Evaluation of the food grade expression systems NICE and pSIP for the production of 2,5-diketo-D-gluconic acid reductase from Corynebacterium glutamicum
© Kaswurm et al.; licensee Springer. 2013
Received: 19 January 2013
Accepted: 21 January 2013
Published: 28 January 2013
2,5-diketo-D-gluconic acid reductase (2,5-DKG reductase) catalyses the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-keto-L-gulonic acid (2-KLG), a direct precursor (lactone) of L-ascorbic acid (vitamin C). This reaction is an essential step in the biocatalytic production of the food supplement vitamin C from D-glucose or D-gluconic acid. As 2,5-DKG reductase is usually produced recombinantly, it is of interest to establish an efficient process for 2,5-DKG reductase production that also satisfies food safety requirements. In the present study, three recently described food grade variants of the Lactobacillales based expression systems pSIP (Lactobacillus plantarum) and NICE (Lactococcus lactis) were evaluated with regard to their effictiveness to produce 2,5-DKG reductase from Corynebacterium glutamicum. Our results indicate that both systems are suitable for 2,5-DKG reductase expression. Maximum production yields were obtained with Lb. plantarum/pSIP609 by pH control at 6.5. With 262 U per litre of broth, this represents the highest heterologous expression level so far reported for 2,5-DKG reductase from C. glutamicum. Accordingly, Lb. plantarum/ pSIP609 might be an interesting alternative to Escherichia coli expression systems for industrial 2,5-DKG reductase production.
KeywordsAscorbic acid 2,5-diketo-D-gluconic acid reductase 2-keto-L-gulonic acid Corynebacterium glutamicum Food–grade Lactic acid bacteria pSIP NICE
The bacterial enzyme 2,5-diketo-D-gluconic acid reductase (2,5-didehydrogluconate reductase; 2,5-DKG reductase; EC 220.127.116.114) is an NAD(P)(H)-dependent oxidoreductase assigned to the aldo-keto reductase (AKR) family ( Ellis 2002 ). 2,5-DKG reductase catalyses the stereo specific reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) at position C-5 to 2-keto-L-gulonic acid (2-KLG), a key intermediate in the production of L-ascorbic acid (Anderson et al. 1985 ). At present, 2,5-DKG reductase is an integral part of several industrial processes designed to synthesize 2-KLG based on the 2,5-diketo-D-gluconic acid pathway (from D-glucose via D-gluconate, 2-keto-D-gluconate and 2,5-diketo-D-gluconate) ( Hancock and Viola 2002 , Bremus et al. 2006 ). An efficient hybrid process for the production of 2-KLG comprising the conversion of D-glucose or D-gluconic acid into 2,5-DKG by Pectobacter cypripedii HEPO1 (DSM 12939) and the subsequent reduction of 2,5-DKG to 2-KLG using 2,5-DKG reductase from Corynebacterium glutamicum was previously developed in our laboratory (Pacher et al. 2008 ). This process involves a commercially available glucose dehydrogenase in order to recycle the costly coenzyme NADPH in situ through oxidation of D-glucose to gluconic acid. An alternative biocatalytic process for 2-KLG production involving 2,5-DKG reductase has been presented by Genencor Inc. (Chotani et al. 2000 ).
The above mentioned processes depend on the heterologous (high-level) expression of the 2,5-DKG reductase gene (dkr), usually achieved with Escherichia coli. However, the utilization of genetically modified organisms (GMOs) to produce enzymes intended for food applications is strictly regulated (Pedersen et al. 2005 , Peterbauer et al. 2011 ). Lipopolysaccharide (endotoxin) production by E. coli is a further obstacle for protein expressions intended for food or medical purposes (Berczi et al. 1966 , Beutler and Rietschel 2003 ). Therefore, laborious and costly measures of down stream processing and quality control are required to comply with the purity and safety specifications for food grade enzymes, as recommended for example by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Food Chemical Codex (FCC). While recombinant (GMO) as well, an attractive alternative is to use expression hosts with the “generally recognized as safe” (GRAS) status, as defined by the US Food and Drug Administration (FDA). Although the reported performance of food grade expression systems is usually low compared to standard expression systems using E. coli (Nguyen et al. 2011a ), an advantage of applying GRAS (i.e., food-grade, Peterbauer et al. 2011 ) expression systems is that the costs to satisfy food safety requirements could be drastically reduced. Accordingly, efforts using lactic acid bacteria (LAB) as expression hosts have gained significance in the last decade (Peterbauer et al. 2011 ). Recently, examples of true food grade host/vector combinations have been presented and applied using the expression systems Lactobacillus plantarum / pSIP (Nguyen et al. 2011a ) and Lactococcus lactis / NICE (Maischberger et al. 2010 ). In both systems, antibiotic resistance marker genes have been replaced by selection markers (pSIP: alr, alanine racemase gene; NICE: lacF, gene encoding the soluble carrier enzyme IIA of the lactose specific phosphotransferase system) complementing corresponding gene deletions in the host chromosomes.
Previous studies on the above mentioned expression systems demonstrated high expression levels with bacterial β-galactosidase genes (Nguyen et al. 2011a , Maischberger et al. 2010 ). However, in these studies, the target genes originated from members of the same taxonomic order (Lactobacillales) as the expression hosts. It is therefore important as well to evaluate the performance of such LAB expression systems with genes of taxonomic distant origin. The aim of the present work was to evaluate the food grade expression systems pSIP and NICE for their capacity to produce the industrially important enzyme 2,5-DKG reductase from C. glutamicum (order Actinomycetales).
Material and methods
Oligonucleotide primers used for PCR amplifications in this study
Restriction enzyme c
P sppA , P sppQ
Bacterial strains and plasmids used in this study a
Strains or plasmids
Reference or source
DMSZ strain 20301
Lactobacillus plantarum WCFS1
a single colony isolated from Lb. plantarum NCIMB8826, which was originally isolated from human saliva (National Collection of Industrial and Marine Bacteria, Aberdeen, U.K.)
Kleerebezem et al. 2003
Lactobacillus plantarum TLG02
WCFS1 derivative, Δalr, D-alanine auxotroph, expression host
Nguyen et al. 2011a
Lactococcus lactis NZ3900
NZ3000 derivative, ΔlacF, pepN::nisRK, selection based on the ability to grow on lactose (lacF), expression host
de Ruyter et al. 1996
MC1000 derivative, D-alanine auxotroph, cloning host
Strych et al. 2001
New England Biolabs
CloneJET™ PCR Cloning Kit
NICE derivative plasmids
Cm r , P nisA
lac F, pNZ8150 derivative containing Lb. reuteri lacLM genes downstream of P nisA
Maischberger et al. 2010
lac F, pTM51R derivative containing the multiple cloning site (from pNZ8150) downstream of P nisA
lac F, pVK51 derivative containing C. glutamicum dkr downstream of P nisA
pSIP derived plasmids
alr, pSIP403 derivative containing Lb. reuteri lacLM controlled by P sppA
Nguyen et al. 2011a
alr, pSIP409 derivative containing Lb. reuteri lacLM controlled by P sppQ
Nguyen et al. 2011a
alr, pSIP603R derivative, lacLM replaced by C. glutamicum dkr controlled by P sppA
alr, pSIP609R derivative, lacLM replaced by C. glutamicum dkr controlled by P sppQ
Construction of NICE-based expression vectors
Construction of pSIP-based expression vectors under control of P sppA and P sppQ
The coding region of dkr was amplified with the primer pair V1/V3 (Table 1). Promoters P sppA (pSIP603R) and P sppQ (pSIP609R) (Nguyen et al. 2011a ), were amplified from the respective plasmid DNA using the primer pairs P1/P2 and P1/P3 (Table 1). The amplified dkr fragment was fused to the promoters P sppA and P sppQ by overlap extension polymerase chain reaction. Each of the two resulting fragments (P sppA :dkr, P sppQ :dkr) was ligated directly to the pJet1.2 blunt-end cloning vector (CloneJET PCR cloning kit; Fermentas GmbH, St. Leon-Rot, Germany) and transformed into chemically competent E. coli NEB 5-α cells (New England Biolabs). The inserts were excised with Spe I and Xho I (restriction sites on primers, Table 1) and ligated to a ~5.5 kb fragment obtained by cleavage of pSIP603R with the same restriction enzymes, resulting in the expression plasmids pSIP603dkr and pSIP609dkr. Following the same procedure, pSIP-based expression vectors containing the complete dkr ORF (designated pSIP603dkr ORF and pSIP609dkr ORF) were constructed. For dkr ORF amplification forward primer 5′-ATGTCTGTTGTGGGTACCGG-3′ and reverse primer V2 (Table 1) were used. After plasmid amplification with E. coli MB2159 (Strych et al. 2001 ), the constructs were electroporated into the D-alanine auxotroph expression host Lb. plantarum TLG02 (Nguyen et al. 2011a ) as described by Josson et al. ( 1989 ) and tranformants were cultivated in de Man, Rogosa and Sharpe broth (MRS medium; Oxoid, Basingstoke, U.K.) at 37°C without agitation. Competent cells of E. coli MB1259 were prepared and transformed according to the method of Inoue et al. ( 1990 ). Cultures of E. coli NEB 5-α and E. coli MB1259 transformants were grown in Luria-Bertani medium (LB; Sambrook et al. 1989 ) at 37°C with constant agitation (200 rpm). For the selection of E. coli NEB 5-α, ampicillin was added to a final concentration of 100 mg mL-1. For cultivation of E. coli MB2159 and Lb. plantarum TLG02 without plasmids, the respective growth media were supplemented with D-alanine (200 μg mL-1).
