WO2019216466A1

WO2019216466A1 - Cyclic dipeptide synthetase

Info

Publication number: WO2019216466A1
Application number: PCT/KR2018/005452
Authority: WO
Inventors: Minkyu KWAK; Rui Liu; Saouk KANG
Original assignee: Seoul National University R&Db Foundation
Priority date: 2018-05-11
Filing date: 2018-05-11
Publication date: 2019-11-14

Abstract

The present invention provides novel cyclic dipeptidase. The cyclic dipeptidase according to present invention could be used to produce antibacterial cyclic dipeptide. By using this cyclic dipeptidase it is possible to make a new microorganism having high antibacterial activity. Preferably, it is possible to give antibacterial activity to a useful food microorganism. With the increase antibacterial activity in the food microorganism, there is less need or even no further antibacterial agents for food preservation. Thus, the present invention could be used to preserve food without further antibiotic additive.

Description

CYCLIC DIPEPTIDE SYNTHETASE

The present invention relate to a novel enzyme, an ATP-dependent cyclic dipeptide synthetase involved in the production of bioactive cyclic dipeptides in Lactobacillus plantarum LBP-K10

In the culture filtrates (CFs) of kimchi-driven isolated Lactobacillus plantarum LBP-K10 grown in modified de Man, Rogosa and Sharpe (MRS without beef extracts) medium-^[1], L-proline-based cyclic dipeptides (CDPs), including cis-cyclo(L-Leu-L-Pro), cis-cyclo(L-Phe-L-Pro), and cis-cyclo(L-Val-L-Pro) confirmed by X-ray crystallography, were elucidated against plant and human pathogenic fungi and influenza virus^[2,3]. Inspired by these findings, a complete set of the CF CDPs of this isolate, consisting of seven proline-based CDPs and one non-proline-containing CDP, was demonstrated by using anion exchange resins coupled with gas chromatography-mass spectroscopy (GC-MS)^[4]. As previous data obtained from preliminary experiments, Lactobacillus CDPs significantly showed the time point with the highest total amount at 72 h occurring concomitantly with the decline in cell numbers. All these evidence gives rise to concerns on the strong selectivity enhancement for CDP isolations exerted by the repeated chromatographic separation of possible racemic substances of the diastereomers decisively in a methylene chloride (MC) extraction-dependent manner^[2-4]. Similar relations between Lactobacillus growth control by secondary metabolites particularly through the CDP biosynthesis^[4] and agr-mediated dual-channel quorum-sensing signaling associated with CDP production, as fully illustrated in Lb. reuteri RC-14 cyclo(l-Phe-l-Pro) and cyclo(l-Tyr-l-Pro)-^[5], seem more attractive when seen as part of the amino acid (AA) utilization entirely derived from complex MRS medium in laboratory experiments. During Lactobacillus or Leuconostoc species growth, they commonly require complex nitrogen sources^[6,7]. Prominently, Lb. plantarum species displayed absolute requirements for typical types of essential proteogenic (isoleucine, leucine, valine, lysine, tryptophan, threonine) and non-essential proteogenic (glutamic acid, cysteine) AAs-[8]. The AA metabolism renders a bulk contribution to physiological events sufficiently amicable to permit protein biosynthesis, pH modulation, metabolic energy/redox balance alteration, and stress resistance by various types of intermediate metabolites, seemingly representative of bacteriocin-like substances and non-peptidyl small compounds^[9,10].

In the culture filtrates (CFs) of kimchi-driven isolated Lactobacillus plantarum LBP-K10 grown in modified de Man, Rogosa and Sharpe (MRS without beef extracts) medium-[1], L-proline-based cyclic dipeptides (CDPs), including cis-cyclo(L-Leu-L-Pro), cis-cyclo(L-Phe-L-Pro), and cis-cyclo(L-Val-L-Pro) confirmed by X-ray crystallography, were elucidated against plant and human pathogenic fungi and influenza virus^[2,3]. Inspired by these findings, a complete set of the CF CDPs of this isolate, consisting of seven proline-based CDPs and one non-proline-containing CDP, was demonstrated by using anion exchange resins coupled with gas chromatography-mass spectroscopy (GC-MS)^[4]. As previous data obtained from preliminary experiments, Lactobacillus CDPs significantly showed the time point with the highest total amount at 72 h occurring concomitantly with the decline in cell numbers. All these evidence gives rise to concerns on the strong selectivity enhancement for CDP isolations exerted by the repeated chromatographic separation of possible racemic substances of the diastereomers decisively in a methylene chloride (MC) extraction-dependent manner^[2-4]. Similar relations between Lactobacillus growth control by secondary metabolites particularly through the CDP biosynthesis^[4] and agr-mediated dual-channel quorum-sensing signaling associated with CDP production, as fully illustrated in Lb. reuteri RC-14 cyclo(l-Phe-l-Pro) and cyclo(l-Tyr-l-Pro)-^[5], seem more attractive when seen as part of the amino acid (AA) utilization entirely derived from complex MRS medium in laboratory experiments. During Lactobacillus or Leuconostoc species growth, they commonly require complex nitrogen sources^[6,7]. Prominently, Lb. plantarum species displayed absolute requirements for typical types of essential proteogenic (isoleucine, leucine, valine, lysine, tryptophan, threonine) and non-essential proteogenic (glutamic acid, cysteine) AAs-^[8]. The AA metabolism renders a bulk contribution to physiological events sufficiently amicable to permit protein biosynthesis, pH modulation, metabolic energy/redox balance alteration, and stress resistance by various types of intermediate metabolites, seemingly representative of bacteriocin-like substances and non-peptidyl small compounds^[9,10].

Considering the broad spectrum of bioactivity of CDPs^[11,12], which bears 2,5-diketopiperazines observed for naturally occurring structural motifs notably those harboring antimicrobial actions^[12], hormone-like quorum sensing circuitry-^[13], and immunosuppressive moiety-^[14], these three-dimensionally defined head to tail dipeptide dimer-ring closure has steadily been established as attractive scaffolds for their modes of bioactivity-^[15]. Despite the elusive nature of in vivo functions of CDPs, they exhibit different biochemical events^[5,16] due to the notified rigid chiral side conformations and membrane diffusing capacity evocative of cross-talk with quorum sensing autoinducers^[17,18]. Although the biosynthetic origins of CDPs have not been proved sufficiently, current studies demonstrated the nonribosomal peptide synthetases (NRPSs) responsible for their assembly, either by dedicated control of biosynthetic pathways or by premature release of dipeptidyl intermediates during the chain elongation^[19-21]. Importantly, AlbC^[22-24], which was found to be an enzyme specifically constructing CDPs, and its eleven homologs entirely characterized^[25-27] catalyzes the production of albonoursin, mycocyclosin^[22,23], siderochrome pulcherrimin^[28-30], nocazine family-^[26], and methylated ditryptophan CDPs-^[25]. The action of certain antibiotics contributes to a CDP biosynthesis through a second dedicated route through using hijacking aminoacylated tRNAs as substrates. Owing to our preliminary works on bioactivity of proline-containing CDPs, we herein hypothesized that the CF AA composition during cultures in mMRS media might render the enzymatic-based CDP-biosynthesizing activity susceptible to all of the components of the CDP pool, which further support to be more dependent on the utilization of the specific AAs, including proline, leucine, and phenylelalanine. We thereby tried to find out a new class of protein CDP synthetase from Lactobacillus isolate, named LbCDPS, verified by the AA-quenching-based enzyme-activity assay primarily using both native gels through the modified AA/ninhydrin mixture without typical markers and consecutive HPLC fractionations. Additionally, some types of enzymatic products driven by Lb. plantarum LBP-K10 LbCDPS in the presence of ATP were isolated by HPLC and confirmed by GC-MS. On the basis of our previous studies regarding the bioactivity of a single CDP^[2,3], we compared the difference of bioactivity between single CDPs purified and enzymatically catalysed products by LbCDPS. Thus, ATP-dependent LbCDPS with enzyme products bearing antibacterial activity was discovered by novelty search from a specific bacterial species radically different from the previously reported members of the CDP synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes.

