JPWO2020018506A5 - - Google Patents

Claims (36)

A method for producing a steviol glycoside product from a steviol glycoside intermediate, comprising:
providing a bacterial cell that intracellularly expresses one or more UDP-dependent glycosyltransferase enzymes (UGT enzymes) ;
Incubating said bacterial cells with a stevia leaf extract or a fraction thereof containing steviol glycoside intermediates and converting said steviol glycoside intermediates into steviol glycoside products by enzymatic transfer of one or more glycosyl groups from the UDP-glucose cofactor. glycosylating; and
recovering the steviol glycoside product;
including
the UGT enzyme glycosylate steviol and steviol glycoside substrates;
Said bacterial cell has the following genetic mutation:
deletion, inactivation, or reduced activity or expression of UDP-sugar hydrolase and one or more UDP-galactose biosynthetic enzymes;
deletion, inactivation, or reduced activity or expression of glucose-6-phosphate isomerase; and
overexpression of phosphoglucomutase and UTP-glucose-1-phosphate uridylyltransferase;
including,
Method. 2. The method of claim 1 , wherein the steviol glycoside intermediate comprises one or more of stevioside, steviol-rubioside, rebaudioside A, dulcoside A, dulcoside B, rebaudioside C, and rebaudioside F. 3. The method of claim 2 , wherein the extract contains stevioside, steviol-rubioside, and rebaudioside A as prominent components. 4. The method of claim 3 , wherein said steviol glycoside product comprises RebM. 3. The method of claim 2 , wherein said steviol glycoside products comprise RebK, RebC+1, and/or RebC+2. 2. The method of claim 1 , wherein the bacterial cell is of the genera Escherichia, Bacillus, Rhodobacter, Zymomonas, or Pseudomonas. 7. The method of claim 6 , wherein the bacterial cell is Escherichia coli, Bacillus subtilis, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis, or Pseudomonas putida. Said bacterial cell is E. 8. The method of claim 7 , which is E. coli. 9. The method of claim 8 , wherein the bacterial cell comprises the following genetic modifications: deletion of ushA and galETKM, deletion of pgi, and overexpression of pgm and galU. 2. The method of claim 1 , wherein said bacterial cell expresses a 1-3' glycosylated UGT enzyme. 11. The method of claim 10 , wherein said 1-3' glycosylating UGT enzyme comprises an amino acid sequence that is at least 90 % identical to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17. 2. The method of claim 1 , wherein said bacterial cell expresses a 1-2' glycosylated UGT enzyme. 2. The method of claim 1 , wherein the bacterial cell expresses a UGT enzyme that glycosylate the C13 hydroxyl of steviol or steviol glycoside substrates . 2. The method of claim 1, wherein the bacterial cell expresses a UGT enzyme that glycosylate the C19 hydroxyl of steviol or steviol glycoside substrates. 2. The method of claim 1 , wherein said bacterial cell expresses 1-3' glycosylated UGT enzymes and 1-2' glycosylated UGT enzymes . 16. The method of claim 15 , wherein said bacterial cell expresses 1-3'glycosylated UGT enzymes, 1-2'glycosylated UGT enzymes, C19 O-glycosylated UGT enzymes, and C13 O-glycosylated UGT enzymes. . SrUGT85C2 (SEQ ID NO: 1) or a derivative thereof with at least 90% sequence identity ; MbUGT1,2 ( SEQ ID NO: 9) or a derivative thereof with at least 90% sequence identity ; No. 2) or a derivative thereof having at least 90% sequence identity therewith; and SrUGT76G1 (SEQ ID NO.3) or a derivative thereof having at least 90% sequence identity therewith, MbUGT1-3_1 (SEQ ID NO.15) or at least 90% thereof. % sequence identity , MbUGT1-3_1.5 (SEQ ID NO: 16) or derivatives thereof with at least 90% sequence identity , and MbUGT1-3_2 (SEQ ID NO: 17) or at least 90% sequence with it. 17. The method of claim 16, expressing an enzyme selected from derivatives thereof with identity . 2. The method of claim 1 , wherein the UGT enzyme is integrated into the chromosome of the bacterial cell or expressed extrachromosomally. 2. The method of claim 1 , wherein the galETKM gene is inactivated, deleted, or reduced in expression. 2. The method of claim 1, wherein said bacterial cell has deletion, inactivation, or reduced activity or expression of trehalose -6-phosphate synthase . 2. The method of claim 1, wherein said bacterial cell has deletion , inactivation, or reduced activity or expression of UDP-glucose 6- dehydrogenase . 2. The method of claim 1 , wherein said bacterial cell has overexpression or increased activity or expression of ycjU (β-phosphoglucomutase) and Bifidobacterium bifidum ugpA . Said bacterial cells are E. 2. The method of claim 1 , having one or more genetic modifications that increase flux into the pentose phosphate pathway (PPP), which is overexpression of E. coli zw f . Said bacterial cell is E . coli pyrH (UMP kinase), E. coli cmk (cytidylate kinase), E. coli adk (adenylate kinase), and E. 2. The method of claim 1 , having one or more genetic modifications that increase UTP production and recycling selected from increased expression or activity of E. coli ndk (nucleoside diphosphate kinase) . The bacterial cell contains upp (uracil phosphoribosyltransferase), pyrH (UMP kinase), and cmk (cytidyllate kinase ); dctA (C4 dicarboxylate/oleate:H+ symporter), pyrE (olelate 2. The method of claim 1, having one or more genetic modifications that increase UDP production selected from overexpression or increased activity of pyrH (UMP kinase), pyrH (UMP kinase), and cmk (cytidyl rate kinase). Method. 2. The bacterial cell of claim 1 , wherein said bacterial cell has one or more genetic modifications to ablate or reduce regulation of glucose uptake, including deletion, inactivation, or reduced expression of the sgrS minor regulatory RNA. the method of. said bacterial cell has reduced conversion of glucose-1-phosphate to TDP-glucose comprising deletion, inactivation, or reduced expression or activity of one or more of the d TDP-glucose pyrophosphorylase genes 2. The method of claim 1 , having one or more genetic modifications that cause wherein said bacterial cell has reduced conversion of glucose-1-phosphate to ADP-glucose comprising deletion, inactivation, or reduced expression or activity of glucose -1-phosphate adenyltransferase 2. The method of claim 1 , having the above genetic modifications. 2. The method of claim 1, wherein the method results in at least 40% by weight conversion of steviol glycoside intermediates to steviol glycoside products. 30. The method of claim 29 , wherein the method provides at least 50% by weight conversion of steviol glycoside intermediates to steviol glycoside products. 2. The method of claim 1 , wherein the method provides at least 75% by weight conversion of steviol glycoside intermediates to steviol glycoside products. 2. The method of claim 1 , wherein a RebM to RebD ratio of at least 5:1 is recovered as said glycosylated product. 2. The method of claim 1 , wherein the method is performed by batch fermentation, continuous fermentation, or semi-continuous fermentation. 34. The method of claim 33 , wherein the method is performed by feed-batch fermentation. 35. The method of claim 34 , wherein said steviol glycoside intermediate is incubated with said bacterial cell for no more than about 72 hours. 3. The method of claim 2, wherein said steviol glycoside product comprises RebD, RebE, RebI or RebB.