Bioproduction of UDP-sugars using a microbial host

Abstract

The present invention relates to compositions and methods for the production of UDP-sugars, including UDP-glucose and UDP-galactose. In particular, the present invention provides engineered cell lines for the production of UDP-sugars, and methods for the production of UDP-sugars using these engineered cell lines. Also provided herein are engineered cells for the production of UDP-glucose, wherein the engineered cells comprise one or more genetic modifications that increase UDP-glucose production by increasing the metabolic flux to the UDP-glucose precursor and / or by reducing carbon losses resulting from the production of by-products.

Description

Technical Field

[0001] I. Field of the Invention The present invention relates to materials and methods comprising engineered cells and cell lines involved in the production of uridine diphosphate (UDP) sugars, including UDP-glucose and UDP-galactose.

Background Art

[0002] II. Background of the Invention Nucleotide sugars are key precursors for all glycosylation reactions and are required for both the synthesis of oligosaccharides and polysaccharides and the glycosylation of proteins and lipids. Among all nucleotide sugars, UDP-sugars are the most important precursors for biomass production in nature. UDP-galactose is used in the synthesis of galactolipids such as monogalactosyldiacylglycerol and digalactosyldiacylglycerol. UDP-galactose is also used as a precursor for galactinol, which is used in the synthesis of polysaccharides together with sucrose.

[0003] Uridine diphosphate glucose (UDP-glucose) is one of the nucleotide sugars widely distributed in the cells of microorganisms, animals, and plants and is used as a glucose donor in the biosynthesis of various glycosides, oligosaccharides, and polysaccharides. Furthermore, uridine-5'-diphosphate-glucose (UDP-glucose) is a fundamentally important molecule in biology, food, biopharmaceuticals, and cosmetic chemistry. Uridine-5'-diphosphate-glucose (UDP-glucose) is one of the key precursors for sugar interconversion, for the formation of disaccharides and polysaccharides, and in amino and nucleotide sugar metabolism. Furthermore, UDP-glucose can be used as a source for other industrially interesting compounds such as antibiotics.

[0004] Although many chemical methods for UDP-glucose synthesis have already been proposed, they create problems of reactivity and selectivity and often require modification of functional groups to protect residues of sugar molecules that should not react and simultaneously expose groups that should react. Furthermore, these chemical reactions often require expensive catalysts and organic solvents. As a result, the chemical synthesis of UDP-glucose is not cost-effective and not environmentally friendly.

Summary of the Invention

[0005] III. Summary of the Invention To move away from chemically derived products, the present invention provides engineered cells for the bioproduction of UDP-sugars. This approach provides an executable route for the rapid, safe, economical, and sustainable production of important groups of molecules. The present invention provides an executable route for the rapid, safe, economical, and sustainable production of UDP-sugars including, but not limited to, UDP-glucose and UDP-galactose. Aspects of the present invention are achieved using engineered cells for the bioproduction of UDP-sugars via engineered host cells that include one or more genetic modifications. As used herein, UDP-sugars are biomanufactured using a modified microbial host. As used herein, engineered cells include one or more genetic modifications that increase the bioproduction of UDP-sugars including UDP-glucose and UDP-galactose.

[0006] Cells engineered for the production of UDP-glucose, wherein the engineered cells comprise one or more genetic modifications that increase UDP-glucose production by increasing the metabolic flux to UDP-glucose precursors and / or by reducing carbon losses resulting from the production of by-products. By way of non-limiting example, the genetic modifications can be modifications to overexpress or underexpress one or more endogenous genes in the engineered host cell, or can be modifications to express one or more non-native genes in the engineered host cell. The engineered cells provided herein can comprise multiple genetic modifications.

[0007] Cells engineered for the production of UDP-galactose, wherein the engineered cells comprise one or more genetic modifications that increase UDP-galactose production by increasing the metabolic flux to UDP-galactose precursors and / or by reducing carbon losses resulting from the production of by-products. By way of non-limiting example, the genetic modifications can be modifications to overexpress or underexpress one or more endogenous genes in the engineered host cell, or can be modifications to express one or more non-native genes in the engineered host cell. The engineered cells provided herein can comprise multiple genetic modifications.

[0008] Cells engineered for the production of UDP-glucose from sucrose, wherein the engineered cells comprise one or more genetic modifications that increase UDP-glucose production by increasing the metabolic flux to UDP-glucose precursors and / or by reducing carbon losses resulting from the production of by-products. Engineered cells are provided herein. By way of non-limiting example, the genetic modification can be a modification to overexpress or underexpress one or more endogenous genes in the engineered host cell or a modification to express one or more non-native genes in the engineered host cell. The engineered cells provided herein can comprise multiple genetic modifications.

[0009] A method for producing UDP-sugar is further provided, which comprises culturing the cells engineered for the production of UDP-sugar provided herein. By way of non-limiting example, the genetic modification can be a modification to overexpress or underexpress one or more endogenous genes in the engineered host cell or a modification to express one or more non-native genes in the engineered host cell. The engineered cells provided herein can comprise multiple genetic modifications.

[0010] In one aspect, the invention provides an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of a carbon source to UDP-sugar via multiple chemical intermediates by the engineered host cell. In certain embodiments, the UDP-sugar is glucose. In certain embodiments, the UDP-sugar is UDP-galactose. In certain embodiments, the chemical intermediate is UDP-glucose and the UDP-sugar is UDP-galactose. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the carbon source is selected from the group consisting of glycerol, glucose, sucrose, galactose, fructose, or any combination thereof.

[0011] In certain embodiments, one or more genetic modifications in the engineered host cell result in increased production of UDP-glucose. In certain embodiments, one or more genetic modifications that result in increased production of UDP-glucose are overexpression of one or more genes selected from the group consisting of (i) the glucose facilitator gene (glf) or a homolog thereof, (ii) glucokinase (glk) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) fructokinase (cscK) or a homolog thereof, and (vi) any combination thereof.

[0012] In certain embodiments, one or more genetic modifications that result in increased production of UDP-glucose are downregulation or deletion of one or more genes selected from the group consisting of: (i) glucose-6-phosphate isomerase (pgi); (ii) glucose-1-phosphate adenylyltransferase (glgC); (iii) UDP-glucose 6-dehydrogenase (ugd); (iv) glucan biosynthesis glucosyltransferase (OpgG); (v) UDP-glucose 4-epimerase (galE); (vi) UDP-sugar hydrolase (ushA); (vii) UTP-glucose-1-phosphate uridylyltransferase (ugp), (viii) phosphoglucomutase (pgm), (ix) (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG), (x) trehalose-6-phosphate synthase (otsA), (xi) glucose-1-phosphatase (agp), and (xii) any combination thereof. In certain embodiments, one or more genetic modifications that result in increased production of UDP-glucose are downregulation or deletion of phosphoglucomutase (pgm). In certain embodiments, the engineered host cell is supplemented with a medium containing glucose. In certain embodiments, the engineered host cell is supplemented with a medium containing fructose.

[0013] In certain embodiments, one or more genetic modifications that result in increased production of UDP-glucose are overexpression of one or more genes selected from the group consisting of: (i) sucrose transporter (cscB) or a homolog thereof; (ii) sucrose synthase (SuSy) or a homolog thereof; (iii) sucrose phosphorylase (SPase) or a homolog thereof; and (iv) any combination thereof. In certain embodiments, the engineered host cell is supplemented with a medium containing sucrose.

[0014] In certain embodiments, one or more genetic modifications in the engineered host cell result in increased production of UDP-glucose. In certain embodiments, one or more genetic modifications that result in increased production of UDP-glucose are overexpression of one or more genes selected from the group consisting of: (i) glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof; (ii) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof; (iii) phosphoribosylpyrophosphate synthetase (prs) or a homolog thereof; (iv) phosphoribosyltransferase (pyrE) or a homolog thereof; (v) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof; (vi) uridylic acid kinase (pyrH) or a homolog thereof; (vii) nucleoside diphosphate kinase (ndk) or a homolog thereof; and (viii) adenylate kinase (adk) or a homolog thereof; and (ix) any combination thereof. In certain embodiments, the engineered host cell is supplemented with a medium containing orotic acid.

[0015] In certain embodiments, one or more genetic modifications in the engineered host cell result in increased production of UDP-glucose.In certain embodiments, one or more genetic modifications in engineered host cells that result in enhanced production of UDP-glucose are overexpression of one or more genes selected from the group consisting of glucose facilitator gene (glf) or its homolog, glucokinase (glk) or its homolog, phosphoglucomutase (pgm) or its homolog, UTP-glucose-1-phosphate uridylyltransferase (galU) or its homolog, sucrose transporter (cscB) or its homolog, sucrose synthase (SuSy) or its homolog, sucrose phosphorylase (SPase) or its homolog, glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog; 6-phosphogluconate dehydrogenase (gnd) or its homolog; phosphoribosyl pyrophosphate synthetase (prs) or its homolog; phosphoribosyl transferase (pyrE) or its homolog; orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, uridylic acid kinase (pyrH) or its homolog, nucleoside diphosphate kinase (ndk) or its homolog, and adenylate kinase (adk) or its homolog, fructokinase (cscK) or its homolog; and / or downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi), glucose-1-phosphate adenylyltransferase (glgC), UDP-glucose 6-dehydrogenase (ugd), glucan biosynthesis glucosyltransferase (OpgG), UDP-glucose 4-epimerase (galE), UDP-sugar hydrolase (ushA), UTP-glucose-1-phosphate uridylyltransferase (ugp), phosphoglucomutase (pgm), (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG), trehalose-6-phosphate synthase (otsA), glucose-1-phosphatase (agp); and / or any combination thereof.In certain embodiments, the engineered host cell is supplemented with a medium comprising orotic acid, sucrose, glucose, fructose, or combinations thereof.

[0016] In certain embodiments, one or more genetic modifications in the engineered host cell result in increased production of UDP-galactose. In certain embodiments, one or more genetic modifications in the engineered host cell that result in enhanced production of UDP-galactose are selected from the group consisting of: (i) overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof; (ii) overexpression of galactokinase (galK) or a homolog thereof; (iii) overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof; and (iv) any combination thereof. In certain embodiments, the engineered host cell is supplemented with a medium comprising galactose.

[0017] In certain preferred embodiments, the one or more genetic modifications in an engineered host cell for increasing the production of UDP-galactose comprise one or more genetic modifications that result in increased production of UDP-glucose.Thus, in certain preferred embodiments, one or more genetic modifications for increasing the production of UDP-galactose are overexpression of one or more genes selected from the group consisting of glucose facilitator gene (glf) or its homolog, glucokinase (glk) or its homolog, phosphoglucomutase (pgm) or its homolog, UTP-glucose-1-phosphate uridylyltransferase (galU) or its homolog, sucrose transporter (cscB) or its homolog, sucrose synthase (SuSy) or its homolog, sucrose phosphorylase (SPase) or its homolog, glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog; 6-phosphogluconate dehydrogenase (gnd) or its homolog; phosphoribosyl pyrophosphate synthetase (prs) or its homolog; phosphoribosyl transferase (pyrE) or its homolog; orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, uridylate kinase (pyrH) or its homolog, nucleoside diphosphate kinase (ndk) or its homolog, and adenylate kinase (adk) or its homolog, overexpression of UDP-glucose 4-epimerase (galE) or its homolog, overexpression of galactokinase (galK) or its homolog, overexpression of galactose-1-phosphate uridylyltransferase (galT) or its homolog; and / or downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi), glucose-1-phosphate adenylyltransferase (glgC), UDP-glucose 6-dehydrogenase (ugd), glucan biosynthesis glucosyltransferase (OpgG), UDP-sugar hydrolase (ushA), UTP-glucose-1-phosphate uridylyltransferase (ugp), phosphoglucomutase (pgm); and / or any combination thereof. In certain embodiments, the engineered host cell is supplemented with a medium containing glucose, galactose, orotic acid, sucrose, or any combination thereof.

[0018] In another aspect, the present invention provides a method for increasing the production of UDP-sugar, comprising providing an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of a carbon source to UDP-sugar via a plurality of chemical intermediates by the engineered host cell. In certain embodiments, the UDP-sugar is glucose. In certain embodiments, the UDP-sugar is UDP-galactose. In certain embodiments, the chemical intermediate is UDP-glucose and the UDP-sugar is UDP-galactose. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the carbon source is selected from the group consisting of glycerol, glucose, sucrose, galactose, fructose, or any combination thereof.

