University of Illinois at Urbana-Champaign, Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2522 VMBSB, 2001 South Lincoln Avenue, Urbana, IL 61802, USA
Received on November 15, 2000; revised on February 14, 2001; accepted on March 2, 2001.
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Abstract |
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Key words: sialic acid/metabolic engineering/Escherichia coli K1/pathway analysis/carbohydrate flux
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Introduction |
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The difficulty in producing some oligosaccharide precursors is especially evident with members of the family of nonulosonates known as the sialic acids. These unique N- or O-substituted derivatives of N-acetylneuraminic acid (Neu5Ac) are ubiquitous in animals of the deuterostome lineage (starfish to humans) but do not seem to be synthesized by most plants, protostomia, protists, Archaea, or eubacteria (Warren, 1994). Despite the limited distribution of sialates in nature, a few (mostly pathogenic) bacterial strains synthesize Neu5Ac de novo and incorporate it into cell surface glycolipids (Vimr et al., 1995
). Of medical importance is Escherichia coli K1, the leading cause of Gram-negative neonatal meningitis in humans, in addition to a frequent etiologic agent of childhood and adult urinary tract infections and septicemia (Silver and Vimr, 1990
). The polysialic acid (PSA) capsule of E. coli K1 inhibits host innate defense mechanisms and provides molecular mimicry of the PSA chains on the neural cell adhesion molecule (NCAM). The discovery of PSA as both virulence factor and regulator of central nervous system development prompted extensive investigations into sialate biosynthesis.
In the mammalian liver, Neu5Ac is synthesized and activated for transfer to glycoconjugate acceptors (R-OH) in five sequential steps involving phosphorylated intermediates (Warren, 1994), beginning with UDP-N-acetylglucosamine (UDP-GlcNAc):
The specificity of sialyltransferases for a given acceptor (step 6) dictates the pattern of sialoglycoconjugates (Neu5Ac-O-R) synthesized by various cells. The bifunctional enzyme, UDP-GlcNAc 2-epimerase/N-acetylmannosamine (ManNAc) kinase (Stasche et al., 1997), catalyzes the first two steps of the pathway, yielding ManNAc-6-phosphate (step 2). Production of CMP-Neu5Ac in step 5 is tightly regulated through allostery, with increasing CMP-Neu5Ac concentrations inhibiting the epimerase that produces ManNAc in step 1 (Kornfeld et al., 1964
). In step 3, ManNAc-6-P is condensed with phosphoenolpyruvate (PEP) to yield Neu5Ac-9-P, with subsequent dephosphorylation by specific or nonspecific phosphatase(s) in step 4 yielding free Neu5Ac. Alternative mechanisms for synthesizing Neu5Ac may involve the direct epimerization of free GlcNAc to ManNAc (Maru et al., 1996
), Neu5Ac recycling from surface glycoconjugates through a potential salvage pathway involving turnover by lysosomal sialidase and the direct enzymatic condensation of ManNAc with pyruvate by Neu5Ac aldolase (Rodriguez-Aparico et al., 1995
).
Irrespective of the exact mechanism(s) used by different eukarya to synthesize sialic acids, synthesis of the activated sialyl donor, CMP-Neu5Ac, by E. coli K1 does not appear to be tightly regulated by allosteric inhibition (Steenbergen and Vimr, 1990; Vimr and Troy, 1985b
). Because E. coli lacks CMP-Neu5Ac hydrolase (Masson and Holbein, 1983
), accumulation of CMP-Neu5Ac in a polymerase-defective genetic background is a physiological dead-end. From a commercial viewpoint, exploitation of this metabolic blockade for targeted overproduction of free Neu5Ac or CMP-Neu5Ac offers alternatives to the chemoenzymatic syntheses of these compounds. A solely fermentative source of CMP-Neu5Ac would obviate the need for expensive starting materials, such as Neu5Ac, PEP, nucleoside phosphates, or recycling enzymes currently required for efficient in vitro syntheses of sialooligosaccharides. Our current results contribute to the understanding of regulation of sialic acid metabolism by the microbial metabolic engineering of intermediate flux through the synthetic pathway.
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Results |
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As indicated in Figure 1, sugars such as GlcNAc, ManNAc, or Fru (not shown) are transported as phosphorylated derivatives by the phosphotransferase uptake system (PTS). Gro (not shown) enters cells by a facilitated diffusion process and therefore would not influence PTS-regulated operons. The modest increases in sialate pool size of EV136 or EV240, when grown in basal medium containing GlcNAc or Fru, but not Gro (Table II), suggest that catabolite repression is not critical for sialate overproduction. Furthermore, there was no difference (P > 0.05) between sialate pools from cells grown with Fru or Glc (data not shown), suggesting that the type of PTS sugar was unimportant to sialate overproduction. We infer from these results that the effect of medium composition on E. coli sialate pool size is largely a function of final cell density, which for basal medium supplemented with Glc or Fru is two to three times greater than that of LB. Therefore, when the carbon supply is plentiful, there appears to be adequate UDP-GlcNAc synthesized to support the essential production of peptidoglycan and LPS while simultaneously allowing overproduction of sialate (Table II). We have not systematically investigated whether sialate pool size is affected by leakage or efflux mechanisms; however, previous results suggest such losses are probably minor (Vimr and Troy, 1985b). We conclude that fermentation in a defined medium produces approximately 20 nmols of CMP-Neu5Ac per A600 per ml. Because growth yields of 30 absorbance units are routinely attainable (Hoffman et al., 1995
), gram quantities of CMP-Neu5Ac should be readily available through simple fermentation of appropriate bacterial strains in defined or complex media.
