Abstract

BackgroundAs a metabolic engineering tool, an adaptive laboratory evolution (ALE) experiment was performed to increase the specific growth rate (µ) in an Escherichia coli strain lacking PTS, originally engineered to increase the availability of intracellular phosphoenolpyruvate and redirect to the aromatic biosynthesis pathway. As result, several evolved strains increased their growth fitness on glucose as the only carbon source. Two of these clones isolated at 120 and 200 h during the experiment, increased their μ by 338 and 373 %, respectively, compared to the predecessor PB11 strain. The genome sequence and analysis of the genetic changes of these two strains (PB12 and PB13) allowed for the identification of a novel strategy to enhance carbon utilization to overcome the absence of the major glucose transport system.ResultsGenome sequencing data of evolved strains revealed the deletion of chromosomal region of 10,328 pb and two punctual non-synonymous mutations in the dhaM and glpT genes, which occurred prior to their divergence during the early stages of the evolutionary process. Deleted genes related to increased fitness in the evolved strains are rppH, aas, lplT and galR. Furthermore, the loss of mutH, which was also lost during the deletion event, caused a 200-fold increase in the mutation rate.ConclusionsDuring the ALE experiment, both PB12 and PB13 strains lost the galR and rppH genes, allowing the utilization of an alternative glucose transport system and allowed enhanced mRNA half-life of many genes involved in the glycolytic pathway resulting in an increment in the μ of these derivatives. Finally, we demonstrated the deletion of the aas-lplT operon, which codes for the main components of the phosphatidylethanolamine turnover metabolism increased the further fitness and glucose uptake in these evolved strains by stimulating the phospholipid degradation pathway. This is an alternative mechanism to its regeneration from 2-acyl-glycerophosphoethanolamine, whose utilization improved carbon metabolism likely by the elimination of a futile cycle under certain metabolic conditions. The origin and widespread occurrence of a mutated population during the ALE indicates a strong stress condition present in strains lacking PTS and the plasticity of this bacterium that allows it to overcome hostile conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0382-6) contains supplementary material, which is available to authorized users.

Highlights

  • As a metabolic engineering tool, an adaptive laboratory evolution (ALE) experiment was performed to increase the specific growth rate (μ) in an Escherichia coli strain lacking phosphotransferase system (PTS), originally engineered to increase the availability of intracellular phosphoenolpyruvate and redirect to the aromatic biosynthesis pathway

  • Detection and description of the genetic changes in the PB12 and PB13 strains Comparative genome analysis of the PB13 strain carried out by Winter Genomics Inc., shows that similar to the PB12 strain genome, several point mutations that are present in the evolved PB12 and PB13 strains were generated during the ALE process compared to the PB11 parental strain

  • The analysis of the point mutations in non-coding regions and the synonymous point mutations that were detected in the PB12 strain [2] and in PB13 are not included in this work because the non-coding point mutations are located outside regulatory regions, and the synonymous mutations unlikely have any significant effect on the physiology of the strains (Additional file 1: Table S1)

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Summary

Introduction

As a metabolic engineering tool, an adaptive laboratory evolution (ALE) experiment was performed to increase the specific growth rate (μ) in an Escherichia coli strain lacking PTS, originally engineered to increase the availability of intracellular phosphoenolpyruvate and redirect to the aromatic biosynthesis pathway. A short-term adaptive laboratory evolution (ALE) process has been performed in our group to increase the diminished growth capacity of an E. coli strain (PB11) lacking the major glucose uptake system, phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). This PTS− strain shows a specific growth rate (μ) of 0.1 h−1 and was generated after ptsHIcrr operon inactivation in the JM101 wild type parental strain that grows with a μ of 0.7 h−1 on glucose as the only carbon source [6,7,8]. Because of this enhanced capacity, mainly the PB12 strain has been used for the overproduction of aromatic compounds with high yields [9,10,11]

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