The first purpose of the present invention is to provide new cylcic dipeptidase.

The second purpose of the present invention is to provide food microorganism enhaced in antibacterial acitiv with the said cyclic dipetidase.

The third purpose of the present invention is to provide method of preparation of a food by using the microorganism with increased antibacterial activity.

For the purpose of above object, one aspect of present invention provides cyclic dipeptidase of Seq. No. 1. The said cyclic peptidase include its functional equivalent. As used herein, the term "functional equivalent" is intended to include amino acid sequence variants having amino acid substitutions in some or all of a cyclic dipeptidase of SEQ ID NO: 1, or amino acid additions or deletions in some of the cyclic dipeptidase.　The amino acid substitutions are preferably conservative substitutions. Examples of the conservative substitutions of naturally occurring amino acids include aliphatic amino acids (Gly, Ala, and Pro), hydrophobic amino acids (Ile, Leu, and Val), aromatic amino acids (Phe, Tyr, and Trp), acidic amino acids (Asp, and Glu), basic amino acids (His, Lys, Arg, Gln, and Asn), and sulfur-containing amino acids (Cys, and Met). The deletions of amino acids are located in a region which is not involved directly in the activity of the cyclic dipeptidase.

According to a second aspect, the present invention provides a gene encoding said cyclic dipeptidase. Preferably, the said gene is a gene comprising nucleotide sequence of SEQ ID NO: 2.

According to a third aspect, the present invention provides a recombinant vector comprising the said gene encoding cyclic dipeptidase. The present invention provides various recombinant vector, for example plasmid, cosmid or phagemid, phage, virus etc. The method of preparation of said recombinant vector has publicly known.

According to a fourth aspect, the present invention provides host cell transformed with said recombinant vector. Host cell suitable for transformation could be, but not limited thereto, prokaryotic cell. Most preferably,　E. coli or Lactobacillus sp. cell is used.

According to a fifth aspect, the present invention provides a method for producing a cyclic dipeptide using said host cell.

According to sixth aspect, the present invention provides a method of preparation of cyclic peptide by using said cyclic peptidase.

According to seventh aspect, the present invention provides a food composition comprising cyclic peptide produced by said cyclic peptidase. The said food could be, but not limited thereof, a food that the said host cell producing said cyclic dipeptide is removed so that it does not include a genetically modified microorganism. The food composition according to present invention has antimicrobial activity arising from the cyclic dipeptide. When used as a food composition, the food composition of the present invention may be mixed with various foods such as beverages, soups, soups, frozen foods, and other processed foods (bread, cookies, jam, candy, gum, tea, functional foods) to be a functional food composition.

Hereinafter, the definition of "health functional food" of the present invention means foods manufactured with functional raw materials or ingredients beneficial to for the human body. A functional food is a food given an additional function (often one related to health-promotion or disease prevention) by adding new ingredients or more of existing ingredients.

The amount of addition of the present invention in a mixed food composition is determined based on the effective amount of ingestion per day for an adult. It is desirably added in an amount of 1 ~ 100g. The method of addition is not particularly limited. It may be added from the beginning with the raw food material. The amount of addition of the present invention could be generally selected from 0.01 to 70 weight % of the total weight of the food composition.

The food composition of present invention could further comprise food additives such as natural carbohydrate or various flavoring agents. The said natural carbohydrate could be monosaccharide, (for example glucose, fructose etc.), disaccharide (for example sucrose, lactose, maltose etc.), polysaccharide (for example dextrin, cyclodextrin etc.) or sugar alcohol (for example sorbitol, erythritol, xylitol etc.). The said flavoring agents could be natural flavoring agents such as taumartin, stevia extract, rebaudioside A, glycyrrhizin etc., and synthetic flavoring agents such as saccharine and aspartame etc. In addition, the food composition of present invention could comprise various nutrients, vitamins, minerals, electrolytes, coloring agents, pectic acid and its salt, alginic acid and its salt, organic acid, protective colloidal thickener, pH adjusting agent, stabilizer, preservative, glycerin, and alcohol. The food composition of the present invention could be added in Lactobacillus drink or paste such as yogurt.

Fig. 1. AA profiles of Lb. plantarum LBP-K10 according to the period of growth phase.

Fig. 2. A strategy for CDPS purification by using activity staining in native gels.

Fig. 3. The enzyme activity was verified by native activity staining.Synthesis of cyclic dipeptide, catalyzed by CDPS, resulted an achromatic band appearing on the blue ninhydrin stained gel (indicated with red arrows).

Fig. 4. HPLC analysis of cyclic dipeptides in reaction solution including enzyme and substrates. Enzyme catalyzed L-Proline/L-Leucine/L-Phenylalanine mixture to generating new substances, which were shown as peaks appeared after 9.7 min with present of ATP (A; Bond black line). However without ATP, Enzyme could not catalyze any reaction in the CDPS/ L-Proline/ L-Leucine/ L- Phenylalanine mixture (A; Dot line). Solutions contained the enzyme only were applied as control (A; Dash line). cis-cyclo(L-Pro-L-Leu) (B;MW 210) and cis-cyclo(L-Pro-L-Phe)(C; MW 244) were examined with HPLC, and the results indicated the existence of two cyclic dipeptide in the Enzyme/ L-Proline/ L-Leucine/ L- Phenylalanine mixture after reaction (Bond black line).

Fig. 5. Purification of enzyme in Lb. plantarum LBP-K10. (A) An achromatic band (red arrow) confirmed the protein existed in fraction 7-11 possessed CDPs synthesis ability (please refer to Fig. 14). The bands, whose level changing corresponding to native staining band in 14, were screen out and indicated with red arrows. (A). Laemmli-stained band of fraction 7-9 (indicated with red arrow in B) presented on SDS-PAGE again, the band from

fraction

7 and 9 could be observed.

Fig. 6. The nucleotide and amino acid sequence of ORF of the cyclic dipeptide synthesis enzyme (NCBI gi: 311821850). A 690 bp sequence of the cyclic dipeptide synthesis enzyme was obtained from NCBI genome database.

Fig. 7. Multiple sequence alignment of cyclic dipeptide synthetase in Lb. plantarum LBP-K10. The amino acid sequence deduced from the Lb. plantarum LBP-K10 cyclic dipeptide synthetase gene was aligned with other species using vector NTT9.0 explorer clustal X program. K10: Lb. plantrum LBP-K10; Lb.n Lb. namurensis; Lb.p : Lb. plantarum; Lb.f : Lb. fabifermentans; St.n : Streptomyces noursei.

Fig. 8. Overproduction and native activity staining of CDPS. Overproduction of CDPS was verified by denaturing electrophoresis (A) and its function was confirmed with native activity staining (B).

Fig. 9. Cyclic dipeptides increased in culture filtrates of recombinant CDPS. To confirm the induction of cyclic dipeptides in transfected E .coli culture, HPLC analysis of culture extracts were performed. The result indicate, the most important functional CDPs of F7(L-Val-L-Pro), F14(L-Leu-L-Pro), F17(L-Phe-L-Pro) increased significantly.

Fig. 10. Overall profiles of the increased production of cyclic dipeptides using HPLC system in CDPS gene transfected bacteria.

Fig. 11. MC extract of transfected E. coli medium showed enhanced antibacterial activity. After transfected with CDPS plasmid, the antimicrobial activity of MC extract from E. coli was increased and was comparable with MCK10. However, the extract from control plasmid transfected or intact E. coli display a weak or negligible effect on the bacteria indicators.

Fig. 12. Purification and activity assay of Recombinant CDPS. After gel filtration with superdex75 column chromatography, recombined CDPS were confirmed on SDS-PAGE (A). Activity of purified CDPS were checked using native staining assay (B).

Fig. 13a. HPLC analysis confirmed CDPS that catalyzed the synthesis of cyclo(Pro-Lys) in vitro. CDPS catalyzes two amino acid pairs, such as L-Proline and L-Lysine, to generate cyclo(Pro-Lys) in vitro. The chromatograms peaks of L-Proline (upper left), L-Lysine (left right), ATP (middle left) and CDPS (lower left) were shown as indicated. In the case of the reaction mixture of L-Proline, L-Lysine and ATP, it did not show CDPs peaks within 10 min (middle right). However, when CDPS was added to the reaction mixture, the biosynthesized cyclo(Pro-Lys) and cyclo(Lys-Lys) (as indicated in the figure) was observed as new peaks ranging from 16.5-16.7 min.

Fig. 13b. HPLC analysis confirmed CDPS that catalyzed the synthesis of cyclo(Pro-Phe) in vitro. CDPS catalyzes two amino acid pairs, such as L-Proline and L-Lysine, to generate cyclo(Pro-Lys) in vitro. L-Proline (upper left), L-Lysine (left right), ATP (middle left) and CDPS (lower left) peaks were shown as indicated. In the case of the reaction mixture of L-Proline, L-Lysine and ATP, it did not show CDPs peaks within 10 min (middle right). However, when CDPS was added to the reaction mixture, the biosynthesized cyclo(Pro-Phe) and cyclo(Phe-Phe) was observed as new peaks ranging from 15.5-26 min.

Fig. 13c. HPLC analysis confirmed CDPS that catalyzed the synthesis of cyclo(Pro-Ser) in vitro. L-Proline (upper left), L-Serine (left right), ATP (middle left) CDPS (lower left) peaks were shown in HPLC chromatogram. The reaction mixture of L-Proline, L-Serine and ATP presented no peaks after ten min (middle right). However, when CDPS was added, a peak of new substance (cyclo(Pro-Ser)) appeared around 15 min.

Fig. 13d. HPLC analysis confirmed CDPS that catalyzed the synthesis of cyclo(Pro-Val) in vitro. L-Proline (upper left), L-Valine (left right), ATP (middle left) CDPS (lower left) peaks were shown in HPLC chromatogram. The reaction mixture of L-Proline, L-Valine and ATP presented no peaks after ten min (middle right). However, when CDPS was added, the reaction mixture of L-Proline, L-Valine, ATP and cyclic dipeptides showed a peak of new substance (which was confirmed as cyclo(Pro-Val) later on around 13 min.

Fig. 13e. HPLC analysis confirmed CDPS that catalyzed the synthesis of cyclo(Pro-Val) in vitro. L-Proline (upper left), L-Leucine (left right), ATP (middle left) CDPS (lower left) peaks were shown in HPLC chromatogram. The reaction mixture of L-Proline, L-Leucine and ATP presented no peaks after ten min (middle right). However, when CDPS was added in, the reaction mixture of L-Proline, L-Leucine, ATP and cyclic dipeptides showed a peak of new substance around 19 min.

Fig. 14. Molecular mass was confirmed with superose 12 gel permeation column (GE Healthcare) for determining the oligomeric state. Superpose 12 gel permeation column used for determine the molecular weight with the standard contrast of aprotinin, cytochrome C, Albumin, and alcohol dehydrogenase (A). According the results of FPLC, the molecular of CDPS was calculated as 99.18KD (C). Purified CDPS was confirmed with 12% SDS-PAGE as 25KD (B).

Fig. 15. Crystals and diffraction images of CDPS. A: The crystals of CDPS. B: The diffraction image of CDPS of X-ray radiation.

Fig. 16. The homotetramer structure of CDPS. As shown in the new cartoon model, CDPS was constructed by four identical subunits.

Fig. 17. The secondary structure of CDPS subunit. For a better view, the subunit A were colored depended on the secondary structure (left panel). Each of the identical subunits contains 6 beta sheets (yellow), surrounded by 7 alpha helix (purple) and 5 3₁₀ helix (blue). Thus, each subunit has 3 layer of alpha/beta/alpha structure.

Fig. 18. A hollow space centered in the structure of CDPS (Arrow). A hollow space centered in the structure of CDPS (indicated by yellow arrow).

Fig. 19. Comparation of cyclic dipeptide synthetase and PGM1 (PDBID: 1YFK). Through PDBj data base, it is confirmed cyclic dipeptide synthetase contains the similar combining site with human PGM1.

METHODS

Chemicals. All of the biochemicals and reagents, including those used for LbCDPS screening and enzymatic products by HPLC analysis, were from Sigma-Aldrich or Fisher Scientific and had sufficient purity (>99.9%) without further purification unless otherwise noticed except for the following experiments as described below.

Cells. Lb. plantarum LBP-K10 from Korean fermented kimchi was identified by 16S rDNA sequencing method and cultured in mMRS as previously noticed^[2]. For the routine growth of Lactobacillus cells, the modified MRS medium without beef extract (2% glucose, 0.5% yeast extract, 0.5% sodium acetate (CH₃COONa), 0.2% ammonium citrate dibasic (C₆H₁₄N₂O₇), 0.2% dipotasium phosphate (K₂HPO₄), 0.01% magnesium sulfate (MgSO₄), 0.005% manganese sulfate (MnSO₄)) and appropriate supplements in liquid broth or 1.8% agar-containing plates were used as described previously-^[1]. Lb. plantarum LBP-K10 were commonly maintained in MRS medium including 1.8% agar. Before growing cells in liquid medium, stock cultures were grown on agar slants and stored at 4 ^oC. Additionally, all antibacterial experiments tested here were performed by using both each single CDP purified from the CF of Lb. plantarum LBP-K10 cultures and the LbCDPS activity-based CDPs synthesized from the combination of AAs. Antibacterial activity against multidrug-resistant bacteria and reference strains was measured every 24 h after seed inoculation and dilution method was used to determine the minimum inhibitory concentration (MIC) of antimicrobial substances-^[31].

Free AA content analysis. The sample preparation for free AA analysis was followed as proposed previously with minor modifications^[32]. The free AA content was determined through an L-8800 high-speed AA analyser (Hitachi, Japan) as described previously^[33]. 0.75 g of lyophilized Lactobacillus CF powder through freeze-dryer was diluted with 10 mL of 3% trichloroacetic acid for 1 h and centrifuged at 10,000 rpm for 15 min. The collected supernatant was filtered with 0.22 μm-cellulose acetate membranes (GE Healthcare, USA). The resulting filtrates were loaded onto an L-8800 high-speed AA analyser (Hitachi, Japan). The chromatographic separation was achieved on an ion exchange column #2622SC PF and the mobile phases used were PF1, PF2, PF3, PF4, PF-RG, R-3, C-1, ninhydrin solution and buffer solution (Wako, Japan). The standard AA solutions, type ANII and type B, were obtained from Wako (Wako-shi, Japan).

Activity assay. The LbCDPS activity was basically employed by both ninhydrin (2,2-Dihydroxyindane-1,3-dione)-Schiff staining technique^[34] through activity staining using native gels and spectrophotometrically at 340 nm or fluorometrically measured by ninhydrin reaction with AAs and amines, both which can cause to form chromophores, particularly using a method for activity staining after native PAGE with modifications ^[35](Shaykh et al. 1983, Friedman 2004). The 50 ml LbCDPS assay mixture for enzyme reactions consisted of 1.0 μg/ml of LbCDPS, 1 mM ATP, and 1 mM each of the AAs, which were the combination of two AAs link together to form CDPs, in 50 mM Tris-HCl, pH 8.0, respectively. Additionally, 0.2% dipotasium phosphate (K₂HPO₄), 0.01% magnesium sulfate (MgSO₄), 0.005% manganese sulfate (MnSO₄) were also added to be minor compounds similar to the enzymatic conditions of Lactobacillus cultures. The LbCDPS activity was fundamentally observed by the native PAGE with substrate-containing assay mixtures, which made it possible even to gel them completely, and thus to obtain stable protein, AA, and amine staining with ninhydrin. Upon experimental principles as described, both cell crude extract and fractions of eluents driven by several types of column chromatographies, the native PAGE was performed and the resulting gels were briefly equilibrated with 50 mM Tris-HCl, pH 8.0 after rinsing in ice-cold sterilized distilled water twice for 10 min, respectively. Then, the gels were soaked with the reactant containing various types of AA pairs equilibrated with 50 mM Tris-HCl, pH 8.0. The resulting gels were stained with 25 mM ninhydrin and further incubated for 2 h. The archromatic bands were observed on native gels. Because the AAs near LbCDPS were dehydrated, condensated and cyclized into CDPs, which lost the amino group, the achromatic bands could be shown as a blue or brown color. In contrast, other parts of the ninhydrin-based stained gels where did not show LbCDPS.

LbCDPS purification from Lb. plantarum LBP-K10. To obtain cellular proteins from cell crude extracts, the three-day cultured Lb. plantarum LBP K-10 was collected by centrifugation at 10,000 ｘ g for 20 min, and the cell pellet was washed twice with ice-cold sterilized distilled water and resuspended in 50 mM Tris-HCl, pH 8.0, containing 1 mM phenylmethanesulfonylfluoride. The cell wall homogenized with sonication using a W-225R Sonicator (Heat Systems-ultrasonics, Inc) with an appropriate amount of lysozyme (Sigma) for 30 s each at a setting of 2.5 for 3 h on ice after repeating 10 times of the thawing and refreezing cell pellet. Unbroken cells and cell debris were removed by centrifugation at 10,000 ｘ g for 30 min. The resulting supernatant containing cytosolic fraction was filtered with 0.22 μm-cellulose acetate membranes used to purify LbCDPS. To isolate LbCDPS by catalysis of the production of CDPs, 150 g (wet mass) of Lb. plantarum LBP-K10 cells in each experiment was prepared as described above. 30% ammonium sulfate ((NH₄)-₂SO₄) was added and stirred in the cold more than 90 min. The precipitate was collected by centrifugation and approximately 550 mg of proteins was sequentially used for column chromatography (GE Healthcare) as follows: anion exchange chromatography with Mono Q ^TM 5/50 GL (MQ-AEC), hydrophobic interaction chromatography with Phenyl Superose ^TM 6 10/300 GL(PS-HIC), Mono Q HR5/5 column (MQ-AEC), gel filtration chromatography with Superdex 200 10/300 GL. All of the chromatographic separation was performed using 50 mM Tris-HCl, pH 8.0, as follows. MQ-AEC: samples were eluted with a linear gradient from 0 to 0.5 M NaCl (0.2 ml/min); PS-HIC: samples were eluted with a linear gradient from 1.5 to 0 M (NH₄)₂SO₄ (0.5 ml/min). In every experimental procedure, the resulting protein fractions bearing a significant enzyme activity were immediately visualized by 12% native-PAGEs using the proposed method as described above. Each fraction was utilized to react with appropriate amount of AAs, ATP, and minor components as described earlier. All solutions were prepared with the deionized and filtrated water (resistivity 18.2-MΩ cm at 298 K Milli-Q, Millipore).

Escherichia coli expression constructs and overproduction of LbCDPS in Eschericia coli. To express LbCDPS in E. coli, a DNA fragment derived from the nucleotide sequence, which corresponds to the deduced the entire AA sequence, was amplified by polymerase chain reaction (PCR) using Lb. plantarum LBP K-10 genomic DNA as a template. A PCR amplification was performed by using the primers as follows: 5'-CATATGGCAAAATTAGTATTGATTCGTCACGGT-3' (NdeI site, forward) and 5'-GGATCCTTATTTGCCTAACTTTTCCTTACCAAG-3 (BamHI site, reverse) (NCBI accession number: CBX85836). PCR amplification was performed with a Biometra thermocycler (Tampa, USA) for 30 cycles using amplification mixture contained 100 ng of genomic DNA, 0.5 μM of primer DNA, 0.2 mM dNTPs, 10ｘ Ex Taq buffer solution, and Taq polymerase (TaKaRa Bio Inc., Japan) 0.025 U/μl. PCR conditions were setted at 30 s of denaturing at 95 degree Celsius, 30 s of annealing at 55 degree Celsius, and 1.5 min of extension at 72degree Celsius. From a 693-base pair nucleotide sequence, primers are amplified by PCR. Sequences of the plasmid constructs and the detailed construction schemes are available upon request, and all of the constructs are sequence-verified. We constructed a plasmid containing an LbCDPS gene that can be excised with NdeI/BamHI. The PCR-amplified fragments corresponding to 0.69 kb NdeI/BamHI-digested fragment was inserted into the pGEM T EASY vector, yielding pCDPS, and was digested with NdeI/BamHI, and the resultant fragment was consecutively cloned into the pET3a (+) vector using TaKaRa's Mix ligation kit (TaKaRa Bio Inc.) the ratio of insert to vector was 5:1 (mole). After the ligation at 4degree Celsius for overnight, the E. coli BL21 (DE3) was used to transform the pET3a (+) vector containing LbCDPS.

Purification of LbCDPS in E. coli. The pET3a (+)-LbCDPS/BL21 (DE3) was incubated in LB, pH 7.4, with 50 μg/mL of ampicillin, while the control group BL21 (DE3) was incubated in LB at 37 degree Celsius to obtain the seed cultures. 1% (v/v) pET3a-CDPS/BL21 (DE3) culture was introduced to the LB added with 50 μg/mL of ampicillin and the reference group BL21 (DE3) in LB, and incubated at 37 degree Celsius approximately for 2-2.5 h until the optical density reached a value of 0.4-0.5 at 600 nm. At the optical density points, isopropyl-beta-d-thiogalactopyranoside (IPTG 1, Sigma, USA) was added at a final concentration of 1 mM and further cultured to induce the LbCDPS protein synthesis for 6 h at 30 degree Celsius. The transformed pET3a-LbCDPS in BL21 (DE3) was cultured, harvested, centrifuged at 8,000 x g at 4 degree Celsius for 20 min. The LbCDPS-overproducing cells were dissolved in lysis buffer, which consists of 50 mM Tris-HCl, pH 8.0, at a ratio of 1:9 (w/v) with 1 mM PMSF, and sonicated on ice for 15 min. After the cell debris was discarded by centrifugation at 10,000 x g at 4 degree Celsius for 20 min, the filtrates was collected in a fresh tube and was used as an extract solution[36]. The quantity of protein was determined by the Bradford method[37] using the Bio-Rad protein assay reagent (Bio-Rad, USA). The resulting filtrates was loaded onto Phenyl sepharose CL-4B equilibrated with 1 M (NH₄)-₂SO₄. After the column was washed with the same buffer, the bound protein was eluted with a reverse linear gradient ranging from 1 to 0 mM (NH₄)-₂SO₄in 50 mM Tris-HCl buffer at a flow rate of 3 mL/min. The resulting fractions were tested by activity staining using ninhydrin as described followed by combining and concentrating by ultrafiltration using PM10 membrane (Amicon). Subsequently, the concentrated enzyme solution was desalted using superdex75 through FPLC system with 50 mM Tris-HCl, pH 8.0. The LbCDPS was further loaded on a DEAE-Sepharose CL-6B equilibrated with 50 mM Tris-HCl, pH 8.0. The enzyme was eluted with a linear gradient of 0 to 1 M NaCl in the same buffer. The purified enzyme was stored at 4 degree Celsius.

Molecular mass determination. The molecular mass of the purified LbCDPS was calculated by using protein standards calitrated with alcochol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), cytochrome c (12.4 kDa), and aprotinin (6.5 kDa), respectively, through gel filteraion chromatography on a superose 12 GL300 (HiLoad 16/60, 1.6 x 10 cm, GE Pharmacia).

