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

Aguilar C, Flores N, Riveros-McKay F, Sahonero-Canavesi D, Carmona SB, Geiger O, Escalante A, Bolívar F - Microb. Cell Fact. (2015)

Bottom Line: During 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.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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), 62210, Cuernavaca, Morelos, Mexico. galnex@ibt.unam.mx.

ABSTRACT

Background: 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. 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.

Results: Genome 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.

Conclusions: During 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.

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Mutation rates test. Mutation rates in the laboratory evolved strains and its parentals were determined by a modified Luria-Delbrück fluctuation test, employing the MSS-MLE and the LC methods  in the estimation of the number of mutants. The mutation rate is higher in the PB12 and PB13 strains as compared to JM101 and PB11 strains. Mutation frequency was also calculated (Freq). All the results have a confidence interval of 95 %. A detailed table is available in the Additional file 2: Table S2
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Fig4: Mutation rates test. Mutation rates in the laboratory evolved strains and its parentals were determined by a modified Luria-Delbrück fluctuation test, employing the MSS-MLE and the LC methods  in the estimation of the number of mutants. The mutation rate is higher in the PB12 and PB13 strains as compared to JM101 and PB11 strains. Mutation frequency was also calculated (Freq). All the results have a confidence interval of 95 %. A detailed table is available in the Additional file 2: Table S2

Mentions: With the enormous stress that the absence of the major glucose transport system represents, a mutagenic response was triggered in the population, which in turn resulted in better adaptation capacities [1, 2, 28]. Therefore, during this ALE process, the deletion of this important chromosomal fragment was originated in the parental PB11 strain. This event implied the appearance of a population with a higher mutation rate than the wild type due to the absence of the mutH gene, which was located in the missing chromosomal region and is involved in DNA repair pathways [29]. The mutation rate in both evolved strains was measured based on the mutation rate in the rpsL locus (streptomycin-resistant mutants). Mutation rates were determined by a modified Luria-Delbrück fluctuation test, employing the Ma-Sandri-Sarkar maximum likelihood method (MSS-MLE) and the Lea-Coulson method of the median (LC) in the estimation of the number of mutants [30–32]. As expected, increased mutation rates were observed in the evolved strains due to the deletion of mutH. However, a 5-fold higher mutation rate was detected in the PB13 strain compared to the PB12 strain (Fig. 4; Additional file 2: Table S2). A PB11ΔReg derivative (which lacks the entire chromosomal fragment deleted in the evolved strains) showed a similar mutation rate to that of the PB12 strain, so it is feasible to propose that the PB13 strain carries at least one additional mutation that increases its mutation rate. Interestingly, the point mutations detected in the PB12 strain were higher than the observed in the PB13 despite having a lower mutation rate. Since all five previously isolated evolved strains did not carry the polA mutation (data not shown), a possible explanation is that the polA mutation, has arisen during the last stages of the evolutionary process, and is responsible for the higher mutation rate in the PB13 strain.Fig. 4


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

Aguilar C, Flores N, Riveros-McKay F, Sahonero-Canavesi D, Carmona SB, Geiger O, Escalante A, Bolívar F - Microb. Cell Fact. (2015)

Mutation rates test. Mutation rates in the laboratory evolved strains and its parentals were determined by a modified Luria-Delbrück fluctuation test, employing the MSS-MLE and the LC methods  in the estimation of the number of mutants. The mutation rate is higher in the PB12 and PB13 strains as compared to JM101 and PB11 strains. Mutation frequency was also calculated (Freq). All the results have a confidence interval of 95 %. A detailed table is available in the Additional file 2: Table S2
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666226&req=5

Fig4: Mutation rates test. Mutation rates in the laboratory evolved strains and its parentals were determined by a modified Luria-Delbrück fluctuation test, employing the MSS-MLE and the LC methods  in the estimation of the number of mutants. The mutation rate is higher in the PB12 and PB13 strains as compared to JM101 and PB11 strains. Mutation frequency was also calculated (Freq). All the results have a confidence interval of 95 %. A detailed table is available in the Additional file 2: Table S2
Mentions: With the enormous stress that the absence of the major glucose transport system represents, a mutagenic response was triggered in the population, which in turn resulted in better adaptation capacities [1, 2, 28]. Therefore, during this ALE process, the deletion of this important chromosomal fragment was originated in the parental PB11 strain. This event implied the appearance of a population with a higher mutation rate than the wild type due to the absence of the mutH gene, which was located in the missing chromosomal region and is involved in DNA repair pathways [29]. The mutation rate in both evolved strains was measured based on the mutation rate in the rpsL locus (streptomycin-resistant mutants). Mutation rates were determined by a modified Luria-Delbrück fluctuation test, employing the Ma-Sandri-Sarkar maximum likelihood method (MSS-MLE) and the Lea-Coulson method of the median (LC) in the estimation of the number of mutants [30–32]. As expected, increased mutation rates were observed in the evolved strains due to the deletion of mutH. However, a 5-fold higher mutation rate was detected in the PB13 strain compared to the PB12 strain (Fig. 4; Additional file 2: Table S2). A PB11ΔReg derivative (which lacks the entire chromosomal fragment deleted in the evolved strains) showed a similar mutation rate to that of the PB12 strain, so it is feasible to propose that the PB13 strain carries at least one additional mutation that increases its mutation rate. Interestingly, the point mutations detected in the PB12 strain were higher than the observed in the PB13 despite having a lower mutation rate. Since all five previously isolated evolved strains did not carry the polA mutation (data not shown), a possible explanation is that the polA mutation, has arisen during the last stages of the evolutionary process, and is responsible for the higher mutation rate in the PB13 strain.Fig. 4

Bottom Line: During 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.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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), 62210, Cuernavaca, Morelos, Mexico. galnex@ibt.unam.mx.

ABSTRACT

Background: 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. 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.

Results: Genome 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.

Conclusions: During 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.

Show MeSH
Related in: MedlinePlus