1,3-Propanediol dehydrogenases in Lactobacillus reuteri: impact on central metabolism and 3-hydroxypropionaldehyde production
© Stevens et al; licensee BioMed Central Ltd. 2011
Received: 31 January 2011
Accepted: 3 August 2011
Published: 3 August 2011
Lactobacillus reuteri metabolizes glycerol to 3-hydroxypropionaldehyde (3-HPA) and further to 1,3-propanediol (1,3-PDO), the latter step catalysed by a propanediol dehydrogenase (PDH). The last step in this pathway regenerates NAD+ and enables therefore the energetically more favourable production of acetate over ethanol during growth on glucose.
A search throughout the genome of L. reuteri DSM 20016 revealed two putative PDHs encoded by ORFs lr_0030 and lr_1734. ORF lr_1734 is situated in the pdu operon encoding the glycerol conversion machinery and therefore likely involved in 1,3-PDO formation. ORF lr_0030 has not been associated with PDH-activity so far. To elucidate the role of these two PDHs, gene deletion mutant strains were constructed. Growth behaviour on glucose was comparable between the wild type and both mutant strains. However, on glucose + glycerol, the exponential growth rate of Δlr_0030 was lower compared to the wild type and the lr_1734 mutant. Furthermore, glycerol addition resulted in decreased ethanol production in the wild type and Δlr_1734, but not in Δlr_0030. PDH activity measurements using 3-HPA as a substrate revealed lower activity of Δlr_0030 extracts from exponential growing cells compared to wild type and Δlr_1734 extracts.
During biotechnological 3-HPA production using non-growing cells, the ratio 3-HPA to 1,3-PDO was approximately 7 in the wild type and Δlr_0030, whereas this ratio was 12.5 in the mutant Δlr_1734.
The enzyme encoded by lr_0030 plays a pivotal role in 3-HPA conversion in exponential growing L. reuteri cells. The enzyme encoded by lr_1734 is active during 3-HPA production by non-growing cells and this enzyme is a useful target to enhance 3-HPA production and minimize formation of the by-product 1,3-PDO.
Lactobacillus reuteri is a heterofermentative lactic acid bacterium (LAB) encountered in a variety of fermented foods like sourdough, meat, and dairy products [1–3]. Furthermore it is a natural inhabitant of the gastro-intestinal (GI) and urogenital tract of humans and other animals [4–8]. Some strains of L. reuteri strains exhibit probiotic properties and were developed as probiotic products .
Most L. reuteri strains produce and excrete reuterin, an antimicrobial compound consisting of hydrated, non-hydrated, and dimeric forms of 3-hydroxypropionaldehyde or 3-HPA [10, 11]. Remarkably, the capability to produce reuterin seems to be absent in most L. reuteri rodent isolates . Reuterin is active against a large range of micro-organisms and is assumed to give L. reuteri a competitive advantage in its ecological niches as e.g. the mammalian GI-tract. Furthermore, 3-HPA produced by the food grade LAB L. reuteri is of industrial interest as it has potential use as food preservative, sanitizing agent, and as precursor for the production of chemicals like acrylic acid and polymers [13, 14]. The reuterin form 3-HPA is produced from glycerol in a vitamin B12-mediated reaction catalysed by a glycerol dehydratase . Synthesis of 3-HPA is a complex multi-protein biological process and takes place in micro-compartments that presumably form a barrier to protect the cytosol against 3-HPA [16, 17]. Biotechnological production of 3-HPA is best-done in a two-stage process, in which biomass is produced in the first stage and glycerol is converted to 3-HPA in the second stage using concentrated cells from stage one. This method results in rapid glycerol conversion and 3-HPA concentrations of up to 235 mM out of 400 mM glycerol . However, apart from 3-HPA, the by-product 1,3-propanediol (1,3-PDO) accumulates in the medium, resulting in lower yields and more complicated purification of 3-HPA.
In this work two genes encoding putative 1,3-propanediol dehydrogenases (PDHs) were identified in the genome of L. reuteri DSM 20016. Mutagenesis of the genes and subsequent physiological analyses revealed that one PDH is mainly active during exponential growth, whereas the other PDH is involved in 3-HPA conversion in non-growing cells.
Material and Methods
Bacterial strains, media, and growth conditions
Strains and plasmids used in this study and their relevant characteristics
Relevant features a
Wild type strain, human intestinal isolate
Wild type strain, human milk isolate
lr_1734 gene replacement (lr_1734::P32cat) derivative of L. reuteri DSM20016, CmR.
lr_0030 gene replacement (lr_0030::P32cat) derivative of L. reuteri DSM20016, CmR.
lr_1734 gene replacement (lr_1734::P32cat) derivative of L. reuteri SD2112, CmR.
CmR, EmR, gene replacement mutagenesis vector.
CmR, EmR, pNZ5319 derivative containing 1.1 kb 3' flanking region of lr_1734.
CmR, EmR, pNZ5319 derivative lr_1734::cat gene replacement mutagenesis vector containing 5'- and 3'-flanking regions of lr_1734.
CmR, EmR, pNZ5319 derivative containing 1.2 kb 5' flanking region of lr_0030.
CmR, EmR, pNZ5319 derivative lr_0030:: cat gene replacement mutagenesis vector containing 5'- and 3'-flanking regions of lr_0030.
DNA manipulations and gene disruption
Molecular cloning and DNA manipulations were essentially performed as described by Sambrook et al. . Large scale plasmid DNA isolations from E. coli were performed using a Maxiprep Kit (Qiagen, Basel, Switzerland). Chromosomal DNA isolation from L. reuteri was performed using a phenol-chloroform extraction method based on a protocol for Lactobacillus plantarum. Restriction enzymes and Phusion-polymerase were obtained from New England Biolabs (Frankfurt am Main, Germany) and T4-ligase from Invitrogen (Basel, Switzerland). Primers were purchased from Microsynth (Balgach, Switzerland).
Construction of plasmids and strains
Primers used in this study
A similar strategy was used for the deletion of ORF lr_0030. The primers 0030-U-5' and 0030-U-3' (Table 2) were used to amplify a 1.2-kb upstream fragment of lr_0030 and this fragment was cloned into the SwaI site of pNZ5319, resulting in pLFB1003. Similarly, a 1.1-kb downstream fragment of lr_0030 was amplified using the primer set 0030-D-5' and 0030-D-3' (Table 2) and this fragment was cloned into the Ecl136II-site of pLFB1003, resulting in the gene replacement vector pLFB1004 for ORF lr_0030. The restriction sites introduced in the primers 0030-D-5' and 0030-D-3' were not used for cloning.
The gene deletion vectors were transformed to L. reuteri as described previously  and plasmid-integrants were selected anaerobically at 37°C on MRS plates complemented with 8 μg/ml chloramphenicol. The anticipated genetic organization after correct gene replacement results in a chloramphenicol resistant but erythromycin sensitive phenotype. Colonies were therefore picked and transferred to MRS plates containing 10 μg/ml erythromycin and grown anaerobically at 37°C overnight. Integrants displaying a chloramphenicol resistant an erythromycin sensitive phenotype were checked by PCR using universal-primers annealing in the cat-gene (primers Con-cat-for and Con-cat-rev, table 2) and a site-specific primer annealing outside of the chromosomal region used for homologous recombination (con- primers, Table 2). Integrants showing the correct phenotype and positive PCR analyses were streaked on MRS with chloramphenicol and a single colony isolate was checked again by PCR (Additional file 1, Figure S1).
Production and purification of 3-hydroxypropionaldehyde
All L. reuteri strains were grown in MRS supplemented with 35 mM glycerol (Sigma-Aldrich, Buchs, Switzerland) at 37°C to an OD600 of approximately 8.0, representing the early stationary phase. To obtain cells with comparable metabolic activity, 10 mM glucose and 20 mM glycerol (end-concentrations) were added to the culture and cells were reactivated for 30 minutes at 37°C. Subsequently, cells were harvested (4,000g, 10', RT) and washed once in 10 mM KPO4-buffer, pH 7.0. Productions were performed in 250 mM aqueous glycerol solution at an OD600 of approximately 60 for one hour. Samples for HPLC were centrifuged (12,000g, 5', 4°C), sterile filtered, and stored at 4°C until further analyses.
To purify 3-HPA, production supernatants were lyophilized and 3-HPA was subsequently purified by chromatography as described previously .
Cell free supernatants were prepared as follows: cells (50 ml) were harvested from an exponentially growing culture at OD600 = 1.0, resuspended in 1/100 volume MRS broth and transferred to a screw-cap tube containing 500 mg zirconium beads. Cells were disrupted by 3 times 30" bead-beat treatments in a fast prep device (MP Biomedicals, Illkirch, France) interspaced by cooling on ice. Cell debris and beads were removed by centrifugation (12,000g, 5', 4°C), and the cell free supernatant containing the cytosolic proteins was transferred to a new tube and stored at 4°C until further use.
Conversion rate of 3-HPA to 1,3-PDO was determined by measuring the decrease of NADH2-absorption at 340 nm in a spectrophotometer UVIKON 810P. The reaction mixture contained 12.5 mM 3-HPA and 480 μM NADH2 (Sigma-Aldrich) in 50 mM Tris-HCl buffer pH 7.2. The reaction was initiated by addition of 20 μl cell free supernatant (approx. 40 μg protein) and specific activity was defined as the decrease of NADH2 in μmol mgprot-1 min-1.
Protein concentrations were determined as described by Bradford .
Determination of metabolic compounds
Concentrations of 3-HPA, glycerol, and 1,3-PDO were determined by HPLC on an Aminex HPX-87H column as described previously . Concentrations of sugars and end-fermentation products were determined on the same column in a separate run with identical settings.
Identification of putative 1,3-PDO dehydrogenases in L. reuteri DSM 20016
Results of BLASTP-search for putative 1,3-propenediol dehydrogenases (PDHs) in L. reuteri DSM 20016
a Exponential (2 n )
a Stationary (2 n )
117 of 348 (33%)
176 of 348 (50%)
133 of 348 (38%)
193 of 348 (55%)
129 of 275 (46%)
181 of 275 (65%)
A genome-wide transcription analysis of L. reuteri cells grown on glucose in the presence of glycerol compared to cells grown without glycerol revealed an about 3 times higher expression of ORF lr_1734 and lr_0030 in the presence of glycerol in both exponential and stationary cells (Table 3, ). In contrast, lr_0321 was down regulated by a factor >4 in the presence glycerol (Table 3), indicating that the corresponding enzyme is not involved in conversion of 3-HPA to 1,3-PDO. Therefore the combined BLAST and transcriptional analyses strongly suggest that L. reuteri DSM 20016 possesses two proteins involved in the conversion of 3-HPA to 1,3-PDO encoded by lr_1734 and lr_0030.
Growth characteristics of two propanediol dehydrogenase mutants
Maximum growth rate of L. reuteri DSM 20016 and its mutant derivatives
No glycerol [h -1 ]
With glycerol [h -1 ]
Significance of increase in growth rate
DSM20016 wild type
0.73 ± 0.01
0.87 ± 0.03
p = 0.02
0.75 ± 0.05
0.84 ± 0.00
p = 0.13
0.79 ± 0.05
0.78 ± 0.03
p = 0.81
Glucose and glycerol consumption and end-product formation in L. reuteri DSM 20016 and its mutant derivatives LFB1001 and LFB1002 grown with and without glycerola
DSM 20016 (wild type)
- 31.8 ± 3.6
36.6 ± 1.8
62.7 ± 7.5
- 32.1 ± 3.6
36.8 ± 3.0
62.8 ± 7.6
- 34.2 ± 0.4
38.6 ± 0.8
60.9 ± 5.8
with 35 mM glycerol
DSM 20016 (wild type)
- 24.8 ± 1.8
- 34.5 ± 3.8
27.1 ± 2.5
20.5 ± 5.1
19.8 ± 0.8
- 24.8 ± 1.6
- 34.5 ± 4.8
26.4 ± 2.3
20.5 ± 3.7
19.1 ± 0.2
- 24.9 ± 2.6
- 34.5 ± 4.0
27.1 ± 2.8
32.2 ± 4.9b
16.3 ± 0.5b
If the wild type and LFB1001 produce less ethanol, they must regenerate NAD+ via another pathway, probably via conversion of 3-HPA to 1,3-PDO. Indeed 1,3-PDO is produced by both strains up to 20 mM (Table 5). Production of 1,3-PDO in LFB1002 was lower (16 mM), which correlates with the observed higher ethanol production by this strain.
Conversion of 3-HPA to 1,3 PDO in the mutant strains
The lower enzymatic activity of LFB1002 extracts from exponential growing cells shows that ORF lr_0030 encodes a gene product involved in 3-HPA conversion in cells exponentially growing in the presence of glycerol.
Role of the PDHs during 3-HPA production
3-HPA and 1,3-PDO production out of 250 mM glycerol by L. reuteri wild type and mutant strainsa
DSM 20016 (wild type)
180.3 ± 11.9
24.7 ± 6.8
200.7 ± 8.2
16.0 ± 1.3
125.7 ± 8.8
16.0 ± 1.0
SD2112 (wild type)
177.6 ± 15.1
15.0 ± 0.3
236.4 ± 2.0
6.8 ± 0.3
L. reuteri SD2112 (ATCC 53608) is commonly used for 3-HPA production [18, 34]. Therefore we constructed a knock-out strain in the lr_1734 homologous of strain SD2112, using the same gene replacement vector as for strain LFB1001. The resulting mutant, designated LFB1003 (Table 1), was applied in 3-HPA-production and the 1,3-PDO and 3-HPA profiles were compared to those in its parental strain SD2112 (Table 6). In parallel to the 3-HPA production profile of DSM 20016 compared to its Δlr_1734 mutant derivative LFB1001, strain LFB1003 produced smaller amounts of 1,3-PDO compared to its parental wild type, an effect even more pronounced compared to DSM 20016 (Table 6). The mutagenesis results in a significant increased 3-HPA/1,3-PDO ratio compared to the wild type strain (p = 0.02). This decrease of 1,3-PDO production in LFB1003 confirms that ORF lr_1734 in DSM 20016 encodes an enzyme involved in 1,3-PDO formation during 3-HPA production by resting cells.
L. reuteri has the unique capability among LAB to produce and excrete large amounts of 3-HPA, an intermediate of the glycerol reductive pathway. Engineering of metabolic pathways via road-blocking is a suitable method for minimizing by-product formation. However, deletion of a gene encoding the undesired enzymatic activity is not always successful as other enzymes might fulfil the deleted function. Indeed we identified more than one gene encoding for conversion of 3-HPA to 1,3-PDO, but deletion of only one of these two ORFs (lr_1734) resulted in decreased formation of the undesired product 1,3-PDO during 3-HPA production.
Glycerol derived 3-HPA can be used as an electron acceptor by L. reuteri, allowing the bacterium to close its NAD+/NADH2-balance and to produce acetate plus one additional ATP (Figure 1). Supplementation of the growth medium with glycerol leads to a higher growth on glucose by L. reuteri, as also observed in this study. Strain LFB1002 (Δlr_0030) did not display an increased growth rate after addition of glycerol, suggesting an impaired NAD+ regeneration via 3-HPA conversion. L. reuteri DSM 20016 can grow up to 3-HPA concentration of 50 mM . The low initial glycerol concentration (35 mM) does not result in such 3-HPA concentrations in the medium, making growth inhibition by accumulation of 3-HPA unlikely. Furthermore, addition of an electron acceptor leads to a shift in the metabolite production from ethanol to acetate [19, 20]. Such a shift was clearly observed in the wild type and LFB1001 but not in LFB1002. Glucose consumption in the presence of glycerol was similar in all three strains (Table 5) and hence the different growth behaviour and fermentation pattern of LFB1002 indicates impaired 3-HPA conversion in this strain. Indeed LFB1002 produced 3 mM less 1,3-PDO compared to the wild type, but the lower production did not correlate with 12 mM higher ethanol production, indicating a redox imbalance. However, samples from early stationary phase were analysed and the activity of the lr_1734 gene product in the late exponential growth phase is probably responsible for the additional 1,3-PDO production. Conversion of 3-HPA by cell-free extracts was similar for the wild type and LFB1001, but clearly lower in LFB1002, showing that 3-HPA conversion is indeed impaired in the latter and that the gene product of lr_0030 plays a major role in the conversion of 3-HPA to 1,3-PDO during exponential growth on glucose in the presence of glycerol. As the phenotypic differences between the wild type and LFB1002 were only observed in the presence and not in the absence of glycerol, our results strongly suggest that lr_0030 is also induced in MRS+glycerol, confirming data obtained in chemically defined medium (Table 3). In contrast, the role of lr_0030 during 3-HPA production seems to be minor, despite the presence of glycerol. The regeneration of NADH via 3-HPA to 1,3-PDO conversion provides only a benefit in the presence of glucose and as glucose is absent during 3-HPA production by resting cells, lr_0030 is probably down regulated.
