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Wilbert Sybesma, Marjo Starrenburg, Michiel Kleerebezem, Igor Mierau, Willem M. de Vos and Jeroen Hugenholtz Appl. Environ. Microbiol. 2003, 69(6):3069. DOI: 10.1128/AEM.69.6.3069-3076.2003.
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and pyrimidines. 6 Increased Production of Folate by Metabolic Engineering of Lactococcus lactis Wilbert Sybesma. 6718 ZA Ede. one-carbon substitution of the pteridine ring. The in vivo function of folate is that of a cofactor that donates one-carbon units in a variety of reactions involved in the de novo biosynthesis of amino acids. 20. The Netherlands. bottom). NIZO Food Research. 2012 by guest Lactococcus lactis is by far the most extensively studied lactic acid bacterium. while the biosynthesis of purines is required for the production of the building block GTP (Fig. The design of rational approaches to metabolic engineering requires a proper understanding of the pathways that are manipulated and the genes involved. All Rights Reserved. predominantly in the polyglutamyl form. 43). a number of speciﬁc enzymatic steps are involved in the ﬁnal assembly of folate and for production of the various folate derivatives. 37). P. and bacteria are able to synthesize folate and can serve as a folate source for the auxotrophic vertebrates.1128/AEM. 32. 3069 Downloaded from http://aem. 20). resulting in high-level production of both natural and novel end products. the pentose phosphate pathway. lactis MG1363: folA. 6. Box 20. June 2003. In the present and previous studies we have used L. Due to the ability of lactic acid bacteria to produce folate (31).2003 Copyright © 2003. lactis was found to increase the extracellular folate production almost 10-fold. Mailing address: Department of Flavor.69. Marjo Starrenburg. For pregnant women. and the number of glutamate residues. Although L. Fax: 31(0)318-650400. most notably anemia and neural tube defects in newborns (33) and mental disorders such as psychiatric syndromes among the elderly and decreased cognitive performance (7. The cloning and overexpression of folA. Folate is a general term for a large number of folic acid derivatives that differ by their state of oxidation. 1.O. p. lactis subsp. Five genes involved in folate biosynthesis were identiﬁed in a folate gene cluster in L. 44) and alanine (18). The Netherlands Received 25 November 2002/Accepted 6 March 2003 The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate. Another complicated pathway is the biosynthesis of folate (13). lactis (3. folB. Igor Mierau. and the shikimate pathway for the production of the folate building block p-aminobenzoate. and over the last decades a number of elegant and efﬁcient genetic tools have been developed for this starter bacterium. E-mail: hugenhol@nizo. Only small amounts of the produced folate are released in the extracellular medium. folate levels in fermented dairy products are higher than those in the corresponding nonfermented dairy products (1). de Vos. The daily recommended intake of dietary folate for an adult is 400 g. American Society for Microbiology. folate that can directly be absorbed in the gastrointestinal tract. 36. 6718 ZB Ede. especially the nisin-controlled expression (NICE) system for controlled heterologous and homologous gene expression in L. These tools are of critical importance in metabolic engineering strategies that aim at inactivation of undesired genes and/or (controlled) overexpression of existing or novel ones. increased the retention of folate in the cell. 3069–3076 0099-2240/03/$08. there are . Vol. 600 g is recommended. while the total folate production increased almost 3-fold. Recent studies done in The Netherlands and Ireland have indicated that folate deﬁciency is common even among various population groups in the developed countries. Folate is an essential nutrient in the human diet. encoding dihydrofolate reductase. cremoris MG1363 for metabolic engineering experiments (3. Nutrition and Natural Ingredients. Many plants.6. The overexpression of folKE in L. 21). No. NIZO Food Research. including diacetyl (8. i.org/ on February 19. fungi. This biosynthesis includes parts of glycolysis. folate is assumed to have protective properties against cardiovascular diseases and several types of cancer (5. lactis IL1403 (4) reveals the existence of a folate gene cluster containing all genes encoding the folate biosynthesis pathway (Fig. 69. folKE. Michiel Kleerebezem. 18. Phone: 31-(0)318-659629. In this respect. and Jeroen Hugenholtz* Wageningen Centre for Food Sciences. These differences are associated with different physicochemical properties which may inﬂuence folate bioavailability. 33). The natural diversity among dairy starter cultures with respect to their capacity to produce folate can be exploited to design new complex starter cultures which yield fermented dairy products with elevated folate levels. In addition. In addition. The gene folKE encodes the biprotein 2-amino-4-hydroxy-6hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I.nl. lactis IL1403 and MG1363 show a high degree of homology on the genome level. purines. top). The controlled combined overexpression of folKE and folC. lactis has proven to be very valuable (9). decreased the folate production twofold. Folate deﬁciency leads to numerous physiological disorders. including women of childbearing age (24. encoding polyglutamyl folate synthetase. Willem M.00 0 DOI: 10..e. preferably combined with knowledge about ﬂuxes and control factors.asm.3069–3076. folP. The annotated genome sequence of L. Most of the metabolic engineering strategies so far applied to lactic acid bacteria are related to primary metabolism and comprise efﬁcient rerouting of the lactococcal pyruvate metabolism to end products other than lactic acid. Metabolic engineering of more complicated pathways involved in secondary metabolism has only recently begun by the engineering of exopolysaccharide production in L. 30. lactis subsp. and folC. * Corresponding author. suggesting a feedback inhibition of reduced folates on folate biosynthesis.APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 1.
L. lactis was grown at 30°C in M17 medium (47) supplemented with 0. When appropriate. Palo Alto. The Netherlands. MICROBIOL. Restriction enzymes and T4 DNA ligase were purchased from Life Technologies BV. For successful application of metabolic engineering in the ﬁnal steps of the complicated biosynthesis pathway of folate in L. oligonucleotides (50 pM) (Table 2). lactis folate gene cluster as identiﬁed in L. (40). Isolation of chromosomal and plasmid DNAs from L. and white arrows represent genes that are not expected to be involved in folate biosynthesis.0 U of Pfx polymerase (Invitrogen.). Bad Oeynhausen. Hatched arrows represent genes involved in folate biosynthesis.5 mM each). Breda. Isolation of E. coli plasmid DNA and standard recombinant DNA techniques were performed as described by Sambrook et al. 1. DNA manipulations and transformations. Several L. PCR ampliﬁcation of DNA and nucleotide sequence analysis.0 to 3. the media contained chloramphenicol (10 g/ml) or kanamycin (50 g/ml). Ampliﬁcation was performed on a Mastercycler (Eppendorf. The results presented here are an important step in the development of fermented foods with increased bioavailable and natural folate. lactis was performed as previously described (11). Germany) with 30 cycles of denaturation at 95°C for 30 s (3 min in the ﬁrst cycle). characterization of the folate gene cluster in this strain is necessary. and 1. Downloaded from http://aem. Thick arrows indicate enzymatic reactions that are controlled in metabolic engineering experiments (see text for details). Sequence analysis of the genes involved in folate biosynthesis was done after ampliﬁcation of a 9-kb DNA fragment ﬂanked by the upstream regions of folA (29) and hom (34) by using primers Fol-F and Hom-R (Table 2) and cloning of the fragment in pCR-BLUNT (Invitrogen). lactis and transformation of plasmid DNA to L. MATERIALS AND METHODS Bacterial strains and plasmids. Hamburg. annealing at 50°C for 30 s.5% (wt/vol) glucose. ENVIRON. lactis genes were ampliﬁed from chromosomal DNA by PCR with 25 ng of DNA in a ﬁnal volume of 50 l containing deoxyribonucleoside triphosphates (0. coli plasmid for nucleotide sequence analysis was performed with JetStar columns by following the instructions of the manufacturer (Genomed. Germany). and elongation at 68°C (Pfx) or 72°C (Taq-Tth) for 1 to 8 min.asm. folKE encodes a bifunctional protein. Great Britain) or Taq-Tth polymerase mix (Clontech. generating pCR-BLUNT-FOL. Escherichia coli was grown at 37°C in tryptone yeast medium (40). media and culture conditions. lactis MG1363. black arrows represent genes involved in folate biosynthesis that are overexpressed in metabolic engineering experiments. considerable differences (28). 2012 by guest FIG. Paisley. The bacterial strains and plasmids used in this study are listed in Table 1. Calif. lactis IL1403. (Top) Chemical structure of tetrahydrofolate and folate biosynthesis pathway.org/ on February 19. APPL.3070 SYBESMA ET AL. lactis MG1363 and L. The generated plasmid was transformed by electroporation . (Bottom) Schematic representation of the L. Large-scale isolation of E.25 to 0.
... Next... encoding dihydrofolate reductase...GAGAAATCAAATCCTCATTCTAGATTAAAATTCC FolA-F .CATACCACTTCTTTTTCGATTTGTAAAGG Hom-R . introducing a HindIII restriction site... The terminator from pepV (17) was ampliﬁed from chromosomal DNA by using primers TpepV-F and TpepV-R (Table 2).... The gene folC.... thereby placing the folKE gene under the control of nisA.GAAGATCTCACCTCTATTTCTAGAATAAAG FolKE2-F. encoding a bifunctional protein predicted to display both folate synthetase and polyglutamyl folate synthetase activities. introducing an SphI restriction site. The ampliﬁcation product includes a ribosome binding site and was digested with KpnI and XbaI.. thereby placing the folA gene under the control of nisA....... the gene folKE was ampliﬁed from chromosomal DNA by using primers FolKE2-F and FolKE2-R (Table 2)..5.. lactis subsp. the gene was cloned in pNZ7010 downstream of folKE.. The reverse primer FolA-R was extended at the 5 end...... The addition of nisin and the subsequent overexpression of genes did not affect the growth characteristics of the engineered strains.. plasmid free MG1363 pepN::nisRK L. Lactococcal plasmid pNZ8048 (25..... pNZ8161 derivative containing a functional lactococcal folKE behind the constitutive pepN promoter Cmr. pNZ8048 derivative containing a functional lactococcal folA gene behind the nisA promoter Cmr.. The gene folKE. thereby placing the folKE gene under the control of the constitutive promoter of pepN. pNZ8048 derivative containing a functional lactococcal folKE gene behind the nisA promoter Cmr. cloning......GGGGTACCGATTCTTGATTAAGTTCTAAG FolC-F . The constitutive promoter from pepN (48) was ampliﬁed from plasmid pNZ1120 (48) by using primers Pcon-F and Pcon-R (Table 2)..asm. coli strain TOP10 Plasmids pCR-blunt pCR-blunt-fol pNZ8048 pNZ8160 pNZ8161 pNZ7010 pNZ7017 pNZ7011 pNZ7012 PNZ7013 PNZ7014 L. generating pNZ7011.. by using primers FolA-ASF and FolA-ASR (Table 2). Nisin induction..... The new plasmid is pNZ7013. plasmid free Cloning host 12 26 4 Invitrogen Kanr Derivative of pCR-blunt carrying lactococcal folate gene cluster Cmr.. lactis...... Sweden)..... generating pNZ8160.GCCCAAAAATTGATTTTGCCATGGTTG Pcon-F . The new plasmid is pNZ7010. The reverse primer Pcon-R was extended at the 5 end.... Oligonucleotides used for DNA ampliﬁcation by PCR Primer Sequencea Fol-F .. The vector contains a nisA promoter and an NcoI cloning site. nisR and nisK....... Both primers were extended at the 5 end...SphI (digested with BglII)..... The cells were induced with different concentrations of nisin A (referred to as nisin) ranging from 0... The new plasmid is pNZ7017. The generation of a plasmid containing a constitutive promoter and a nisin-inducible promoter separated by a terminator (pNZ8161) was as follows...ATACATGCATGCAAACAACTTATTTAAGCATGGG FolKE2-R ...... Both primers were extended at the 5 end... derivative of pNZ8048 carrying terminator of pepV Cmr..1 to 5 ng/ml.... introducing an NcoI restriction site resulting in a slight modiﬁcation of the mature gene (Table 2). was ampliﬁed from chromosomal DNA by using primers FolA-F and FolA-R (Table 2). An overnight culture of L... encoding a protein predicted to display dihydropteroate synthase activity.... and harvested for further analysis....... cremoris..... generating pNZ7012... was ampliﬁed from chromosomal DNA by using primers FolP-F and FolP-R (Table 2)..CGGGATCCTTATGAACTTGCAAAATAAG TpepV-R .......... derivative of pNZ8160 carrying constitutive promoter of pepN Cmr.. introducing a BamHI restriction site..... coli TOP10 (Invitrogen).GAATGGTACCTTTAGGAGGTCTTTTATGAAAATCTTAGAAC FolP-R .. introducing a KpnI restriction site and an XbaI restriction site... The ampliﬁcation product was digested with BglII and BamHI and cloned in pNZ8160 (digested with BamHI).TCTCTAGACTACTTTTCTTTTTTCAAAAATTCACG FolP-F........ encoding a bifunctional protein predicted to display both 2-amino4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I activities.. and they are constitutively expressed under the control of the nisR promoter...org/ on February 19.... lactis subsp....GGAATTCCATGGTTATTGGAATATGGGCAGAAG