Method of production of monosaccharides

Abstract

The present invention is directed towards genetic modification of native gene encoding for D-tagatose 3-epimerase and rhamnose isomerase to substantially increase the expression level of these enzymes and use of the enzymes in a process to produce rare monosaccharides such as psicose and allose. 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 sequence comprising SEQ ID NO: 1 encoding D-tagatose 3-epimerase; or a modified polynucleotide sequence comprising SEQ ID NO: 2 encoding rhamnose isomerase.

2. The modified polynucleotide sequence according to claim 1 comprising SEQ ID NO: 2 encoding rhamnose isomerase.

3. The modified polynucleotide sequence according to claim 1 comprising SEQ ID NO: 1 encoding D-tagatose 3-epimerase.

4. The modified polynucleotide sequence according to claim 1 present in an expression construct.

5. The modified polynucleotide sequence according to claim 4, wherein SEQ ID NO: 1 and SEQ ID NO: 2 are operably linked to a T7 promoter.

6. The modified polynucleotide sequence according to claim 4, wherein the modified polynucleotide sequence encodes D tagatose 3-epimerase.

7. The modified polynucleotide sequence according to claim 4, wherein the modified polynucleotide sequence encodes rhamnose isomerase.

8. A host cell comprising the expression construct of claim 4.

9. The host cell of claim 8, wherein the host cell is a prokaryotic host cell.

10. A process of production of recombinant D-tagatose 3-epimerase or rhamnose isomerase, said process comprising the steps of: 1. culturing host cell transformed with an expression construct comprising SEQ ID NO: 1 or SEQ ID NO: 2 in a suitable medium in presence of IPTG or lactose for a period in the range of 2-3 hours, 2. isolation of expressed protein from the host cells by conventional method, and 3. purifying the recombinant proteins using chromatographic techniques.

11. The method according to claim 10, wherein the method is a method of producing D-tagatose 3-epimerase.

12. The method according to claim 10, wherein the method is a method of producing rhamnose isomerase.

13. A process of overproduction of rare monosaccharides from fructose, said process comprising the steps of: 1. culturing host cells transformed with an expression construct comprising SEQ ID NO 1: and SEQ ID NO: 2 in a separate suitable medium in presence of IPTG or lactose for a period in the range of 2-3 hours to produce D-tagatose 3-epimerase and rhamnose isomerase respectively, 2. isolating the expressed protein from the host cells by conventional method, and purifying the isolated protein using chromatographic techniques, 3. immobilizing D-tagatose 3-epimerase and rhamnose isomerase thus produced in the previous step in a suitable matrix, 4. contacting fructose with immobilized D-tagatose 3-epimerase for a period in the range of 5 to 10 hours to produce psicose, and 5. contacting D-psicose produced in the previous step with immobilized rhamnose isomerase for a period in the range of 6-12 hours to produce D-allose.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Schematic view of a gene construct generated for expression of D-tagatose 3-epimerase in E. coli

(2) A: D-tagatose 3-epimerase encoding sequence (DTE) was cloned into pET11a using NdeI and BamHI sites. D-Tagatose 3-epimerase (DTE) 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: D-Tagatose 3-epimerase encoding sequence (DTE) was cloned into pET23a using BamHI and HindIII sites. D-Tagatose 3-epimerase (DTE) 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 rhamnose isomerase in E. coli

(5) A: Rhamnose isomerase encoding sequence (RHI) was cloned into pET11a using NdeI and BamHI sites. Rhamnose isomerase (RHI) 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: Rhamnose isomerase encoding sequence (RHI) was cloned into pET23a using BamHI and HindIII sites. Rhamnose isomerase (RHI) 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.

(7) FIG. 3: Expression analysis of recombinant D-tagatose 3-epimerase in E. coli.

(8) A. Control and recombinant E. coli cells [JM109 carrying pET11-DTE] 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 12% 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. Lane 1 and 2: Host cell lysate un-induced and induced. Lane 3 and 4: Recombinant strain uninduced and induced. Immuno-detection was carried our using protein specific antibodies.

(10) FIG. 4: Expression analysis of recombinant rhamnose isomerase in E. coli.

(11) A. Control and recombinant E. coli cells [JM109 carrying pET11-RHI] were induced for protein expression by addition of 0.2 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.

(12) B. Identity analysis of recombinant protein by Western blot analysis. Lane 1 and 2: Host cell lysate un-induced and induced. Lane 3 and 4: Recombinant strain uninduced and induced. Immuno-detection was carried our using protein specific antibodies.

(13) FIG. 5: HPLC analysis of recombinant D-tagatose 3-epimerase 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 (B) were confirmed with commercially available Fructose (Sigma Aldrich) (A) and Psicose as substrate and product standards, respectively.

(15) FIG. 6: HPLC analysis of recombinant rhamnose isomerase 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 (A) were confirmed with commercially available Psicose (Sigma Aldrich) (B) and Allose as substrate and product standards, respectively.

(17) FIG. 7: Analysis of purified DTEase

(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 DTEase; Lane 5: Column 2 purified DTEase.

(19) B. Identity analysis of recombinant protein by Western blot analysis. Lane 1 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.