Expression of 2,5-DKG reductase with food–grade vectors
Batch cultivations of LAB with food grade vectors were performed in computer-controlled stirred reactors (6 × 0.5 L) of the HT-Multifors system (Infors HT, Bottmingen, Switzerland). Comparative studies without and with pH control (pH 6.5) were performed. Culture pH was maintained by automated addition of sterile NaOH (1 M). To ensure homogenous distribution of the culture broth with limited oxygen transfer, a low agitation speed of 80 rpm was used. All experiments were performed in triplicate.
Inocula for the batch cultivations were prepared by transferring 20 μl of a frozen stock culture to 200 mL fresh medium (M17 for L. lactis; MRS for Lb. plantarum) and incubation at 30°C without shaking. After 12 hours, the cells were transferred to the bioreactor already containing the corresponding medium to reach an optical density at 600 nm (OD600) of ∼0.1. Expression was induced at an OD600 of 0.35 ± 0.03. For the induction of Lb. plantarum harbouring pSIP alr-based vectors, the synthetic peptide pheromone SppIP (Eijsink et al. 1996 ) (25 ng mL-1; CASLO Laboratory, Lyngby, Denmark) was used. To induce the NICE expression system with L. lactis NZ3900, nisin ( Mierau and Kleerebezem 2005 ), a 34 amino acid lantibiotic bacteriocin, was applied at a final concentration of 10 ng mL-1. In parallel to the induced cultures, noninduced negative controls were included to determine background activities and to calculate the induction factors (the quotient of specific activity obtained under induced conditions and the activity obtained under noninduced conditions). All experiments were carried out at 30°C for 20 hours following induction.
Off-line analysis of parameters
Samples were taken in appropriate time intervals during the fermentations to monitor the growth of bacterial cultures by measuring OD600 and wet cell weight (WCW) after centrifugation at 15,000 × g for 15 min at 4°C. 2,5-DKG reductase activities and the total intracellular protein concentrations were determined in order to evaluate the expression levels. For that purpose, bacterial cells were harvested from 5 mL of culture by centrifugation at 3,220 × g for 10 min at 4°C, washed with Bis-Tris buffer (50 mM, pH 6.5) and resuspended in 500 μL of the same buffer. The cells were mechanically disrupted through bead beating with ∼1 g glass beads (average diameter of 0.5 mm) using a Precelly 24 glass bead mill (PEQLAB Biotechnologie GmbH, Erlangen, Germany). The cell-free crude extracts obtained after 10 min centrifugation at 9,000 × g (4°C) were used for 2,5-DKG reductase activity assays and determination of protein concentrations.
2,5-DKG reductase activity assay was performed spectrophotometrically as previously described (Kaswurm et al. 2012 ). One unit of 2,5-DKG reductase activity is defined as the enzyme quantity required to reduce 1 μmol of 2,5-DKG per min under assay conditions, which is equivalent to the production of 1 μmol of NADP+ per min (Kaswurm et al. 2012 ). Protein concentrations were determined by the dye binding method of Bradford (Bradford, 1976 ) using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories Inc.). Bovine serum albumin (BSA), in concentrations of 0.1 – 1.0 mg mL-1, was used for the standard calibration curve. All assays were performed in triplicate, and the data are expressed as mean values ± standard deviation (SD).
SDS-PAGE was performed with a PerfectBlue standard vertical gel electrophoresis system (PEQLAB Biotechnologie GmbH) using 5% stacking gels and 10% separating gels. Samples were prepared according to method of Laemmli ( Laemmli 1970 ) and loaded in aliquots of 10 μL per line onto gel. Protein bands were stained using Coomassie blue R250. Precision Plus Protein™ Standard (Bio-Rad Laboratories Inc.) was used as molecular mass standard.