HPLC. Active or CDP-containing fractions exerted by in vitro enzyme-catalyzed reactions were lyophilized and extracted with MC. After the elimination of methylene chloride by evaporation, the resultant was dissolved in distilled water and filtrated with a 0.22 μm-cellulose acetate membrane (Milli-Q, Millipore). Filtered samples of both the CDPs produced by LbCDPS or CDPs purified from Lactobacillus cultures were separated using a semi-preparative Agilent 1200 series HPLC system (Agilent, USA) with a semi-preparative Hypersil octadecyl silica C18 reverse-phase column (9.4 x 250 mm, Agilent, USA) and the ChemStation HPLC software as proposed previously[2]. The mobile phase was 67.0% water, 3.0% acetonitrile and 30.0% methanol for 45 min using UV absorbance with wavelengths at 210, 260 and 280 nm, for observing the corresponding chromatograms, respectively. Each fraction was collected and concentrated by lyophilization to obtain a powder.

2D-LC MS/MS analysis. The gel spots visualized by Coomassie Blue R-250 staining were excised, followed by destaining and reduction/alkylation with 10 mM and 50 mM iodoacetamide[38]. Following the dehydration of gel slices with acetonitrile, two volumes of freshly prepared trypsin (20 ng/mL in 25 mM NH₄HCO₃) were added and incubated at 37 degree Celsius for 18 h. Peptides were extracted with 10 mL of 0.1% trifluoroacetic acid/50% acetonitrile solution in two consecutive steps, dried through a vacuum centrifuge, and re-dissolved in 10 mL of 0.1% trifluoroacetic acid. Mass analysis and protein identification were carried out on a ProteomeX LTQ 2D-LC-MS/MS spectrometer (Sinco, Inc.). The AA sequences were aligned using Clustal 2.1.

Crystallization and structure determination by X-ray crystallography. The LbCDPS was crystallized by the hanging-drop vapor-diffusion method using 24-well costa plates at 295 K. The initial crystallization screening of LbCDPS was performed by the micro-batch method with Crystal ScreenI/II, Index, SaltRx. Natrix, Cryo, MembFac kits (Hampton Research, USA) and Wizard I, II screening soultions (Emerald BioSystems). Droplets composed of 1uL protein solution and equal volume of crystallization screening solution were loaded under layer of 1:1 mixture of silicon oil and paraffin oil in 72-well HLA plates (Nunc) and equilibrated at 295 K. Serval bundles of rod-shaped crystals were produced under the condition containing 100 mM HEPES, pH 7.5, 50 mM magnesium chloride and 30% polyethyleneglycol 550 (PEG 550) in three days. Then the crystallization condition was optimized with hanging-drop vapor diffusion method using 24-well well culture plate by adjusting from 30 % PEG 550 to 16 % PEG550. Finally, single crystal was made in droplets containing 1 μL of protein sample (20 mg/ml) and an equal volume of precipitant solution containing 50 mM MgCl₂, 100 mM HEPES, pH 7.5, and 17% PEG550. The droplets were equilibrated against 400 μL of the same precipitant solution at 295 K and crystals grew to maximum size in 7 days. The selenomethionyl LbCDPS was crystallized by the same procedures as the crystallization of native LbCDPS with a precipitant solution containing 50 mM MgCl₂, 100 mM HEPES, pH 7.5, and 17% PEG 550. Native LbCDPS single crystals were soaked with 5 mM substrates (ATP, L-proline and phenylalaine) in crystallization condition to solve the cofactor binding complex structure. The selenomethionyl LbCDPS was crystallized according to the same procedures as the crystallization of native LbCDPS with a precipitant solution containing 50 mM MgCl₂, 100 mM HEPES, pH 7.5, and 17% PEG 550. Native CDPS single crystals are soaked with 5 mM substrates (ATP, L-proline and phenylalaine) in crystallization condition to solve the cofactor binding complex structure.

Single crystals of LbCDPS were mounted using a nylon loop (50 μm Mounted CryoLoop, Hampton Research) for data collection and were cooled to 100 K using a Cryostream cooler (Oxford Cryosystems) without additional cryoprotectant. A 1.89 angstrom resolution native data set was collected at a wavelength of 1.1 angstrom using an ADSC Quantum 210 CCD on beamline 6C at Pohang Light Source (PLS), Republic of Korea. A total of 360 frames of 1 degree oscilation were collected with the crystal-to-detector distance set to 150 mm. A 2.30 angstrom resolution SAD data set was collected at a wavelength of 20.8 angstrom using an ADSC Quantum 270 CCD on beamline 7A of PLS, Republic of Korea. A total of 999 frames of 1 degree oscillation were collected with the crystal-to-detector distance set to 120 mm.

Antimicrobial assays of CDPs produced by recombinant CDPS

Antimicrobial activity was investigated by using disk diffusion assay^[39]. Culture supernatant of recombinant E. coli were used and spotted on 6 mm paper disk (Toyo Roshi kaisha, 1td). Multidrug-resistant bacteria strains, used as indicator strains, was inoculated onto 1 % of the suitable molten agar. The spotted disk paper was putted on the agar plate and incubated 24 h at suitable temperature. Antimicrobial activity was estimated by inhibition zone diameter (mm).

AA utilization conveyed by Lactobacillus CFs is seemingly altered by bacterial cell growth accompanying CDP biosynthesis

Lactobacillus strains are usaually confronted with various metabolic events, including nutritional, environmental and oxidative stresses, during fermentation prominently due to both their nutritionally fastidious culture requirements, which were elaborated or altered for their limited biosynthetic capabilities^[40]. Thus, such nutrients needed for cell growth are commonly reconstituted with the host in vivo or with the supplementation of complex subtances, including peptone and yeast extract to culture medium in vitro^[41]. In the case of the AAs, they can contribute to Lactobacillus growth, fermentative activity and lactic acid production^[42,43]. The AA profile during Lactobacillus growth was preferentially examined in relying upon this reason, whether the AAs consumed by cells might reflect CDP production linked to AA content change of CFs. An precedented proline decrease through AA analyzer was addressed peculiarly in CFs approximately ranging from 60 to 72 h, which were highlighted by maximal CDP production periods^[4] whereas most of other free AAs, including the essential or non-eesential proteogenic AAs, were significantly elevated between 60 h and 72 h following the maintenance of steady state contents during cell growth (Fig. 1).

This free AA profile coincided with the result that Lb. reuteri growth was contingent on the explicit the presence or absence of exogenous glutathione in MRS medium ^[44]. Additionally, these established AA values of Lactobacillus CFs can be fundamental data for undertaking further experiments regarding the presence of the proline-based CDP^[4] biosynthesizing-CDPS.

The coupled assay by using both the activity staining and consecutive chromatographic separation is promised by ninhydrin reaction on native gels.

A schematic hierarchy of ninhydrin reactions bearing colored chromophores on native gels with when using ninhydrin-positive compounds to characterize biomolecules, including AAs, peptides, and proteins, was introduced for a new activity staining method based on the function of basicities and steric connditions of α-amino groups (Fig. 2).

An activity stain for the detection of LbCDPS in cell crude extracts or eluents from column chromatography was promised to follow the incubation of the gel with substrates, including several L-AAs, in buffer conditions of varying different types of pHs in the presence or absence of ATP and minor compounds for the enzymatic activity as like in the Lactobacillus cultures (Fig. 2). Additionally, stoichiometrically ninhydrin-derived chromophores, including Ruhemann's purple (2-(1,3-dioxoindan-2-yl) iminoindane-1,3-dione) and hydrindantin (2,2'-dihydroxy-1H,1' H-2,2'-biindene-1,1',3,3'(2H,2' H)-tetrone) during the ninhydrin test for amines, following treating 2 mM of ninhydrin solution were commonly assumed to be a key reaction inermediates or end-products (Fig. 2).

CDPS activity and its verification

We examined the existence of CDPS by its synthesis ability in vivo. First, the crude extract from Lb. plantarum LBP-K10 was eluted with 300 mM NaCl through HPLC system and collected every hour, totally 11 fractions were obtained. Then, linear gradient of NaCl were performed for each fraction. After native SDS-PAGE loaded with each fraction was incubated with proline/Leucine or proline /phenylalanine with or without ATP, the gel was stained with ninhydrin. As our expectation, achromatic band could be observed on the blue stained gel in the ATP supplanted conditions (Fig. 3).We then tried to verify the generation of cyclic dipeptides. We applied all fraction of extract from Lb. plantarum LBP-K10 mix with amino acids and ATP. HPLC analysis confirmed the synthesis of cis-cyclo(L-Pro-L-Leu) and cis-cyclo(L-Pro-L-Phe). These results indicated the existence of CDPS in Lb. plantarum LBP-K10 conditioned medium as well as provided the clue that actively of CDPS was ATP dependent (Fig. 4C).