ORF lr_1734 is situated in the glycerol utilization pdu operon. The organization of the pdu operon in L. reuteri is highly similar to that in Salmonella species , and therefore the lr_1734 gene product is likely involved in conversion of 3-HPA to 1,3-PDO. However, deletion of this gene had only limited impact on growth performance of L. reuteri in the presence of glycerol, whereas no altered end-product formation was observed. This is a puzzling observation, since lr_1734 is presumably transcriptionally coupled to the glycerol dehydratase genes (encoded by pduCDE). However, lr_1734 is located approximately 6 kB downstream of the pduE gene and polar effects could have a negative impact on the mRNA abundance of lr_1734. Consequently, limited cellular amounts of the encoded enzyme cannot cope with the abundance of glycerol dehydratase, resulting in limited NAD+ regeneration and probably accumulation of toxic 3-HPA in the cell. Continuation of the metabolic flux therefore necessitates the activity of the second enzyme encoded by lr_0030 is necessary.
Alternatively, the product of lr_0030 could have a preference for NADP. During exponential growth the phosphoketolase pathway produces NADPH and co-factor regeneration could be more efficient if an NADP preferring 1,3-propanediol dehydrogenase is expressed. The enzymatic activities revealed clearly lower NADH conversion in the lr_0030 mutant compared to the wild type, showing that Lr_0030 can use NADH as substrate. However, NADPH oxidation by Lr_0030 is likely and the exact mechanism of regeneration of oxidizing equivalents during growth of L. reuteri in the presence of glycerol remains to be elucidated. Indeed LFB1002 produced 3 mM less 1,3-PDO compared to the wild type, although a high increase in ethanol production of 12 mM was recorded. This data suggests a redox imbalance. Samples from early stationary phase were analysed and data suggest that the activity of the lr_1734 gene product in the late exponential growth phase is likely responsible for additional 1,3-PDO production.
Lr_0030 is almost 3 times higher expressed in the presence of glycerol compared to growth without glycerol, whereas the effect of glycerol addition on lr_1734 expression was less notable (Table 3). The higher expression of lr_0030 correlates with the predominant role of the protein in 3-HPA to 1,3-PDO conversion during growth. The identification of this second 1,3-propanediol dehydrogenase provides new insight in glycerol conversion by L. reuteri.
Apart from its role as a substrate for regeneration of NAD+, 3-HPA is an anti-microbial compound assumed to play an important role in the ecology of L. reuteri. Although the mechanism of 3-HPA toxicity is partly unravelled [35, 36], little is known about the self-defence mechanisms of the producer strains. Conversion of 3-HPA to 1,3-PDO might be a detoxification reaction for the cells. However, no differences in 3-HPA sensitivity was observed between the wild type and the two mutants (data not shown), suggesting that the function of the two enzymes encoded by lr_1734 and lr_0030 is restricted to 1,3-PDO conversion for NAD+.
Our data provide new insight in the genes and enzymes involved in the conversion of 3-HPA to 1,3-PDO during growth on glucose plus glycerol in L. reuteri. We identified a gene product so far not associated with 3-HPA conversion and showed that the corresponding enzyme plays a pivotal role in 3-HPA conversion in exponential growing L. reuteri cells. Another gene product was shown to be involved in 3-HPA conversion in non-growing cells in a biotechnological production. The activities of this enzyme might be useful for future metabolic engineering processes to enhance production of both 3-HPA and 1,3-PDO.
Our study suggests that expression of paralogous genes enables fine-tuning of the metabolic flux in the cell and that different gene products are active under different circumstances as e.g. different growth conditions. These findings imply that the selection of targets in metabolic engineering strategies should be based on not only the primary biological function as annotated in the genome, but also on activity patterns of the corresponding enzyme.
The research presented in this paper was partly funded by the German "Fachagentur für Nachwachsende Rohstoffe e.V." and the German "Bundesministerium für Ernährung Landwirtschaft und Verbraucherschutz". We thank Michiel Kleerebezem (TI food and Nutrition) for the gift of the pNZ5319 plasmid.
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