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Production of L-carnitine by secondary metabolism of bacteria.

Bernal V, Sevilla A, Cánovas M, Iborra JL - Microb. Cell Fact. (2007)

Bottom Line: The use of different cell environments, such as growing, resting, permeabilized, dried, osmotically stressed, freely suspended and immobilized cells, to maintain enzymes sufficiently active for L-carnitine production is discussed in the text.Moreover, the combined application of both bioprocess and metabolic engineering has allowed a deeper understanding of the main factors controlling the production process, such as energy depletion and the alteration of the acetyl-CoA/CoA ratio which are coupled to the end of the biotransformation.Furthermore, the profiles of key central metabolic activities such as the TCA cycle, the glyoxylate shunt and the acetate metabolism are seen to be closely interrelated and affect the biotransformation efficiency.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology B and Immunology, Campus of Espinardo, University of Murcia, E-30100, Spain. jliborra@um.es.

ABSTRACT
The increasing commercial demand for L-carnitine has led to a multiplication of efforts to improve its production with bacteria. The use of different cell environments, such as growing, resting, permeabilized, dried, osmotically stressed, freely suspended and immobilized cells, to maintain enzymes sufficiently active for L-carnitine production is discussed in the text. The different cell states of enterobacteria, such as Escherichia coli and Proteus sp., which can be used to produce L-carnitine from crotonobetaine or D-carnitine as substrate, are analyzed. Moreover, the combined application of both bioprocess and metabolic engineering has allowed a deeper understanding of the main factors controlling the production process, such as energy depletion and the alteration of the acetyl-CoA/CoA ratio which are coupled to the end of the biotransformation. Furthermore, the profiles of key central metabolic activities such as the TCA cycle, the glyoxylate shunt and the acetate metabolism are seen to be closely interrelated and affect the biotransformation efficiency. Although genetically modified strains have been obtained, new strain improvement strategies are still needed, especially in Escherichia coli as a model organism for molecular biology studies. This review aims to summarize and update the state of the art in L-carnitine production using E. coli and Proteus sp, emphasizing the importance of proper reactor design and operation strategies, together with metabolic engineering aspects and the need for feed-back between wet and in silico work to optimize this biotransformation.

No MeSH data available.


Related in: MedlinePlus

Simplified model for the interaction of L-carnitine production pathway with central metabolism of E. coli strains. The main pathways involved (and their codifying genes) are shown. Central metabolism: AceK (aceK), isocitrate dehydrogenase phosphatase/kinase; ACK (ackA), acetate kinase; ACS (acs), acetyl-CoA synthetase; ICDH (icd), isocitrate dehydrogenase; ICL (aceA), isocitrate lyase; ICLR (iclR), repressor of the glyoxylate shunt; MDH (maeB), malate dehydrogenase; ME (sfcA), malic enzyme; PEPCK (pckA), phosphoenolpyruvate carboxykinase; PFL (pflB), pyruvate:formate lyase; PTA (pta), phosphotransacetylase; FumR (frdABCD), fumarate reductase. L(-)-carnitine pathway: CaiB: carnitine:CoA transferase; CaiC: betaine:CoA ligase; CaiD, enoyl-CoA hydratase; CaiT, carnitine/crotonobetaine/γ-butyrobetaine transporter. Pathway regulators are shown in red. Steps which are not functional under anaerobiosis are shown with grey arrows. Adapted from [30].
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Figure 5: Simplified model for the interaction of L-carnitine production pathway with central metabolism of E. coli strains. The main pathways involved (and their codifying genes) are shown. Central metabolism: AceK (aceK), isocitrate dehydrogenase phosphatase/kinase; ACK (ackA), acetate kinase; ACS (acs), acetyl-CoA synthetase; ICDH (icd), isocitrate dehydrogenase; ICL (aceA), isocitrate lyase; ICLR (iclR), repressor of the glyoxylate shunt; MDH (maeB), malate dehydrogenase; ME (sfcA), malic enzyme; PEPCK (pckA), phosphoenolpyruvate carboxykinase; PFL (pflB), pyruvate:formate lyase; PTA (pta), phosphotransacetylase; FumR (frdABCD), fumarate reductase. L(-)-carnitine pathway: CaiB: carnitine:CoA transferase; CaiC: betaine:CoA ligase; CaiD, enoyl-CoA hydratase; CaiT, carnitine/crotonobetaine/γ-butyrobetaine transporter. Pathway regulators are shown in red. Steps which are not functional under anaerobiosis are shown with grey arrows. Adapted from [30].

Mentions: In the case of carnitine production, the link or connection between central and secondary metabolisms was seen to rely on the level of cofactors [22,30]. In fact, energy depletion and an altered acetyl-CoA/CoA ratio were coupled to the end of the biotransformation. Moreover, the regulatory profiles of key central metabolic activities involved in the regulation of the acetyl-CoA/CoA ratio, such as the TCA cycle, the glyoxylate shunt and the acetate metabolism were closely interrelated and exercised a control on the biotransformation efficiency [26,30,104] (Fig. 5). Moreover, to determine control points in the metabolism of production strains, we analysed the dynamic evolution of the central metabolism after perturbations affecting the level of carbon sources, biotransformation substrate or electron acceptors. The results underlined the fact that both ATP levels and the availability of free CoA depended on cellular metabolism under production conditions [100]. Furthermore, suboptimal energetic state of E. coli upon high cell density cultivation conditions limited maximum productivity because of the low level of betaine activation. In addition, changes in gene expression, as reflected by altered levels of enzyme activity and external metabolite fluxes, were a result of the rapid modification of metabolite pools after pulses, revealing the ability of bacterial metabolism to redirect fluxes in order to restore the steady state conditions.


