posted on 2023-12-23, 14:29authored byMahsa Babaei, Philip Tinggaard Thomsen, Marc Cernuda Pastor, Michael Krogh Jensen, Irina Borodina
Identification of
metabolic engineering targets is a fundamental
challenge in strain development programs. While high-throughput (HTP)
genetic engineering methodologies capable of generating vast diversity
are being developed at a rapid rate, a majority of industrially interesting
molecules cannot be screened at sufficient throughput to leverage
these techniques. We propose a workflow that couples HTP screening
of common precursors (e.g., amino acids) that can be screened either
directly or by artificial biosensors, with low-throughput targeted
validation of the molecule of interest to uncover nonintuitive beneficial
metabolic engineering targets and combinations hereof. Using this
workflow, we identified several nonobvious novel targets for improving p-coumaric acid (p-CA) and l-DOPA
production from two large 4k gRNA libraries each deregulating 1000
metabolic genes in the yeast Saccharomyces cerevisiae. We initially screened yeast cells transformed with gRNA library
plasmids for individual regulatory targets improving the production
of l-tyrosine-derived betaxanthins, identifying 30 targets
that increased intracellular betaxanthin content 3.5–5.7 fold.
Hereafter, we screened the targets individually in a high-producing p-CA strain, narrowing down the targets to six that increased
the secreted titer by up to 15%. To investigate whether any of the
six targets could be additively combined to improve p-CA production further, we created a gRNA multiplexing library and
subjected it to our proposed coupled workflow. The combination of
regulating PYC1 and NTH2 simultaneously
resulted in the highest (threefold) improvement of the betaxanthin
content, and an additive trend was also observed in the p-CA strain. Lastly, we tested the initial 30 targets in a l-DOPA producing strain, identifying 10 targets that increased the
secreted titer by up to 89%, further validating our screening by proxy
workflow. This coupled approach is useful for strain development in
the absence of direct HTP screening assays for products of interest.