Mechanism of sialate overproduction
To determine if the overproduction of Neu5Ac in neuS mutants is dependent on a functionally intact biosynthetic system (Figure 1), we analyzed the sialate pool in the triple mutant EV239. This mutant has defects in NanA, NeuS, and sialate synthase (NeuB), suggesting either that the Neu5Ac peak detected in this strain (Figure 2) arose from an alternate synthetic pathway or, less likely, a compound unrelated to Neu5Ac eluted at the same time as authentic Neu5Ac. The first possibility was considered more likely for two reasons. First, EV36 extracts contained no detectable sialate (Figure 2), suggesting that the peak in EV239 was actually Neu5Ac. Second, it was possible we were observing a phenotype of the synthase mutant that was being magnified by the simultaneous polysialyltransferase and aldolase deficiencies in EV239. Others (Rodriguez-Aparico et al., 1995) concluded that E. coli K1 relies on NanA to synthesize Neu5Ac by catalyzing the aldol condensation of pyruvate with ManNAc, suggesting that the NeuB-independent peak detected in extracts of EV239 may have arisen from residual nanA expression. However, when EV239 was grown in defined medium, Neu5Ac was no longer detected (Figure 3 and Table II). Because LB contains trace sialate (Steenbergen et al., 1992
), we concluded that the peak detected in EV239 grown on LB (Figure 2) resulted from intracellular concentration of exogenous Neu5Ac by the sialic acidspecific permease, NanT (Figure 1).
NanA limits the accumulation of free Neu5Ac
Assuming our proposed model of sialate biosynthesis in Figure 1 is correct, it should be possible to overproduce free Neu5Ac by truncating the biosynthetic pathway at neuA. Although the absence of NanA appeared to have little effect on increasing the sialate pools in neuS strains (Table II), a defective neuA gene should allow increased sialate accumulation until full induction of nanA is reached. The metabolic consequence of this phenotype (aldolase induction) is predicted to limit the endogenous sialate pool size. As shown in Figure 4, NanR negatively regulates the nan operon by acting as a repressor that is inactivated on binding free Neu5Ac (Plumbridge and Vimr, 1999). Therefore, on full induction of the nan operon, NanA would convert the available sialate pool to ManNAc and, ultimately, back to Fru-6-P (Figure 1). However, in a nanA neuA double mutant, active aldolase would not exist and the sialate pool should be stabilized.
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Discussion |
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Current methods for the chemoenzymatic synthesis of sialooligosaccharides require specific precursors and enzymes to generate CMP-Neu5Ac (Liu et al., 1992; Gilbert et al., 1998
). Fermentation has obvious advantages of simplicity and nominal cost in comparison to traditional organic synthetic methods and potentially obviates the need for CMP-Neu5Ac regeneration in vitro. More important, by expressing the appropriate sialyltransferase in tandem with CMP-Neu5Ac overproduction, it should be possible to engineer E. coli that synthesize virtually any sialylated oligosaccharide by coexpression of the relevant acceptor. The large number of completely sequenced microbial genomes and availability of E. coli with modified sialometabolic pathways should greatly facilitate engineering sialooligosaccharide synthesis in bacteria.
Mechanism of sialic acid biosynthesis
Our current results demonstrate the obligatory requirement of NeuB for sialate biosynthesis in E. coli. Others failed to detect this activity in vitro, suggesting that NanA instead of NeuB was the sole biosynthetic source of sialate in E. coli (Rodriguez-Aparico et al., 1995; Ferrero et al., 1996
). Although earlier studies contradicted this suggestion (Vimr and Troy, 1985b
; Steenbergen et al., 1992
; Vimr, 1992
), our current results (Figure 3) allow unambiguous rejection of the hypothesis that NanA normally functions biosynthetically in E. coli. This conclusion, together with direct biochemical support (Vann et al., 1997
), demonstrates the versatility and power of experimentally coupling the relevant physiology of amino sugar metabolism to systematic genetic and biochemical analyses of sialic acid synthesis.
