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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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Adaptive laboratory experiment. a Isolation of evolved strains from a continuous culture of the PB11 from Aguilar et al. [2]. The arrows indicate the isolation time for various strains including PB12 and PB13 strains. Dotted line indicates the end of the batch culture and the start of the continuous culture; numbers indicate dilution rates (D = h−1) as follows: 1 for D = 0.4, 2 for D = 0.6 and 3 for D = 0.8. b Chromosomal gene organization in the parental wild type JM101 strain and in the laboratory evolved PB12 and PB13 strains. c PCR test for the chromosomal deletion in the evolved strains isolated at D = 0.4, 0.6 and 0.8, where the absence of the 10,328 bp chromosomal DNA fragment can be observed in 6 strains. Line 1, (M) molecular weight marker; lines 2 and 3, amplification of the chromosomal region in the JM101 and PB11 strains controls respectively (12 kb); lines 4–9, amplification of the 1.9 kb from the chromosomal region in the six strains isolated during the continuous culture as follows: line 4 and 5, PB12 and the second strain isolated at D = 0.4, line 6 and 7, first and second strains isolated at D = 0.6, line 8 and 9, first strain isolated at D = 0.8 and PB13 strain; line 10, (M) molecular weight marker
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Fig1: Adaptive laboratory experiment. a Isolation of evolved strains from a continuous culture of the PB11 from Aguilar et al. [2]. The arrows indicate the isolation time for various strains including PB12 and PB13 strains. Dotted line indicates the end of the batch culture and the start of the continuous culture; numbers indicate dilution rates (D = h−1) as follows: 1 for D = 0.4, 2 for D = 0.6 and 3 for D = 0.8. b Chromosomal gene organization in the parental wild type JM101 strain and in the laboratory evolved PB12 and PB13 strains. c PCR test for the chromosomal deletion in the evolved strains isolated at D = 0.4, 0.6 and 0.8, where the absence of the 10,328 bp chromosomal DNA fragment can be observed in 6 strains. Line 1, (M) molecular weight marker; lines 2 and 3, amplification of the chromosomal region in the JM101 and PB11 strains controls respectively (12 kb); lines 4–9, amplification of the 1.9 kb from the chromosomal region in the six strains isolated during the continuous culture as follows: line 4 and 5, PB12 and the second strain isolated at D = 0.4, line 6 and 7, first and second strains isolated at D = 0.6, line 8 and 9, first strain isolated at D = 0.8 and PB13 strain; line 10, (M) molecular weight marker

Mentions: The adaptive capacity of bacteria can be exploited by metabolic engineering to enhance a particular characteristic in a microorganism [1, 2, 5]. 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 [6–8]. Despite the low growth capacity using glucose as the unique carbon source in the PB11 strain, this strategy diverts a large proportion of the phosphoenolpyruvate (PEP) to the aromatic biosynthesis pathway. However, because of its diminished growth rate, PB11 is not useful as an industrial production strain. Therefore, an adaptive laboratory evolution (ALE) experiment was carried out with this strain by growing in a fermentor with glucose as the only carbon source fed at progressively higher rates (Fig. 1a). During this process, spontaneous mutants that grew faster on glucose were isolated. Further characterization of two strains obtained at 120 h (PB12) and 200 h (PB13) during the fermentation process showed increased μs of 338 and 373 %, respectively, compared to the parental PB11 strain (Fig. 1a) [2, 6–8]. Because of this enhanced capacity, mainly the PB12 strain has been used for the overproduction of aromatic compounds with high yields [9–11].Fig. 1


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)

Adaptive laboratory experiment. a Isolation of evolved strains from a continuous culture of the PB11 from Aguilar et al. [2]. The arrows indicate the isolation time for various strains including PB12 and PB13 strains. Dotted line indicates the end of the batch culture and the start of the continuous culture; numbers indicate dilution rates (D = h−1) as follows: 1 for D = 0.4, 2 for D = 0.6 and 3 for D = 0.8. b Chromosomal gene organization in the parental wild type JM101 strain and in the laboratory evolved PB12 and PB13 strains. c PCR test for the chromosomal deletion in the evolved strains isolated at D = 0.4, 0.6 and 0.8, where the absence of the 10,328 bp chromosomal DNA fragment can be observed in 6 strains. Line 1, (M) molecular weight marker; lines 2 and 3, amplification of the chromosomal region in the JM101 and PB11 strains controls respectively (12 kb); lines 4–9, amplification of the 1.9 kb from the chromosomal region in the six strains isolated during the continuous culture as follows: line 4 and 5, PB12 and the second strain isolated at D = 0.4, line 6 and 7, first and second strains isolated at D = 0.6, line 8 and 9, first strain isolated at D = 0.8 and PB13 strain; line 10, (M) molecular weight marker
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Adaptive laboratory experiment. a Isolation of evolved strains from a continuous culture of the PB11 from Aguilar et al. [2]. The arrows indicate the isolation time for various strains including PB12 and PB13 strains. Dotted line indicates the end of the batch culture and the start of the continuous culture; numbers indicate dilution rates (D = h−1) as follows: 1 for D = 0.4, 2 for D = 0.6 and 3 for D = 0.8. b Chromosomal gene organization in the parental wild type JM101 strain and in the laboratory evolved PB12 and PB13 strains. c PCR test for the chromosomal deletion in the evolved strains isolated at D = 0.4, 0.6 and 0.8, where the absence of the 10,328 bp chromosomal DNA fragment can be observed in 6 strains. Line 1, (M) molecular weight marker; lines 2 and 3, amplification of the chromosomal region in the JM101 and PB11 strains controls respectively (12 kb); lines 4–9, amplification of the 1.9 kb from the chromosomal region in the six strains isolated during the continuous culture as follows: line 4 and 5, PB12 and the second strain isolated at D = 0.4, line 6 and 7, first and second strains isolated at D = 0.6, line 8 and 9, first strain isolated at D = 0.8 and PB13 strain; line 10, (M) molecular weight marker
Mentions: The adaptive capacity of bacteria can be exploited by metabolic engineering to enhance a particular characteristic in a microorganism [1, 2, 5]. 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 [6–8]. Despite the low growth capacity using glucose as the unique carbon source in the PB11 strain, this strategy diverts a large proportion of the phosphoenolpyruvate (PEP) to the aromatic biosynthesis pathway. However, because of its diminished growth rate, PB11 is not useful as an industrial production strain. Therefore, an adaptive laboratory evolution (ALE) experiment was carried out with this strain by growing in a fermentor with glucose as the only carbon source fed at progressively higher rates (Fig. 1a). During this process, spontaneous mutants that grew faster on glucose were isolated. Further characterization of two strains obtained at 120 h (PB12) and 200 h (PB13) during the fermentation process showed increased μs of 338 and 373 %, respectively, compared to the parental PB11 strain (Fig. 1a) [2, 6–8]. Because of this enhanced capacity, mainly the PB12 strain has been used for the overproduction of aromatic compounds with high yields [9–11].Fig. 1

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