CRAPS: Chromosomal-Repair-Assisted Pathway Shuffling in Yeast

A fundamental challenge of metabolic engineering involves
assembling
and screening vast combinations of orthologous enzymes across a multistep
biochemical pathway. Current pathway assembly workflows involve combining
genetic parts ex vivo and assembling one pathway
configuration per tube or well. Here, we present CRAPS, Chromosomal-Repair-Assisted Pathway Shuffling, an in vivo pathway engineering technique
that enables the self-assembly of one pathway configuration per cell.
CRAPS leverages the yeast chromosomal repair pathway and utilizes
a pool of inactive, chromosomally integrated orthologous gene variants
corresponding to a target multistep pathway. Supplying gRNAs to the
CRAPS host activates the expression of one gene variant per pathway
step, resulting in a unique pathway configuration in each cell. We
deployed CRAPS to build more than 1000 theoretical combinations of
a four-step carotenoid biosynthesis network. Sampling the CRAPS pathway
space yielded strains with distinct color phenotypes and carotenoid
product profiles. We anticipate that CRAPS will expedite strain engineering
campaigns by enabling the generation and sampling of vast biochemical
spaces.