Adaptive evolution of Escherichia coli K-12 MG1655 during growth on a Nonnative carbon source, L-1,2-propanediol

Abstract

Laboratory adaptive evolution studies can provide key information to address a wide range of issues in evolutionary biology. Such studies have been limited thus far by the inability of workers to readily detect mutations in evolved microbial strains on a genome scale. This limitation has now been overcome by recently developed genome sequencing technology that allows workers to identify all accumulated mutations that appear during laboratory adaptive evolution. In this study, we evolved Escherichia coli K-12 MG1655 with a nonnative carbon source, l-1,2-propanediol (l-1,2-PDO), for approximately 700 generations. We found that (i) experimental evolution of E. coli for approximately 700 generations in 1,2-PDO-supplemented minimal medium resulted in acquisition of the ability to use l-1,2-PDO as a sole carbon and energy source so that the organism changed from an organism that did not grow at all initially to an organism that had a growth rate of 0.35 h(-1); (ii) six mutations detected by whole-genome resequencing accumulated in the evolved E. coli mutant over the course of adaptive evolution on l-1,2-PDO; (iii) five of the six mutations were within coding regions, and IS5 was inserted between two fuc regulons; (iv) two major mutations (mutations in fucO and its promoter) involved in l-1,2-PDO catabolism appeared early during adaptive evolution; and (v) multiple defined knock-in mutant strains with all of the mutations had growth rates essentially matching that of the evolved strain. These results provide insight into the genetic basis underlying microbial evolution for growth on a nonnative substrate.

FIG. 1.

Metabolic pathway and fuc regulon for l-fucose and l-1,2-PDO. (A) Metabolic pathway for l-fucose and l-1,2-PDO. In E. coli, fucose metabolism is initiated by the sequential actions of a permease (encoded by fucP), an isomerase (encoded by fucI), a kinase (encoded by fucK), and an aldolase (encoded by fucA). The aldolase catalyzes cleavage of fuculose-1-phosphate to dihydroxyacetone phosphate and l-lactaldehyde. Under aerobic respiratory conditions, the l-lactaldehyde is further oxidized by a series of enzymes to pyruvate, which subsequently enters central metabolism. Under anaerobic fermentative conditions, the l-lactaldehyde is reduced to l-1,2-PDO by oxidoreductase (encoded by fucO). (B) Genetic organization of the fuc regulon. The fuc regulon for l-fucose uptake and metabolism consists of two divergent operons, fucAO and fucPIKUR.

FIG. 2.

Evolutionary trajectory and phenotype characterization of l-1,2-PDO-evolved E. coli populations. (A) Evolutionary trajectory. The growth rates of the evolved population and cell division are expressed as a function of time of evolution. The final average growth rate was 0.35 ± 0.04 h⁻¹. The amounts of glycerol added with 1,2-PDO are indicated by gray bars. The total number of cell divisions for the entire period of adaptation is also shown. (B) Phenotype characterization. Three populations at the end point of adaptive evolution were used to determine the l-1,2-PDO uptake rate, the acetate secretion rate, and the biomass yield. Each population stored at −80°C was grown in batch culture under the same adaptive evolution conditions. Metabolites were detected by HPLC. •, l-1,2-PDO uptake rate; ▪, acetate secretion rate; ▴, biomass yield. DW, dry weight.

FIG. 3.

Timeline showing the temporal order of appearance of acquired mutations in the eBOP12 population. Sanger sequencing was used to screen the mutations that we found after whole-genome resequencing of the eBOP12-6 clone. The first day that a mutation was identified is indicated by an arrowhead. Once a mutation was present, it was found on all subsequent days of the experiment. The line shows the growth rate trajectory over the course of experimental evolution. *ylbE1 indicates a synonymous mutation in ylbE1.

FIG. 4.

Growth rates of site-directed mutants. The gene gorging method was used to introduce mutations individually and in combination into WT and GC strain backgrounds. For each mutant of the WT and GC strains, the designations of the mutated genes are indicated. The growth rate recovery was calculated by dividing the growth rate of the mutant by the growth rate of the evolved eBOP12-6 clone in minimal medium containing l-1,2-PDO. A single mutation of fucO or the IS5 insertion did not allow the WT or GC strain to grow in minimal medium containing l-1,2-PDO.

FIG. 5.

Relative transcription levels of ilvEA and fucAO determined by real-time quantitative PCR. The GC strain was grown on minimal medium containing glycerol (GG), and the eBOP12-6 clone was cultured on minimal medium containing glycerol (PG) or minimal medium containing l-1,2-PDO (PP). The ilvE and ilvA gene and fucAO operon were upregulated in the eBOP12-6 clone cultured on minimal medium containing glycerol compared to the GC strain grown on minimal medium containing glycerol. When the eBOP12-6 clone cultured on minimal medium containing l-1,2-PDO was compared to the eBOP12-6 strain grown on minimal medium containing glycerol, the fucAO operon was still upregulated, suggesting that the fucAO operon was constitutively expressed in the evolved E. coli regardless of the carbon source.