FolA-R ...... The new plasmid is pNZ7014.. introducing a multiple cloning site including a BglII restriction site... lactis strains MG1363 NZ9000 IL1403 E..... . Bacterial strains and plasmids Strain or plasmid Relevant characteristics INCREASED FOLATE PRODUCTION IN LACTOCOCCUS LACTIS 3071 Source or reference L..... The forward primer FolKE-F was extended at the 5 end.. The reverse primer TpepV-R was extended at the 5 end. The reverse primer FolKE-R was extended at the 5 end.VOL. 26) is a translational fusion vector used in nisin-controlled expression systems.... introducing a KpnI restriction site... Primer sequences were obtained from published sequence data for folA (29) and hom (34) and by subsequent primer walking....... The ampliﬁcation product was digested with NcoI and HindIII and cloned in pNZ8048 (digested with NcoI and HindIII)...GAAGATCTGTCGACCTGCAGTAGACAGTTTTTTTAATAAG Pcon-R. Next.GCCTCAAGCTTCATGGTTGTTTCACTTTTTC FolA-ASF..GAAGAGGTACCAGAAGAGTTTAAAAAGTATTATCG FolC-R...... inducible expression vector carrying nisA promoter Cmr. L...CGATCCCGGGAAGCCCTGTGCCAACTGTCC FolKE-F.... The ampliﬁcation product was digested with NcoI and KpnI and cloned in pNZ8048 (digested with NcoI and KpnI).. Folate was analyzed in cell extracts and fermentation broth.. was ampliﬁed from chromosomal DNA by using primers FolKE-F and FolKE-R (Table 2).... 2003 TABLE 1... introducing an NcoI restriction site resulting in a slight modiﬁcation of the mature gene (Table 2).GAGGGGTACCTATGATAATTGGAATATGG FolA-ASR . except for the orientation..... respectively.... was ampliﬁed from chromosomal DNA by using the primers FolC-F and FolC-R (Table 2).... pNZ8048 derivative containing a functional lactococcal folP gene behind the nisA promoter Cmr...CATGCCATGGGGCAAACAACTTATTTAAGCATGG FolKE-R . The forward primer folA-F was extended at the 5 end. In NZ9000 the genes for a nisin response regulator and a nisin sensor. lactis strain NZ9000 (26) was used as a host for the plasmids described in Table 1. incubated for 2 h. The forward primer Pcon-F was extended at the 5 end. The ampliﬁcation product was digested with SphI and cloned in pNZ8161 (digested with SphI).. The cloning of the antisense RNA of the gene encoding dihydrofolate reductase was achieved in a way similar to that described for folA. and sequencing were performed twice in independent experiments. pNZ8048 derivative containing a functional lactococcal folKE and folC gene behind the nisA promoter Cmr. Ampliﬁcation.. The ampliﬁcation product includes a ribosome binding site and was digested with KpnI and XbaI and cloned behind folKE in pNZ7010....CGGGATCCGCATGCCTTCTCCTAAATATTCAGTATTAA TpepV-F.. The nucleotide sequence of the ampliﬁed folate gene cluster was determined by automatic double-stranded DNA sequence analysis with a MegaBACE DNA analysis system (Amersham Pharmacia Biotech AB. lactis NZ9000 harboring pNZ8048 or one of the plasmids described above was diluted (1:100) in GM17 supplemented with chloramphenicol and grown to an optical density at 600 nm (OD600) of 0.. Construction of plasmids and transformation of strains....... The gene folA. The gene folP.ATACATGCATGCGATTCTTGATTAAGTTCTAAG a Underlined nucleotides represent modiﬁcations with regard to the mature gene.... Uppsala.... and overproduction of Downloaded from http://aem. 69. introducing a KpnI restriction site and an XbaI restriction site.. The forward primer TpepV-F was extended at the 5 end..... are stably integrated at the pepN locus in the chromosome.. introducing BamHI and SphI restriction sites.. generating pNZ8161... introducing a BglII restriction site........ The ampliﬁcation product was digested with BglII and BamHI and cloned in pNZ8048.... pNZ8048 derivative containing antisense RNA of a lactococcal folA gene behind the nisA promoter Invitrogen This study 26 This study This study This study This study This study This study This study This study to E. 2012 by guest TABLE 2.. Both primers were extended at the 5 end... Differences in both DNA sequences were reanalyzed after independent ampliﬁcation and cloning of the regions ﬂanking the ambiguous sequences.
