Deletion of the 2-acyl-glycerophosphoethanolamine cycle improve glucose metabolism in Escherichia coli strains employed for overproduction of aromatic compounds

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.

The Role of the <b><i>ydiB</i></b> Gene, Which Encodes Quinate/Shikimate Dehydrogenase, in the Production of Quinic, Dehydroshikimic and Shikimic Acids in a PTS^- Strain of <b><i>Escherichia coli</i></b>

“…The same reproducible expression level of this gene was detected in all the strains in the conditions (EXP and STA growth phases) in which bacteria were grown and sampled from SA-producing cultures since this is the most important characteristic that a reference gene should have in accordance with the MIQE guidelines. Values for ihfB gene detected for the utilized strains demonstrate the stability of the expression of this reference gene in all the analyzed derivatives for the used conditions in this report and also in previous reports (data not shown) [Flores et al, 2005[Flores et al, , 2007[Flores et al, , 2008Aguilar et al, 2012Aguilar et al, , 2015.…”

Trends in the Use of Proper Methods for Estimating Mutation Rates in Fluctuation Experiments

“…Genotoxicity studies exhibited the highest prevalence of inappropriate methods' usage (88.9%), largely due to the dominant influence of the Ames test in this field, as we correctly anticipated. Interestingly, only two articles in the genotoxicity category reported the use of appropriate methods, and they are from groups mostly working in the areas of DNA repair and in evolutionary genetics [123,155]. Stacked areas depict the proportion of the selected studies employing specific methods or method families.…”

Enhanced Production of Pterostilbene in Escherichia coli Through Directed Evolution and Host Strain Engineering

“…Shen et al, 2020). Aguilar et al (2012Aguilar et al ( , 2015 reported that deletion of the 2-acyl-glycerophosphoethanolamine cycle allows fast growth in glucose and improves the production of aromatic compounds in E. coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). The 2-acylglycerophosphoethanolamine cycle comprises 12 contiguous genes (rppH, ygdT, mutH, ygdQ, ygdR, tas, lplT, aas, omrB, and part of ptsP and galR, 10,328 bp).…”

“…The 2-acylglycerophosphoethanolamine cycle comprises 12 contiguous genes (rppH, ygdT, mutH, ygdQ, ygdR, tas, lplT, aas, omrB, and part of ptsP and galR, 10,328 bp). This growth advantage may be the increase of mRNA levels of glycolytic genes due to RppH pyrophosphohydrolase activity deficiency and the increased glucose uptake through GalP because of the lack of GalR (Aguilar et al, 2012(Aguilar et al, , 2015. The starting strain E. coli TYR-30 in this study is a derivative of E. coli DOPA-30N which is lacking PTS (Wei et al, 2016).…”