In mammals, the bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase is likely the pivotal biosynthetic enzyme for synthesis of ManNAc-6-P in vivo. It is noteworthy that in E. coli, the putative ManNAc kinase (NanK) is genetically and functionally separate from the UDP-GlcNAc epimerase (NeuC). NanK would thus appear to function exclusively in sialic acid degradation as a product of the nan operon, whereas its mammalian counterpart appears to be a purely biosynthetic enzyme. The low yet potentially significant similarity between the bacterial and mammalian ManNAc kinases (Table I) may support an ancient origin of sialate metabolic enzymes. However, because sialate biosynthesis is a sporadic trait in microbes, the possibility that biosynthesis of this sugar evolved late remains tenable. Entertaining the speculation that microbes may have acquired some of their biosynthetic and catabolic machinery from eukaryotes (Hoyer et al., 1992), it will be interesting to compare whether select unicellular eukaryotes such as Candida albicans, which reportedly synthesizes sialic acid (Soares et al., 2000
), utilizes the prokaryotic or mammalian synthetic mechanism.
In Neisseria meningitidis group B, the ortholog of neuC (siaA) is reported to be a GlcNAc-6-P to ManNAc-6-P epimerase (Petersen et al., 2000). Though biosynthesis of Neu5Ac in meningococci (as well as in E. coli) appears to require unphosphorylated ManNAc as the hexosamine sialate precursor (Figure 1), it has been postulated that N. meningitidis uses specific or nonspecific phosphatase(s) to produce ManNAc (Petersen et al., 2000
). Despite the homology between neuC and siaA, neuC and its mammalian homolog use UDP-GlcNAc as ManNAc precursor, whereas SiaA may recognize GlcNAc-6-P. However, GlmS produces GlcN-6-P instead of GlcNAc-6-P, with GlcN-6-P subsequently isomerized by GlmM and converted to UDP-GlcNAc via the bifunctional GlmU (Figure 1). This microbial pathway for the production of UDP-GlcNAc implies that there is little intracellular GlcNAc-6-P in the absence of an exogenous supply of free GlcNAc. If the proposed function of SiaA is correct, there must be an alternative source of GlcNAc-6-P in N. meningitidis. Pending purification of NanK, it should become straightforward to chemoenzymatically synthesize radiolabeled ManNAc-6-P for detection of the predicted specific or nonspecific phosphatase(s) in N. meningitidis or other organisms.
Conclusion
We have quantified the overproduction of free Neu5Ac and CMP-Neu5Ac under defined growth conditions and shown how sialate aldolase diverts the flux of intermediates away from the synthetic pathway. Recently, Betenbaugh and his associates cloned and expressed the mammalian equivalent of neuB in insect cells (Lawrence et al., 2000). The transformed cells were shown to synthesize Neu5Ac when provided with an exogenous source of ManNAc, but did not produce CMP-Neu5Ac. By capitalizing on the ability of genetically engineered E. coli to synthesize PSA, we have shown that simply growing the appropriate nonpathogenic strain in defined medium attains overproduction of sialoglycoconjugate precursor synthesis. It should be possible to further engineer this system for synthesis of more complex sialosides.
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Materials and methods |
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Sample preparation
After harvesting cells (2.55.0 ml) by centrifugation, culture supernatants were discarded and the tube sides wiped to remove as much residual medium as possible. Water was added to give a tenfold concentration of the starting culture, and the A600 of samples determined spectrophotometrically after appropriate dilution. With our instrument (Beckman DU 640), an A600 of 1.0 corresponds to 6.25 x 108 cells/ml. Cultures were frozen at 20°C, thawed, and then sonicated briefly to disrupt bacteria. After cell disruption, TCA was added to 10% final concentration and samples chilled on ice for 1 h. The copious precipitate that formed after incubation was removed by centrifugation; the supernatant was dried under vacuum in a Savant SpeedVac.
Sialic acid analysis
For quantitation of Neu5Ac, lyophilized samples of hydrolyzed culture products of known volume were reconstituted in 250 µl of deionized water and subjected to ultrafiltration (Ultrafree-MC 100,000 NMWL Filter Unit, Millipore). Neu5Ac analysis was carried out with a Dionex model DX-300 high-performance liquid chromatography system equipped with pulsed amperometric detection. Isocratic runs were partitioned using a Carbopac PA-1 column in 0.1 M NaOH0.05 M sodium acetate at a flow rate of 1.0 ml/min. Elution times and concentrations were compared with a standard curve generated from commercial Neu5Ac (Sigma). Relative detector responses shown in Figure 2 and 3 are in nA. Unless indicated otherwise, data were normalized to express the concentration of sialate per unit of A600 per ml of culture.
Statistical analyses
Differences in sialate accumulation between E. coli strains EV136 and EV240 were analyzed by the t-test. Differences in sialate accumulation among E. coli EV5 and three isogenic transductants (Table III) were analyzed by analysis of variance using contrasts to compare the transductants to EV5. P-values < 0.05 were considered significant.
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Acknowledgment |
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Abbreviations |
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Footnotes |
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References |
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