, Laura M; Cornish, Virginia W (2013) Gene assembly and combinatorial libraries in S. cerevisiae via reiterative recombination. Methods Mol Biol 978:187-203