Title: Iterative screening methodology enables isolation of strains with improved properties for a FACS-based screen and increased L-DOPA production

Abstract

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 distinctmore » reaction.« less

Authors:

Savitskaya, Judy; Protzko, Ryan J.; Li, Francesca-Zhoufan; Arkin, Adam P.; Dueber, John E.

• Search DOE PAGES for author "Dueber, John E."
• Search DOE PAGES for ORCID "0000-0001-8404-7153"

Publication Date:

Tue Apr 09 00:00:00 EDT 2019

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC)

OSTI Identifier:

1619472

Alternate Identifier(s):

OSTI ID: 1559205

Grant/Contract Number:  

BioDesign; AC02-05CH11231

Resource Type:

Published Article

Journal Name:

Scientific Reports

Additional Journal Information:

Journal Name: Scientific Reports Journal Volume: 9 Journal Issue: 1; Journal ID: ISSN 2045-2322

Publisher:

Nature Publishing Group

Country of Publication:

United Kingdom

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES