Abstract
Optimizing microbial hosts for the large-scale production of valuable metabolites often requires multiple mutations and modifications to the host’s genome. We describe a three-round screen for increased L-DOPA production in S. cerevisiae using FACS enrichment of an enzyme-coupled biosensor for L-DOPA. Multiple rounds of screening were enabled by a single build of a barcoded in vitro transposon-mediated disruption library. New background strains for screening were built for each iteration using results from previous iterations. The same in vitro transposon-mediated disruption library was integrated by homologous recombination into new background strains in each round of screening. Compared with creating new transposon insertions in each round, this method takes less time and saves the cost of additional sequencing to characterize transposon insertion sites. In the first two rounds of screening, we identified deletions that improved biosensor compartmentalization and, consequently, improved our ability to screen for L-DOPA production. In a final round, we discovered that deletion of heme oxygenase (HMX1) increases total heme concentration and increases L-DOPA production, using dopamine measurement as a proxy. We further demonstrated that deleting HMX1 may represent a general strategy for P450 function improvement by improving activity of a second P450 enzyme, BM3, which performs a distinct reaction.
Highlights
Metabolic engineering studies aim to increase production of valuable molecules using microbial hosts
Barcode sequencing (BarSEQ) demonstrated acceptable uniformity of coverage for the 4,785 unique ORF deletions reported to be present in the deletion collection (Supplementary Fig. S1)[26]
UPtag and DNtag barcodes as well as revised barcode sequences from Eason et al were included in analysis, but 541 ORF deletions still could not be identified by barcode sequencing
Summary
Metabolic engineering studies aim to increase production of valuable molecules using microbial hosts. A common strategy is to create a library of DNA mutations and use a screen or selection to isolate library members with increased production. The strategy uses plasmids containing sgRNA and donor DNA sequences to produce Cas9-mediated deletions at specified genomic positions[3] Another strategy for creating gene knockout libraries in new background strains is to use transposon-disruption[4,5]. Www.nature.com/scientificreports generation with transposons, each round would require the researcher to generate a new strain library and characterize the insertion sites of the transposons. Insertional mutations enriched through selection or screening can be rapidly identified by PCR amplification and sequencing the associated barcode (BarSEQ) This enables iteration to identify a multi-insertion strain with improved performance. Using our iterative method for introducing transposon insertion mutations, we were able to identify mutations that improve our single-cell, fluorescence-activated cell sorting (FACS) screening methodology in the first and second rounds, and a mutation that increases L-DOPA production in our third round
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