[0019] In certain embodiments, the method comprises one or more genetic modifications in an engineered host cell that result in increased production of UDP-glucose. In certain embodiments, the one or more genetic modifications that result in increased production of UDP-glucose are overexpression of one or more genes selected from the group consisting of (i) the glucose facilitator gene (glf) or a homolog thereof, (ii) glucokinase (glk) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, and (v) any combination thereof. In certain embodiments, the one or more genetic modifications that result in increased production of UDP-glucose are downregulation or deletion of one or more genes selected from the group consisting of (i) glucose-6-phosphate isomerase (pgi); (ii) glucose-1-phosphate adenylyltransferase (glgC); (iii) UDP-glucose 6-dehydrogenase (ugd); (iv) glucan biosynthesis glucosyltransferase (OpgG); (v) UDP-glucose 4-epimerase (galE); (vi) UDP-sugar hydrolase (ushA); (vii) UTP-glucose-1-phosphate uridylyltransferase (ugp), (viii) phosphoglucomutase (pgm), (ix) (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG), (x) trehalose-6-phosphate synthase (otsA), (xi) glucose-1-phosphatase (agp), and (xii) any combination thereof. In certain embodiments, the one or more genetic modifications that result in increased production of UDP-glucose are downregulation or deletion of phosphoglucomutase (pgm). In certain embodiments, the engineered host cell is supplemented with a medium containing glucose. In certain embodiments, the engineered host cell is supplemented with a medium containing fructose.

[0020] In certain embodiments, the method comprises one or more genetic modifications that result in increased production of UDP-glucose, and the one or more genetic modifications that result in increased production of UDP-glucose are overexpression of one or more genes selected from the group consisting of (i) a sucrose transporter (cscB) or a homolog thereof, (ii) a sucrose synthase (SuSy) or a homolog thereof, (iii) a sucrose phosphorylase (SPase) or a homolog thereof, and (iv) any combination thereof. In certain embodiments, the engineered host cell is supplemented with a medium containing sucrose.

[0021] In certain embodiments, the method comprises one or more genetic modifications in an engineered host cell that result in increased production of UDP-glucose. In certain embodiments, the one or more genetic modifications that result in increased production of UDP-glucose are overexpression of one or more genes selected from the group consisting of (i) glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof; (ii) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof; (iii) phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof; (iv) phosphoribosyl transferase (pyrE) or a homolog thereof; (v) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (vi) uridylate kinase (pyrH) or a homolog thereof, (vii) nucleoside diphosphate kinase (ndk) or a homolog thereof, and (viii) adenylate kinase (adk) or a homolog thereof, and (ix) any combination thereof. In certain embodiments, the engineered host cell is supplemented with a medium containing orotic acid.

[0022] In certain embodiments, the method comprises one or more genetic modifications in a engineered host cell that result in increased production of UDP-glucose.In certain embodiments, one or more genetic modifications in an engineered host cell that result in enhanced production of UDP-glucose are overexpression of one or more genes selected from the group consisting of glucose facilitator gene (glf) or its homolog, glucokinase (glk) or its homolog, phosphoglucomutase (pgm) or its homolog, UTP-glucose-1-phosphate uridylyltransferase (galU) or its homolog, sucrose transporter (cscB) or its homolog, sucrose synthase (SuSy) or its homolog, sucrose phosphorylase (SPase) or its homolog, glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog; 6-phosphogluconate dehydrogenase (gnd) or its homolog; phosphoribosyl pyrophosphate synthetase (prs) or its homolog; phosphoribosyl transferase (pyrE) or its homolog; orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, uridylate kinase (pyrH) or its homolog, nucleoside diphosphate kinase (ndk) or its homolog, and adenylate kinase (adk) or its homolog, fructokinase (cscK) or its homolog; and / or downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi), glucose-1-phosphate adenylyltransferase (glgC), UDP-glucose 6-dehydrogenase (ugd), glucan biosynthesis glucosyltransferase (OpgG), UDP-glucose 4-epimerase (galE), UDP-sugar hydrolase (ushA), UTP-glucose-1-phosphate uridylyltransferase (ugp), phosphoglucomutase (pgm), (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG), trehalose-6-phosphate synthase (otsA), glucose-1-phosphatase (agp); and / or any combination thereof.In certain embodiments, the engineered host cell is supplemented with a medium comprising orotic acid, sucrose, glucose, fructose, or combinations thereof.

[0023] In certain embodiments, the method comprises one or more genetic modifications in the engineered host cell that result in increased production of UDP-galactose. In certain embodiments, the one or more genetic modifications in the engineered host cell that result in enhanced production of UDP-galactose are selected from the group consisting of (i) overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof, (ii) overexpression of galactokinase (galK) or a homolog thereof, (iii) overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof, and (iv) any combination thereof. In certain embodiments, the engineered host cell is supplemented with a medium comprising galactose.

[0024] In certain preferred embodiments, the method provides that one or more genetic modifications in an engineered host cell for increasing the production of UDP - galactose comprise one or more genetic modifications that result in increased production of UDP - glucose. Thus, in certain preferred embodiments, one or more genetic modifications for increasing the production of UDP - galactose are overexpression of a glucose facilitator gene (glf) or a homolog thereof, glucokinase (glk) or a homolog thereof, phosphoglucomutase (pgm) or a homolog thereof, UTP - glucose - 1 - phosphate uridylyltransferase (galU) or a homolog thereof, sucrose transporter (cscB) or a homolog thereof, sucrose synthase (SuSy) or a homolog thereof, sucrose phosphorylase (SPase) or a homolog thereof, glucose - 6 - phosphate 1 - dehydrogenase (zwf) or a homolog thereof; 6 - phosphogluconate dehydrogenase (gnd) or a homolog thereof; phosphoribosylpyrophosphate synthetase (prs) or a homolog thereof; phosphoribosyltransferase (pyrE) or a homolog thereof; orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, uridylic acid kinase (pyrH) or a homolog thereof, nucleoside diphosphate kinase (ndk) or a homolog thereof, and adenylate kinase (adk) or a homolog thereof; overexpression of UDP - glucose 4 - epimerase (galE) or a homolog thereof, overexpression of galactokinase (galK) or a homolog thereof, overexpression of galactose - 1 - phosphate uridylyltransferase (galT) or a homolog thereof, overexpression of fructokinase (cscK) or a homolog thereof, selected from the group consisting of one or more genes;and / or downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi), glucose-1-phosphate adenylyltransferase (glgC), UDP-glucose 6-dehydrogenase (ugd), glucan biosynthesis glucosyltransferase (OpgG), UDP-sugar hydrolase (ushA), UTP-glucose-1-phosphate uridylyltransferase (ugp), phosphoglucomutase (pgm), (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG), trehalose-6-phosphate synthase (otsA), glucose-1-phosphatase (agp); and / or selected from any combination thereof. In certain embodiments, the engineered host cell is supplemented with a medium comprising glucose, galactose, orotate, sucrose, or any combination thereof.;

[0025] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of a carbon source through multiple chemical intermediates by the engineered host cell to produce UDP-glucose. In certain embodiments, the one or more genetic modifications are overexpression of a glucose facilitator gene (glf) or a homolog thereof. In certain embodiments, the one or more genetic modifications are overexpression of a glucokinase (glk) or a homolog thereof. In certain embodiments, the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for overexpressing one or more endogenous genes in the engineered host cell; (ii) one or more modifications for underexpressing one or more endogenous genes in the engineered host cell; (iii) one or more genetic modifications expressing one or more non-native genes in the engineered host cell; and (iv) combinations thereof. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the one or more genetic modifications are overexpression of one or more genes selected from the group consisting of: (i) glucokinase (glk) or a homolog thereof, (ii) glucose facilitator gene (glf) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) orotate phosphoribosyltransferase (pyrE) or a homolog thereof, (vi) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (vii) uridylic acid kinase (pyrH) or a homolog thereof, (viii) nucleoside diphosphate kinase (ndk) or a homolog thereof, (ix) adenylate kinase (adk) or a homolog thereof, (x) fructokinase (cscK) or a homolog thereof, and (xi) any combination thereof.In certain embodiments, one or more genetic modifications are overexpressions of one or more genes selected from the group consisting of: (i) the glucose facilitator gene (glf) or a homolog thereof; (ii) glucokinase (glk) or a homolog thereof; (iii) phosphoglucomutase (pgm) or a homolog thereof; (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof; (v) glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof; (vi) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof; (vii) phosphoribosylpyrophosphate synthetase (prs) or a homolog thereof; and (viii) any combination thereof. In certain embodiments, one or more genetic modifications are selected from the group consisting of: (i) overexpression of glucokinase (glk) or a homolog thereof; (ii) overexpression of the glucose facilitator gene (glf) or a homolog thereof; and (iii) any combination thereof.

[0026] In certain embodiments, one or more genetic modifications are overexpression of phosphoglucomutase (pgm) or its homologs. In certain embodiments, one or more genetic modifications are overexpression of UTP-glucose-1-phosphate uridylyltransferase (galU) or its homologs. In certain embodiments, one or more genetic modifications are overexpression of orotate phosphoribosyltransferase (pyrE) or its homologs. In certain embodiments, one or more genetic modifications are overexpression of orotidine 5’ phosphate decarboxylase (pyrF) or its homologs. In certain embodiments, the engineered cells are supplemented with a medium containing orotic acid. In certain embodiments, one or more genetic modifications are overexpression of uridylic acid kinase (pyrH) or its homologs. In certain embodiments, one or more genetic modifications are overexpression of nucleoside diphosphate kinase (ndk) or its homologs. In certain embodiments, one or more genetic modifications are overexpression of adenylate kinase (adk) or its homologs. In certain embodiments, one or more genetic modifications are overexpression of fructokinase (cscK) or its homologs. In certain embodiments, overexpression of fructokinase (cscK) results in phosphorylation of fructose to produce fructose-6-phosphate. In certain embodiments, one or more genetic modifications are downregulation or deletion of UDP-glucose 4-epimerase (galE) to prevent the production of UDP-galactose. In certain embodiments, one or more genetic modifications are downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd) to prevent the conversion of UDP-glucose to UDP-glucuronic acid. In certain embodiments, one or more genetic modifications are downregulation or deletion of glucose-1-phosphate adenylyltransferase (glgC).In certain embodiments, one or more genetic modifications are downregulation or deletion of glucan - synthesizing glucosyltransferase (OpgG) that prevents UDP - glucose consumption to form membrane - derived oligosaccharides (MDO). In certain embodiments, one or more genetic modifications are downregulation or deletion of glucose - 6 - phosphate isomerase (pgi). In certain embodiments, one or more genetic modifications are overexpression of glucose - 6 - phosphate 1 - dehydrogenase (zwf) or its homolog. In certain embodiments, one or more genetic modifications are overexpression of 6 - phosphogluconate dehydrogenase (gnd) or its homolog. In certain embodiments, one or more genetic modifications are overexpression of phosphoribosylpyrophosphate synthetase (prs) or its homolog. In certain embodiments, one or more genetic modifications are overexpression of sucrose transporter (cscB) or its homolog. In certain embodiments, one or more genetic modifications are overexpression of sucrose synthase (SuSy) or its homolog. In certain embodiments, one or more genetic modifications are overexpression of sucrose phosphorylase (SPase) or its homolog. In certain embodiments, the engineered host cell is supplemented with a medium containing glucose. In certain embodiments, one or more genetic modifications are deletion or downregulation of UDP - sugar hydrolase (ushA). In certain embodiments, one or more genetic modifications are deletion or downregulation of UTP - glucose - 1 - phosphate uridylyltransferase (ugp). In certain embodiments, one or more genetic modifications are downregulation or deletion of (heptosyl)lipopolysaccharide α - 1,3 - glucosyltransferase (WaaG). (Heptosyl)lipopolysaccharide α - 1,3 - glucosyltransferase (WaaG) uses UDP - glucose as a substrate for lipopolysaccharide synthesis.Thus, in certain embodiments, downregulation of (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG) increases the yield of UDP-glucose. In certain embodiments, one or more genetic modifications are downregulation or deletion of trehalose-6-phosphate synthase (otsA). Trehalose-6-phosphate synthase (otsA) catalyzes the biosynthesis of trehalose from UDP-glucose. Thus, in certain embodiments, downregulation of trehalose-6-phosphate synthase (otsA) increases the yield of UDP-glucose. In certain embodiments, one or more genetic modifications are downregulation or deletion of glucose-1-phosphatase (agp). Glucose-1-phosphatase (agp) is a periplasmic protein that hydrolyzes phosphate from glucose-1-phosphate and other substrates. Thus, in certain embodiments, downregulation or deletion of glucose-1-phosphatase (agp) increases the availability of glucose-1-phosphate for UDP-glucose production and increases the yield of UDP-sugars.

[0027] In certain embodiments, the engineered host cell is supplemented with a medium containing glucose. In certain embodiments, the engineered host cell is supplemented with a medium containing sucrose. In certain embodiments, the engineered host cell is supplemented with a medium containing fructose.

