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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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Related in: MedlinePlus

Accumulation of free fatty acids in different E. coli strains. In vivo labeling of E. coli using [14C]acetate and after thin-layer chromatographic separation of lipids was performed. The accumulation of free fatty acids of the different ΔfadDE. coli JM101, PB11, PB12 and PB13 derivative strains at 24, 48 and 72 h of growth were determined. The experiments were made by triplicate. In all strains lacking the fadD gene, a progressive increase of free fatty acid was observed over time due to a lack of consumption. The PB11ΔfadD and PB13ΔfadD strains reached 18 and 33 % of the total lipidic species after 72 h. Interestingly, the PB12ΔfadD reached around 8 % of free fatty acids while the JM101ΔfadD reached about 12 % in 72 h of growth. The control strains are capable to consume completely the generated free fatty acids during the growth
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Fig6: Accumulation of free fatty acids in different E. coli strains. In vivo labeling of E. coli using [14C]acetate and after thin-layer chromatographic separation of lipids was performed. The accumulation of free fatty acids of the different ΔfadDE. coli JM101, PB11, PB12 and PB13 derivative strains at 24, 48 and 72 h of growth were determined. The experiments were made by triplicate. In all strains lacking the fadD gene, a progressive increase of free fatty acid was observed over time due to a lack of consumption. The PB11ΔfadD and PB13ΔfadD strains reached 18 and 33 % of the total lipidic species after 72 h. Interestingly, the PB12ΔfadD reached around 8 % of free fatty acids while the JM101ΔfadD reached about 12 % in 72 h of growth. The control strains are capable to consume completely the generated free fatty acids during the growth

Mentions: The relative amounts of free fatty acids in E. coli reflect a steady state situation defined by formation and consumption of free fatty acids in a given physiological condition. In FadD-deficient mutants, consumption of free fatty acids is eliminated [39] and therefore a more precise estimate of free fatty acid formation can be obtained. To test the utilization of the phospholipid degradation pathway in the PB12 and PB13 evolved strains, the fadD gene was inactivated in the different genetic backgrounds. The FadD protein catalyzes the esterification of fatty acids into metabolically active CoA thioesters concomitant with their transport [36], which in this condition are derived from phospholipid degradation (Figs. 6, 7). Inactivation of fadD in the PB12 and PB13 evolved strains decreased the μ in these derivatives (Table 2), indicating an important role of this protein in the evolved strains. Therefore, the content of free fatty acids was determined in all the derivative strains. Surprisingly, the JM101ΔfadD and the PB11ΔfadD strain increased the relative amount of free fatty acids up to 12 and 18 %, respectively after 72 h, suggesting that the degradation pathway is also active in both JM101 and PB11 strains. However, this characteristic does not represent an advantage for the growth of these strains because JM101 is not limited by carbon consumption and in the PB11 the 2-acyl-GPE cycle is present. However, a 33 % increase in the free fatty acid concentration after 72 h in the PB13ΔfadD was observed (Fig. 6), confirming the utilization of this degradation pathway in PB13 and suggesting that the lack of the major membrane phospholipid turnover cycle allowed for more efficient carbon metabolism in this strain. The decrease in the growth rate of the ΔfadD and the ΔglpT mutant strains (Table 2) indicates that this accumulation is due to the PtdEtn degradation pathway. This strategy results in a concomitant optimization of the carbon sources, probably through the elimination of this turnover cycle, which in the PTS− scenario results in a new metabolic capacity to assimilate PtdEtn. This last proposition is supported by the increase in μ in the PB11 strain when the aas-lplT operon is inactivated (Fig. 5a). In the PB12ΔfadD strain, the FA accumulation was approximately 8 % after 72 h (Fig. 6) and the growth decreased by 27 % (Table 2), suggesting that the growth reduction in this strain is due to a different mechanism that could block the pathway rather than a reduction in the re-assimilation products from phospholipid degradation. Therefore, it is feasible that the regulation for this pathway in the PB12 strain works in a slightly different manner. In that sense, non-synonymous point mutations in yjjU and rssA (which code for predicted esterases) are present in the PB12 strain [2]. Functional and mutational predictions on the proteins coded by these genes in order to evaluate its contribution in the fatty acid accumulation behavior of this strain were carried out (data not shown), however the results are not conclusive.Fig. 6