2D-LC-MS/MS of Lb. plantarum LBP-K10 CDPS

According the results mentioned above, we confirmed the synthesis active native activity staining of fraction 5-11, which present a strong activity in figure 3. The synthesis capability was increased and reach the strongest activity in fraction 9. Afterwards, the activity was gradually decrease (Fig. 3). Then, proteins fraction 5-11 were separated on SDS page for examine the candidate protein worked as CDPS. We proposed the synthesis activities should presented the concentration of CDPS proteins. Interestingly, the band indicated by red arrows present the same trends with the synthesis activity indicated as the band shown in A (Fig. 5).The red arrow indicated band (fraction 7-9) were cut and run on SDS-PAGE after stained with laemmli-staining method. Band of

fraction

7 and 9 could be observed (Fig. 6B). Mass analysis and protein identification were carried out with the purified CDPS from fraction 9 band represented in Figure 5A on ProteomeX LTQ spectrometer (Sinco, Inc). The purified CDPS like protein, could be identified as a polypeptide consisted of 230 amino acids with a calculated molecular mass 26.1 kDa (Table 1). A 690 bp of sequence from NCBI genome database could be obtained (Figs. 6 and 7). However, only an accession number as 11821150 was assigned. We then named this CDPS-like protein as cyclic dipeptide synthetase (CDPS) in this study.

Table 1 Results from 2D LC-MS analysis of purified CDPS

Activity of recombinant CDPS

CDPS overexpression in pET3a (+) system in E. coli was firstly confirmed on SDS page stained with coomassie blue (Fig. 8). After the native gel loaded with CDPS was incubated for 4 h with several kinds of amino acids in the presence of ATP, the achromatic band was observed by ninhydrin staining (Fig. 8B).To confirm the overexpression of CDPS could result the generation of cyclic dipeptides, HPLC was applied for analyzing the total cyclic dipeptides produced by this gene transfected bacteria. According to the broad spectra of HPLC result, the expression of three L-Proline-based cyclic dipeptides, cis-cyclo (L-Val-L-Pro), cis-cyclo(L-Leu-L-Pro),cis-cyclo(L-Phe-L-Pro), was increased significantly (Fig. 9). After transfected with CDPS plasmid, the content of these fractions were remarkably increased up to 2.05-, 2.33- and 2.56-fold (Figs. 9 and 10). To confirm the E. coli transfected with pET3a-CDPS plasmid are capable of producing functional CDPS, disk diffusion assay was performed following the same method employed for Lb. plantrum LBP-K10 as mentioned above. As shown in Figure 11, CDPS transfected E. coli culture filtrates displayed antibacterial activity against gram positive and gram negative bacteria whereas control group did not. These data strongly suggested that CDPS transfection in E. coli results the functional target CDPS expression.

Detection of cyclic dipeptides produced by purified enzyme

To confirm the recombined over expressed CDPS was functional, cyclic dipeptide synthesis activity was tested in vitro or in vivo. CDPS were purified with superdex 75 chromatogramn and collected every hour. Each fraction run on SDS-page the over expressed 25 kDa band were confirmed. Consistant with our previous results, F9 fraction contains the most enriched CDPs (Fig. 12A). Synthesis function analysis followed by Ninhydrin staining of purified CDPS were performed as described as before. Through the enzyme reaction and consecutive activity staining, the active bands where CDPS located showed transparent grey color while the background generally present blue color (Fig. 12B). The presence of cyclic dipeptides synthesized by purified enzyme in vitro was further confirmed by HPLC. In HPLC analysis conditions, the impurity or solvent peaks were detected within ten min. Moreover, the retention time of cyclic dipeptides stands were always shown up longer than 10 min. Therefore, the peaks which were detected by HPLC chromatogram with a retention time more than 10 min were regarded as cyclic dipeptides or other byproducts through the enzyme reactions. These results indicate that the predicted cyclic dipeptide fractionation driven by the enzyme reactions could be performed. Because the optimal temperature for bacteria biological activities is 37 degree Celsiusand the activity of CDPS was ATP-dependent, pH valve of the reaction solution might be the most important factor to be concerned in the reaction conditions. Therefore, several pH conditions were intensively examined using each amino acid combinations including L-Pproline and L-Lysine, L-Proline and L-Phenylalanine, L-Proline and L-Serine. The amount of the cyclic dipeptides produced by enzyme reactions in the in vitro condition was measured by HPLC chromatograms. Interestingly, the most efficient pH values for enzyme activity were delicately different by the amino acids combination-dependent manner. Therefore, CDPS activity was significantly higher in pH 8 than in other pH. pH 8 was the most optimal reaction pH condition for the production of cyclo(Pro-Lys) and cyclo(Pro-Val) with the highest peak of cyclic dipeptide presented on the HPLC spectra (Fig. 13a, Fig. 13d) contrast to the low activity at pH 6 for producing cyclo(Pro-Ser) (Fig. 13c). When comparing to reference experiments, HPLC chromatograms explained that CDPS could contribute to the generation each cyclic dipeptide such as Pro-Lys, Pro-Phe, Pro-Val, Pro-Ser and Pro-Leu) (Fig. 13).

To confirm the peaks in HPLC represented are cyclic dipeptides, we collected each compound according each peak, and then subjected them for GC/MS analysis using electron ionization and chemical ionization. With the reference we have obtained from the data base and the compound isolated from lactic acid before, we concluded that cyclo(Phe-Phe) and cyclo(Pro-Phe) could be generated in the reaction system including L-Proline and L-Phenylalaline (Table 2), cyclo(Pro-Lys) and cyclo(Lys-Lys) could be found in the reaction system including L-Proline and L-Lysine (Table 3), cyclo(Pro-Ser) in the system including L-Proline and L-Serine (Table 4), cyclo(Pro-Val) in the system including L-Proline and L-Valine. However, we could not exclude the possibility of the existing of other kind of cyclic dipeptides, which could be undetectable due to small amount or inefficiency ionization.

Table 2. Mass analysis of products reacted with L-Proline and L-Phenylalanine

Table 3. Mass analysis using EI and CI by GC-MS of products reacted with L-Proline and L-Lysine

Table 4. Mass analysis using EI and CI by GC-MS of products reacted with L-Proline and L-Valine

Table 5. Mass analysis using EI and CI by GC-MS of products reacted with L-Proline and L-Serine

Table 6. Mass analysis using EI and CI by GC-MS of products reacted with L-Proline and L-Leucine

Molecular mass of CDPS in solution

To confirm the oligomeric state, purified CDPS protein (Fig. 14) was applied on Superose 12 10/300 GL column (GE Healthare) with molecular mass standards. A standard curve was generated by plotting the logarithm of melecular mass of standard proteins against their Kav, where kav= (Ve-Vo)/(Vt-Vo): Ve, elution volume; Vo, void volume; Vt, total ved volume. Kav of CDPS determined by using the same column was compared to the profile of protein standards; predicted molecular mass of CDPS is 99.18 kDa (Kav=0.5), indicating that CDPS exists as tetramer in solution (Fig. 14C).

Structural determination of CDPS

To better understand the structure of CDPs, X-ray analysis was performed. Crystal native data sets and Se-Met substituted were collected at 100 K with area detector Systems Corporation (ADSC) Quantum 210 charge-coupled device area detector system at BL-7A at Pohang Light Source (PLS), South Korea. Diffraction data were processed and scaled with program DENZO and SCALEPACK -[45]. Crystals belongs to the monoclinic space group, c2, with unit-cell parameters a=234.63, b=63.60, c=70.43angstrom, and β=94.17. The solvent content of crystal was 39.3% when the asymmetric unit is assumed to contain one molecule ^[46].

