Polynucleotide for recombinant expression of sucrose isomerase

Abstract

The present invention is directed towards genetic modification of native gene encoding for sucrose isomerase and isomaltulose synthase to substantially increase the expression level of these enzymes and use of said enzymes in a process to produce rare disaccharides such as isomaltulose and trehalulose. Also disclosed in the present invention is expression constructs comprising the modified genes and a host cells to express the same.

Claims

1. A modified polynucleotide encoding a sucrose isomerase, wherein the modified polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1.

2. An expression construct comprising the modified polynucleotide according to claim 1.

3. The expression construct according to claim 2, wherein the expression construct further comprises a T7 promoter and the modified polynucleotide is operably linked to the T7 promoter.

4. An isolated host cell comprising the expression construct of claim 2.

5. The host cell according to claim 4, wherein the host cell is a prokaryotic host cell.

6. A process for production of sucrose isomerase, said process comprising the steps of: 1. culturing host cell of claim 4 in a suitable medium in the presence of IPTG or lactose for 2-3 hours to produce the sucrose isomerase, 2. isolating the sucrose isomerase from the host cell, and 3. purifying the sucrose isomerase using chromatographic techniques.

7. A process for the production of trehalulose from sucrose, said process comprising the steps of: 1. culturing the host cell of claim 4 in a suitable medium in the presence of IPTG or lactose for 2-3 hours to produce the sucrose isomerase, 2. isolating the sucrose isomerase from the host cell, and purifying the sucrose isomerase using chromatographic techniques, 3. immobilizing the purified sucrose isomerase in a suitable matrix, and 4. contacting sucrose with the immobilized sucrose isomerase for a period in the range of 4 to 14 hours to produce trehalulose.

8. The process according to claim 7, wherein the sucrose is contacted with the immobilized sucrose isomerase for a period in the range of 4 to 6 hours to produce trehalulose.

9. The process according to claim 7, wherein the sucrose is contacted with the immobilized sucrose isomerase for a period in the range of 8 to 14 hours.

10. The process according to claim 7, wherein 90% of total sugar produced is trehalulose.

11. The process according to claim 7, wherein the suitable medium is a defined medium.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Schematic view of a gene construct generated for expression of sucrose isomerase in E. coli

(2) A: Sucrose isomerase (SI) was cloned into pET11a using NdeI and BamHI sites. Sucrose isomerase (SI) gene is flanked by BglII, XbaI and NdeI at 5end, and BamHI at 3end. During cloning procedure NheI site was removed. The properties of plasmid are: T7 promoter, T7 terminator and Ampicillin resistance marker.

(3) B: Sucrose isomerase encoding sequence (SI) was cloned into pET23a using BamHI and HindIII sites. Sucrose isomerase (SI) gene is flanked by BglII, XbaI, NdeI, NheI and BamHI at 5end, and HindIII, NotI and XhoI at 3end. During cloning procedure EcoRI, SacI and SalI sites were removed. The properties of plasmid are: T7 promoter, T7 terminator, Epitope tag: 6HIS and Ampicillin resistance marker.

(4) FIG. 2: Schematic view of a gene construct generated for expression of isomaltulose synthase in E. coli

(5) A: Isomaltulose synthase encoding sequence (IS) was cloned into pET11a using NdeI and BamHI sites. Isomaltulose synthase (IS) gene is flanked by BglII, XbaI and NdeI at 5end, and BamHI at 3end. During cloning procedure NheI site was removed. The properties of plasmid are: T7 promoter, T7 terminator and Ampicillin resistance marker.

(6) B: Isomaltulose synthase encoding sequence (IS) was cloned into pET15b using NdeI and BamHI sites. Isomaltulose synthase (IS) gene is flanked by NcoI and NdeI at 5end and HindIII at 3end. During cloning procedure XhoI site was removed. The properties of plasmid are: T7 promoter, T7 terminator, Epitope tag: 6HIS and Ampicillin resistance marker.

(7) FIG. 3: Expression analysis of recombinant sucrose isomerase in E. coli.

(8) A. Control and recombinant E. coli cells [JM109 carrying pET11-SI] were induced for protein expression by addition of 0.5 mM IPTG into media. Cells were lysed and supernatant and pellet fractions were subjected to 10% SDS-PAGE. Control strain: Lane 1 and 2 are uninduced and induced total cell lysate. Recombinant strain: Lane 3 and 4 are uninduced and induced total cell lysate. Cell fractions of recombinant strains: Lane 6 and 7 are uninduced cell supernatant and pellet, Lane 8 and 9 are two hrs induced supernatant and pellet, Lane 10 and 11 are four hrs induced supernatant and pellet. Abbreviations are: M: Protein molecular weight marker and kDa=Kilo Dalton.