[0028] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of glucose to uridine diphosphate galactose (UDP-galactose) via a plurality of chemical intermediates by the engineered host cell. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the one or more genetic modifications are: (i) one or more modifications to overexpress one or more endogenous genes in the engineered host cell; (ii) one or more modifications to underexpress one or more endogenous genes in the engineered host cell; (iii) one or more genetic modifications express one or more non-native genes in the engineered host cell; and (iv) at least one genetic modification selected from the group consisting of combinations thereof. In certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof; (ii) overexpression of galactokinase (galK) or a homolog thereof; (iii) overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof; and (iv) any combination thereof. In certain embodiments, the one or more genetic modifications is overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof. In certain embodiments, the one or more genetic modifications is overexpression of galactokinase (galK) or a homolog thereof. In certain embodiments, the one or more genetic modifications is overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof. In certain embodiments, the modifications recited herein may be combined with the modifications recited in any other aspect of the present invention. In certain embodiments, the modifications recited herein may be combined with one or more modifications recited for the conversion of chemical intermediates to UDP-glucose.In certain embodiments, the engineered host cell is supplemented with a medium containing galactose. In certain embodiments, the engineered host cell is supplemented with a medium containing fructose.

[0029] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of sucrose to uridine diphosphate glucose (UDP-glucose) by the engineered host cell. In certain embodiments, the genetic modification is selected from the group consisting of (i) heterologous expression of a sucrose transporter (cscB) or a homolog thereof, (ii) heterologous expression of a sucrose synthase (SuSy) or a homolog thereof, (iii) downregulation or deletion of UDP-glucose 4-epimerase (galE), (iv) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd), (v) downregulation or deletion of a glucan biosynthesis glucosyltransferase (OpgG), and (vi) any combination thereof. In certain embodiments, the engineered cell comprises a heterologous sucrose transporter (cscB) or a homolog thereof, a heterologous sucrose synthase (SuSy) or a homolog thereof, and a genetic modification selected from the group consisting of (i) downregulation or deletion of UDP-glucose 4-epimerase (galE), (ii) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd), (iii) downregulation or deletion of a glucan biosynthesis glucosyltransferase (OpgG), and (iv) any combination thereof. In certain embodiments, the genetic modification is selected from the group consisting of (i) expression of a phosphoribosyltransferase (pyrE) or a homolog thereof, (ii) expression of an orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (iii) expression of a uridylic acid kinase (pyrH) or a homolog thereof, (iv) expression of a nucleoside diphosphate kinase (ndk) or a homolog thereof, (v) expression of an adenylate kinase (adk) or a homolog thereof, and (vi) any combination thereof. In certain embodiments, the modifications recited herein may be combined with the modifications recited in any other aspect of the present invention.

[0030] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of sucrose to uridine diphosphate glucose (UDP-glucose) by the engineered host cell. In certain embodiments, the genetic modifications are selected from the group consisting of: (i) heterologous expression of a sucrose transporter (cscB) or a homolog thereof; (ii) heterologous expression of a sucrose phosphorylase (SPase) or a homolog thereof; (iii) overexpression of UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof; (iv) downregulation or deletion of UDP-glucose 4-epimerase (galE); (v) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd); (vi) downregulation or deletion of a glucan biosynthesis glucosyltransferase (OpgG); (vii) downregulation or deletion of phosphoglucomutase (pgm); and (viii) any combination thereof. In certain embodiments, the engineered cell comprises heterologous expression of a sucrose transporter (cscB) or a homolog thereof, heterologous expression of a sucrose phosphorylase (SPase) or a homolog thereof, overexpression of UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, and genetic modifications selected from the group consisting of: (i) downregulation or deletion of UDP-glucose 4-epimerase (galE); (ii) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd); (iii) downregulation or deletion of a glucan biosynthesis glucosyltransferase (OpgG); and (iv) any combination thereof. In certain embodiments, the genetic modifications are selected from the group consisting of: (i) expression of phosphoribosyltransferase (pyrE) or a homolog thereof; (ii) expression of orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof; (iii) expression of uridylic acid kinase (pyrH) or a homolog thereof; (iv) expression of nucleoside diphosphate kinase (ndk) or a homolog thereof; (v) expression of adenylate kinase (adk) or a homolog thereof; and (vi) any combination thereof.In certain embodiments, the modifications recited herein may be combined with the modifications recited in any other aspect of the invention.

[0031] In another aspect, the present invention provides a method for increasing the production of uridine diphosphate glucose (UDP-glucose), the method comprising providing an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of glucose to UDP-glucose via a plurality of chemical intermediates by the engineered host cell. In certain embodiments, the one or more genetic modifications are: (i) one or more modifications for overexpressing one or more endogenous genes in the engineered host cell; (ii) one or more modifications for underexpressing one or more endogenous genes in the engineered host cell; (iii) one or more genetic modifications that express one or more non-native genes in the engineered host cell; and (iv) at least one genetic modification selected from the group consisting of combinations thereof. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the one or more genetic modifications are overexpression of one or more genes selected from the group consisting of: (i) glucokinase (glk) or a homolog thereof; (ii) glucose facilitator gene (glf) or a homolog thereof; (iii) phosphoglucomutase (pgm) or a homolog thereof; (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof; (v) phosphoribosyltransferase (pyrE) or a homolog thereof; (vi) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof; (vii) uridylic acid kinase (pyrH) or a homolog thereof; (viii) nucleoside diphosphate kinase (ndk) or a homolog thereof; (ix) adenylate kinase (adk) or a homolog thereof; (x) fructokinase (cscK) or a homolog thereof; and (xi) any combination thereof.In certain embodiments, one or more genetic modifications are overexpressions of one or more genes selected from the group consisting of: (i) glucokinase (glk) or a homolog thereof; (ii) glucose facilitator gene (glf) or a homolog thereof; (iii) phosphoglucomutase (pgm) or a homolog thereof; (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof; (v) glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof; (vi) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof; (vii) phosphoribosylpyrophosphate synthetase (prs); and (viii) any combination thereof. In certain embodiments, one or more genetic modifications are selected from the group consisting of: (i) overexpression of glucokinase (glk) or a homolog thereof; (ii) overexpression of glucose facilitator gene (glf) or a homolog thereof; and (iii) any combination thereof. In certain embodiments, one or more genetic modifications are overexpression of phosphoglucomutase (pgm) or a homolog thereof. In certain embodiments, one or more genetic modifications are overexpression of UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof. In certain embodiments, one or more genetic modifications are overexpression of fructokinase (cscK) or a homolog thereof. In certain embodiments, one or more genetic modifications are overexpression of orotate phosphoribosyltransferase (pyrE). In certain embodiments, one or more genetic modifications are overexpression of orotidine 5'-phosphate decarboxylase (pyrF). In certain embodiments, the engineered host cell is supplemented with a medium containing orotic acid. In certain embodiments, one or more genetic modifications are overexpression of uridylic acid kinase (pyrH). In certain embodiments, one or more genetic modifications are overexpression of nucleoside diphosphate kinase (ndk).In certain embodiments, one or more genetic modifications are overexpression of adenylate kinase (adk). In certain embodiments, one or more genetic modifications are downregulation or deletion of UDP-glucose 4-epimerase (galE) to prevent conversion of UDP-glucose to UDP-galactose. In certain embodiments, one or more genetic modifications are downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd) to prevent conversion of UDP-glucose to UDP-glucuronic acid. In certain embodiments, one or more genetic modifications are downregulation or deletion of glucose-1-phosphate adenylyltransferase (glgC). In certain embodiments, one or more genetic modifications are downregulation or deletion of glucan biosynthesis glucosyltransferase (mdoA) to prevent conversion of UDP-glucose to membrane-derived oligosaccharide (MDO). In certain embodiments, one or more genetic modifications are downregulation or deletion of glucose-6-phosphate isomerase (pgi). In certain embodiments, one or more genetic modifications are downregulation or deletion of (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG). (Heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG) uses UDP-glucose as a substrate for lipopolysaccharide synthesis. Thus, in certain embodiments, downregulation of (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG) increases the yield of UDP-glucose. In certain embodiments, one or more genetic modifications are downregulation or deletion of trehalose-6-phosphate synthase (otsA). Trehalose-6-phosphate synthase (otsA) catalyzes the biosynthesis of trehalose from UDP-glucose. Thus, in certain embodiments, downregulation of trehalose-6-phosphate synthase (otsA) increases the yield of UDP-glucose. In certain embodiments, one or more genetic modifications are downregulation or deletion of glucose-1-phosphatase (agp).Glucose-1-phosphatase (agp) is a periplasmic protein that hydrolyzes phosphate from glucose-1-phosphate and other substrates. Thus, in certain embodiments, downregulation or deletion of glucose-1-phosphatase (agp) increases the availability of glucose-1-phosphate for UDP-glucose production and enhances the yield of UDP-sugar. In certain embodiments, one or more genetic modifications are overexpression of glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog. In certain embodiments, one or more genetic modifications are overexpression of 6-phosphogluconate dehydrogenase (gnd) or its homolog. In certain embodiments, one or more genetic modifications are overexpression of phosphoribosylpyrophosphate synthetase (prs) or its homolog. In certain embodiments, the engineered host cell comprises overexpression of (i) glucokinase (glk) or its homolog, (ii) glucose facilitator gene (glf) or its homolog, (iii) phosphoglucomutase (pgm) or its homolog, (iv) UTP-glucose-1-phosphate-uridylyltransferase (galU) or its homolog, (v) phosphoribosyltransferase (pyrE) or its homolog, (vi) orotidine 5’ phosphate decarboxylase (pyrF) or its homolog, (vii) uridylic acid kinase (pyrH) or its homolog, (viii) nucleoside diphosphate kinase (ndk) or its homolog, (ix) adenylate kinase (adk) or its homolog, (x) fructokinase (cscK) or its homolog, and (xi) any combination thereof.In certain embodiments, the engineered host cell comprises overexpression of (i) glucokinase (glk) or a homolog thereof, (ii) glucose facilitator gene (glf) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof, (vi) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof, and (vii) phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof. In another aspect, the present invention provides a method for increasing the production of uridine diphosphate galactose (UDP-galactose), the method comprising providing an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of glucose and / or galactose to uridine diphosphate galactose (UDP-galactose) via a plurality of chemical intermediates by the engineered host cell. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) one or more modifications for overexpressing one or more endogenous genes in the engineered host cell; (ii) one or more modifications for underexpressing one or more endogenous genes in the engineered host cell; (iii) one or more genetic modifications that express one or more non-native genes in the engineered host cell; and (iv) combinations thereof. In certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof, (ii) overexpression of galactokinase (galK) or a homolog thereof, (iii) overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof, and (iv) any combination thereof.In certain embodiments, one or more genetic modifications are overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof. In certain embodiments, one or more genetic modifications are overexpression of galactokinase (galK) or a homolog thereof. In certain embodiments, one or more genetic modifications are overexpression of hydroxyproline O-galactosyltransferase (galT) or a homolog thereof. In certain embodiments, the genetic modification is selected from the group consisting of (i) expression of phosphoribosyltransferase (pyrE) or a homolog thereof, (ii) expression of orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (iii) expression of uridylic acid kinase (pyrH) or a homolog thereof, and (iv) any combination thereof. In certain embodiments, the modifications recited herein may be combined with the modifications recited in any other aspect of the invention. In certain embodiments, the engineered host cell is supplemented with a medium containing galactose.

[0032] In another aspect, the present invention provides a method for increasing the production of UDP-glucose, comprising providing an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of sucrose to uridine diphosphate glucose (UDP-glucose) by the engineered host cell. In certain embodiments, the genetic modifications are selected from the group consisting of: (i) heterologous expression of a sucrose transporter (cscB) or a homolog thereof; (ii) heterologous expression of a sucrose phosphorylase (SPase) or a homolog thereof; (iii) overexpression of UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof; (iv) downregulation or deletion of UDP-glucose 4-epimerase (galE); (v) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd); (vi) downregulation or deletion of glucan biosynthesis glucosyltransferase (OpgG); (vii) downregulation or deletion of phosphoglucomutase (pgm); and (viii) any combination thereof. In certain embodiments, the engineered cell comprises heterologous expression of a sucrose transporter (cscB) or a homolog thereof, heterologous expression of a sucrose phosphorylase (SPase) or a homolog thereof, overexpression of UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, and genetic modifications selected from the group consisting of: (i) downregulation or deletion of UDP-glucose 4-epimerase (galE); (ii) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd); (iii) downregulation or deletion of glucan biosynthesis glucosyltransferase (OpgG); and (iv) any combination thereof. In certain embodiments, the genetic modifications are selected from the group consisting of: (i) expression of phosphoribosyltransferase (pyrE) or a homolog thereof; (ii) expression of orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof; (iii) expression of uridylic acid kinase (pyrH) or a homolog thereof; (iv) expression of nucleoside diphosphate kinase (ndk) or a homolog thereof; (v) expression of adenylate kinase (adk) or a homolog thereof; and (vi) any combination thereof.In certain embodiments, the modifications recited herein may be combined with the modifications recited in any other aspect of the invention.