5. lactis folA contains an identiﬁed promoter region (29) and that folKE.1 M sodium acetate (pH 4. It appears that. folE. a 9-kb DNA fragment ﬂanked by folA. folP. To measure intraand extracellular folate concentrations. Folate was released from the cells and from folate binding proteins by incubating the samples at 100°C for 5 min.8)–1% ascorbic acid.4. It has been reported previously that in L. 10 min.org/ on February 19. overproduction of the same biprotein and polyglutamyl folate synthetase.asm. Its nucleotide sequence was determined and revealed the presence of nine open reading frames. lactis IL1403 (4). and folC are cotranscribed in a multicistronic operon (45).5% (vol/vol) concentration of the clariﬁed human plasma solution was added to the folate samples. The genes clpX and ysxC may be involved in stress responses (22). 0.8 with human plasma (Sigma-Aldrich Chemie. was ampliﬁed from the genome of L. in L. lactis MG1363 and IL1403 is 89%.) and centrifugation (20. overproduction of dihydrofolate reductase.1 M sodium acetate buffer (pH 4.17). predicted to encode an ATP binding protein for ClpP. ranging from 73% identity for ylgG to 98% for both clpX and dukB. predicted to encode folate synthetase/folyl polyglutamate synthetase (EC 6.1 M 2-mercaptoethanol– 0. which was determined to be optimal for maximum folate release. The cells were washed with 0. APPL. predicted to encode GTP cyclohydrolase I (EC 3. in contrast to the case for many other microorganisms. predicted to encode neopterine aldolase (EC 4. Coomassie brilliant blue-stained gel after SDS-PAGE of cell extracts from cultures induced with nisin.5% sodium ascorbate and cleared from precipitates by centrifugation (10. A 1% yeast extract medium solution (Difco. encoding dihydrofolate reductase. 0. The analysis of total folate concentration. which corresponds to the combined molecular masses of the predicted enzymes encoded by folK and folE. is the ﬁrst enzyme in the folate biosynthesis pathway (Fig. washed.). 4. Based upon the genetic organization of a folate gene cluster in L. encoding dihydrofolate reductase (EC 1. Analysis of intra. lactis IL1403 showed that the two strains have an identical genetic organization. Molecular markers are indicated on the right. predicted to encode a deoxynucleoside kinase (EC 2. N.. encoded by folKE. Consequently. RESULTS Sequencing and annotation of a folate gene cluster.Y. and folC.3072 SYBESMA ET AL. Compared to that in a noninduced strain. encoded by folC. Analysis of the nucleotide sequence of the putative folK and folE genes could identify neither a stop codon at the end of the putative folK gene nor a start codon at the beginning of the putative folE gene. Twenty to 100 l of the cell extract was used to measure dihydrofolate reductase activity as described previously (38). total folate concentrations can be measured only after deconjugation of the polyglutamyl tails in samples containing folate derivatives with more than three glutamyl residues. 2). or pNZ7014 was grown and induced with nisin as described previously. the heating inactivates the folate-producing bacteria and prevents their interference in the microbiological folate assay. and resuspended in 1 ml of buffer (10 mM KPO4.16). predicted to encode dihydropteroate synthase (EC 2.5.1 mM dithiothreitol.1. At an OD600 of 2.1. Forty milliliters of a culture of L.3). 2 min). both cells and supernatant were recovered from a full-grown cell culture (5 ml) after centrifugation (12.000 g. The Coomassie brilliant blue-stained gel revealed one intense protein band with an apparent molecular mass of 40 kDa.2. the overexpression of folKE in strain NZ9000 harboring pNZ7010 caused an increased concentration of extracellular folate from . a DNA sequence comprising both genes was fused to the nisA promoter of pNZ8048. folB.6. Md. 3. The overall amino acid identity of these nine putative proteins between the two L.8)–1% ascorbic acid and resuspended in 5 ml of the same buffer.and extracellular folate concentrations. The intense band was absent in a noninduced strain (Fig.2. Becton Dickinson Microbiology Systems.113). Dihydrofolate reductase activity.1 mM EDTA [pH 7. and a 2. 20°C). MICROBIOL. Folate was quantiﬁed by using a Lactobacillus casei microbiological assay (19). 