Codon usage analysis
The fraction of usage of each codon of the C. glutamicum dkr gene by L. lactis subsp. cremoris MG1363 and Lb. plantarum WCFS1 (Kleerebezem et al. 2003 ), was predicted with the Graphical Codon Usage Analyser (Fuhrmann et al. 2004 ) and the results are presented as relative adaptiveness values. The codon usage table of L. lactis subsp. cremoris MG1363 is estimated based on 2572 CDS’s (739646 codons) and that of Lb. plantarum WCFS1 based on 3057 CDS’s (934462 codons) (Nakamura et al. 2000 ).
Expression of the C. glutamicum dkr gene
Maximum 2,5-DKG reductase activities in cell free extracts of induced and noninduced L. lactis NZ3900 and Lb. plantarum TLG02 cultures
Not pH regulated cultivations of L. lactis and Lb. plantaruma
Volumetric activity (U L -1 fermentation broth)
Specific activity (U mg -1 protein)
Induction factor b
L. lactis NZ3900/pVK51dkr
81.6 ± 9.3
16.0 ± 0.24
0.191 ± 0.022
0.038 ± 0.001
102 ± 5.8
21.7 ± 2.0
0.232 ± 0.022
0.054 ± 0.002
104 ± 2.75
23.8 ± 2.2
0.243 ± 0.017
pH regulated cultivations (pH 6.5) of L. lactis and Lb. plantarum a
Volumetric activity (U L -1 fermentation broth)
Specific activity (U mg -1 protein)
Induction factor b
L. lactis NZ3900/pVK51dkr
114 ± 1.9
14.6 ± 2.0
0.188 ± 0.001
0.022 ± 0.003
226 ± 5.9
27.2 ± 0.56
0.264 ± 0.026
0.032 ± 0.001
262 ± 1.7
30.2 ± 1.2
0.308 ± 0.016
0.033 ± 0.004
With L. lactis NZ3900, the highest volumetric activities (82 U L-1 fermentation broth) were obtained 4 hours after induction. L. lactis reached a maximum OD600 of approx. 4 and a WCW of 3.6 g L-1. Lb. plantarum (with both pSIP603 and pSIP609) reached a maximum OD600 of just above 9 (WCW approx. 10 g L-1) and displayed maximum 2,5-DKG reductase activities of approximately 100 U L-1 after 8 hours of induction. The results of expressions without pH control (Figure 3) indicate that acid formation is the limiting factor for 2,5-DKG reductase production: In all cases, the volumetric activities of 2,5-DKG reductase decreased after reaching their maximum values, concomitant with a decrease of pH, levelling off at approximately pH 4. This is also evident on SDS-PAGE (Figure 2), as the intensities of the protein bands corresponding to 2,5-DKG reductase (31 kDa) decrease during continued fermentation.
The highest production levels of 2,5-DKG reductase were obtained with the system Lb. plantarum/pSIP609, resulting in 104 U L-1 without pH regulation and 262 U L-1 with pH control at 6.5. Although formation of recombinant 2,5-DKG reductase by Lb. plantarum (both pSIP603 and pSIP609) was higher than with L. lactis, the induction factors did not differ significantly because of slightly higher basal expression of noninduced Lb. plantarum TLG02 cells. It can be concluded that some basal 2,5-DKG reductase expression, caused by “leakage” of the corresponding promoters, occured in noninduced Lb. plantarum TLG02 cells (Table 3). Additional experiments using wild type Lb. plantarum WCFS1 (ancestral strain of TLG02, see Table 2) (Kleerebezem et al. 2003 ) were performed in MRS medium under equal conditions as described above, but without induction. The highest 2,5-DKG reductase activities detected were 11.8 ± 0.8 U L-1 without pH regulation and 13.6 ± 0.9 U L-1 with pH control at 6.5. Database research using the BLASTp algorithm (NCBI Database; http://www.ncbi.nlm.nih.gov/; Altschul et al. 1997 ) revealed the presence of several putative oxidoreductases in the Lb. plantarum WCFS1 genome with up to 47% amino acid sequence identities with dkr. This circumstance might be an explanation for the recorded 2,5-DKG reductase background activities as well. Putative aldo/keto reductases with up to 48% amino acid sequence identities to dkr could also be identified in the published genome of L. lactis MG1363 (ancestral strain of L. lactis NZ3900) (de Ruyter et al. 1996 ).