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)

Accumulation of free fatty acids in different E. coli strains. In vivo labeling of E. coli using [14C]acetate and after thin-layer chromatographic separation of lipids was performed. The accumulation of free fatty acids of the different ΔfadDE. coli JM101, PB11, PB12 and PB13 derivative strains at 24, 48 and 72 h of growth were determined. The experiments were made by triplicate. In all strains lacking the fadD gene, a progressive increase of free fatty acid was observed over time due to a lack of consumption. The PB11ΔfadD and PB13ΔfadD strains reached 18 and 33 % of the total lipidic species after 72 h. Interestingly, the PB12ΔfadD reached around 8 % of free fatty acids while the JM101ΔfadD reached about 12 % in 72 h of growth. The control strains are capable to consume completely the generated free fatty acids during the growth
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Accumulation of free fatty acids in different E. coli strains. In vivo labeling of E. coli using [14C]acetate and after thin-layer chromatographic separation of lipids was performed. The accumulation of free fatty acids of the different ΔfadDE. coli JM101, PB11, PB12 and PB13 derivative strains at 24, 48 and 72 h of growth were determined. The experiments were made by triplicate. In all strains lacking the fadD gene, a progressive increase of free fatty acid was observed over time due to a lack of consumption. The PB11ΔfadD and PB13ΔfadD strains reached 18 and 33 % of the total lipidic species after 72 h. Interestingly, the PB12ΔfadD reached around 8 % of free fatty acids while the JM101ΔfadD reached about 12 % in 72 h of growth. The control strains are capable to consume completely the generated free fatty acids during the growth
Mentions: The relative amounts of free fatty acids in E. coli reflect a steady state situation defined by formation and consumption of free fatty acids in a given physiological condition. In FadD-deficient mutants, consumption of free fatty acids is eliminated [39] and therefore a more precise estimate of free fatty acid formation can be obtained. To test the utilization of the phospholipid degradation pathway in the PB12 and PB13 evolved strains, the fadD gene was inactivated in the different genetic backgrounds. The FadD protein catalyzes the esterification of fatty acids into metabolically active CoA thioesters concomitant with their transport [36], which in this condition are derived from phospholipid degradation (Figs. 6, 7). Inactivation of fadD in the PB12 and PB13 evolved strains decreased the μ in these derivatives (Table 2), indicating an important role of this protein in the evolved strains. Therefore, the content of free fatty acids was determined in all the derivative strains. Surprisingly, the JM101ΔfadD and the PB11ΔfadD strain increased the relative amount of free fatty acids up to 12 and 18 %, respectively after 72 h, suggesting that the degradation pathway is also active in both JM101 and PB11 strains. However, this characteristic does not represent an advantage for the growth of these strains because JM101 is not limited by carbon consumption and in the PB11 the 2-acyl-GPE cycle is present. However, a 33 % increase in the free fatty acid concentration after 72 h in the PB13ΔfadD was observed (Fig. 6), confirming the utilization of this degradation pathway in PB13 and suggesting that the lack of the major membrane phospholipid turnover cycle allowed for more efficient carbon metabolism in this strain. The decrease in the growth rate of the ΔfadD and the ΔglpT mutant strains (Table 2) indicates that this accumulation is due to the PtdEtn degradation pathway. This strategy results in a concomitant optimization of the carbon sources, probably through the elimination of this turnover cycle, which in the PTS− scenario results in a new metabolic capacity to assimilate PtdEtn. This last proposition is supported by the increase in μ in the PB11 strain when the aas-lplT operon is inactivated (Fig. 5a). In the PB12ΔfadD strain, the FA accumulation was approximately 8 % after 72 h (Fig. 6) and the growth decreased by 27 % (Table 2), suggesting that the growth reduction in this strain is due to a different mechanism that could block the pathway rather than a reduction in the re-assimilation products from phospholipid degradation. Therefore, it is feasible that the regulation for this pathway in the PB12 strain works in a slightly different manner. In that sense, non-synonymous point mutations in yjjU and rssA (which code for predicted esterases) are present in the PB12 strain [2]. Functional and mutational predictions on the proteins coded by these genes in order to evaluate its contribution in the fatty acid accumulation behavior of this strain were carried out (data not shown), however the results are not conclusive.Fig. 6

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