(20) FIG. 8: Analysis of purified RHIase

(21) 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 RHIase; Lane 5: Column 2 purified RHIase.

(22) B. Identity analysis of recombinant protein by Western blot analysis. Lane 1 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.

(23) FIG. 9: Activity of a D-tagatose 3-epimerase against reaction pH and reaction temperature. The reaction mixture containing fructose and purified DTEase were incubated at different pH (A) and temperature (B) as indicated. After bioconversion the reaction was stopped by boiling the reaction mixture at 95 C. The reaction mixtures were subject to HPLC analysis to confirm the residual substrate and product formation with appropriate standards. The product peaks were confirmed with commercially available Fructose (Sigma Aldrich) and Psicose as substrate and product standards

(24) FIG. 10: Activity of a rhamnose isomerase against reaction pH and reaction temperature. The reaction mixture containing Psicose and purified RHIase were incubated at different pH (A) and temperature (B) as indicated. After bioconversion the reaction was stopped by boiling the reaction mixture at 95 C. The reaction mixtures were subject to HPLC analysis to confirm the residual substrate and product formation with appropriate standards. The product peaks were confirmed with commercially available Psicose (Sigma Aldrich) and allose as substrate and product standards

(25) FIG. 11: Sequence alignment analysis of modified gene sequence with native gene sequence encoding for D-tagatose 3-epimerase.

(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 cichorii ST-24 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 compared to native gene sequence.

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

(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 Pseudomonas stutzeri 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 23% of nucleotides were changed compared to native gene sequence.

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 D-Tagatose 3-epimerase (DTE) 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 rhamnose isomerase 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-DTE and pUC57-RHI constructs. Cloned gene sequence was confirmed by sequence analysis.

(32) The DNA fragment encoding for D-tagatose 3-epimerase was PCR amplified using gene specific primers, and sub cloned into pET11a using NdeI and BamHI restriction enzyme sites to generate pET11-DTE (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-DTE-HIS construct expressing D-tagatose 3-epimerase with C-terminal 6Histidine tag. The recombinant plasmid carrying D-tagatose 3-epimerase gene (pET11-DTE and pET23-DTE) was confirmed by restriction digestion analysis and followed by DNA sequencing.

(33) The DNA fragment encoding for rhamnose isomerase was PCR amplified using gene specific primers, and sub cloned into pET11a using NdeI and BamHI restriction enzyme sites to generate pET11-RHI (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 pET23a (FIG. 2B) using BamHI and HindIII restriction enzymes to generate pET15-RHI-HIS construct expressing rhamnose isomerase with C-terminal 6Histidine tag. The recombinant plasmid carrying D-tagatose 3-epimerase gene (pET11-RHI and pET15-RHI) was confirmed by restriction digestion analysis and followed by DNA sequencing.

Example 2

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

(35) For D-tagatose 3-epimerase

(36) Recombinant plasmid DNA (pET11-DTE) 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 Rhamnose Isomerase

(38) Recombinant plasmid DNA (pET11-RHI) 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, D-Tagatose 3-Epimerase and Rhamnbose Isomerase

(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 30-14 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 D-tagatose 3-epimerase and rhamnose isomerase activity and purity by SDS-PAGE (FIGS. 7 and 8). The purification yield, activity recovery and fold purification for D-tagatose 3-epimerase and rhamose isomerase 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.

Example 5

(43) Immobilization of Enzymes:

(44) The same protocol was followed for DTEase and RHIase. Partially purified or purified DTEase and RHIase 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 DTEase or RHIase 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

(45) Production of Rare Monosaccharide

(46) Production of Psicose by Recombinant DTEase

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

(48) Production of Psicose form Fructose was carried out by using 140 units of immobilized DTE enzymes with 100 g/l, 200 g/l and 400 g/l Fructose solution was used in 20 mM Tris buffer, 5 mM MnCl.sub.2 (pH 8.0) at 50 C.

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

(50) 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 Allose in this process. Bioconversion of Psicose from Fructose was observed to be 25%.

(51) Production of Allose by Recombinant RHIase

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

(53) Production of Allose from Psicose was carried out by using 25 units of immobilized RHIase with 15 g/l, 30 g/l and 60 g/l Psicose solution was used in 20 mM Tris buffer, 5 mM MnCl.sub.2 (pH 8.0) at 60 C.

(54) The sugar solution passed through cation and anion exchange resins to remove salt and ions present in buffer solutions.

(55) 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 Psicose in this process. Bioconversion of Allose form Psicose was observed to be 17%.

(56) Advantage of the Present Invention:

(57) The genetic modification of the native gene encoding for D-tagatose 3-epimerase and rhamose isomerase proposed by the present invention results into an increase in expression level in the range of 14% to 18% and 11% to 14% of the total cellular protein.

(58) The recombinant enzymes thus produced by the claimed process appears to be active than the native one and the fact is established from the sugar conversion data. The present invention has used 140 units of Dtase for the conversion of fructose to psicose within a period of 8 hours. In the prior art researcher had used 1000 to 3000 units of Dtase for the conversion of fructose to psicose. Moreover the time taken for the conversion was 30 to 90 hours (U.S. Pat. No. 5,679,562, U.S. Pat. No. 5,811,271).