[0033] In another aspect, the present invention provides a method for increasing the production of uridine diphosphate glucose (UDP-glucose) from sucrose, the method comprising providing an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of sucrose to UDP-glucose by the engineered host cell. In certain embodiments, the genetic modifications are selected from the group consisting of: (i) heterologous expression of a sucrose transporter (cscB) or a homolog thereof; (ii) heterologous expression of a sucrose synthase (SuSy) or a homolog thereof; (iii) downregulation or deletion of UDP-glucose 4-epimerase (galE); (iv) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd); (v) downregulation or deletion of a glucan biosynthesis glucosyltransferase (OpgG); and (vi) any combination thereof. In certain embodiments, the engineered cell comprises a heterologous sucrose transporter (cscB) or a homolog thereof, a heterologous sucrose synthase (SuSy) or a homolog thereof, and a genetic modification selected from the group consisting of: (i) downregulation or deletion of UDP-glucose 4-epimerase (galE); (ii) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd) or a homolog thereof; (iii) downregulation or deletion of a glucan biosynthesis glucosyltransferase (OpgG); and (iv) any combination thereof. In certain embodiments, the engineered cell comprises one or more genetic modifications selected from the group consisting of: (i) expression of a phosphoribosyltransferase (pyrE) or a homolog thereof; (ii) expression of an orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof; (iii) expression of a uridylic acid kinase (pyrH) or a homolog thereof; and (iv) any combination thereof. In certain embodiments, the engineered cell is supplemented with a medium comprising orotic acid, sucrose, fructose, and / or glucose. IV. BRIEF DESCRIPTION OF THE DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]

Figure 1

[0035]

Figure 2

[0036] V. DETAILED DESCRIPTION OF THE INVENTION This application provides engineered cells for producing UDP - sugars, including but not limited to UDP - glucose and / or UDP - galactose, cultures containing the engineered cells, and methods for producing UDP - sugars, including but not limited to UDP - glucose and / or UDP - galactose. As used herein, the term "precursor" can refer to any intermediate present in the biosynthetic pathway that leads to the production of UDP - glucose and / or UDP - galactose. UDP - sugar precursors can include, but are not limited to, glucose, galactose, sucrose, fructose - 6 - phosphate (F6P), glucose - 6 - phosphate (G6P), 6 - phosphogluconic acid, ribose - 5 - phosphate (R5P), 5 - phosphoribosyl 1 - pyrophosphate (PRPP), orotidine 5'-phosphate (OMP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), glucose - 1 - phosphate (glucose - 1 - P), galactose - 1 - phosphate (galactose - 1 - P), uridine diphosphate glucuronic acid (UDP - glucuronic acid), orotic acid, and ADP - α - D - glucose.

[0037] Cells engineered for the production of UDP-sugars can have one or more modifications including, but not limited to, downregulation, disruption or deletion of endogenous genes, upregulation of endogenous genes, and introduction of exogenous genes.

[0038] The term "not naturally occurring" is intended to mean, when used with respect to an enzyme, that the nucleic acid or polypeptide contains at least one genetic change not normally found in a naturally occurring polypeptide or nucleic acid sequence. Naturally occurring nucleic acids and polypeptides may be referred to as "wild-type" or "original". A host cell, organism or microorganism containing at least one genetic modification generated by human intervention may also be referred to as "not naturally occurring", "engineered", "genetically engineered" or "recombinant".

[0039] Host cells, organisms, or microorganisms engineered to express or overexpress a gene or nucleic acid sequence, or to overexpress an enzyme or polypeptide, are genetically engineered via recombinant DNA techniques to contain a gene or nucleic acid sequence that does not naturally encode the enzyme or polypeptide, or to express an endogenous gene at a level that exceeds its level of expression in an unmodified cell. By way of non-limiting example, a host cell, organism, or microorganism engineered to express or overexpress a gene or nucleic acid sequence, or to overexpress an enzyme or polypeptide, can have any modification that affects the coding sequence of the gene, the location of the gene on the chromosome, or the regulatory elements associated with the gene. Overexpression of a gene can also be by increasing the copy number of the gene in a cell or organism. Similarly, a host cell, organism, or microorganism engineered to underexpress a gene, nucleic acid sequence, or to have a reduced expression of a gene, nucleic acid sequence, or to underexpress an enzyme or polypeptide can have any modification that affects the coding sequence of the gene, the location of the gene on the chromosome, or the regulatory elements associated with the gene. Specifically, gene disruption is included, and gene disruption includes any insertion, deletion, or sequence variation to or of a gene or part of a gene that affects the expression of the gene or the activity of the encoded polypeptide. Gene disruption includes "knockout" mutations that eliminate the expression of a gene. Modifications for underexpressing a gene also include modifications to the regulatory region of the gene that can reduce the expression of the gene.

[0040] The terms "exogenous" or "heterologous" are intended to mean that the molecule or activity referred to has been introduced into the host microorganism. The molecule can be introduced, for example, by introduction of the coding nucleic acid into the host genetic material, such as by integration into the host chromosome, or as extrachromosomal genetic material introduced on a vehicle such as a plasmid. Thus, the term "endogenous" refers to the molecule or activity referred to that is naturally present in the host.

[0041] Genes or nucleic acid sequences can be introduced stably or transiently into host cells using techniques well known in the art, including but not limited to conjugation, electroporation, chemical transformation, transduction, and transfection. Optionally, for heterologous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as N-terminal mitochondrial or other targeting signals, which can be removed prior to transformation of the prokaryotic host cell if desired. Furthermore, to achieve optimized expression of a protein, the gene can be subjected to codon optimization using techniques well known in the art.

[0042] The percent identity (% identity) between two sequences is determined when the sequences are aligned for maximum homology. Algorithms well known to those of skill in the art, such as Align, BLAST, Clustal Omega, etc., can compare and determine raw sequence similarity or identity and also determine the presence or significance of gaps in the sequences that can be assigned weights or scores. Such algorithms are also known in the art and are similarly applicable for determining the similarity or identity of nucleotide or amino acid sequences and can be useful in identifying orthologs of a gene of interest. Additional sequences added to a polypeptide sequence, including but not limited to immunodetection tags, purification tags, localization sequences (present or absent), etc., do not affect the % identity.

[0043] A homolog is one or more genes that have the same or identical functions in different organisms. Genes that are orthologous can encode proteins having sequence similarity with an amino acid sequence identity of about 45% to 100%, more preferably about 60% to 100%. Multiple genes can be considered orthologs if they share a sufficient amount of three-dimensional structure similarity, but not necessarily sequence similarity, to indicate that the genes have evolved from a common ancestor to an extent where primary sequence similarity cannot be identified. Paralogs are genes that are related by duplication within a genome and can evolve new functions, even if related to the original function.

[0044] Engineered cells for producing UDP-glucose can contain an exogenous nucleic acid sequence encoding glf (glucose facilitator) activity or a homolog thereof, an exogenous nucleic acid sequence encoding glk (glucokinase) activity or a homolog thereof, an exogenous nucleic acid sequence encoding pgm (phosphoglucomutase) activity or a homolog thereof, an exogenous nucleic acid sequence encoding galU (UTP-glucose-1-phosphate uridylyltransferase) activity or a homolog thereof, and an exogenous nucleic acid sequence encoding pyrE (phosphoribosyltransferase) activity, an exogenous nucleic acid sequence encoding pyrF (orotidine 5'-phosphate decarboxylase) or a homolog thereof, an exogenous nucleic acid sequence encoding pyrH (uridylic acid kinase) activity or a homolog thereof, an exogenous nucleic acid sequence encoding ndk (nucleoside diphosphate kinase) activity or a homolog thereof, an exogenous nucleic acid sequence encoding adk (adenylate kinase) activity or a homolog thereof, an exogenous nucleic acid sequence encoding zwf (glucose-6-phosphate 1-dehydrogenase) activity or a homolog thereof, an exogenous nucleic acid sequence encoding gnd (6-phosphogluconate dehydrogenase) activity or a homolog thereof, an exogenous nucleic acid sequence encoding prs (phosphoribosyl pyrophosphate synthetase) activity or a homolog thereof, an exogenous nucleic acid sequence encoding cscB (sucrose transporter) activity or a homolog thereof, an exogenous expressed nucleic acid sequence encoding SuSy (sucrose synthase) activity or a homolog thereof, and an exogenous expressed nucleic acid sequence encoding SPase (sucrose phosphorylase enzyme) activity or a homolog thereof.Optionally, the engineered cell can further comprise downregulation or deletion of UDP-glucose 4-epimerase (galE) activity, downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd) activity, downregulation or deletion of glucose-1-phosphate adenylyltransferase (glgC) activity, downregulation or deletion of glucosyltransferase (OpgG) activity, downregulation or deletion of glucose-6-phosphate isomerase (pgi) activity, downregulation or deletion of UDP-sugar hydrolase (ushA) activity, downregulation or deletion of UTP-glucose-1-phosphate uridylyltransferase (ugp) activity, and downregulation or deletion of phosphoglucomutase (pgm) activity.

[0045] The glucose facilitator gene or glucose transporter gene (glf) can be, for example, a member of the major facilitator superfamily or sugar transporter (TC2.A.1.1) that facilitates glucose uptake by the cell. An exemplary glucose facilitator gene can be glf from Zymomonas mobilis (NCBI accession ID: AAA27691.1). Glf homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 and having the activity of transporting glucose into the cell are also contemplated for use in the engineered cells provided herein.

[0046] Glucokinase (glk) can be a member of the hexokinase family of proteins that, for example, phosphorylates glucose to produce glucose-6-phosphate. An exemplary glucokinase (glk) is Zymomonas mobilis (EC: 2.7.1.2, NCBI reference number: AAA27694.1). Glk homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 and having the activity of glucokinase to produce glucose-6-phosphate from glucose are also contemplated for use in the engineered cells provided herein.

[0047] Phosphoglucomutase (pgm) catalyzes the conversion of glucose 6-phosphate to glucose 1-phosphate. An exemplary phosphoglucomutase (pgm) can be pgm from Escherichia coli (EC: 5.4.2.2, NCBI accession AAC73782.1). Pgm having SEQ ID NOs: 4-22, pgm homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 3-22 and having the activity to catalyze the conversion of glucose 6-phosphate to glucose 1-phosphate are also contemplated for use in the engineered cells provided herein.

[0048] UTP-glucose-1-phosphate uridylyltransferase (galU) is an enzyme that synthesizes UDP-glucose from glucose-1-phosphate and UTP. Exemplary UTP-glucose-1-phosphate uridylyltransferase (galU) can be galU from E. coli (EC: 2.7.7.9, NCBI accession EEW2752841.1). galU having SEQ ID NOs: 23-39 or 72-74 is also contemplated for use in the engineered cells provided herein. ugpA from Bifidobacterium bifidum (NCBI accession WP_021648042.1) is also contemplated for use in the engineered cells provided herein. ugpA having SEQ ID NOs: 41-47 is also contemplated for use in the engineered cells provided herein. galU or ugpA homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 23-47 or 72-74 and having the activity to synthesize UDP-glucose from glucose-1-phosphate are also contemplated for use in the engineered cells provided herein.

[0049] Orotidine 5'-phosphate decarboxylase (pyrE) is an enzyme that catalyzes the transfer of a ribosyl phosphate group from 5-phosphoribose 1-diphosphate to orotic acid, resulting in the formation of orotidine monophosphate (OMP). An exemplary orotidine 5'-phosphate decarboxylase can be pyrE (NCBI accession WP_000806177.1) from Enterobacteriaceae. Also contemplated for use in the engineered cells provided herein are pyrE, having SEQ ID NO: 49, and pyrE homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 48-49 and having activity to catalyze phosphoribose-1-diphosphate (PRPP) to orotidine monophosphate (OMP).

[0050] Orotidine-5'-phosphate decarboxylase (pyrF) is an enzyme that catalyzes the conversion of orotidine monophosphate (OMP) to uridine monophosphate (UMP). An exemplary orotidine-5'-phosphate decarboxylase (pyrF) can be pyrF from E. coli (EC: 2.4.2.10, NCBI accession WP_110991478.1). Also contemplated for use in the engineered cells provided herein are pyrF, having SEQ ID NO: 51, and pyrF homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 50-51 and having activity to catalyze orotidine monophosphate (OMP) to uridine monophosphate (UMP).

