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

[Image: see text] 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.