Production of L-carnitine by secondary metabolism of bacteria

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.

Figure 1

L-carnitine metabolism in bacteria. A) Schematic representation of the main pathways for L(-)-carnitine metabolization in bacteria. Shaded and striped reactions correspond to the pathways observed in Enterobacteria and Acinetobacter strains, respectively. Reactions in the dashed box correspond to Pseudomonas strains. Enzymes or "systems" involved: 1, γ-butyrobetaine hydroxylase; 2, L-carnitine dehydrogenase; 3, L-carnitine dehydratase; 3a, "carnitinyl-CoA hydrolase"; 4, crotonobetaine reductase; 4a, "γ-butyrobetaine dehydrogenase"; 5, monooxygenase; 6, "carnitine racemase"; 7, D-carnitine dehydrogenase. Adapted from [4]. B) Complete anaerobic biotransformation pathway for trimethylammonium compounds in E. coli. Adapted from [30]. Abbreviations: Crot, crotonobetaine; γ-BB, γ-butyrobetaine, L-Car, L-carnitine; D-Car, D-carnitine; Gly, glycine; 3-dehydro-Car, 3-dehydrocarnitine; Me₃N, trimethylamine; Mal, malate; Suc, succinate; CaiT, L-carnitine/γ-butyrobetaine/crotonobetaine protein transporter; CaiA, CaiB, crotonobetaine reduction reaction; CaiB, CoA transferase; CaiC, L-carnitine/γ-butyrobetaine/crotonobetaine CoA ligase; CaiD, enoyl-CoA hydratase or carnitine racemase activity.

Figure 2

L-carnitine transport systems in E. coli strains. A) Main characteristics of L-carnitine transporters: CaiT: carnitine/crotonobetaine/γ-butyrobetaine antiporter [20]; ProU and ProP, osmotic stress related transporters [6,69]. B) Effect of permeabilizers on cell envelope and outer membrane Adapted from [76].

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].

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.

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].

Figure 6

L-carnitine production with genetically engineered E. coli cells: effect of gene overexpression on the production of L-carnitine in E. coli. Experiments were performed in batch anaerobic systems in L-Broth (black bars) and L-Broth supplemented with 2 g·L^-1 fumarate (grey bars). Overproduction of CaiT, CaiB and CaiC was performed using pBAD24 as expression vector and E. coli LMG194 [F^- ΔlacX74 galE galK thi rpsL ΔphoA (PvuII) Δara714 leu::Tn10] as expression host. Control experiments with the non genetically engineered overexpressing E. coli O44K74 strain are shown for comparison. Adapted from [26,72].

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].