Investigation of an alternative "dkr gene variant"
In this and previous studies (Kaswurm et al. 2012 , Pacher 2006 ) the dkr gene was cloned and expressed such that the third in-frame ATG codon of the complete open reading frame (ORF) (GenBank accession JQ407590.1) was used as translation start (Figure 1). This is a consequence of previous experiments conducted in our laboratory that demonstrated the presence of two protein bands with distinct electrophoretic mobilities (both identified as dkr gene products by MALDI-TOF analysis) when the complete dkr ORF (His6-tagged) was expressed with an E. coli expression system ( Pacher 2006 ). Sequence analysis of the dkr gene shows a region with a high concentration of purine bases (GAG GAA GAG), located downstream of the first initiation codon (between position 36 and 54, see Figure 1), which may have been recognized as an alternative ribosomal binding site by E. coli. Subsequent experiments (E. coli) using the third ATG codon as translational start (Figure 1) resulted in the presence of only a single discernable band on SDS-PAGE and especially, higher expression yields than with the complete ORF. Interestingly, the automated gene product annotations (i.e., predicted start of translation) of the currently available coding sequences of the dkr gene from C. glutamicum (GenBank, 99% sequence identities to JQ407590.1; BLASTn; Altschul et al. 1997 ), differ in the above discussed respect, whereas either the first, second or third ATG codon are predicted as putative translation start sites (accession nos. CAF21024.1.; BAF55246.1; CCH25497.1 and BAB99752.1).
Following these considerations, the expression of the complete dkr ORF was investigated with all presented LAB systems/variants as well (see results in Additional file 1, Additional file 2, Additional file 3, Additional file 4). Interestingly, in contrast to E. coli, only a single protein band was visible on SDS-PAGE (see Additional file 1). However, the yields of 2,5-DKG reductase activities (in terms of both volumetric and specific activities) achieved by expression of the complete dkr ORF were significantly lower than by expression of the "dkr gene" (starting at the third ATG codon in frame) with all systems (see Additional file 2, Additional file 3, Additional file 4).
A possible explanation for the improved expression charateristics of "dkr" compared to the complete ORF may be indicated by codon usage analysis: Compared to L. lactis subsp. cremoris MG1363 and Lb. plantarum WCFS1, the mean difference of the codon usage in ORF of dkr gene from C. glutamicum was 39.6% and 36.7% for the complete ORF and 40.1% and 37.1% for dkr gene, respectively. Additionally, according to the codon usage table of L. lactis subsp. cremoris MG1363 (Additional file 5) an analysis of usage of the first 50 codons of the complete ORF and dkr gene, shows that 10 codons (ORF) and 8 codons (dkr), respectively can be considered “rare codons” (i.e. codons used in less than 20% of the cases) or “very rare codons” (i.e. codons used in less than 10% of the cases). Conversely, for Lb. plantarum there is no rare codon with a low fraction of usage within the first 50 codons of both ORF and dkr gene from C. glutamicum (Additional file 6).
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. 2002a, b , Powers 1996 , Sanli et al. 2004 , 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. 1985 , Grindley et al. 1988 , Wührer 2006 ). 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. 1988 ).
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. 2012 ). 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. 2005 ). 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., 2011 ). 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 sppA and P sppQ , 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. 2011a ), 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. 2005 ).
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. 2011b , Friehs 2004 , 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 ( Fuglsang 2003 ). In addition, design of fermentation medium and further optimization of cultivation conditions using a well reasoned strategy ( Kennedy and Krouse 1999 , Berlec et al. 2008 ) 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.
Alanine racemase gene
- Cmr :
5-DKG reductase: 2,5-diketo-D-gluconic acid reductase
5-DKG: 2,5-diketo-D-gluconic acid
Food and Drug Administration
Genetically modified organism
Lactic acid bacteria
- lacF :
The soluble carrier enzyme IIA encoding gene
- lacLM :
Overlapping genes encoding β-galactosidase
Nicotinamide adenine dinucleotide phosphate (reduced form)
- NADP+ :
Nicotinamide adenine dinucleotide phosphate (oxidized form)
Nisin controlled gene expression
Open reading frame
- P nisA :
Promoter nisin A
- P sppA P sppQ :
The bacteriocin promoters in the spp gene cluster.
Herbert Michlmayr was supported by the Austrian Science Fund (FWF project 20246-B11). We thank the Austrian Academic Exchange Service (ÖAD) for scholarship support of Tien-Thanh Nguyen. We thank Dr. Martin, from Edinburgh Napier University, for kindly supplying E. coli MB2159. We are grateful to Clemens Peterbauer for critically reading the manuscript.
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