Method of production of monosaccharides
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.
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.