Genome-scale promoter engineering by coselection MAGE

Abstract

Multiplex automated genome engineering (MAGE) uses short oligonucleotides to scarlessly modify genomes; however, insertions >10 bases are still inefficient but can be improved substantially by selection of highly modified chromosomes. Here we describe 'coselection' MAGE (CoS-MAGE) to optimize biosynthesis of aromatic amino acid derivatives by combinatorially inserting multiple T7 promoters simultaneously into 12 genomic operons. Promoter libraries can be quickly generated to study gain-of-function epistatic interactions in gene networks.

Fig 1. Co-selection MAGE strategy for enriching highly modified genomes

a, Cells undergoing cycles of MAGE were enriched by a co-selection stage through phenotypic selection of a revertible genomic locus (i.e. malK, galK, cat or bla) and used for subsequent cycles of MAGE in an iterative fashion. Twelve genomic operons were targeted for insertion of the T7 promoter sequence upstream of the first relevant open reading frame (ORF). b, Enhanced T7 insertion frequency near the cat locus was observed only when the population was co-selected by chloramphenicol for cat (blue lines) in comparison to without co-selection (orange lines) after two (dotted lines) and four (solid lines) MAGE cycles as determined by MASC-PCR (Online Methods). c, Biosynthesis of tryptophan and indole derivatives indigo and indirubin. Intermediate products are shown in orange and the first gene in each relevant operon shown in black. Targeted genes that are not first in the operon are shown in gray. d, Each stage of CoS-MAGE involves 4 cycles of MAGE followed by screening of 96 colonies to find the most modified mutant. Population frequency of colonies with 0 to 3 new T7p insertions for each co-selection stage are shown for the corresponding conditions. Co-selection enriches for highly modified genomes, which are used in the subsequent CoS-MAGE stages with a reduced oligo pool to target the remaining sites. The most enriched strains at the end of each CoS stage are noted (H46, H63, H65, H77).

Fig 2. Indigo production from synthetic T7p regulated operons

Strain E7N is an E2N-derivative that contains the bfmo-encoding pJ401 plasmid. Strain E47N is an E7N derivative with inactivated trpR repression and absent allosteric feedback through aroF(P148L), trpE(M293T), and aroG(D146N). Strains H1-H80 are T7p derivatives of E47N. The top bar graph shows relative change in growth rate for each strain normalized to E7N, Normalized percent values for each strain are calculated by (y−x)/y where x is the doubling time of each strain and y is doubling time of the E7N wild-type. n = 3 for each strain, error bars, s.e.m. The bottom bar graph shows indigo production based on measured indirubin levels (Online Methods). Color coding reflects grouping by the number of T7 insertions. Genotypes for each clone are marked by black boxes and ordered by their relative location along the biosynthesis pathway (ppsA at the top is proximal and trpE at the bottom is distal). The gray box of strain H70 corresponds to an insert in aroH with a mutated T7 sequence (TAATACACTCAGTATGGG).