[0051] Uridylate kinase (pyrH) is an enzyme that catalyzes the conversion of uridine monophosphate (UMP) to uridine diphosphate (UDP). An exemplary uridylate kinase (pyrH) can be pyrH from E. coli (EC: 2.7.4.22, NCBI accession WP_000224573.1). Also contemplated for use in the engineered cells provided herein are pyrH, having SEQ ID NO: 53, and pyrH homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 52 - 53 and having the activity to catalyze the conversion of uridine monophosphate (UMP) to uridine diphosphate (UDP).

[0052] Nucleoside diphosphate kinase (ndk) is an enzyme that catalyzes the conversion of uridine diphosphate (UDP) to uridine triphosphate (UTP). An exemplary nucleoside diphosphate kinase (ndk) can be ndk from Enterobacteriaceae (EC: 2.7.4.6, NCBI accession WP_000963837.1). Also contemplated for use in the engineered cells provided herein are ndk, having SEQ ID NO: 55, and ndk homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 54 - 55 and having the activity to catalyze the conversion of uridine diphosphate (UDP) to uridine triphosphate (UTP).

[0053] Adenylate kinase (adk) is an enzyme that catalyzes the interconversion of various adenosine phosphates (ATP, ADP, AMP). An exemplary adenylate kinase (adk) is adk derived from E. coli (EC: 2.7.4.3, NCBI accession AAC73576.1). Also contemplated for use in the engineered cells provided herein are adk, having SEQ ID NO: 57, and adk homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 56 - 57 and having the activity to catalyze the interconversion of various adenosine phosphates (ATP, ADP, and AMP).

[0054] Glucose - 6 - phosphate 1 - dehydrogenase (zwf) is an enzyme that catalyzes the oxidation of glucose 6 - phosphate to 6 - phosphoglucono - δ - lactone. An exemplary glucose - 6 - phosphate 1 - dehydrogenase (zwf) can be zwf derived from E. coli (EC: 1.1.1.49, NCBI accession UGE34297.1). Also contemplated for use in the engineered cells provided herein are zwf homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 58 and having the activity to catalyze the oxidation of glucose 6 - phosphate to 6 - phosphoglucono - δ - lactone.

[0055] 6-Phosphogluconate dehydrogenase (gnd) is an enzyme that catalyzes the oxidative decarboxylation of 6-phosphogluconate to ribose 5-phosphate and CO2, accompanied by the simultaneous reduction of NADP to NADPH. Exemplary 6-phosphogluconate dehydrogenase (gnd) can be gnd from Enterobacteriaceae (EC: 1.1.1.44, NCBI accession WP_000043484.1). Also contemplated for use in the engineered cells provided herein are gnd homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 59 and having the activity to catalyze the oxidative decarboxylation of 6-phosphogluconate to ribose-5-phosphate and CO2, accompanied by the simultaneous reduction of NADP to NADPH.

[0056] PRPP synthetase (prs) is an enzyme that catalyzes the conversion of ribose-5-phosphate to PRPP via the transfer of a pyrophosphoryl group from ATP to PRPP. Exemplary PRPP synthetase (prs) can be prs from Enterobacteriaceae (EC: 2.7.6.1, NCBI accession WP_001298109.1). Also contemplated for use in the engineered cells provided herein are prs homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 60 and having the activity to catalyze the conversion of ribose-5-phosphate to PRPP via the transfer of a pyrophosphoryl group from ATP to PRPP.

[0057] Sucrose permease (cscB) is responsible for the transport of sucrose into the cell. An exemplary sucrose permease (cscB) can be cscB (EC: 2.7.1.69, NCBI accession WP_001197025.1) from the Enterobacteriaceae family. Also contemplated for use in the engineered cells provided herein are cscB homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 61 and having the activity of transporting sucrose intracellularly.

[0058] Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and nucleoside diphosphate to fructose and nucleotide (NDP)-glucose. An exemplary sucrose synthase (SuSy) can be SuSy from Acidithiobacillus caldus (E.C. 2.4.1.13, NCBI accession WP_004872341.1). Also contemplated for use in the engineered cells provided herein are SuSy homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 62 and having the activity of the reversible conversion of sucrose and nucleoside diphosphate to fructose and nucleotide (NDP)-glucose.

[0059] Sucrose phosphorylase (SPase) catalyzes the reversible phosphorolytic cleavage of sucrose into alpha-D-glucose 1-phosphate (Glc1P) and D-fructose. An exemplary sucrose phosphorylase can be sucP from Bifidobacterium adolescentis (E.C. 2.4.1.7, NCBI accession WP_011742626.1). Also contemplated for use in the engineered cells provided herein are SPase homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 63 and having activity for the reversible phosphorolytic cleavage of sucrose into alpha-D-glucose 1-phosphate (Glc1P) and D-fructose.

[0060] UDP-glucose 4-epimerase (galE) is an enzyme that catalyzes the reversible conversion of UDP-galactose to UDP-glucose via a mechanism involving the transient reduction of NAD. An exemplary UDP-glucose 4-epimerase (galE) can be galE from E. coli (EC: 5.1.3.2, NCBI accession AAV80748.1). Also contemplated herein for overexpression, downregulation or deletion in the engineered cells provided herein are galE homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 64 and having activity for the reversible conversion of UDP-galactose to UDP-glucose via a mechanism involving the transient reduction of NAD.

[0061] UDP-glucose 6-dehydrogenase (ugd) is an enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. An exemplary UDP-glucose 6-dehydrogenase (ugd) can be ugd from E. coli (EC: 1.1.1.22, NCBI accession WP_089615770.1). Also contemplated for downregulation or deletion in the engineered cells provided herein are ugd homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 65 and having activity to catalyze the reversible conversion of UDP-glucose to UDP-glucuronic acid.

[0062] Glucose-1-phosphate adenylyltransferase (glgC) is an enzyme that catalyzes the conversion of glucose-1-phosphate to ADP-α-D-glucose. An exemplary glucose-1-phosphate adenylyltransferase (glgC) can be glgC from E. coli (EC: 2.7.7.27, NCBI accession WP_097472330.1). Also contemplated for use in downregulation or deletion in the engineered cells provided herein are glgC homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 66 and having activity to catalyze the reversible conversion of glucose-1-phosphate to ADP-α-D-glucose.

[0063] Glucan biosynthesis glucosyltransferase (OpgG) is an enzyme involved in the conversion of UDP-glucose to MDO. Exemplary glucan biosynthesis glucosyltransferase (OpgG) can be OpgG from Enterobacteriaceae (NCBI accession WP_001343212.1). Also contemplated for use in the engineered cells provided herein are OpgG homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 67 and having activity to catalyze the reversible conversion of UDP-glucose to MDO.

[0064] Glucose-6-phosphate isomerase (pgi) catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. Exemplary glucose-6-phosphate isomerase (pgi) can be pgi from E. coli (EC: 5.3.1.9, NCBI accession AAC76995.1). Also contemplated for downregulation or deletion in the engineered cells provided herein are pgi homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 68 and having activity to catalyze the reversible conversion of glucose-6-phosphate to fructose-6-phosphate.

[0065] UDP-sugar hydrolase (ushA) is an enzyme having UDP-sugar hydrolase activity. An exemplary UDP-sugar hydrolase (ushA) can be ushA from E. coli (EC: 3.1.3.5, NCBI accession WP_000771748.1). Also contemplated herein are ushA homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 69 and having activity to hydrolyze UDP-sugar for down-regulation or deletion in the engineered cells provided herein. Also contemplated herein are down-regulation or deletion in the engineered cells provided herein.

[0066] In addition to the modifications in the engineered cell lines discussed above, the present invention further includes engineered cells for producing UDP-galactose by overexpressing a nucleic acid sequence encoding UDP-glucose 4-epimerase (galE) activity, a nucleic acid sequence encoding galactokinase (galK) activity, and a nucleic acid sequence encoding galactose-1-phosphate uridyltransferase (galT).

[0067] Galactokinase (galK) catalyzes the formation of galactose-6-phosphate from galactose. An exemplary galactokinase (galK) can be galK from E. coli (EC: 2.7.1.6, NCBI accession WP_000053415.1). Also contemplated herein are galK homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 70 and having activity to catalyze the formation of galactose-6-phosphate from galactose for use in the engineered cells provided herein.

[0068] Galactose-1-phosphate uridylyltransferase (galT) catalyzes the formation of UDP-galactose from galactose-1-phosphate. An exemplary galactose-1-phosphate uridylyltransferase (galT) can be galT from E. coli (EC: 2.7.7.12, NCBI accession WP_000191497.1). Also contemplated for use in the engineered cells provided herein are galT homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 71 and having the activity to catalyze the formation of UDP-galactose from galactose-1-phosphate.

[0069] Fructokinase (cscK) phosphorylates fructose to produce fructose-6-phosphate. An exemplary fructokinase (cscK) can be cscK from E. coli (EC 2.7.1.4, accession WP_001274885.1). Also contemplated for use in the engineered cells provided herein are cscK homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 75 and having the activity to catalyze the formation of fructose-6-phosphate from fructose.

[0070] (Heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG) uses UDP-glucose as a substrate for lipopolysaccharide synthesis. An exemplary (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG) can be WaaG from E. coli (EC2.4.AAC76655.1). Also contemplated for use in the engineered cells provided herein are WaaG homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 76 and having activity to catalyze lipopolysaccharide synthesis from UDP-glucose.

[0071] Trehalose-6-phosphate synthase (otsA) catalyzes the biosynthesis of trehalose from UDP-glucose. An exemplary trehalose-6-phosphate synthase (otsA) can be otsA from E. coli (EC2.4.1.15, AAC74966.1). Also contemplated for use in the engineered cells provided herein are otsA homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 77 and having activity to catalyze lipopolysaccharide synthesis from UDP-glucose.

[0072] Glucose-1-phosphatase (agp) is a periplasmic protein that hydrolyzes phosphate from glucose-1-phosphate and other substrates. Exemplary glucose-1-phosphatase (agp) can be agp from E. coli (EC 3.1.3.10, accession AAC74087.1). Also contemplated for use in the engineered cells provided herein are agp homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 78 and having activity to hydrolyze phosphate from glucose-1-phosphate.

[0073] The present invention further provides an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of sucrose to uridine diphosphate glucose (UDP-glucose) by the engineered host cell. The engineered host cell comprises an exogenous nucleic acid sequence encoding a sucrose transporter (cscB) activity or a homolog thereof and an exogenous expressed nucleic acid sequence encoding a sucrose synthase (SuSy) activity or a homolog thereof. The engineered host cell further comprises, optionally, downregulation or deletion of UDP-glucose 4-epimerase (galE), downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd), downregulation or deletion of glucan biosynthesis glucosyltransferase (mdoA), downregulation or deletion of glucose-1-phosphate adenylyltransferase (glgC), and downregulation or deletion of glucose-6-phosphate isomerase (pgi), including but not limited to, genetic modifications. In certain embodiments, the engineered cell comprises a heterologous sucrose transporter (cscB) or a homolog thereof, a heterologous sucrose synthase (SuSy) or a homolog thereof, and (i) downregulation or deletion of UDP-glucose 4-epimerase (galE) or a homolog thereof, (ii) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd) or a homolog thereof, (iii) downregulation or deletion of glucan biosynthesis glucosyltransferase (mdoA) or a homolog thereof, and (iv) genetic modifications selected from the group consisting of any combination thereof. In certain embodiments, the modifications recited herein can be combined with the modifications recited in any other aspect of the present invention. In certain embodiments, the modifications recited herein can be combined with one or the modifications recited for the conversion of chemical intermediates to UDP-glucose.

[0074] Figure 2 of the present invention provides an overview of genetic modifications to an engineered host cell for increasing the production of UDP-sugars, including UDP-glucose and UDP-galactose.

[0075] As described herein, the present invention provides an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of a carbon source to UDP-sugar via a plurality of chemical intermediates by the engineered host cell. In certain embodiments, the UDP-sugar is glucose. In certain embodiments, the UDP-sugar is UDP-galactose. In certain embodiments, the chemical intermediate is UDP-glucose and the UDP-sugar is UDP-galactose. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the carbon source is selected from the group consisting of glycerol, glucose, sucrose, galactose, fructose, or any combination thereof.

