The majority of the so far published studies concerned with the heterologous expression of dkr genes (Corynebacterium sp.) were focussed on 2,5-DKG reductase optimization by site-directed mutagenesis and the kinetic characterisation of the obtained mutants after expression in E. coli, rather than the optimization of expression yields (Banta et al.
, Sanli et al.
Banta and Anderson 2002
). However, Erwinia species (Erwinia herbicola and Erwinia citreus) that naturally accumulate 2,5-DKG from D-glucose have been used as expression host for dkr as well, and have been employed in the one-step production of 2-KLG (Anderson et al.
, Grindley et al.
). The expression degree of dkr in Erwinia strains was evaluated through the production titer of 2-KLG, and the highest productivity rate of 6.6 g L-1 d-1 was achieved with Erwinia citreus, mutant strain ER1026 (Grindley et al.
The focus of the present study was to determine the value of two recently developed LAB based food-grade expression systems for the production of 2,5-DKG reductase. The best results (judged by enzyme activity in the crude extract) were obtained with Lb. plantarum/pSIP609. Interestingly, the corresponding production yields were in the same range as those previously obtained by dkr expression with E. coli/pET21d (approx. 200 U L-1 fermentation broth) (Kaswurm et al.
). Additionally, this is the highest expression level so far reported for this enzyme and shows that LAB systems are suitable for dkr expression as well. However, it needs to be critically discussed whether LAB systems could compete with E. coli in an industrial production process. Considering the current costs of the required growth media (at the time of writing: MCHGly medium approx. 3 € per liter; MRS medium approx. 9 €), the estimated costs for 2,5-DKG reductase production with Lb. plantarum would be at least 3 fold compared to E. coli. A strong argument to employ food grade expression systems however is that such, the costs to satisfy food safety requirements may be significantly reduced (Mierau et al.
). Although the options presented here do not represent "self-clones" and have therefore to be considered as GMO, the use of gram positive expression hosts is still highly attractive because lipopolysaccharide formation can be avoided such, which might indeed reduce the costs for downstream processing and quality assurance required for "food grade" enzymes. In addition, the here applied food grade expression systems do not contain potentially harmful, transferable antibiotic resistence markers (Peterbauer et al.,
). Since vitamin C is an important and widely used food supplement, expression of 2,5-DKG reductase with such food grade systems could indeed represent an interesting option.
In this regard, it is important to note that research on LAB expression systems is still in progress, and it can reasonable be expected that expression efficiencies of such systems will be much improved over the next years. An important aspect to improve a particular system is the choice of the inducible promotor, which was also indicated in the present study: Heterologous expression levels (Table 3) of the C. glutamicum dkr gene with Lb. plantarum (pSIP603, pSIP609), clearly indicate that the expression characteristics of the same system can be significantly influenced by the used promotor (P
, respectively), as pSIP609 showed improved expression levels compared to pSIP603 in all cases. These data stand in contrast to the results recently published by Nguyen and co-workers (Nguyen et al.
), who found no significant differences between pSIP603 and pSIP609 comparing the levels of β-galactosidase expressions. However, our results are in excellent accordance with those obtained for the β-glucuronidase (GusA) from E. coli and aminopeptidase N (PepN) from L. lactis expressed with Lb. plantarum NC8 harbouring corresponding pSIP based vectors with erythromycin resistance (Sørvig et al.
Further strategies recently discussed involve the increase of plasmid copy numbers and optimization of mRNA secondary structure in the translational initiation region (TIR) (Nguyen et al.
Ganoza and Louis 1994
). Another important aspect is to analyse the codon usage preference among organisms used as expression systems. Accordingly, by modification of the target gene towards the set of codons that the host organism (L. lactis. or Lb. plantarum) naturally uses in its highly expressed genes, the risk of tRNA depletion during translation can be minimized and hence the heterologous expression by lactic acid bacteria could be further optimized (
). In addition, design of fermentation medium and further optimization of cultivation conditions using a well reasoned strategy (
Kennedy and Krouse 1999
, Berlec et al.
) could contribute to multiple increases of cell densities and expression productivities.
In conclusion, with dkr from C. glutamicum as example, our results confirm that LAB expression systems such as NICE and pSIP are indeed attractive candidates for high level protein production and may gain further interest for industrial purposes in the near future.