Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa, and Instituto de Biologia Experimental e Tecnológica, Rua da Quinta Grande, 6, Apt 127, 2780-156 Oeiras, Portugal1
Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK2
Author for correspondence: Helena Santos. Tel: +351 21 4469829. Fax: +351 21 4428766. e-mail: santos{at}itqb.unl.pt
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ABSTRACT |
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Keywords: mannitol catabolism, L. lactis, in vivo 13C-NMR
Abbreviations: FBP, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12); LDH, L-lactate dehydrogenase (EC 1.1.1.27); LDHd, lactate-dehydrogenase-deficient; Mtl1P, mannitol 1-phosphate; Mtl1PDH, mannitol-1-phosphate dehydrogenase (EC 1.1.1.17); PEP, phosphoenolpyruvate; PFK, 6-phosphofructokinase (EC 2.7.1.11); 3-PGA, 3-phosphoglycerate; PK, pyruvate kinase (EC 2.7.1.40); PTSMtl, mannitol phosphotransferase system
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INTRODUCTION |
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Recently, we reported the production of mannitol and Mtl1P during glucose metabolism in cell suspensions of a lactate-dehydrogenase-deficient (LDHd) strain of Lactococcus lactis (Neves et al., 2000 ). This metabolic peculiarity was rationalized as an alternative way to regenerate NAD+ in the absence of the pivotal enzyme LDH. Interestingly, after glucose depletion, mannitol was taken up from the medium and converted mainly to ethanol. To the best of our knowledge, there are no reports in the literature concerning the ability of L. lactis to use mannitol as a substrate for growth; being more reduced than glucose, mannitol metabolism implies the formation of an extra NADH molecule that has to be reoxidized downstream of the pyruvate node.
Although mannitol is not important as a substrate in dairy sources, its presence in food products is desirable, since it can be converted in the human gut to short-chain fatty acids, which presumably confer protection against the development of colon cancer (van Munster & Nagengast, 1993 ). Furthermore, it is a low-calorie sweetener that can replace sucrose (Furia, 1972
) and a scavenger of free hydroxyl radicals (Rozenberg-Arska et al., 1985
). Therefore, the production of mannitol by L. lactis could be exploited to obtain healthier foods. Mannitol has also been shown to act as an osmolyte (Kets et al., 1996
; Luxo et al., 1993
) and as a protector of L. lactis cells when subjected to drying (Efiuvwevwere et al., 1999
). In spite of its physiological and biotechnological interest, mannitol metabolism has not been investigated in lactic acid bacteria, except for the oral pathogens (Yamada, 1987
). A greater understanding of the pathways and regulatory mechanisms involved in mannitol metabolism is a requisite for the design of mannitol-overproducing strains.
In this work, we studied mannitol metabolism in L. lactis MG1363 and a derivative LDHd strain; growth parameters as well as intracellular metabolite pools were determined. The presence of mannitol led to a strong induction of Mtl1PDH activity and to the accumulation of Mtl1P and the production of high amounts of ethanol and formate. The pools of intracellular metabolites, Mtl1P, fructose 1,6-bisphosphate (FBP), 3-phosphoglycerate (3-PGA) and PEP in mannitol- or glucose-grown cells were monitored non-invasively by 13C-NMR.
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METHODS |
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Quantification of fermentation products.
Samples (5 ml) of the LDHd or MG1363 cultures grown in medium containing mannitol or glucose were taken at different growth stages, centrifuged (2000 g, 5 min, 4 °C) and supernatant solutions were stored at -20 °C until analysis by HPLC using a refractive index detector (LKB2142). Glucose, mannitol, acetate, ethanol, formate, lactate, acetoin and 2,3-butanediol were quantified using an HPX-87H anion exchange column (Bio-Rad) at 60 °C, with 5 mM H2SO4 as the elution fluid and a flow rate of 0·5 ml min-1 (Hugenholtz & Starrenburg, 1992 ).
Preparation of ethanol extracts and determination of intracellular phosphorylated metabolites by 31P-NMR.
Ethanol extracts of the LDHd and MG1363 strains were prepared as described previously by Ramos et al. (2001) . The dried extracts were dissolved in 4 ml H2O containing 5 mM EDTA and 2·5% (v/v) 2H2O (final pH approximately 7·2). Assignment of resonances and quantification of phosphorylated metabolites was based on previous studies (Ramos & Santos, 1996
; Ramos et al., 2001
) or by spiking the NMR samples with the suspected pure compounds. The reported values for intracellular phosphorylated compounds are means of two independent growth experiments and the accuracy varied from 10 to 15%.
In vivo NMR experiments and quantification of metabolites.