1A). while the response to longer-chain polyglutamyl folate (more than three glutamyl residues) decreases markedly in proportion to chain length (46). Increased band intensities are indicated by asterisks. and ysxC and ylgG. Moreover. lactis the enzymes 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I are produced as one bifunctional protein and are encoded by one gene. all of which have the same orientation. Sparks. The remaining genes that were identiﬁed in the gene cluster are clpX. folK. 0°C). The deconjugation reaction mixture was prepared as follows: 1 g of human plasma was diluted in 5 ml of 0. encoded by folA. cells were harvested. and triglutamyl folate. encoding homoserine dehydrogenase. 2. predicted to encode 2-amino-4hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC 2. Increased extracellular folate production by overexpression of folKE. overproduction of biprotein 2-amino4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The supernatant was diluted 1:1 with 0. ylgG. A cell extract was made by addition of 1 g of silica beads to the cell suspension followed by disruption of the cells in an FP120 Fastprep cell disrupter (Savant Instruments Inc.25). lactis strain NZ9000.5. pNZ7013.12/6.7.0]). Nucleotide sequence accession number.000 g. and cell extracts were prepared for SDS-PAGE.3. control strain. here designated folKE. 2. Sequence comparison with the genome of L. both encoding unknown proteins. proteins was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (3).3.000 g. part of the biprotein encoded by folKE. Zwijndrecht. 2012 by guest FIG.7. The Netherlands) as a source for -glutamyl hydrolase activity. folP. lactis MG1363. except for folA (29). was done after enzymatic deconjugation of the folate samples for 4 h at 37°C and pH 4. To verify the nature of folK and folE. containing almost exclusively polyglutamyl folates.15).2. and introduced in L.3). Only ﬁve or six genes in the folate gene cluster appeared to be involved in folate biosynthesis: folA. Downloaded from http://aem. lactis strains is 90%. The nucleotide identity of the folate gene clusters in L. The standard deviation of the microbiological assay varied between 0 and 15%. ENVIRON. diglutamyl folate. dukB.1. including polyglutamyl folate. with a previously determined total folate content was used as a positive control for actual deconjugation. 10 min. lactis NZ9000 harboring pNZ8048. Lanes: 1. The nucleotide sequence data reported in this paper have been submitted to the GenBank database under accession number AY156932. Holbrook. In the latter strain the sequence of the genes involved in folate biosynthesis was not yet known. GTP cyclohydrolase I. The microbiological folate assay has nearly equal responses to monoglutamyl folate. and hom.1.5. Cells were induced with nisin. generating pNZ7010.
Folate concentrations in different L. the gene folA. folB. lactis MG1363. similar to what was observed with overexpression of only folKE.and folC-overexpressing strain. or pNZ7013 (overexpressing folA). indicating that the polyglutamyl folate was not excreted by the cells (Fig. 2003 INCREASED FOLATE PRODUCTION IN LACTOCOCCUS LACTIS 3073 FIG. at an OD600 of approximately 2. Cell extracts of strain NZ9000 harboring pNZ7014. encoding dihydropteroate synthase. Error bars indicate standard deviations. lactis strains harboring pNZ8048 (empty vector). the induction of strain NZ9000 harboring pNZ7013 resulted in production of the enzyme with a predicted molecular mass of 15 kDa at a level that could be visualized by SDS-PAGE (Fig. encoding the bifunctional protein folate synthetase and polyglutamyl folate synthetase. 4). encoding dihydrofolate reductase. The cloning and overexpression of the area Downloaded from http://aem. The extra. white hatched bars. Under inducing conditions. The constitutive expression of folKE behind the constitutive promoter of pepN that could be achieved in NZ9000 harboring pNZ7017 resulted in the same increase of folate production as observed by using the NICE system (results not shown). intracellular folate production after deconjugation. encoding the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. lactis strains overexpressing folA showed a more-than-1. The overexpression of folA caused a twofold decrease in folate production compared to that in a control strain or a noninduced strain (Fig. pNZ7011 (overexpressing folKE and folC). was also overexpressed. starting at the 5 end with the complementary sequence of the stop codon of folA and ﬁnishing at the 3 end with the complementary start codon of the gene. approximately 10 to 80 ng/ml. pNZ7014. a small but reproducible increase of approximately 20% in the total folate production was observed compared to that in a control strain (results not shown). It can be concluded that by overexpression of folKE. or triglutamyl folate. After deconjugation of the cell extracts. 