The four selenium sites were located (data not shown), and phase refinement was done by using the programs SOLVE and RESOLVE ^[47,48]. Initial phasing and model building was with Se-Met CDPS MAD data set. Model building was done using the program COOT software [49] and refined with REFMAC5 [50] from the CCP4 program suite (Collaborative Computational Project, 1994). Single crystal of CDPS was made in 50 mM MgCl _2,100 mM HEPES pH 7.5 and 17 % PEG550 (Fig. 15) The native CDPS structure was refined to R_work of 39.3 and R_free of 11.6 in the resolution range of 5.0-2.08 angstrom. The refinement statistics of the native CDPS structure are summarized in Table 7.

Table 7. Data collection and refinement statistics of CDPS

a. The number in parentheses is for the outer shell.

b. R_sym=Σ_hΣ_I/Σ_h,_i-I_h/Σ_hΣ_II_h,iwhere I_h is the mean intensity of the I observations of symmetry related reflections of h.

c. R=Σ/F_o-F_c/ΣF_O, where F_o=F_p, and F_c is the calculated protein structure factor from the atomic model. R_free was calculated with 10% of the reflections.

d. R=Σ/F_o-F_c/ΣF_O, where F_o=F_p, and F_c is the calculated protein structure factor from the atomic model. R_free was calculated with 10% of the reflections.

Overall Structural of Recombined CDPS

According to the ribbon model of the CDPS as shown as Figure 16, CDPS was composed as a tetramer structure with 4 identical subunits. In terms of the secondary structure, the subunit has 3 main layers of alpha/beta/alpha (Fig. 17). It contains a mixed bate sheet of 6 strands with one strand existing as an anti-parallel strand to the rest. Totally 7 alpha helix and 5 3₁₀ helix surround the beta sheet layer. Four subunits polymerized together symmetrically and form a hollow space in the center (Fig. 18). The similar binding site were searched through PDBj database (http://pdbj.org/giraf/). Interestingly the result reveal the sequence of CDPS are comparable with human phosphoglycerate mutase 1 (PGM1), a member of alkaline phosphatase superfamily, with a similarity score of 74 % (Fig. 19). Noticeably, except PGM enzyme of yeast which is a homotetramer of mass 110,000 kDa, PGM enzymes are usually homodimer molecular. Thus, we confirmed CDPS isolated from lactic acid bacterial is a novel member of alkaline phosphatase superfamily.

Discussion

Unwiring the structure and function of CDPS is pivotal for revealing the physiological processing of CDPs synthesis in microorganism as well as inventing in vitro bioreactor for producing functional CDPs. In this study, we identified a kind of new CDPS protein which displayed the capability of synthesis antibiotic CDPs, especially proline based CDPs. When transferring this gene into E. coli, functional CDPS could be obtained. Moreover, with HPLC confirmation, we successfully defined an in vitro reactions system which consisted with CDPS, amino acid, ATP in PBS buffer solutions. We figured out that, in 37 degree Celsius and ATP supplement, CDPS could synthesize the target CDPs. However, compare with the in vivo system, the synthesizing efficacy was quite low. This may indicate that for higher yield of CDPs, other auxiliary molecular may be needed besides more optimal physiochemical conditions should be defined. Moreover, we noticed CDPS especially catalyze the synthesis of CDPs with proline based DZK ring structure. We further analyzed the 3 dimensional structure of CDPS by X-ray crystallographic analysis (data not shown). We are now working for identify the catalytic site in order to reveal the synthesis process in more detail. As a conclusion, it was firstly reported the CDPS like function of an unnamed protein (gi311821850), and successfully development the in vitro CDPs synthesis conditions based on the newly found CDPS protein, CDPS. These studies will contribute to producing CDPs in vitro through biosynthesis pathway and to discover physiology mechanisms about functional CDPs synthesis pathway in microorganism.

References

[1] De Man, J.C., Rogosa, M. and Sharpe, M.E. (1960). A medium for the cultivation of Lactobacilli. J. Appl. Microbiol. 23, 130-135.

[2] Kwak, M.-K., Liu, R., Kwon, J.-O., Kim, M.-K., Kim, A.H. and Kang, S.-O. (2013). Cyclic dipeptides from lactic acid bacteria inhibit proliferation of the influenza A virus. J. Microbiol. 51, 836-843.

[3] Kwak, M.-K., Liu, R., Kim, M.-K., Moon, D., Kim, A.H., Song, S.-H. and Kang, S.-O. (2014). Cyclic dipeptides from lactic acid bacteria inhibit the proliferation of pathogenic fungi. J. Microbiol. 52, 64-70.

[4] Kwak, M.-K., Liu, R., Kim, A.H. and Kang, S.-O. (2016). Antibiotic cyclic dipeptides produced by Lactobacillus plantarum LBP-K10 with activity against multidrug-resistant bacteria, pathogenic fungi and influenza A virus. Front. Microbiol. (Submitted)

[5] Li, J., Wang, W., Xu, S.X., Magarvey, N.A. and McCormick, J.K. (2011). Lactobacillus reuteri-produced cyclic dipeptides quench agr-mediated expression of toxic shock syndrome toxin-1 in staphylococci. Proc. Natl. Acad. Sci. USA 108, 3360-3365.

[6] Amoroso, M.J., Saguir, F.M. and Manca de Nadra, M.C. (1993). Variation of nutritional requirements of Leuconostoc oenos by organic acids. J. Int. Sci. Vigne. Vin. 27, 135-144.

[7] Elli, M., Zink, R., Reniero, R. and Morelli, L. (1999). Growth requirements of Lactobacillus johnsonii in skim and UHT milk. Int. Dairy J. 9, 507-513.

[8] Saguir, F.M. and Manca de Nadra, M.C. (2007). Improvement of a chemically defined medium for the sustained growth of Lactobacillus plantarum: nutritional requirements. Curr. Microbiol. 54, 414-418.

[9] Fernadez, M. and Zuniga, M. (2006). Amino acid catabolic pathways of lactic acid bacteria. Crit. Rev. Microbiol. 32, 155-183.

[10] Rouse, S. and van Sinderen, D. (2008). Bioprotective potential of lactic acid bacteria in malting and brewing. J. Food Prot. 71, 1724-1733.

[11] Prasad, C. (1995). Bioactive cyclic dipeptides. Peptides 16, 151-164.

[12] Huang, R., Zhou, X., Xu, T., Yang, X. and Liu, Y. (2010). Diketopiperazines from marine organisms. Chem. Biodivers. 7, 2809-2829.

[13] Bellezza, I., Peirce, M.J. and Minelli, A. (2014). Cyclic dipeptides: from bugs to brain. Trends Mol. Med. 20, 551-558.

[14] Waring, P. and Beaver, J. (1996). Gliotoxin and related epipolythiodioxopiperazines. Gen. Pharmacol. 27, 1311-1316.

[15] Borthwick, A.D. (2012). 2,5-Diketopiperazines: synthesis, reactions, medicinal chemistry, and bioactive natural products. Chem. Rev. 112, 3641-3716.

[16] Ortiz-Castro, R., Diaz-Perez, C., Martinez-Trujillo, M., del Rio, R.E., Campos-Garcia, J. and Lopez-Bucio, J. (2011). Transkingdom signaling based on bacterial cyclodipeptides with auxin activity in plants. Proc. Natl. Acad. Sci. USA 108, 7253-7258.

[17] Degrassi, G., Aguilar, C., Bosco, M., Zahariev, S., Pongor, S. and Venturi, V. (2002). Plant growth-promoting Pseudomonas putida WCS358 produces and secretes four cyclic dipeptides: Cross-talk with quorum sensing bacterial sensors. Curr. Microbiol. 45, 250-254.

[18] Holden, M.T. et al. (1999). Quorum-sensing cross talk: Isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other gram-negative bacteria. Mol. Microbiol. 33, 1254-1266.

