ArticleScientific reports2019
Iterative screening methodology enables isolation of strains with improved properties for a FACS-based screen and increased L-DOPA production.
Article in Scientific reports, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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17 citing papers in PubMed, 37 citations in OpenAlex.
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- Effective use of biosensors for high-throughput library screening for metabolite production.Journal of industrial microbiology & biotechnology · 2021Review
- De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories.Nature communications · 2021Article
- Sorting for secreted molecule production using a biosensor-in-microdroplet approach.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Transportome-wide engineering of Saccharomyces cerevisiae.Metabolic engineering · 2021Article
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- Investigation of Bar-seq as a method to study population dynamics of Saccharomyces cerevisiae deletion library during bioreactor cultivation.Microbial cell factories · 2020Article
- Bidirectional titration of yeast gene expression using a pooled CRISPR guide RNA approach.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
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- Emerging molecular biology tools and strategies for engineering natural product biosynthesis.Metabolic engineering communications · 2020Review
- Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps.Nature communications · 2020Article
- Integrating enzyme evolution and high-throughput screening for efficient biosynthesis of L-DOPA.Journal of industrial microbiology & biotechnology · 2019Article
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Authors and funding
5 authors at 2 institutions in 1 country.
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No grant is acknowledged in the PubMed record.
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.
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