[0076] As described in FIG. 2, one or more genetic modifications in the engineered host cell result in increased production of UDP-glucose. As described in FIG. 2, one or more genetic modifications in the engineered host cell that result in enhanced production of UDP-glucose are the overexpression of one or more genes selected from the group consisting of glucose facilitator gene (glf) or its homolog, glucokinase (glk) or its homolog, phosphoglucomutase (pgm) or its homolog, UTP-glucose-1-phosphate uridylyltransferase (galU) or its homolog, sucrose transporter (cscB) or its homolog, sucrose synthase (SuSy) or its homolog, sucrose phosphorylase (SPase) or its homolog, glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog; 6-phosphogluconate dehydrogenase (gnd) or its homolog; phosphoribosyl pyrophosphate synthetase (prs) or its homolog; phosphoribosyl transferase (pyrE) or its homolog; orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, uridylate kinase (pyrH) or its homolog, nucleoside diphosphate kinase (ndk) or its homolog, and adenylate kinase (adk) or its homolog; and / or the downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi), glucose-1-phosphate adenylyltransferase (glgC), UDP-glucose 6-dehydrogenase (ugd), glucan biosynthesis glucosyltransferase (OpgG), UDP-glucose 4-epimerase (galE), UDP-sugar hydrolase (ushA), UTP-glucose-1-phosphate uridylyltransferase (ugp), phosphoglucomutase (pgm); and / or any combination thereof. As described in FIG. 2, the engineered host cell is supplemented with a medium containing orotic acid, sucrose, glucose, or a combination thereof.

[0077] As described in Figure 2, one or more genetic modifications in the engineered host cell result in increased production of UDP-galactose. One or more genetic modifications in the engineered host cell that result in enhanced production of UDP-galactose are selected from the group consisting of: (i) overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof; (ii) overexpression of galactokinase (galK) or a homolog thereof; (iii) overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof; and (iv) any combination thereof. As described in Figure 2, the engineered host cell is supplemented with a medium containing galactose.

[0078] As described in Figure 2, one or more genetic modifications in an engineered host cell to increase the production of UDP-galactose include one or more genetic modifications that result in increased production of UDP-glucose.Thus, as depicted in Figure 2, one or more genetic modifications to increase the production of UDP-galactose are the overexpression of one or more genes selected from the group consisting of the glucose facilitator gene (glf) or its homolog, glucokinase (glk) or its homolog, phosphoglucomutase (pgm) or its homolog, UTP-glucose-1-phosphate uridylyltransferase (galU) or its homolog, sucrose transporter (cscB) or its homolog, sucrose synthase (SuSy) or its homolog, sucrose phosphorylase (SPase) or its homolog, glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog; 6-phosphogluconate dehydrogenase (gnd) or its homolog; phosphoribosyl pyrophosphate synthetase (prs) or its homolog; phosphoribosyl transferase (pyrE) or its homolog; orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, uridylic acid kinase (pyrH) or its homolog, nucleoside diphosphate kinase (ndk) or its homolog, and adenylate kinase (adk) or its homolog, UDP-glucose 4-epimerase (galE) or its homolog, overexpression of galactokinase (galK) or its homolog, overexpression of galactose-1-phosphate uridylyltransferase (galT) or its homolog; and / or downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi), glucose-1-phosphate adenylyltransferase (glgC), UDP-glucose 6-dehydrogenase (ugd), glucan biosynthesis glucosyltransferase (OpgG), UDP-sugar hydrolase (ushA), UTP-glucose-1-phosphate uridylyltransferase (ugp), phosphoglucomutase (pgm); and / or any combination thereof. As depicted in Figure 2, the engineered host cell is supplemented with a medium containing glucose, galactose, orotic acid, sucrose, or any combination thereof.

[0079] Figure 1 of the present specification provides an exemplary embodiment that illustrates a combination of modifications to an E. coli host genome that includes deletions and overexpressions of enzymes from other organisms to reproduce the biological production pathway described in that figure.

[0080] The present invention provides an engineered host cell comprising one or more genetic modifications (as shown in Figure 1, described in Example 1 herein, and recited herein in the present application) that result in the enzymatic conversion of glucose to UDP-glucose via a plurality of chemical intermediates by the engineered host cell. As shown in Figure 1, in certain embodiments, one or more of the genetic modifications result in an increased metabolic flux to UDP-glucose precursors. As shown in Figure 1, in certain embodiments, one or more of the genetic modifications cause a decrease in the formation of by-products. As shown in Figure 1, in certain embodiments, the genetic modifications are selected from the group consisting of: (i) the glucose facilitator gene (glf) or a homolog thereof; (ii) glucokinase (glk) or a homolog thereof; (iii) phosphoglucomutase (pgm) or a homolog thereof; (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof; (v) phosphoribosyltransferase (pyrE) or a homolog thereof; (vi) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof; (vii) uridylate kinase (pyrH) or a homolog thereof; (viii) nucleoside diphosphate kinase (ndk) or a homolog thereof; (ix) adenylate kinase (adk) or a homolog thereof; and (x) any combination thereof. In certain embodiments, one or more of the genetic modifications is an overexpression of fructokinase (cscK) or a homolog thereof.

[0081] As shown in FIG. 1, in certain embodiments, one or more genetic modifications are overexpression of one or more genes selected from the group consisting of: (i) a glucose facilitator gene (glf) or a homolog thereof; (ii) a glucokinase (glk) or a homolog thereof; (iii) a phosphoglucomutase (pgm) or a homolog thereof; (iv) a UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof; (v) a glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof; (vi) a 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof; (vii) a phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof; and (viii) any combination thereof.

[0082] As shown in FIG. 1, in certain embodiments, one or more genetic modifications are overexpression of: (i) a glucose facilitator gene (glf) or a homolog thereof; (ii) a glucokinase (glk) or a homolog thereof; and (iii) any combination thereof, selected from the group consisting of. In certain embodiments, one or more genetic modifications are overexpression of a phosphoglucomutase (pgm) or a homolog thereof. In certain embodiments, one or more genetic modifications are overexpression of a UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof.

[0083] As shown in FIG. 1, in certain embodiments, one or more genetic modifications are overexpression of an orotate phosphoribosyltransferase (pyrE) or a homolog thereof. In certain embodiments, one or more genetic modifications are overexpression of an orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof. In certain embodiments, a medium containing orotate is also provided. In certain embodiments, one or more genetic modifications are overexpression of a uridylic acid kinase (pyrH) or a homolog thereof.

[0084] As shown in FIG. 1, in certain embodiments, one or more genetic modifications are overexpression of nucleoside diphosphate kinase (ndk) or its homolog. In certain embodiments, one or more genetic modifications are overexpression of adenylate kinase (adk) or its homolog.

[0085] As shown in FIG. 1, in certain embodiments, one or more genetic modifications are downregulation or deletion of UDP-glucose 4-epimerase (galE). As shown in FIG. 1, in certain embodiments, one or more genetic modifications are downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd) to prevent the conversion of UDP-glucose to UDP-glucuronic acid.

[0086] As shown in FIG. 1, in certain embodiments, one or more genetic modifications are downregulation or deletion of glucose-1-phosphate adenylyltransferase (glgC).

[0087] As shown in FIG. 1, in certain embodiments, one or more genetic modifications are downregulation or deletion of glucan biosynthesis glucosyltransferase (OpgG) to prevent the conversion of UDP-glucose to membrane-derived oligosaccharide (MDO).

[0088] As shown in FIG. 1, in certain embodiments, one or more genetic modifications are downregulation or deletion of glucose-6-phosphate isomerase (pgi).

[0089] As shown in FIG. 1, in certain embodiments, one or more genetic modifications are overexpression of glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog.

[0090] As shown in Figure 1, in certain embodiments, one or more genetic modifications are overexpression of 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof.

[0091] As shown in Figure 1, in certain embodiments, one or more genetic modifications are overexpression of phosphoribosylpyrophosphate synthetase (prs) or a homolog thereof.

[0092] As shown in Figure 1, in certain embodiments, the engineered host cell comprises overexpression of (i) glucose facilitator gene (glf) or a homolog thereof, (ii) glucokinase (glk) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) phosphoribosyltransferase (pyrE) or a homolog thereof, (vi) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (vii) uridylic acid kinase (pyrH) or a homolog thereof, (viii) nucleoside diphosphate kinase (ndk) or a homolog thereof, and (ix) adenylate kinase (adk) or a homolog thereof.

[0093] As shown in Figure 1, in certain embodiments, the engineered host cell comprises overexpression of (i) glucose facilitator gene (glf) or a homolog thereof, (ii) glucokinase (glk) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof, (vi) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof, and (vii) phosphoribosylpyrophosphate synthetase (prs) or a homolog thereof.

[0094] Here, aspects of the present invention will be described with reference to FIG. 1.

[0095] Step 1: Conversion of glucose to glucose-6-phosphate. The glucose facilitator gene (glf) or its homolog, and glucokinase (glk) or its homolog are overexpressed. Additionally, a phosphotransferase enzyme or its homolog may be overexpressed.

[0096] Step 2: Conversion of glucose-6-phosphate to glucose-1-phosphate. Phosphoglucomutase (pgm) or its homolog is overexpressed.

[0097] Step 3: Conversion of glucose-1-phosphate and UTP to UDP-glucose. UTP-glucose-1-phosphate-uridylyltransferase (galU) or its homolog is overexpressed.

[0098] Step 4: Conversion of 5-phosphoribosyl 1-pyrophosphate (PRPP) to uridine monophosphate (UMP). Orotate phosphoribosyltransferase (pyrE) or its homolog and orotidine 5'-phosphate decarboxylase (pyrF) or its homolog are overexpressed. In certain embodiments, this step includes supplementation with orotic acid.

[0099] Step 5: Conversion of uridine monophosphate (UMP) to uridine diphosphate (UDP). Uridylate kinase (pyrH) or its homolog is overexpressed.

[0100] Step 6: Conversion of uridine diphosphate (UDP) to uridine triphosphate (UTP). Nucleoside diphosphate kinase (ndk) or its homolog and / or adenylate kinase (adk) or its homolog are overexpressed.

[0101] Step 7: Conversion of UDP-glucose to UDP-galactose. UDP-glucose 4-epimerase (galE) is downregulated or deleted to prevent the production of UDP-galactose.

[0102] Step 8: Conversion of UDP-glucose to UDP-glucuronic acid. UDP-glucose 6-dehydrogenase (ugd) is downregulated or deleted to prevent the conversion of UDP-glucose to UDP-glucuronic acid.

[0103] Step 9: Conversion of glucose-1-phosphate to ADP-α-D-glucose Glucose-1-phosphate adenylyltransferase (glgC) is downregulated or deleted to prevent the conversion of UDP-glucose to ADP-α-D-glucose.

[0104] Step 10: Conversion of UDP-glucose to membrane-derived oligosaccharide (MDO). Glucan biosynthesis glucosyltransferase (OpgG) is downregulated or deleted to prevent the conversion of UDP-glucose to membrane-derived oligosaccharide (MDO), including the conversion to ADP-α-D-glucose.

[0105] Step 11: Conversion of glucose-6-phosphate to fructose-6-phosphate. Glucose-6-phosphate isomerase (pgi) is downregulated or deleted to prevent glucose from being metabolized via the glycolytic pathway.

[0106] Step 12: Conversion of glucose-6-phosphate to 6-phosphogluconic acid. Glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog can be overexpressed.

[0107] Step 13: Conversion of 6-phosphogluconic acid to ribose-5-phosphate (R5P). 6-Phosphogluconate dehydrogenase (gnd) or its homolog can be overexpressed.

[0108] Step 14: Conversion of ribose-5-phosphate (R5P) to 5-phosphoribosyl 1-pyrophosphate (PRPP). Phosphoribosyl pyrophosphate synthetase (prs) or its homolog can be overexpressed.

[0109] In certain preferred embodiments, the engineered host cell comprises one or more genetic modifications listed above in steps 1-3 and 4-6. In these preferred embodiments, the engineered host cell may comprise overexpression of (i) the glucose facilitator gene (glf) or a homolog thereof, (ii) glucokinase (glk) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) phosphoribosyltransferase (pyrE) or a homolog thereof, (vi) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (vii) uridylic acid kinase (pyrH) or a homolog thereof, (viii) nucleoside diphosphate kinase (ndk) or a homolog thereof, and (ix) adenylate kinase (adk) or a homolog thereof.

[0110] In certain preferred embodiments, the engineered cell line comprises one or more genetic modifications listed above in steps 1-3 and 12-14. In these preferred embodiments, the engineered host cell may comprise overexpression of (i) the glucose facilitator gene (glf) or a homolog thereof, (ii) glucokinase (glk) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof, (vi) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof, and (vii) phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof.

