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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

L-carnitine production with genetically engineered E. coli cells: effect of the deletion of pta, acs, aceA, aceK and iclR on the production of L-carnitine by E. coli BW25113 [rrnB3 ΔlacZ4787 hsdR514Δ(araBAD)567 Δ(rhaBAD)568 rph-1]. The construction of deletion mutants is described in [93]. Experiments were performed in batch anaerobic systems in L-Broth (black bars) and L-Broth supplemented with 2 g·L-1 fumarate (grey bars). Adapted from [26].
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Figure 7: L-carnitine production with genetically engineered E. coli cells: effect of the deletion of pta, acs, aceA, aceK and iclR on the production of L-carnitine by E. coli BW25113 [rrnB3 ΔlacZ4787 hsdR514Δ(araBAD)567 Δ(rhaBAD)568 rph-1]. The construction of deletion mutants is described in [93]. Experiments were performed in batch anaerobic systems in L-Broth (black bars) and L-Broth supplemented with 2 g·L-1 fumarate (grey bars). Adapted from [26].

Mentions: These results have been experimentally validated, since by increasing the levels of CaiB and CaiT proteins in the low-producing E. coli LMG194 laboratory strain, L-carnitine production could be increased 3–4-fold (Fig 6) [72]. Nevertheless, the effect of CaiC overproduction, enhancing L-carnitine yield 50-fold in E. coli LMG194 (a 2–3-fold increase compared with the overproducing E. coli O44K74 strain), was not predicted by this model [26]. The main reason was that at the time that the model was built, the kinetic characteristics of this protein were not known [18]. This fact underlines an important concern: the construction of meaningful models strongly depends on the completeness and goodness of the data available. Moreover, a continuous feed-back between in silico and in vivo experimentation is necessary for the application of Metabolic Engineering and Systems Biology approaches to living systems [72]. In this context, L-carnitine production with genetically engineered E. coli cells with certain central metabolism encoding enzymes deleted, such as pta (phosphotransferase), acs (acetylCoA synthetase), aceA (isocitrate lyase), aceK (isocitrate dehydrogenase) and iclR (aceA inhibition), has been determined (Fig. 7). The results have important implications for the model built to represent this biotransformation, underlining that proper expression of the central metabolism, affecting balanced cofactor levels (acetyl-CoA/CoA in this case), influences secondary metabolism pathways [26].


Production of L-carnitine by secondary metabolism of bacteria.

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

L-carnitine production with genetically engineered E. coli cells: effect of the deletion of pta, acs, aceA, aceK and iclR on the production of L-carnitine by E. coli BW25113 [rrnB3 ΔlacZ4787 hsdR514Δ(araBAD)567 Δ(rhaBAD)568 rph-1]. The construction of deletion mutants is described in [93]. Experiments were performed in batch anaerobic systems in L-Broth (black bars) and L-Broth supplemented with 2 g·L-1 fumarate (grey bars). Adapted from [26].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: L-carnitine production with genetically engineered E. coli cells: effect of the deletion of pta, acs, aceA, aceK and iclR on the production of L-carnitine by E. coli BW25113 [rrnB3 ΔlacZ4787 hsdR514Δ(araBAD)567 Δ(rhaBAD)568 rph-1]. The construction of deletion mutants is described in [93]. Experiments were performed in batch anaerobic systems in L-Broth (black bars) and L-Broth supplemented with 2 g·L-1 fumarate (grey bars). Adapted from [26].
Mentions: These results have been experimentally validated, since by increasing the levels of CaiB and CaiT proteins in the low-producing E. coli LMG194 laboratory strain, L-carnitine production could be increased 3–4-fold (Fig 6) [72]. Nevertheless, the effect of CaiC overproduction, enhancing L-carnitine yield 50-fold in E. coli LMG194 (a 2–3-fold increase compared with the overproducing E. coli O44K74 strain), was not predicted by this model [26]. The main reason was that at the time that the model was built, the kinetic characteristics of this protein were not known [18]. This fact underlines an important concern: the construction of meaningful models strongly depends on the completeness and goodness of the data available. Moreover, a continuous feed-back between in silico and in vivo experimentation is necessary for the application of Metabolic Engineering and Systems Biology approaches to living systems [72]. In this context, L-carnitine production with genetically engineered E. coli cells with certain central metabolism encoding enzymes deleted, such as pta (phosphotransferase), acs (acetylCoA synthetase), aceA (isocitrate lyase), aceK (isocitrate dehydrogenase) and iclR (aceA inhibition), has been determined (Fig. 7). The results have important implications for the model built to represent this biotransformation, underlining that proper expression of the central metabolism, affecting balanced cofactor levels (acetyl-CoA/CoA in this case), influences secondary metabolism pathways [26].

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