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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

Physiological state of E. coli strains during continuous L-carnitine production: A) High density cell-recycle reactor using E. coli O44K74; B) Continuous stirred tank reactor with κ-carrageenan gel immobilized E. coli K38 pT7-5KE32 cells. Bars represent the amount of viable (low bar), depolarized (middle bar) and dead cells (top bar) [57,82] whereas lines represent dry cell weight.
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Figure 4: Physiological state of E. coli strains during continuous L-carnitine production: A) High density cell-recycle reactor using E. coli O44K74; B) Continuous stirred tank reactor with κ-carrageenan gel immobilized E. coli K38 pT7-5KE32 cells. Bars represent the amount of viable (low bar), depolarized (middle bar) and dead cells (top bar) [57,82] whereas lines represent dry cell weight.

Mentions: Given the importance of the cell state in the determination of final bioprocess yield, cells have been classified into viable intact, viable but non-culturable and dead cells [86]. Thus, for optimization purposes, research on the ideal cell state for a given biotransformation needs to be carried out. Double fluorescent staining of cells with propidium iodide and bis-oxonol [86] allows us to determine population kinetics in bioreactors. In resting cell systems, cell physiology decays rapidly as a result of the lack of culture medium [82]. In addition, trimethylammonium compounds, which are osmoprotectant [70], have a protecting effect on cells during the biotransformation. Surprisingly, cell state upon high cell density cultivation in cell recycle reactors was characterized by high levels of viability (Fig. 4A, less than 15% of depolarized cells in the steady state) [82]. When cells were immobilized in κ-carrageenan gel beads, a high accumulation of damaged cells was observed (less than 10–15% of cells retaining membrane polarity in the steady state) (Fig. 4B) [57]. This result is of great importance, since it underlines the fact that cells with somewhat uncertain viability can also be active producers as a result of maintaining the carnitine metabolism still active.


Production of L-carnitine by secondary metabolism of bacteria.

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

Physiological state of E. coli strains during continuous L-carnitine production: A) High density cell-recycle reactor using E. coli O44K74; B) Continuous stirred tank reactor with κ-carrageenan gel immobilized E. coli K38 pT7-5KE32 cells. Bars represent the amount of viable (low bar), depolarized (middle bar) and dead cells (top bar) [57,82] whereas lines represent dry cell weight.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Physiological state of E. coli strains during continuous L-carnitine production: A) High density cell-recycle reactor using E. coli O44K74; B) Continuous stirred tank reactor with κ-carrageenan gel immobilized E. coli K38 pT7-5KE32 cells. Bars represent the amount of viable (low bar), depolarized (middle bar) and dead cells (top bar) [57,82] whereas lines represent dry cell weight.
Mentions: Given the importance of the cell state in the determination of final bioprocess yield, cells have been classified into viable intact, viable but non-culturable and dead cells [86]. Thus, for optimization purposes, research on the ideal cell state for a given biotransformation needs to be carried out. Double fluorescent staining of cells with propidium iodide and bis-oxonol [86] allows us to determine population kinetics in bioreactors. In resting cell systems, cell physiology decays rapidly as a result of the lack of culture medium [82]. In addition, trimethylammonium compounds, which are osmoprotectant [70], have a protecting effect on cells during the biotransformation. Surprisingly, cell state upon high cell density cultivation in cell recycle reactors was characterized by high levels of viability (Fig. 4A, less than 15% of depolarized cells in the steady state) [82]. When cells were immobilized in κ-carrageenan gel beads, a high accumulation of damaged cells was observed (less than 10–15% of cells retaining membrane polarity in the steady state) (Fig. 4B) [57]. This result is of great importance, since it underlines the fact that cells with somewhat uncertain viability can also be active producers as a result of maintaining the carnitine metabolism still active.

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