[0111] In certain preferred embodiments, the engineered cell line comprises one or more genetic modifications as listed above in steps 1-3, 4-6 and 12-14. In these preferred embodiments, the engineered host cell may comprise overexpression of (i) a glucose facilitator gene (glf) or a homolog thereof, (ii) glucokinase (glk) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) phosphoribosyltransferase (pyrE) or a homolog thereof, (vi) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (vii) uridylic acid kinase (pyrH) or a homolog thereof, (viii) nucleoside diphosphate kinase (ndk) or a homolog thereof, (ix) adenylate kinase (adk) or a homolog thereof, (x) glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof, (xi) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof, and (xii) phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof.

[0112] In another aspect, the present invention provides a method for increasing the production of uridine diphosphate glucose (UDP-glucose) by utilizing the engineered host cells described in this example.

[0113] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications (as shown in, described in, and recited herein in the present application as described above in FIG. 1) that result in the enzymatic conversion of glucose and / or galactose to UDP-galactose via multiple chemical intermediates by the engineered host cell.

[0114] As shown in Figure 1, in another aspect, the present invention provides an engineered host cell that results in the enzymatic conversion of glucose and / or galactose to UDP-galactose via a plurality of intermediates by the engineered host cell (as shown in Figure 1, described in Example 1 herein, and recited above in this application), comprising one or more genetic modifications. As shown in Figure 1, in certain embodiments, in addition to all of the genetic modifications listed above, one or more of the genetic modifications results in an increased metabolic flux to the UDP-galactose precursor. As shown in Figure 1, in certain embodiments, one or more of the genetic modifications causes a reduction in the formation of by-products.

[0115] As shown in Figure 1, in certain embodiments, one or more of the genetic modifications is selected from the group consisting of: (i) overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof; (ii) overexpression of galactokinase (galK) or a homolog thereof; (iii) overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof; and (iv) any combination thereof.

[0116] As shown in Figure 1, in certain embodiments, one or more of the genetic modifications is overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof.

[0117] As shown in Figure 1, in certain embodiments, one or more of the genetic modifications is overexpression of galactokinase (galK) or a homolog thereof. In certain embodiments, a supplemental substance comprising galactose is also provided.

[0118] As shown in Figure 1, in certain embodiments, one or more of the genetic modifications is overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof.

[0119] As shown in Figure 1, in another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of glucose to UDP-glucose via multiple intermediates by the engineered host cell (as shown in Figure 1, described in Example 1 herein, and recited hereinabove in the present application).

[0120] Here, aspects of the present invention will be described with reference to Figure 1.

[0121] Step 1: Conversion of glucose to glucose-6-phosphate. The glucose facilitator gene (glf) or its homolog and / or glucokinase (glk) or its homolog are overexpressed. Additionally, a phosphotransferase enzyme or its homolog may be overexpressed.

[0122] Step 2: Conversion of glucose-6-phosphate to glucose-1-phosphate. Phosphoglucomutase (pgm) or its homolog is overexpressed.

[0123] Step 3: Conversion of glucose-1-phosphate and UTP to UDP-glucose. UTP-glucose-1-phosphate uridylyltransferase (galU) or its homolog is overexpressed.

[0124] Step 4: Conversion of 5-phosphoribosyl 1-pyrophosphate (PRPP) to uridine monophosphate (UMP). Orotate phosphoribosyltransferase (pyrE) or its homolog and / or orotidine 5'-phosphate decarboxylase (pyrF) or its homolog are overexpressed. In certain embodiments, a supplemental substance containing orotic acid is also provided.

[0125] Step 5: Conversion of uridine monophosphate (UMP) to uridine diphosphate (UDP). Uridylate kinase (pyrH) or its homolog is overexpressed.

[0126] Step 6: Conversion of uridine diphosphate (UDP) to uridine triphosphate (UTP).

[0127] Nucleoside diphosphate kinase (ndk) or its homolog and / or adenylate kinase (adk) or its homolog is overexpressed.

[0128] Step 7: Conversion of UDP-glucose to UDP-galactose. UDP-glucose 4-epimerase (galE) or its homolog is overexpressed.

[0129] Step 8: Conversion of UDP-glucose to UDP-glucuronic acid. UDP-glucose 6-dehydrogenase (ugd) is downregulated or deleted to prevent the production of UDP-glucuronic acid.

[0130] Step 9: Conversion of glucose-1-phosphate to ADP-α-D-glucose. Glucose-1-phosphate adenylyltransferase (glgC) is downregulated or deleted to prevent the production of UDP-glucuronic acid.

[0131] Step 10: Conversion of UDP-glucose to membrane-derived oligosaccharide (MDO). Glucan biosynthesis glucosyltransferase (OpgG) is downregulated or deleted to prevent the formation of membrane-derived oligosaccharide (MDO) containing ADP-α-D-glucose.

[0132] Step 11: Conversion of glucose-6-phosphate to fructose-6-phosphate. Glucose-6-phosphate isomerase (pgi) is downregulated or deleted to prevent glucose from being metabolized via the glycolytic pathway.

[0133] Step 12: Conversion of glucose-6-phosphate to 6-phosphogluconic acid. Glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog may be overexpressed.

[0134] Step 13: Conversion of 6-phosphogluconate to ribose-5-phosphate (R5P). 6-Phosphogluconate dehydrogenase (gnd) or its homolog can be overexpressed.

[0135] Step 14: Conversion of ribose-5-phosphate (R5P) to 5-phosphoribosyl 1-pyrophosphate (PRPP). Phosphoribosyl pyrophosphate synthetase (prs) or its homolog can be overexpressed.

[0136] Step 15: Conversion of galactose to galactose-1-phosphate. Galactokinase (galK) or its homolog is overexpressed. In certain embodiments, a supplemental substance containing galactose is also provided.

[0137] Step 16: Conversion of galactose-1-phosphate to UDP-galactose. Galactose-1-phosphate uridylyltransferase (galT) or its homolog is overexpressed.

[0138] In certain preferred embodiments, the engineered host cell comprises one or more of the genetic modifications recited above in steps 1-3, 4-7 and 15-16. In these preferred embodiments, the engineered host cell has overexpression of (i) the glucose facilitator gene (glf) or a homolog thereof, (ii) glucokinase (glk) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) orotate phosphoribosyltransferase (pyrE) or a homolog thereof, (vi) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (vii) uridylic acid kinase (pyrH) or a homolog thereof, (viii) nucleoside diphosphate kinase (ndk) or a homolog thereof, (ix) adenylate kinase (adk) or a homolog thereof, (x) overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof, (xi) overexpression of galactokinase (galK) or a homolog thereof, and (xii) overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof may be included.

[0139] In certain preferred embodiments, the engineered cell line comprises one or more of the genetic modifications listed above in steps 1-3, 7 and 12-16. In these preferred embodiments, the engineered host cell comprises overexpression of (i) a glucose facilitator gene (glf) or a homolog thereof, (ii) glucokinase (glk) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof, (vi) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof, (vii) phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof, (viii) UDP-glucose 4-epimerase (galE) or a homolog thereof, (ix) galactokinase (galK) or a homolog thereof, and (x) galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof. In certain embodiments, a supplemental substance comprising galactose is also provided.

[0140] In certain preferred embodiments, the engineered cell line comprises one or more of the genetic modifications listed above in steps 7, 15 and 16. In these preferred embodiments, the engineered host cell comprises overexpression of (i) UDP-glucose 4-epimerase (galE or a homolog thereof), (ii) galactokinase (galK) or a homolog thereof, and (iii) galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof. In certain embodiments, a supplemental substance comprising galactose is also provided.

[0141] In another aspect, the present invention provides a method for increasing the production of uridine diphosphate galactose (UDP-galactose) by utilizing the engineered host cell described in this example.

[0142] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of sucrose to UDP-glucose via multiple chemical intermediates by the engineered host cell (as shown in FIG. 1 and described hereinabove in the present application).

[0143] In certain embodiments, the genetic modifications in the engineered host cell are selected from the group consisting of: (i) heterologous expression of a sucrose transporter (cscB) or a homolog thereof; (ii) heterologous expression of a sucrose synthase (SuSy) or a homolog thereof; (iii) downregulation or deletion of UDP-glucose 4-epimerase (galE); (iv) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd); (v) downregulation or deletion of a glucan biosynthesis glucosyltransferase (OpgG); and (vi) any combination thereof.

[0144] In certain embodiments, the engineered host cell comprises a heterologous sucrose transporter (cscB), a heterologous sucrose synthase (SuSy), and genetic modifications selected from the group consisting of: (i) downregulation or deletion of UDP-glucose 4-epimerase (galE); (ii) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd); (iii) downregulation or deletion of a glucan biosynthesis glucosyltransferase (OpgG); and (iv) any combination thereof.

[0145] As shown in FIG. 1, in another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications (as shown in FIG. 1, described in Example 1 herein, and described hereinabove in this application) that result in the enzymatic conversion of sucrose to UDP-glucose by the engineered host cell. Further, as described in FIG. 1, the present invention provides that the engineered host cell results in the enzymatic conversion of sucrose to UDP-glucose via sucrose phosphorylase (SPase) or a homolog thereof and / or sucrose synthase (SuSy) or a homolog thereof. Accordingly, the present invention provides for the overexpression of sucrose phosphorylase (SPase) or a homolog thereof and / or sucrose synthase (SuSy) or a homolog thereof to increase the conversion of sucrose to UDP-glucose.

[0146] Here, aspects of the present invention will be described with reference to FIG. 1.

[0147] Step 1: Conversion of glucose to glucose-6-phosphate. The glucose facilitator gene (glf) or a homolog thereof, and / or the glucokinase gene (glk) or a homolog thereof is overexpressed. Further, a phospho transferase enzyme or a homolog thereof may be overexpressed.

[0148] Step 2: Conversion of glucose-6-phosphate to glucose-1-phosphate. Phosphoglucomutase (pgm) or a homolog thereof is overexpressed.

[0149] Step 3: Conversion of glucose-1-phosphate and UTP to UDP-glucose. UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof is overexpressed.

[0150] Step 4: Conversion of 5-phosphoribosyl 1-pyrophosphate (PRPP) to uridine monophosphate (UMP). Orotidine phosphoribosyl transferase (pyrE) or its homolog and / or orotidine 5'-phosphate decarboxylase (pyrF) are overexpressed. In certain embodiments, a supplemental substance containing orotic acid is also provided.

[0151] Step 5: Conversion of uridine monophosphate (UMP) to uridine diphosphate (UDP). Uridylate kinase (pyrH) or its homolog is overexpressed.

[0152] Step 6: Conversion of uridine diphosphate (UDP) to uridine triphosphate (UTP).

[0153] Nucleoside diphosphate kinase (ndk) or its homolog and / or adenylate kinase (adk) or its homolog are overexpressed.

[0154] Step 7: Conversion of UDP-glucose to UDP-galactose. UDP-glucose 4-epimerase (GalE) is downregulated or deleted to prevent the conversion of UDP-glucose to UDP-galactose.

[0155] Step 8: Conversion of UDP-glucose to UDP-glucuronic acid. UDP-glucose 6-dehydrogenase (ugd) is downregulated or deleted to prevent the conversion of UDP-glucose to UDP-glucuronic acid.

[0156] Step 9: Conversion of glucose-1-phosphate to ADP-α-D-glucose. Glucose-1-phosphate adenylyltransferase (glgC) is downregulated or deleted to prevent the conversion (converstion) of UDP-glucose to UDP-glucuronic acid.

[0157] Step 10: Conversion of UDP-glucose to membrane-derived oligosaccharide (MDO). The glucan biosynthesis glucosyltransferase (OpgG) is downregulated or deleted to prevent the conversion of UDP-glucose to membrane-derived oligosaccharide (MDO), including the conversion to ADP-α-D-glucose.

[0158] Step 11: Conversion of glucose-6-phosphate to fructose-6-phosphate. The glucose-6-phosphate isomerase (pgi) is downregulated or deleted to prevent glucose from being metabolized via the glycolytic pathway.

[0159] Step 12: Conversion of glucose-6-phosphate to 6-phosphogluconic acid. The glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog can be overexpressed.

[0160] Step 13: Conversion of 6-phosphogluconic acid to ribose-5-phosphate (R5P). The 6-phosphogluconate dehydrogenase (gnd) or its homolog can be overexpressed.

[0161] Step 14: Conversion of ribose-5-phosphate (R5P) to 5-phosphoribosyl 1-pyrophosphate (PRPP).

[0162] The phosphoribosyl pyrophosphate synthetase (prs) or its homolog can be overexpressed.

[0163] In another aspect, the present invention provides a method for increasing the production of uridine diphosphate glucose (UDP-glucose) by using the engineered host cells described in this example.