(9) B. Identity analysis of recombinant protein by Western blot analysis. Lane1 and 2: Host cell lysate un-induced and induced. Lane 3 and 4: Recombinant strain un-induced and induced. Immuno-detection was carried our using protein specific antibodies.

(10) FIG. 4: Expression analysis of recombinant isomaltulose synthase in E. coli.

(11) A. Control and recombinant E. coli cells [JM109 carrying pET11-IS] were induced for protein expression by addition of 0.5 mM IPTG into media. Cells were lysed and supernatant and pellet fractions were subjected to 10% SDS-PAGE. Control strain: Lane 1 and 2 are uninduced and induced total cell lysate. Recombinant strain: Lane 3 and 4 are uninduced and induced total cell lysate. Cell fractions of recombinant strains: Lane 6 and 7 are uninduced cell supernatant and pellet, Lane 8 and 9 are two hrs induced supernatant and pellet. Abbreviations are: M: Protein molecular weight marker and kDa=Kilo Dalton.

(12) B. Identity analysis of recombinant protein by Western blot analysis. Lane1 and 2: Host cell lysate un-induced and induced. Lane 3 and 4: Recombinant strain un-induced and induced. Immuno-detection was carried our using protein specific antibodies.

(13) FIG. 5: HPLC analysis of recombinant sucrose isomerase activity for substrate to product conversion.

(14) The reaction mixtures were subjected to HPLC analysis to confirm the residual substrate and product formation. The product peaks were confirmed with commercially available sucrose and isomaltulose, trehalulose as substrate and product standards, respectively.

(15) FIG. 6: HPLC analysis of recombinant isomaltulsoe synthase activity for substrate to product conversion.

(16) The reaction mixtures were subject to HPLC analysis to confirm the residual substrate and product formation. The product peaks were confirmed with commercially available Sucrose and isomaltulose as substrate and product standards, respectively.

(17) FIG. 7: Analysis of purified SIase

(18) A. Different fractions and purified protein were separated on 12% SDS-PAGE and stained by coomassie brilliant blue R250. Loading pattern are Lane 1: Marker; Lane 2: Total cell Lysate; Lane 3: Cell lyste before loading in column 1; Lane 4: Column 1 purified SIase; Lane 5: Column 2 purified SIase.

(19) B. Identity analysis of purified recombinant protein by Western blot analysis. Lane 1: Marker; Lane 2: Total cell Lysate; Lane 3: Cell lyste before loading in column 1; Lane 4: Column 1 purified SIase; Lane 5: Column 2 purified SIase. Immuno-detection was carried our using protein specific antibodies.

(20) FIG. 8: Analysis of purified ISase

(21) A. Different fractions and purified protein were separated on 12% SDS-PAGE and stained by coomassie brilliant blue R250. Lane 1: Molecular weight marker, Lane 2 to 6: Fractions 1 to 7, Lane 9: Crude cell lysate.

(22) B. Identity analysis of purified recombinant protein by Western blot analysis. Lane 1: Total cell Lysate, Lane 2 and 3: Supernatant and Cell lyste of recombinant cell lysate, Lane 4: Purified ISase. Immuno-detection was carried our using protein specific antibodies.

(23) FIG. 9: Activity of a sucrose isomerase against reaction pH and reaction temperature. The reaction mixture containing sucrose and purified SIase were incubated at different pH and temperature as indicated. Bioconversion reaction stopped by boiling the reaction mixture at 95 C. HPLC analysis of the reaction mixture confirmed the residual substrate and product formation. The product peaks were confirmed with commercially available sucrose and isomaltulose, trehalulose as substrate and product standards

(24) FIG. 10: Activity of an isomaltulose synthase against reaction pH and reaction temperature. The reaction mixture containing sucrose and purified ISase were incubated at different pH and temperature(s) as indicated. The bioconversion the reaction stopped by boiling the reaction mixture at 95 C. HPLC analysis confirmed the residual substrate and product formation. The product peaks were confirmed with commercially available sucrose and isomaltulose as substrate and product standards

(25) FIG. 11: Sequence alignment analysis of modified gene sequence with native gene sequence encoding for sucrose isomerase.