3). Under inducing conditions. 3). The plasmid generated. Furthermore. 4). However. transcribing antisense RNA of folA. The simultaneous overexpression of folKE and folC (NZ9000 harboring pNZ7011) could be visualized by SDS-PAGE (Fig. and folC. 2). To gain further insight into folate biosynthesis control in L. 3). pNZ7010 (overexpressing folKE). 2). no differences in folate concentration or folate distribution were observed compared to the overexpression of only folKE (results not shown). the folKEoverexpressing strain displayed a minor increase in intracellular folate production compared to a control strain or noninduced strain NZ9000 harboring pNZ7010. 69.000-fold increase in dihydrofolate reductase activity compared to a control strain (Fig. In contrast to the folate produced by the overexpression of folKE only. lactis NZ9000. The intracellular folate concentration was measured by analyzing cell extracts for the presence of folate. 3. 3). White bars. the majority of the extra folate produced was present as intracellular folate in the folKE. Increased intracellular folate production by combined overexpression of folate genes. The overexpression of both genes resulted in a more than twofold increase in total folate production. the intracellular folate concentrations were about 80 ng/ml in both strains (Fig. In contrast. was transformed into L. The total folate production by L. The complementary sequence of the coding strand of folA was cloned under the control of the nisin promoter nisA in pNZ8048. extracellular folate production. encoding dihydrofolate reductase.5. no increased folate concentrations were detected. Folate concentrations were determined at the end of growth. lactis.asm. no difference in folate concentration was measured with or without deconjugase treatment.5. The majority of the extra folate produced is present as extracellular mono-.and intracellular folate distribution is assumed to be controlled by the ratio of monoand polyglutamyl folates (35). showed a twofold decrease in dihydrofolate reductase activity (Fig. folK and folE. and analysis of the folate gene cluster in L. Altered folate production by overexpression of folA or antisense folA. differences were detected in the folate distribution. 2012 by guest .VOL. indicating that the overexpression of folKE and folC had no signiﬁcant effect on the amount of polyglutamyl folates with more than three glutamate residues (Fig. di-. the folate production is more than doubled compared to that of a control strain or noninduced NZ9000 harboring pNZ7010. The overexpression of folKE and folP. The intracellular folate distribution and the relative amount of polyglutamyl folates remained unchanged. sequencing. black bars. After deconjugation of the intracellular folate. total folate production. folP. The folate samples were enzymatically deconjugated with human plasma in order to determine whether part of the extracellular folate was present as polyglutamyl folate with more than three glutamate residues that could not be measured by the microbiological assay. lactis was determined by combining the extraand intracellular folate concentrations. In a similar way as described previously. predicted to encode dihydropteroate synthase. the extracellular folate concentration measured for the control strain NZ9000 harboring pNZ8048 was not affected by induction with nisin (Fig. To conﬁrm the effect of the transcription of folA antisense RNA. Strains were induced with 0 ( ) or 2 ( ) ng of nisin per ml at an OD600 of 0. In a similar experiment we studied the effect of the production of antisense RNA encoding dihydrofolate reductase. was achieved by inducing strain NZ9000 harboring pNZ7012. the enzymatic activity of dihydrofolate reductase was determined. black hatched bars. 3). However. predicted to encode dihydroneopterin aldolase. However.org/ on February 19. DISCUSSION We have described successful metabolic engineering of the ﬁnal part of the complicated biosynthetic pathway of folate biosynthesis and the cloning. The enzyme responsible for the synthesis of polyglutamyl folate is polyglutamyl folate synthetase encoded by folC. cell extracts of L. Homology studies with nonredundant databases show that the folate gene cluster contains folA. intracellular folate production.
