PASIV: A Pooled Approach-Based Workflow to Overcome Toxicity-Induced Design of Experiments Failures and Inefficiencies

We present here a
newly developed workflowwhich we have
called PASIVdesigned to provide a solution to a practical
problem with design of experiments (DoE) methodology: i.e., what can
be done if the scoping phase of the DoE cycle is severely hampered
by burden and toxicity issues (caused by either the metabolite or
an intermediary), making it unreliable or impossible to proceed to
the screening phase? PASIVstanding for pooled approach, screening,
identification, and visualizationwas designed so the (viable)
region of interest can be made to appear through an interplay between
biology and software. This was achieved by combining multiplex construction
in a pooled approach (one-pot reaction) with a viability assay and
with a range of bioinformatics tools (including a novel construct
matching tool). PASIV was tested on the exemplar of the lycopene pathwayunder
stressful constitutive expressionyielding a region of interest
with comparatively stronger producers.