Cells were grown as described above on medium containing mannitol or glucose and harvested in the mid-exponential growth phase and suspended in 50 mM potassium phosphate buffer (pH 6·5) to a protein concentration of approximately 13 mg protein ml-1. In vivo NMR experiments were performed using the on-line system described previously (Neves et al., 1999 ). [1-13C]Glucose (20 mM) or [1-13C]mannitol (19 mM) were supplied to the cell suspension and the time-course for their consumption, product formation and intracellular metabolite pools was monitored in vivo. When the substrate was exhausted and no changes in the resonances of intracellular metabolites were observed, an NMR sample extract was prepared as reported previously (Neves et al., 1999
, 2002
). The end products, lactate, acetoin, acetate, 2,3-butanediol, ethanol and formate, were quantified in the NMR sample extract by 1H-NMR in a Bruker AMX300. The concentration of intermediates that remained inside the cells (pyruvate, aspartate, succinate, alanine) was determined in fully relaxed 13C spectra of the NMR sample extracts as described by Neves et al. (2002)
.
For quantification of the intracellular metabolites, correction factors were determined, allowing the conversion of resonance areas into concentrations. The correction factor for FBP (0·73±0·04) was obtained as described previously (Neves et al., 1999 ); a factor of 0·65±0·03 was determined for mannitol and Mtl1P as reported by Neves et al. (2000)
. Metabolite concentrations were calculated using a value of 2·9 µl (mg protein)-1 for the intracellular volume (Poolman et al., 1987
). The concentration limit for detection of intracellular metabolites under the conditions used to acquire in vivo spectra (30 s total acquisition time) was 34 mM. The values shown are means of two to four experiments and the accuracy varied between 2 (end products) and 15% in the case of intracellular metabolites with concentrations below 5 mM.
NMR spectroscopy.
13C or 31P NMR spectra were acquired at 125·77 or 202·45 MHz on a Bruker DRX500 spectrometer. All in vivo experiments were run using a quadruple nuclei probe head at 30 °C, as described previously (Neves et al., 1999 ). For the quantitative analysis of NMR sample extracts by 13C-NMR, a repetition delay of 60·5 s was used. The 31P-NMR spectra of the ethanol extracts were obtained as described by Ramos et al. (2001)
. For the determination of LDH activity in cell extracts, lactate production was monitored by 1H-NMR using a pulse width of 6 µs (90° flip angle) and a recycle delay of 3·1 s. For the quantification of lactate, formate was used as a concentration standard, the recycle delay was increased to 45·7 s and 96 transients were acquired. Carbon and phosphorus chemical shifts were referred to the resonances of external methanol or external 85% H3PO4, designated at 49·3 p.p.m. and 0·0 p.p.m., respectively.
Enzyme activity measurements.
The extracts used for measurement of enzyme activities were prepared from cells harvested in the mid-exponential growth phase (Neves et al., 2000 ). Enzyme activities were assayed in a spectrophotometer (Beckman DU70), equipped with a cell compartment thermostated at 30 °C, in a total volume of 1 ml. One unit of enzyme activity was defined as the amount of enzyme catalysing the conversion of 1 µmol substrate min-1 under the experimental conditions used.
LDH and pyruvate kinase (PK) were assayed as described by Garrigues et al. (1997) . The forward (Mtl1P
F6P) and reverse (F6P
Mtl1P) reactions catalysed by Mtl1PDH were assayed as reported previously (Neves et al., 2000
). Phosphofructokinase (PFK) activity was measured by the method of Fordyce et al. (1982)
. LDH activity in the LDHd strain grown on mannitol was measured by 1H-NMR by monitoring the rate of lactate production after the addition of sodium pyruvate (20 mM) to a reaction mixture containing 35 mM Tris/HCl buffer, pH 7·2, 2·5 mM MgCl2, 0·6 mM NADH, 3 mM FBP (activator of LDH) and 25% (v/v) 2H2O. Anaerobic conditions were used to avoid NADH oxidation by the NADH oxidases present in the cell extracts.
Chemicals.
[1-13C]Glucose (99% enrichment) and [1-13C]mannitol (99%) were obtained from Campro Scientific. Formic acid (sodium salt) and methylphosphonic acid were purchased from Merck and Aldrich, respectively. All other chemicals were reagent grade.
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RESULTS |
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(ii) Growth yields and energetics. In both strains studied, growth on mannitol led to the synthesis of less biomass (Fig. 1) and was characterized by lower specific growth (µmax) and substrate consumption (
) rates than those observed in glucose-containing medium (Table 1
). A similar biomass yield, i.e. the growth yield relative to the substrate consumed, was determined for the LDHd construct growing on glucose or mannitol and MG1363 growing on glucose; in contrast, a considerably lower value was found for the latter strain with mannitol as energy source. The global yields of ATP were calculated from the fermentation products assuming that all ATP was synthesized by substrate-level phosphorylation. In both strains, the higher ATP yield in mannitol-grown cells reflected the increased acetate production per mole substrate consumed, when compared to glucose-grown cells. Consequently, the biomass yield relative to ATP (YATP), in g biomass (mol ATP)-1, was higher on glucose when compared to mannitol, showing that the latter is a poorer substrate for growth. The YATP values determined with glucose as energy source were equivalent to those reported previously for L. lactis (Nóvak et al., 1997
).
