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

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.

Iterative screening methodology enables isolation of strains with improved properties for a FACS-based screen and increased L-DOPA production Authors Judy Savitskaya, Ryan J. Protzko, Francesca-Zhoufan Li, Adam P. Arkin, John E. Dueber*

Supplementary Information
Supplementary Figure S1: Barcode read count distribution for pooled deletion collection. The read counts for 4785 ORFs were binned and plotted as a histogram. For each ORF, the read counts for the UPTAG and DNTAG were both recorded, and the maximum of these two values is shown here. This is to account for mutations or poor sequencing for some barcodes. 510 strains (10.7% of the library) did not have any UPTAG or DNTAG read counts, which is consistent with previous re-sequencing efforts 1 . The median read count is 2,636. A majority of the strains (63.6%) have a read count within a factor of 2 (1,318) or a multiple of 2 (5,272) of the median count.  Figure S2: Δima5 did not increase betaxanthin production. Δima5 was enriched in the first round of sorting. Similar to QDR2, IMA5 encodes a multi-drug family transporter that is regulated by PDR8. However, reconstruction of the IMA5 deletion in a clean background strain shows no improvement in fluorescence over the control. We conclude that IMA5 was a false positive. Schematic demonstrates the location of markers, DNA barcode, and priming sites on the transposon. The transposon is integrated into a pool of plasmid vectors containing fragments of the S. cerevisiae genome. Gateway cloning is used to move transposondisrupted genome fragments to a different vector backbone in order to remove transpositions in the backbone and add SceI homing endonuclease recognition sites for linearization. After linearization by SceI, transposon-disrupted genome fragments are integrated into any S. cerevisiae strain by transformation and homologous recombination.

Supplementary Dataset 1
We aliquoted cultures of the 4785 unique strains in the deletion collection into a common pool. SupplementaryFile1.csv gives the sequencing counts for each member of the deletion collection in the pool. The "UPTAG_list" and "DNTAG_list" columns contain the barcodes known to represent the ORF from the original publiation of the deletion collection and from the revised barcodes reported by Eason et al. who used sequencing to identify mutations in the barcodes. The "UPTAG_counts" column contains the sum of counts for all barcodes in the "UPTAG_list". Similarly, the "DNTAG_counts" column contains the sum of counts for all barcodes in the "DNTAG_list".

Supplementary Dataset 2
Transposon insertion sites were identified using RB-TnSEQ 2 . This file lists DNA features (ORFs, expressed RNAs). For each feature, "scaffoldID" identifies the chromosome number, "strand" indicates if the feature is located on the positive or negative strand, "begin" specifies the first base in the feature relative to the chromosome start, and "end" specifies the last base in the feature relative to the chromosome start. All features and sequence information is taken from the Saccharomyces Genome Database S288C sequencing data. The "barcodes" column contains the list of barcodes for transposons that inserted into the genome. The "relLoc" column gives the position of the transposon insertion, relative to the first base in the feature ("begin" if the feature is on the (+) strand, "end" if the feature is on the (-) strand).

Supplementary Table S1: Plasmids
All plasmids were constructed using the yeast toolkit mo-clo system previously described by our lab 3 . All plasmid backbones contained ColE1 replication origins.