(26) Modified gene sequence (represented as modified) (SEQ ID NO:1) was subjected to sequence alignment with native gene sequence (represented as native) (SEQ ID NO:3) of Pseudomonas mesoacidophila MX45 using multiple sequence alignment tool (ClustalW2). The nucleotides of modified gene sequence were marked as (.) and homology shared to native sequence was marked as (*). In the modified gene 22% of nucleotides were changed and compared to native gene sequence, in addition 66 nucleotides (22 codons) were removed after ATG start codon which codes for 22 amino acid predicted signal sequence in P. mesoacidophila MX-45.

(27) FIG. 12: Sequence alignment analysis of modified gene sequence with native gene sequence encoding for isomaltulose synthase.

(28) Modified gene sequence (represented as modified) (SEQ ID NO:2) was subjected to sequence alignment with native gene sequence (represented as native) (SEQ ID NO:4) of Pantoea dispersa UQ68J using multiple sequence alignment tool (ClustalW2). The nucleotides of modified gene sequence were marked as (.) and homology shared to native sequence was marked as (*). In the modified gene 20% of nucleotides were changed and compared to native gene sequence, in addition 96 nucleotides (32 codons) were removed after ATG start codon which codes for 22 amino acid predicted signal sequence in Pantoea dispersa UQ68J.

EXAMPLES

(29) The following examples are given by way of illustration, which should not be construed to limit the scope of the invention.

Example 1

(30) Gene Construction

(31) Gene encoding for sucrose isomerase (SI) was modified for enhanced expression in Escherichia coli was synthesized using gene synthesis approach. The modified gene sequence is represented as SEQ ID NO 1. Similar modification was done to increase the expression of isomaltulose synthase in E. coli as represented in SEQ ID NO 2. Both sequence ID NOs 1 and 2 were cloned in to pUC57 using EcoRV restriction enzyme site to generate pUC57-SI and pUC57-IS constructs. Cloned gene sequence was confirmed by sequence analysis.

(32) The DNA fragment encoding for sucrose isomerase was PCR amplified using gene specific primers, and sub cloned into pET11a using NdeI and BamHI restriction enzyme sites to generate pET11-SI (FIG. 1A). In addition the coding region was PCR amplified without stop codon using gene specific primers and sub cloned into E. coli expression vector pET23a (FIG. 1B) using BamHI and HindIII restriction enzymes to generate pET23-SI-HIS construct expressing sucrose isomerase with C-terminal 6Histidine tag. The recombinant plasmid carrying sucrose isomerase gene (pET11-SI and pET23-SI) was confirmed by restriction digestion analysis and followed by DNA sequencing.

(33) The DNA fragment encoding for isomaltulose synthase was PCR amplified using gene specific primers, and sub cloned into pET11a using NdeI and BamHI restriction enzyme sites to generate pET11-IS (FIG. 2A). In addition the coding region was PCR amplified without stop codon using gene specific primers and sub cloned into E. coli expression vector pET15b (FIG. 2B) using NdeI and HindIII restriction enzymes to generate pET15-IS-HIS construct expressing isomaltulose synthase with C-terminal 6Histidine tag. The recombinant plasmid carrying isomaltulose synthase gene (pET11-IS and pET15-IS) was confirmed by restriction digestion analysis and followed by DNA sequencing.

Example 2

(34) Development of Recombinant E. coli with Gene Constructs

(35) For Sucrose Isomerase

(36) Recombinant plasmid DNA (pET11-SI) was transformed into E. coli expression host JM109 by electro transformation method and grown on Luria-Bertani (LB) agar plates containing ampicillin (50 g/ml). Individual colonies were picked and grown on LB liquid or defined media containing ampicillin (75 g/ml) for overnight at 37 C. Overnight culture was re-inoculated into 0.1 OD.sub.600 in LB liquid or defined media without ampicillin and grown up to 0.6 OD.sub.600 and the cells were induced for protein expression by addition of 0.5 mM of IPTG (Isopropyl -D-1-thiogalactopyranoside) and incubated at 37 C. An aliquot of E. coli culture was collected at different time points. The cell lysate was subjected to SDS-PAGE and Western blot analysis to verify the protein expression (FIG. 3).