As a consequence. folP. The same cells showed a 20% increase in folate production. see references 14. the overexpression of folA decreases the total folate production approximately 2-fold. since this enzyme in B. Folate gene clusters have previously been identiﬁed in some related microorganisms. may exist as a single gene. MICROBIOL. the majority of the extra folate produced remains intracellular. 39. be achieved only by combining folKE overexpression with altered expression of other folate biosynthesis genes. folC. but in several microorganisms. lactis is intracellularly accumulated. the retention of folate in the cell is decreased. encoding 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase. This demonstrates not only that functional expression of folate biosynthesis genes can also be achieved by using a constitutive promoter but also that a further increase in folate production can. pNZ7013 (overexpressing folA) (hatched bars). Streptococcus pneumoniae. the intracellular polyglutamyl folate may not be available for absorption by the gastrointestinal tract of the consumer if the folate is not released by the (mostly dead) microorganisms. FIG. The use of an inducible promoter system enables study of the effect of various expression levels of the folate biosynthesis enzymes. However. Furthermore. and ylgG. and Rickettsia conorii. The other genes present in the folate gene cluster. It can be assumed that the increase of extracellular folate by overexpression of folKE is due to an increased production of folate with a short polyglutamyl tail.3074 SYBESMA ET AL. in contrast. Almost all of the extra folate produced by overexpression of folKE is excreted into the environment. such as monoglutamyl folate. which is necessary for retention of folate within the cell. APPL. a second folC gene was identiﬁed outside the folate gene cluster. Error bars indicate standard deviations. in food fermentations the use of constitutive promoters is preferred. 2012 by guest . and 16). clpX. The decrease in folate production by overexpression of folA. Overexpression of the ﬁrst enzyme of a biosynthetic pathway (GTP cyclohydrolase I) can be a successful strategy to increase the ﬂux through the pathway. 4. To further improve our knowledge about the suggested effect of tetrahydrofolate on total folate production. ysxC. The activity of these deconjugases is susceptible to inhibition by various constituents found in some foods (2. Moreover. and folK are clustered. pneumoniae. the enzymatic capacity of folate synthetase/polyglutamyl folate synthetase is not sufﬁcient to transform all extra produced folate into the polyglutamyl form. pneumoniae and Lactobacillus plantarum. while folE and folC are far apart in the genome (42). Vinnicombe and Derrick (49) report an inhibiting effect of tetrahydrofolate on dihydropteroate synthase in S. This conﬁrms that an increased capacity of folate synthetase leads to increased retention of folate in the cell due to an increased enzymatic capacity to elongate the glutamyl tail of the extra folate produced by the overexpression of folKE. and only a minor part of the folate is secreted by the cells. and folK are clustered together with genes involved in p-aminobenzoate synthesis.asm. The gene folE. We suggest that by the increased ﬂux through the folate biosynthesis pathway. The cloning of folKE behind the constitutive promoter of pepN resulted in the same increase of folate production as observed by using NICE. In S. folB. ﬁve. comprising folK and folE showed the existence of a bifunctional protein encoded by only one gene. More than 90% of the intracellular folate pool is present in the polyglutamyl form with four. Clostridium perfringens. In Lactobacillus plantarum. lactis strains harboring pNZ8048 (empty vector) (white bars). when folKE and folC are simultaneously overexpressed. encoding GTP cyclohydrolase I. conﬁrming the presumable controlling effect of the folA gene product.g.org/ on February 19. The NICE system was used to induce overexpression of genes involved in folate biosynthesis.. are not likely to be involved in folate biosynthesis. e. Chlamydia muridarum. or pNZ7014 (overexpressing antisense RNA of folA) (black bars). presumably. may indicate a feedback inhibition of its reaction product. was always identiﬁed as an independent gene. folK forms a biprotein with either folB (neopterin aldolase) or folP (dihydropteroate synthase). ENVIRON. folP. folE. although the enzyme production levels were clearly lower. 15. Effect of nisin concentration on dihydrofolate reductase activity measured in cell extracts of L. and six glutamyl residues (unpublished results). Only monoglutamyl folate derivatives can be directly absorbed in the human gut. 41). In Downloaded from http://aem. At least three of the genes from the folate gene cluster appeared to be involved in controlling folate biosynthesis and folate distribution in L. Chlamydia trachomatis. due to the overexpression of folKE. 35). One of the suggested functions of polyglutamylation is retention of folate within the cell (20. but in a different order (23. Comparative genome analysis with nonredundant databases reveals that the gene folK. tetrahydrofolate. It has been established that the bioavailability of monoglutamyl folate is higher than that of polyglutamyl folate (for reviews. The combined overexpression of folKE and folC favors the intracellular accumulation of folate. Enzymatic activity was decreased approximately twofold in cells expressing folA antisense RNA. Most of the folate produced by L. However. Polyglutamyl folates are available for absorption and metabolic utilization only after enzymatic deconjugation in the small intestine by a mammalian deconjugase enzyme. on one of the other enzymes involved in folate biosynthesis. lactis: controlled overexpression of folKE increases the extracellular folate production almost 10-fold and the total folate production almost 3-fold. subtilis has a low turnover and is not regulated by feedback inhibition (10). encoding dihydrofolate reductase. folB. In Bacillus subtilis. 27). folKE. GTP cyclohydrolase I seems to be a good target for overexpression. we used the NICE system to produce the antisense RNA of folA and we measured the dihydrofolate reductase activity in vitro. To further analyze the observed controlling effect of folA. we are working on the substitution of the folA promoter in the chromosome with the nisin-inducible promoter nisA.
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