[19] Healy, F.G., Wach, M., Krasnoff, S.B., Gibson, D.M. and Loria, R. (2000). The txtAB genes of the plant pathogen Streptomyces acidiscabies encode a peptide synthetase required for phytotoxin thaxtomin A production and pathogenicity. Mol. Microbiol. 38, 794-804.

[20] Schultz, A.W. et al. (2008). Biosynthesis and structures of cyclomarins and cyclomarazines, prenylated cyclic peptides of marine actinobacterial origin. J. Am. Chem. Soc. 130, 4507-4516.

[21] Stachelhaus, T., Mootz, H.D., Bergendahl, V. and Marahiel, M.A. (1998). Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain. J. Biol. Chem. 273, 22773-22781.

[22] Gondry, M., Lautru, S., Fusai, G., Meunier, G., Menez, A. and Genet, R. (2001). Cycli^c dipeptide oxidase from Streptomyces noursei. Isolation, purification and partial characterization of a novel, amino acyl alpha,beta-dehydrogenase. Eur. J. Biochem. 268, 1712-1721.

[23] Gondry, M. et al. (2009). Cyclodipeptide synthases are a family of tRNA-dependent peptide bond-forming enzymes. Nat. Chem. Biol. 5, 414-420.

[24] Sauguet, L. et al. (2011). Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis. Nucleic Acids Res. 39, 4475-4489.

[25] Giessen, T.W., von Tesmar, A.M. and Marahiel, M.A. (2013). A tRNA-dependent two-enzyme pathway for the generation of singly and doubly methylated ditryptophan 2,5-diketopiperazines. Biochemistry 52, 4274-4283.

[26] Giessen, T.W., von Tesmar, A.M. and Marahiel, M.A. (2013). Insights into the generation of structural diversity in a tRNA-dependent pathway for highly modified bioactive cyclic dipeptides. Chem. Biol. 20, 828-838.

[27] Belin, P., Moutiez, M., Lautru, S., Seguin, J., Pernodet, J.L. and Gondry, M. (2012). The nonribosomal synthesis of diketopiperazines in tRNA-dependent cyclodipeptide synthase pathways. Nat. Prod. Rep. 29, 961-979.

[28] Bonnefond, L., Arai, T., Sakaguchi, Y., Suzuki, T., Ishitani, R. and Nureki, O. (2011). Structural basis for nonribosomal peptide synthesis by an aminoacyl-tRNA synthetase paralog. Proc. Natl. Acad. Sci. USA 108, 3912-3917.

[29] Cryle, M.J., Bell, S.G. and Schlichting, I. (2010). Structural and biochemical characterization of the cytochrome P450 CypX (CYP134A1) from Bacillus subtilis: A cyclo-L-leucyl-L-leucyl dipeptide oxidase. Biochemistry 49, 7282-7296.

[30] Tang, M.R., Sternberg, D., Behr, R.K., Sloma, A. and Berka, R.M. (2006). Use of transcriptional profiling and bioinformatics to solve production problems. Eliminating red pigment production in a Bacillus subtilis strain producing hyaluornic acid. Ind. Biotechnol. 2, 66-74.

[31] Panagou, E.Z., Tassou, C.C., Saravanos, E.K. and Nychas, G.J. (2007). Application of neural networks to simulate the growth profile of lactic acid bacteria in green olive fermentation. J. Food Prot. 70, 1909-1916.

[32] Zhang, Y.C., Zhang, L.W., Tuo, Y.F., Guo, C.F., Yi, H.X., Li, J.Y., Han, X. and Du, M. (2010). Inhibition of Shigella sonnei adherence to HT-29 cells by lactobacilli from Chinese fermented food and preliminary characterization of S-layer protein involvement. Res. Microbiol. 161, 667-672.

[33] Kim, M.-Y. et al. (2009). Comparison of free amino acid, carbohydrates concentrations in Korean edible and medicinal mushrooms. Food Chem. 113, 386-393.

[34] van Dalen, J.P. and van Duijn, P. (1971). Quantitative aspects of the ninhydrin Schiff reaction on amino cellulose films and tissue sections. Histochemie 26, 180-189.

[35] Friedman, M. (2004). Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences. J. Agric. Food Chem. 52, 385-406.

[36] Cao, W., Zhou, Y., Ma, Y., Luo, Q. and Wei, D. (2005). Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli. Protein Expr. Purif. 40, 404-410.

[37] Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

[38] Shevchenko, A. et al. (1996). A strategy for identifying gel-separated proteins in sequence databases by MS alone. Biochem. Soc. Trans. 24, 893-896.

[39] Felten, A., Grandry, B., Lagrange, P.H. and Casin, I. (2002). Evaluation of three techniques for detection of low-level methicillin-resistant Staphylococcus aureus (MRSA): a disk diffusion method with cefoxitin and moxalactam, the Vitek 2 system, and the MRSA-screen latex agglutination test. Journal of clinical microbiology 40, 2766-2771.

[40] Reiter, R. and Oram, J.D. (1962). Nutritional studies on cheese starters. 1. Vitamin and amino acid requirements of single strain starters. J. Dairy Res. 29, 63-68.

[41] Chamba, J.F., Duong, C., Fazel, A. and Prost, F. (1994). in Bacteries lactiques-Aspects Fondamentaux. Vol. 1 (De Roisart, H., and Luquet, F. M., eds.), Lavoisier, Paris.

[42] Konova, N.J., Stepanova, O.N. and Akimova, A.A. (1986). DeriVdteS of amino acids as intensifiers in the doughmaking process. Promyshlennost 2, 32.

[43] Desmazeaud, M.J. and Hermier, J.H. (1972). Isolation and amino acid composition of casein peptides stimulating growth if Streptococcus thermophilus. Eur. J. Biochem. 28, 190-198.

[44] Lee, K., Kim, H.J., Rho, B.S., Kang, S.K. and Choi, Y.J. (2011). Effect of glutathione on growth of the probiotic bacterium Lactobacillus reuteri. Biochemistry (Mosc) 76, 423-426.

[45] Otwinowski, Z. and Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Macromolecular Crystallography, Part A. 1997; 276: 307-326ed.^eds)

[46] Matthews, B.W. (1968). Solvent content of protein crystals. Journal of molecular biology 33, 491-497.

[47] Terwilliger, T.C. (1999). Reciprocal-space solvent flattening. Acta Crystallographica Section D: Biological Crystallography 55, 1863-1871.

[48] Terwilliger, T.C. and Berendzen, J. (1999). Discrimination of solvent from protein regions in native Fouriers as a means of evaluating heavy-atom solutions in the MIR and MAD methods. Acta Crystallographica Section D: Biological Crystallography 55, 501-505.

[49] Emsley, P., Lohkamp, B., Scott, W.G. and Cowtan, K. (2010). Features and development of Coot. Acta Crystallographica Section D: Biological Crystallography 66, 486-501.

[50] Murshudov, G.N., Vagin, A.A. and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallographica Section D: Biological Crystallography 53, 240-255.

Claims

Cyclic dipeptidase of SEQ. No: 1.
A gene encoding the cyclic dipeptidase according to claim 1.
The gene according to claim 2, wherein the said gene is a gene comprising the nucleotide sequence of SEQ. No: 2.
A recombinant vector comprising the gene according to any one of claims 2-3.
A host cell transformed with the recombinant vector according claim 4.
The host cell according to claim 5, wherein the host cell is a prokaryotic cell.
A method of preparation of cyclic dipeptide comprising the step of culturing the host cell according to claim 5.
A method of preparation of cyclic dipeptide by using cyclic dipeptidase according to claim 1.
The method according to claim 8, wherein the cyclic dipeptide is one or more of cyclic dipeptides selected from the groups of cis-cyclo (L-Val-L-Pro), cis-cyclo (L-Leu-L-Pro) and cis-cyclo (L-Phe-L-Pro).
A food comprising the cyclic dipeptide according to claim 8.
The food according to claim 10, the food is one that the host cell which produce cyclic dipeptide is removed.