(37) For Isomaltulose Synthase

(38) Recombinant plasmid DNA (pET11-IS) was transformed into E. coli expression host JM109 by electro transformation method and grown on Luria-Bertani (LB) agar plates containing ampicillin (50 g/ml). Individual colonies were picked and grown on LB liquid or defined media containing ampicillin (75 g/ml) for overnight at 37 C. Overnight culture was re-inoculated into 0.1 OD.sub.600 in LB liquid or defined media without ampicillin and grown up to 0.6 OD.sub.600 and the cells were induced for protein expression by addition of 0.5 mM of IPTG (Isopropyl -D-1-thiogalactopyranoside) and incubated at 37 C. An aliquot of E. coli culture was collected at different time points. The cell lysate was subjected to SDS-PAGE and Western blot analysis to verify the protein expression (FIG. 4).

Example 3

(39) Production of Enzymes, Namely, Sucrose Isomerase and Isomaltulose Synthase

(40) For large scale production of the above enzymes same protocols were followed. The medium used comprises no components of animal origin. The components of the medium were 4.0 g/L di-ammonium hydrogen phosphate, 13.3 g/L potassium dihydrogen phosphate and 1.7 g/L citric acid, 28 g/L glucose, 1.2 g/L MgSo4.7H2O, 45 mg/L Thiamine HCL, 1 g/L CoCl2.6H2O, 6 g/L MnCl2.4H2O, 0.9 g/L CuSo4.5H2O, 1.2 g/L H3BO3, 0.9 g/L NaMoO4, 13.52 g/L Zn (CH3COO), 40 g/L Fe-Citrate and 14.1 g/L EDTA. Liquor ammonia was used as an alkali and nitrogen source. The temperature of the fermentation was maintained at 37 C. at a pH 6.9 and oxygen level was maintained not less than 40%, throughout the fermentation. The fermentation process at 2 L scale yields 30-40 g/l biomass.

Example 4

(41) Purification of Enzymes

(42) After completion of the fermentation the cells were centrifuged at 5000 g for 10 min and resuspend in 20 mM Tris-EDTA (TE) buffer, pH 8.0. The cells were lysed using the cell disruptor at 25 KPsi twice and the resulted cell lysate was clarified by centrifugation. The crude cell-free extract obtained from the supernatant following centrifugation at 27 000 g for 30 min at 4 C. was used for the purification. Clarified crude cell lysate was applied onto a Q-Sepharose column (GE, Healthcare) pre-equilibrated with 20 mM Tris-HCl buffer pH 8.0 and washed with five column volume of same buffer containing 100 mM NaCl. The bound proteins were eluted with NaCl gradient (0.1-0.4 M) in the same buffer, followed by step elution with 0.5 M and 1M NaCl wash in the same buffer. Fractions were collected and tested for sucrose isomerase and isomaltulose synthase activity and purity by SDS-PAGE (FIGS. 7 and 8). The purification yield, activity recovery and fold purification for sucrose isomerase and isomaltulose synthase were shown in Table 1 and Table 2, respectively. Fractions containing the purified protein were dialyzed against 20 mM Tris pH 8.0 for 16 hours at 4 C. and concentrated by ultrafiltration using Centricon YM-10 devices (Millipore) prior to immobilization or stored with 20% glycerol at 20 C.

(43) TABLE-US-00001 TABLE 1 Purification table for Sucrose isomerase Total Total Protein Activity S. Volume Protein Protein IU/ Activity Yield Fold Recovery No Sample (ml) (mg/ml) (mg) mg (IU) (%) Purification (%) 1 Crude 50 45.13 2256.5 46.94 105920 100 1 100 2 Loading 63 30.4 1915.2 55.15 105623 84.8 1.1 99.7 3 Purified 112 1.54 172.35 550 94792 8.99 9.97 89.5

(44) TABLE-US-00002 TABLE 2 Purification table for Isomaltulose synthase Total Specific Total Protein Activity Volume Protein Protein activity activity yield Fold of recovery Steps Sample (ml) (mg/ml) (mg) (IU/mg) (IU) (%) purification (%) Cell lysate Loading 60 7.443 446.57 397.3 177425 100 1 100 Purfication Pooled 85 4.204 357.26 442 157908 80 1.25 89 fractions

Example 5

(45) Immobilization of Enzymes:

(46) The same protocol was followed for SIase and ISase. Partially purified or purified SIase and ISase were dialyzed against 20 mM Tris buffer (pH 8.0) for 16 hours at 4 C. followed by mixing with equal volume of 4% sodium alginate (final concentration of sodium alginate was 2% w/v). The SIase or ISase containing sodium alginate solution was dropped by a surgical needle into chilled 0.2 M CaCl.sub.2 solution with constant stirring. Immobilized beads were kept in CaCl.sub.2 overnight at 4 C., followed by water wash and kept on a blotting paper for drying at 4 C. Protein retention was found to be 85% w/v with 2% w/v of sodium alginate.