Production of L-carnitine by secondary metabolism of bacteria.

Bernal V, Sevilla A, Cánovas M, Iborra JL - Microb. Cell Fact. (2007)

Simplified model for the interaction of L-carnitine production pathway with central metabolism of E. coli strains. The main pathways involved (and their codifying genes) are shown. Central metabolism: AceK (aceK), isocitrate dehydrogenase phosphatase/kinase; ACK (ackA), acetate kinase; ACS (acs), acetyl-CoA synthetase; ICDH (icd), isocitrate dehydrogenase; ICL (aceA), isocitrate lyase; ICLR (iclR), repressor of the glyoxylate shunt; MDH (maeB), malate dehydrogenase; ME (sfcA), malic enzyme; PEPCK (pckA), phosphoenolpyruvate carboxykinase; PFL (pflB), pyruvate:formate lyase; PTA (pta), phosphotransacetylase; FumR (frdABCD), fumarate reductase. L(-)-carnitine pathway: CaiB: carnitine:CoA transferase; CaiC: betaine:CoA ligase; CaiD, enoyl-CoA hydratase; CaiT, carnitine/crotonobetaine/γ-butyrobetaine transporter. Pathway regulators are shown in red. Steps which are not functional under anaerobiosis are shown with grey arrows. Adapted from [30].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2131755&req=5

Figure 5: Simplified model for the interaction of L-carnitine production pathway with central metabolism of E. coli strains. The main pathways involved (and their codifying genes) are shown. Central metabolism: AceK (aceK), isocitrate dehydrogenase phosphatase/kinase; ACK (ackA), acetate kinase; ACS (acs), acetyl-CoA synthetase; ICDH (icd), isocitrate dehydrogenase; ICL (aceA), isocitrate lyase; ICLR (iclR), repressor of the glyoxylate shunt; MDH (maeB), malate dehydrogenase; ME (sfcA), malic enzyme; PEPCK (pckA), phosphoenolpyruvate carboxykinase; PFL (pflB), pyruvate:formate lyase; PTA (pta), phosphotransacetylase; FumR (frdABCD), fumarate reductase. L(-)-carnitine pathway: CaiB: carnitine:CoA transferase; CaiC: betaine:CoA ligase; CaiD, enoyl-CoA hydratase; CaiT, carnitine/crotonobetaine/γ-butyrobetaine transporter. Pathway regulators are shown in red. Steps which are not functional under anaerobiosis are shown with grey arrows. Adapted from [30].
Mentions: In the case of carnitine production, the link or connection between central and secondary metabolisms was seen to rely on the level of cofactors [22,30]. In fact, energy depletion and an altered acetyl-CoA/CoA ratio were coupled to the end of the biotransformation. Moreover, the regulatory profiles of key central metabolic activities involved in the regulation of the acetyl-CoA/CoA ratio, such as the TCA cycle, the glyoxylate shunt and the acetate metabolism were closely interrelated and exercised a control on the biotransformation efficiency [26,30,104] (Fig. 5). Moreover, to determine control points in the metabolism of production strains, we analysed the dynamic evolution of the central metabolism after perturbations affecting the level of carbon sources, biotransformation substrate or electron acceptors. The results underlined the fact that both ATP levels and the availability of free CoA depended on cellular metabolism under production conditions [100]. Furthermore, suboptimal energetic state of E. coli upon high cell density cultivation conditions limited maximum productivity because of the low level of betaine activation. In addition, changes in gene expression, as reflected by altered levels of enzyme activity and external metabolite fluxes, were a result of the rapid modification of metabolite pools after pulses, revealing the ability of bacterial metabolism to redirect fluxes in order to restore the steady state conditions.

Bottom Line: The use of different cell environments, such as growing, resting, permeabilized, dried, osmotically stressed, freely suspended and immobilized cells, to maintain enzymes sufficiently active for L-carnitine production is discussed in the text.Moreover, the combined application of both bioprocess and metabolic engineering has allowed a deeper understanding of the main factors controlling the production process, such as energy depletion and the alteration of the acetyl-CoA/CoA ratio which are coupled to the end of the biotransformation.Furthermore, the profiles of key central metabolic activities such as the TCA cycle, the glyoxylate shunt and the acetate metabolism are seen to be closely interrelated and affect the biotransformation efficiency.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology B and Immunology, Campus of Espinardo, University of Murcia, E-30100, Spain. jliborra@um.es.

ABSTRACT
The increasing commercial demand for L-carnitine has led to a multiplication of efforts to improve its production with bacteria. The use of different cell environments, such as growing, resting, permeabilized, dried, osmotically stressed, freely suspended and immobilized cells, to maintain enzymes sufficiently active for L-carnitine production is discussed in the text. The different cell states of enterobacteria, such as Escherichia coli and Proteus sp., which can be used to produce L-carnitine from crotonobetaine or D-carnitine as substrate, are analyzed. Moreover, the combined application of both bioprocess and metabolic engineering has allowed a deeper understanding of the main factors controlling the production process, such as energy depletion and the alteration of the acetyl-CoA/CoA ratio which are coupled to the end of the biotransformation. Furthermore, the profiles of key central metabolic activities such as the TCA cycle, the glyoxylate shunt and the acetate metabolism are seen to be closely interrelated and affect the biotransformation efficiency. Although genetically modified strains have been obtained, new strain improvement strategies are still needed, especially in Escherichia coli as a model organism for molecular biology studies. This review aims to summarize and update the state of the art in L-carnitine production using E. coli and Proteus sp, emphasizing the importance of proper reactor design and operation strategies, together with metabolic engineering aspects and the need for feed-back between wet and in silico work to optimize this biotransformation.

No MeSH data available.


Related in: MedlinePlus