Yeast Pathway Kit: A Method for Metabolic Pathway Assembly with Automatically Simulated Executable Documentation

We
have developed the Yeast Pathway Kit (YPK) for rational and
random metabolic pathway assembly in Saccharomyces
cerevisiae using reusable and redistributable genetic
elements. Genetic elements are cloned in a suicide vector in a rapid
process that omits PCR product purification. Single-gene expression
cassettes are assembled in vivo using genetic elements
that are both promoters and terminators (TP). Cassettes sharing genetic
elements are assembled by recombination into multigene pathways. A
wide selection of prefabricated TP elements makes assembly both rapid
and inexpensive. An innovative software tool automatically produces
detailed self-contained executable documentation in the form of pydna
code in the narrative Jupyter notebook format to facilitate planning
and sharing YPK projects. A d-xylose catabolic pathway was
created using YPK with four or eight genes that resulted in one of
the highest growth rates reported on d-xylose (0.18 h–1) for recombinant S. cerevisiae without adaptation. The two-step assembly of single-gene expression
cassettes into multigene pathways may improve the yield of correct
pathways at the cost of adding overall complexity, which is offset
by the supplied software tool.