Example 6

(47) Production of Rare Disaccharides

(48) Production of Trehalulose by Recombinant SIase

(49) The optimization of process parameters for the production of trehalulose was carried out with varying pH and temperature, which were used for the production of trehalulose. Results are shown in FIG. 9.

(50) Production of trehalulose form sucrose was carried out by using 110 units of immobilized SI enzymes with 10%, 20%, 30% and 40% sucrose solution in 20 mM Sodium Acetate, 10 mM CaCl.sub.2 buffer pH 6.5 at 14 C.

(51) The sugar solution was subjected to cation and anion exchange resins to remove salt and ions present in buffer solutions.

(52) The sugar solution was concentrated using rotary vacuum evaporator system and subsequently passed through a column packed with activated charcoal, in order to remove the color. The purity of the product was analyzed by HPLC (FIG. 5) and ions contaminations were analyzed in ion chromatography (Dionex). Physico-chemical properties and purity of the product were carried out using standard techniques to confirm the safety aspects of produced trehalulose in this process. Bioconversion of trehalulose from sucrose was observed to be 92%.

(53) TABLE-US-00003 TABLE 3 Production of trehalulose by immobilized recombinant sucrose isomerase Substrate Immobilized trehalulose:isomaltulose:sucrose ratio (%) enzyme (U) after 6 hours of bioconversion 10 110 92:08:00 20 110 88:06:06 30 110 83:03:14 40 110 76:02:22 50 110 55:00:45

(54) Production of Isomaltulose by Recombinant ISase

(55) The optimization of process parameters for the production of isomaltulose was carried out with varying pH and temperature, which were used for the production isomaltulose. Results are shown in FIG. 10.

(56) Production of isomaltulose from sucrose was carried out by using 110 units of immobilized ISase with 100 g/l, 200 g/l and 400 g/l sucrose solution was used in 20 mM Tris buffer, 5 mM MnCl.sub.2 (pH 8.0) at 35 C.

(57) The sugar solution was subjected to cation and anion exchange resins to remove salt and ions present in buffer solutions.

(58) The sugar solution was concentrated using rotary vacuum evaporator system and subsequently passed through a column packed with activated charcoal, in order to remove the color. The purity of the product was analyzed by HPLC (FIG. 6) and ions contaminations were analyzed in ion chromatography (Dionex). Physico-chemical properties and purity of the product were carried out using standard techniques to confirm the safety aspects of produced isomaltulose in this process. Bioconversion of isomaltulose form sucrose was observed 83%.

(59) TABLE-US-00004 TABLE 4 Production of isomaltulsoe by immobilized recombinant isomaltalose synthase Substrate Immobilized Isomaltulose:trehalulose:sucrose/glucose/ (%) enzyme (U) fructose ratio after 14 hours of bioconversion 10 140 91.5:1.5:7.0 20 140 90.0:1.2:8.8 30 140 87.0:0.6:12.4 40 140 83.0:00:17.0 50 140 60.0:00:40.0

Advantage of the Invention

(60) The genetically modified genes encoding for sucrose isomerase and isomaltulose synthase is capable of expressing 14% to 19% more of the total cellular protein as compared to native gene.

(61) The enzyme produced by the present process appears to be more active as the enzyme requirement and time required for sugar conversion substantially lower than the native enzymes.

Non-Patented Literature

(62) Nagai, Y. T., Sugitani, and K. Tsuyuki. 1994, Characterization of alpha-glucosyltransferase from Pseudomonals mesoacidophila producting trehalulose. Biosci. Biotechnol. Biochem. 58:1789-1793.

(63) Watzlawick, H., Mattes, R. 2009. Gene cloning, protein characterization, and alteration of product selectivity for the trehalulose hydrolase and trehalulose synthase from Pseudomonas mesoacidophila MX-45. Appl. Environ. Microbiol. 75:7026-7036.

(64) Wu, L., Birch, R. G. 2004. Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis. J. Appl. Microbiol. 97: 93-103.