Biological devices and methods of use thereof to produce steviol glycosides
Described herein are devices and methods for increasing the production of steviol glycosides, which have industrial and economic value. The steviol glycosides produced by the devices and methods disclosed herein do not require the ultra purification that is common in conventional or commercial methods and do not have a bitter aftertaste, making them better suited as flavor-enhancing additives to food, pharmaceutical, and nutritional supplement products.
1 . A vector comprising a DNA construct comprising the following genetic components:
a) a gene that expresses hexokinase having SEQ ID NO: 2 or at least 90% homology thereto;
b) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO: 4 or at least 90% homology thereto;
c) a gene that expresses 1-deoxy- D -xylulose-5-phosphate synthase having SEQ ID NO: 5 or at least 90% homology thereto;
d) a gene that expresses mevalonate-5-kinase having SEQ ID NO: 6 or at least 90% homology thereto;
e) a gene that expresses isopentenyl pyrophosphate isomerase having SEQ ID NO: 7 or at least 90% homology thereto;
f) a gene that expresses mevalonate pyrophosphate decarboxylase having SEQ ID NO: 8 or at least 90% homology thereto; and
g) a gene that expresses UDP-glycosyltransferase 76G1 having a His 155Leu mutation and SEQ ID NO: 12 or at least 90% homology thereto;
wherein the vector is a plasmid.
2 . The vector of claim 1 , wherein the DNA construct further comprises a gene that expresses a heat shock protein.
3 . The vector of claim 2 , wherein the gene that expresses a heat shock protein expresses HSP70.
4 . The vector of claim 3 , wherein the gene that expresses HSP70 is SEQ ID NO: 3 or at least 70% homology thereto.
5 . The vector of claim 1 , wherein the DNA construct comprises from 5′ to 3′ the following genetic components in the following order: (a) the gene that expresses mevalonate-5-kinase; (b) the gene that expresses isopentenyl pyrophosphate isomerase; (c) the gene that expresses mevalonate pyrophosphate decarboxylase; (d) the gene that expresses hexokinase; (e) a gene that expresses HSP70; (f) the gene that expresses geranylgeranyl pyrophosphate synthase 2; (g) the gene that expresses a UDP-glycosyltransferase; and (h) the gene that expresses 1-deoxy- D -xylulose-5-phosphate synthase.
6 . The vector of claim 1 , wherein the vector has SEQ ID NO: 14.
7 . The vector of claim 1 , wherein the plasmid is pWLneo, pSV2cat, pOG44, pXT1, pSG, pSVK3, pBSK, pBSKII, pYES, pYES2, pUC, or pUC19.
8 . A biological device comprising host cells transformed with the vector of claim 1 .
9 . A method for producing one or more steviol glycosides comprising growing the biological device of claim 8 for a time sufficient to produce the steviol glycoside.
10 . A method for producing one or more steviol glycosides from plant cells, the method comprising contacting the plant cells with the biological device claim 8 .