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In vitro measurements of enzyme activities
Specific activities of relevant enzymes were measured in crude extracts obtained from mid-exponential cultures of mannitol-grown LDHd and MG1363 (Table 2) and were compared to the activities measured in glucose-grown cells reported previously (Neves et al., 2000
). LDH activity [10·5 U (mg protein)-1] in the MG1363 strain grown on mannitol was lower than that reported for glucose-grown cells [16·1 U (mg protein)-1]. Furthermore, the lactate-producing activity determined in extracts of the LDHd construct was reduced 40-fold upon growth on mannitol. This low LDH activity was measured by 1H-NMR in extracts of mannitol-grown cultures, since the sensitivity of the standard spectrophotometric method coupled to NADH oxidation was too low to allow detection. Interestingly, PFK and PK, enzymes encoded by the same operon as LDH, were reduced by about 1·4-fold in the LDHd strain grown on mannitol when compared to glucose-grown cultures (Neves et al., 2000
).
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DISCUSSION |
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A significant production of lactate by the LDHd strain was observed under all conditions tested. This construct possesses two dysfunctional truncated copies of the ldh gene. One is under the control of the las promoter and the other has no promoter unless there is readthrough from the integrated plasmid (Gasson et al., 1996 ). The activity measured is either due to one of these copies, or more likely, to a distinct LDH, since four genes showing sequence homology with ldh genes from other organisms are present in the genome of L. lactis IL1403 (Bolotin et al., 2001
). The latter hypothesis is probably correct, since an LDH-deficient strain obtained by a single cross-over deletion of the ldh gene in L. lactis MG1614 still produced lactate and a different LDH was isolated from that strain (Gaspar, P., Coelho, P., Neves, A. R., Shearman, C., Gasson, M. J., Ramos, A. & Santos, H., unpublished results). Interestingly, the lactate-producing activity measured in extracts of mannitol-grown cells was 40-fold lower when compared to glucose-grown cells, but the mechanisms underlying this phenomenon, also observed in MG1363 extracts although to a lesser extent (1·5-fold reduction of LDH activity), are unknown. LDH and PK activities are also lower in L. lactis cells grown on galactose when compared to glucose, and this was believed to be due to reduced transcriptional activation of the las operon by the carbon catabolite protein A, CcpA (Luesink et al., 1998
). It was suggested that this could be a consequence of lower intracellular G6P and FBP, which were found to enhance the binding of CcpA to DNA in Bacillus subtilis (Deutscher et al., 1995
; Miwa et al., 1997
). However, a different explanation must hold for L. lactis, since we have found higher FBP levels in mannitol-grown cells than in glucose-grown cells (Fig. 2
).
Also intriguing is the poor utilization of mannitol for growth of MG1363 when compared to LDHd, which exhibits reasonably good growth yields on this substrate (Fig. 1). This suggests that the disruption of the ldh gene per se induced the expression of genes implicated in mannitol transport and metabolism in the LDHd strain. Mtl1PDH, a key enzyme for mannitol utilization, was enhanced by at least 34-fold (compare activities of glucose-grown cells in Table 2
), and the glucose-grown parental strain was unable to utilize mannitol (not shown), whereas comparable consumption rates were observed in the LDHd strain regardless of the growth substrate.
A striking feature in the composition of the end products derived from mannitol by the LDHd strain was the considerable accumulation of ethanol, with 50% of the pyruvate being converted to this alcohol. This is the expected response to the higher pressure to oxidize NADH, since ethanol provides the most efficient pathway for the disposal of reducing power. What is surprising is the minor production of lactate during growth of MG1363 in mannitol despite the very high activity of LDH (Table 2), the main site for NADH oxidation in this organism during the utilization of more natural substrates, such as glucose. At present, the drastic change of the carbon flux distribution at the pyruvate node remains elusive.
Given the potential impact of mannitol in the development of healthier food products, the synthesis of mannitol in L. lactis is a desirable metabolic trait. The insight into mannitol and glucose metabolism gained from the present work discloses the LDHd strain as an adequate genetic background to proceed with a metabolic engineering approach aimed at the enhancement of the in situ production of mannitol. The construction of a mannitol overproducer would have to take into account the ability of L. lactis to use mannitol as energy source. Since the uptake of mannitol is mediated by a PEP:PTS (Bolotin et al., 2001 ; Monedero et al., 2001
) we propose that such a strain could be obtained by knocking out the mtlA gene encoding the transport protein (EIIMtl). Work is in progress in our team to attain this goal.
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ACKNOWLEDGEMENTS |
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Received 18 March 2002;
revised 28 June 2002;
accepted 8 July 2002.