Limits...
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

Effect of different transport engineering strategies on L-carnitine production. Permeabilization of Proteus sp. (A) and E. coli O44K74 (B) cells using detergents and organics. L-carnitine production by E. coli O44K74 under salt stress conditions (C). Bar represents productivity and lines the yield. PEI stands for polyethylenimine. Adapted from [76,79].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2131755&req=5

Figure 3: Effect of different transport engineering strategies on L-carnitine production. Permeabilization of Proteus sp. (A) and E. coli O44K74 (B) cells using detergents and organics. L-carnitine production by E. coli O44K74 under salt stress conditions (C). Bar represents productivity and lines the yield. PEI stands for polyethylenimine. Adapted from [76,79].

Mentions: As previously seen in many other biotechnologically relevant systems, the substrate (crotonobetaine) uptake and product (L-carnitine) efflux are controlled by diffusion through both the outer membrane (OM) and cell envelope, together with the membrane transport system itself (CaiT) (Fig 2B) [77]. Cell damage and integrity during permeabilization has been assessed by fluorescent probe uptake assays (such as 1-phenylnaphthylamine or NPN) [78], protein cell leakage, transmission electron microscopy (cell structural alterations) and transport assays with L [N-methyl14C]carnitine (specific carrier transport studies) [76]. Both E. coli and Proteus sp. cells were treated for different times and at different permeabilizer concentrations, during batch cell growth. After permeabilization treatment, the culture broth was centrifuged and cells were resuspended in the biotransformation medium (crotonobetaine in phosphate buffer) for 48 h. For Proteus sp., Triton X-100 resulted in higher conversion and productivity values than those of the control (Fig. 3A). An increase in L-carnitine molar yield of more than 50% was achieved in both growing and resting biotransformation media [79]. For E. coli O44K74, an almost 100% increase in yield was observed (Fig. 3B) [76], with polyethylenimine (PEI) being the best permeabilizer. Transmission electron microscopy of Proteus sp. and E. coli cells after permeabilization demonstrated that the cell wall was affected while the membrane remained intact, meaning that crotonobetaine/L-carnitine transporter could work faster and far more efficiently (Fig. 2B).


Production of L-carnitine by secondary metabolism of bacteria.

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

Effect of different transport engineering strategies on L-carnitine production. Permeabilization of Proteus sp. (A) and E. coli O44K74 (B) cells using detergents and organics. L-carnitine production by E. coli O44K74 under salt stress conditions (C). Bar represents productivity and lines the yield. PEI stands for polyethylenimine. Adapted from [76,79].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effect of different transport engineering strategies on L-carnitine production. Permeabilization of Proteus sp. (A) and E. coli O44K74 (B) cells using detergents and organics. L-carnitine production by E. coli O44K74 under salt stress conditions (C). Bar represents productivity and lines the yield. PEI stands for polyethylenimine. Adapted from [76,79].
Mentions: As previously seen in many other biotechnologically relevant systems, the substrate (crotonobetaine) uptake and product (L-carnitine) efflux are controlled by diffusion through both the outer membrane (OM) and cell envelope, together with the membrane transport system itself (CaiT) (Fig 2B) [77]. Cell damage and integrity during permeabilization has been assessed by fluorescent probe uptake assays (such as 1-phenylnaphthylamine or NPN) [78], protein cell leakage, transmission electron microscopy (cell structural alterations) and transport assays with L [N-methyl14C]carnitine (specific carrier transport studies) [76]. Both E. coli and Proteus sp. cells were treated for different times and at different permeabilizer concentrations, during batch cell growth. After permeabilization treatment, the culture broth was centrifuged and cells were resuspended in the biotransformation medium (crotonobetaine in phosphate buffer) for 48 h. For Proteus sp., Triton X-100 resulted in higher conversion and productivity values than those of the control (Fig. 3A). An increase in L-carnitine molar yield of more than 50% was achieved in both growing and resting biotransformation media [79]. For E. coli O44K74, an almost 100% increase in yield was observed (Fig. 3B) [76], with polyethylenimine (PEI) being the best permeabilizer. Transmission electron microscopy of Proteus sp. and E. coli cells after permeabilization demonstrated that the cell wall was affected while the membrane remained intact, meaning that crotonobetaine/L-carnitine transporter could work faster and far more efficiently (Fig. 2B).

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