[0164] The following Table 1 provides a list of the sequences of exemplary enzymes of the present invention. Table 1: Exemplary Enzyme Sequences of the Present Invention

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

[0165] References and citations to other documents such as patents, patent applications, patent gazettes, academic journals, books, papers, web content, publicly accessible databases, etc. are made throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes. Equivalents

[0166] In addition to what is shown and described herein, various modifications of the invention and many further embodiments thereof will become apparent to those skilled in the art from the entire contents of this document, including references to scientific and patent literature cited herein. The subject matter of this specification, in its various embodiments and their equivalents, includes important information, exemplification and guidance that can be adapted for the practice of the invention.

Claims

**Claim 1** An engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of a carbon source to UDP-sugar via a plurality of chemical intermediates by the engineered host cell. **Claim 2** The engineered host cell according to claim 1, wherein the UDP-sugar is UDP-glucose. **Claim 3** The engineered host cell according to claim 1, wherein the UDP-sugar is UDP-galactose. **Claim 4** The engineered host cell according to claim 1, wherein the chemical intermediate is UDP-glucose and the UDP-sugar is UDP-galactose. **Claim 5** The engineered host cell according to claim 1, wherein the engineered host cell is E. coli. **Claim 6** The engineered host cell according to claim 2, wherein the one or more genetic modifications is overexpression of one or more genes selected from the group consisting of (i) glucose facilitator gene (glf) or its homolog, (ii) glucokinase (glk) or its homolog, (iii) phosphoglucomutase (pgm) or its homolog, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or its homolog, (v) fructokinase (cscK) or its homolog, and (vi) any combination thereof. **Claim 7** The engineered host cell according to claim 2, wherein the one or more genetic modifications is overexpression of one or more genes selected from the group consisting of (i) sucrose transporter (cscB) or its homolog, (ii) sucrose synthase (SuSy) or its homolog, (iii) sucrose phosphorylase (SPase) or its homolog, and (iv) any combination thereof. **Claim 8** One or more genetic modifications are downregulation or deletion of one or more genes selected from the group consisting of (i) glucose-6-phosphate isomerase (pgi), (ii) glucose-1-phosphate adenylyltransferase (glgC), (iii) UDP-glucose-6-dehydrogenase (ugd), (iv) glucan biosynthesis glucosyltransferase (OpdG), (v) UDP-glucose-4-epimerase (galE), (vi) UDP-sugar hydrolase (ushA), (vii) UTP-glucose-1-phosphate uridylyltransferase (ugp), (viii) phosphoglucomutase (pgm), (ix) (heptosyl) lipopolysaccharide α-1,3-glucosyltransferase (WaaG), (x) trehalose-6-phosphate synthase (otsA), (xi) glucose-1-phosphatase (agp), and (xii) any combination thereof, the engineered host cell according to claim 2, 6 or 7.

9. The engineered host cell according to claims 6-8, wherein the cell is supplemented with glucose, fructose and / or sucrose.

10. One or more genetic modifications are overexpression of one or more genes selected from the group consisting of (i) glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog; (ii) 6-phosphogluconate dehydrogenase (gnd) or its homolog; (iii) phosphoribosylpyrophosphate synthetase (prs) or its homolog; (iv) phosphoribosyltransferase (pyrE) or its homolog; (v) orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, (vi) uridylic acid kinase (pyrH) or its homolog, (vii) nucleoside diphosphate kinase (ndk) or its homolog, and (viii) adenylate kinase (adk) or its homolog, and (ix) any combination thereof, the engineered host cell according to claims 2-4.

11. The engineered host cell according to claims 2-4, wherein the engineered host cell is supplemented with orotic acid.

12. One or more of the one or more genetic modifications are Overexpression of one or more genes selected from the group consisting of glucose facilitator gene (glf) or its homolog, glucokinase (glk) or its homolog, phosphoglucomutase (pgm) or its homolog, UTP-glucose-1-phosphate uridylyltransferase (galU) or its homolog, sucrose transporter (cscB) or its homolog, sucrose synthase (SuSy) or its homolog, sucrose phosphorylase (SPase) or its homolog, glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog; 6-phosphogluconate dehydrogenase (gnd) or its homolog; phosphoribosyl pyrophosphate synthetase (prs) or its homolog; phosphoribosyl transferase (pyrE) or its homolog; orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, uridylic acid kinase (pyrH) or its homolog, nucleoside diphosphate kinase (ndk) or its homolog, (viii) adenylate kinase (adk) or its homolog, and (ix) fructokinase (cscK) or its homolog; and / or Downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi), glucose-1-phosphate adenylyltransferase (glgC), UDP-glucose 6-dehydrogenase (ugd), glucan biosynthesis glucosyltransferase (OpgG), UDP-glucose 4-epimerase (galE), UDP-sugar hydrolase (ushA), UTP-glucose-1-phosphate uridylyltransferase (ugp), phosphoglucomutase (pgm), (heptosyl) lipopolysaccharide α-1,3-glucosyltransferase (WaaG), trehalose-6-phosphate synthase (otsA), glucose-1-phosphatase (agp); and / or Any combination thereof The engineered host cell according to claim 2, selected from

13. The engineered cell according to claim 2, wherein the engineered host cell is supplemented with a medium containing orotic acid, fructose, glucose and / or sucrose.

14. The one or more genetic modifications are selected from the group consisting of (i) overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof, (ii) overexpression of galactokinase (galK) or a homolog thereof, (iii) overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof, and (iv) any combination thereof, for the engineered host cell according to claim 3 or 4.

15. The engineered host cell according to claim 3, 4 or 14, wherein the engineered host cell is supplemented with a medium containing galactose.

16. The one or more genetic modifications are glucose facilitator gene (glf) or a homolog thereof, glucokinase (glk) or a homolog thereof, phosphoglucomutase (pgm) or a homolog thereof, UTP-glucose-1-phosphate-uridylyltransferase (galU) or a homolog thereof, sucrose transporter (cscB) or a homolog thereof, sucrose synthase (SuSy) or a homolog thereof, sucrose phosphorylase (SPase) or a homolog thereof, glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof; 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof; phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof; phosphoribosyl transferase (pyrE) or a homolog thereof; orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, uridylic acid kinase (pyrH) or a homolog thereof, nucleoside diphosphate kinase (ndk) or a homolog thereof, and (viii) adenylate kinase (adk) or a homolog thereof; overexpression of one or more genes selected from the group consisting of overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof, overexpression of galactokinase (galK) or a homolog thereof, overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof, fructokinase (cscK) or a homolog thereof; and / or Downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi), glucose-1-phosphate adenylyltransferase (glgC), UDP-glucose 6-dehydrogenase (ugd), glucan biosynthesis glucosyltransferase (OpG), UDP-sugar hydrolase (ushA), UTP-glucose-1-phosphate uridylyltransferase (ugp), phosphoglucomutase (pgm), (heptosyl) lipopolysaccharide α-1,3-glucosyltransferase (WaaG), trehalose-6-phosphate synthase (otsA), glucose-1-phosphatase (agp); and / or any combination thereof The engineered host cell according to claim 3, 4 or 14, selected from

17. The engineered host cell according to claim 3, 4, 14 or 16, wherein the engineered host cell is supplemented with a medium containing galactose, orotic acid, glucose, fructose and / or sucrose.

18. The engineered host cell according to claim 1, wherein the carbon source is selected from the group consisting of (i) glycerol, (ii) glucose, (iii) sucrose, (iv) galactose, (v) fructose, and (vi) any combination thereof.

19. A method for increasing the production of UDP-sugar, comprising providing an engineered host cell comprising one or more genetic modifications that result in the enzymatic conversion of a carbon source to UDP-sugar via a plurality of chemical intermediates by the engineered host cell.

20. The method according to claim 19, wherein the UDP-sugar is glucose.

21. The method according to claim 19, wherein the UDP-sugar is UDP-galactose.

22. The method according to claim 19, wherein the chemical intermediate is UDP-glucose and the UDP-sugar is UDP-galactose.

23. The method according to claim 19, wherein the engineered host cell is E. coli.

24. The method according to claim 20, wherein said one or more genetic modifications is overexpression of one or more genes selected from the group consisting of (i) a glucose facilitator gene (glf) or a homolog thereof, (ii) glucokinase (glk) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridylyltransferase (galU) or a homolog thereof, (v) fructokinase (cscK) or a homolog thereof, and (vi) any combination thereof.

25. The method according to claim 20, wherein said one or more genetic modifications is overexpression of one or more genes selected from the group consisting of (i) a sucrose transporter (cscB) or a homolog thereof, (ii) sucrose synthase (SuSy) or a homolog thereof, (iii) sucrose phosphorylase (SPase), and (iv) any combination thereof.

26. The method according to claim 20, 24 or 25, wherein said one or more genetic modifications is downregulation or deletion of one or more genes selected from the group consisting of (i) glucose-6-phosphate isomerase (pgi); (ii) glucose-1-phosphate adenylyltransferase (glgC); (iii) UDP-glucose 6-dehydrogenase (ugd); (iv) glucan biosynthesis glucosyltransferase (OpgG); (v) UDP-glucose 4-epimerase (galE); (vi) UDP-sugar hydrolase (ushA); (vii) UTP-glucose-1-phosphate uridylyltransferase (ugp), (viii) phosphoglucomutase (pgm), (ix) (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG), (x) trehalose-6-phosphate synthase (otsA), (xi) glucose-1-phosphatase (agp), and (xii) any combination thereof.

27. The method according to claim 24 to 26, wherein said cells are supplemented with glucose, fructose and / or sucrose.

28. The one or more genetic modifications described above are (i) glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog; (ii) 6-phosphogluconate dehydrogenase (gnd) or its homolog; (iii) phosphoribosyl pyrophosphate synthetase (prs) or its homolog; (iv) phosphoribosyl transferase (pyrE) or its homolog; (v) orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, (vi) uridylic acid kinase (pyrH) or its homolog, (vii) nucleoside diphosphate kinase (ndk) or its homolog, and (viii) adenylate kinase (adk) or its homolog, and (ix) overexpression of one or more genes selected from the group consisting of any combination thereof, according to the method described in claims 20-22.

29. The method described in claims 20-22, wherein the engineered host cell is supplemented with orotic acid.

30. The one or more genetic modifications described above are glucose facilitator gene (glf) or its homolog, glucokinase (glk) or its homolog, phosphoglucomutase (pgm) or its homolog, UTP-glucose-1-phosphate-uridylyltransferase (galU) or its homolog, sucrose transporter (cscB) or its homolog, sucrose synthase (SuSy) or its homolog, sucrose phosphorylase (SPase) or its homolog, glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog; 6-phosphogluconate dehydrogenase (gnd) or its homolog; phosphoribosyl pyrophosphate synthetase (prs) or its homolog; phosphoribosyl transferase (pyrE) or its homolog; orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, uridylic acid kinase (pyrH) or its homolog, nucleoside diphosphate kinase (ndk) or its homolog, (viii) adenylate kinase (adk) or its homolog, and fructokinase (cscK) or its homolog, overexpression of one or more genes selected from the group consisting of; and / or Downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi); glucose-1-phosphate adenylyltransferase (glgC); UDP-glucose 6-dehydrogenase (ugd); glucan biosynthesis glucosyltransferase (OpdG); UDP-glucose 4-epimerase (galE); UDP-sugar hydrolase (ushA); UTP-glucose-1-phosphate uridylyltransferase (ugp), (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG), trehalose-6-phosphate synthase (otsA), glucose-1-phosphatase (agp); and / or any combination thereof The method according to claim 20, selected from

31. The method according to claim 20, wherein the engineered host cell is supplemented with a medium containing orotic acid, glucose, fructose and / or sucrose.

32. The method according to claim 21 or 22, wherein the one or more genetic modifications are selected from the group consisting of (i) overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof, (ii) overexpression of galactokinase (galK) or a homolog thereof, (iii) overexpression of galactose-1-phosphate uridylyltransferase (galT) or a homolog thereof, and (iv) any combination thereof.

33. The method according to claim 21, 22 or 32, wherein the engineered host cell is supplemented with a medium containing galactose.

34. The one or more genetic modifications are Overexpression of one or more genes selected from the group consisting of glucose facilitator gene (glf) or its homolog, glucokinase (glk) or its homolog, phosphoglucomutase (pgm) or its homolog, UTP-glucose-1-phosphate uridylyltransferase (galU) or its homolog, sucrose transporter (cscB) or its homolog, sucrose synthase (SuSy) or its homolog, sucrose phosphorylase (SPase) or its homolog, glucose-6-phosphate 1-dehydrogenase (zwf) or its homolog; 6-phosphogluconate dehydrogenase (gnd) or its homolog; phosphoribosyl pyrophosphate synthetase (prs) or its homolog; phosphoribosyl transferase (pyrE) or its homolog; orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, uridylic acid kinase (pyrH) or its homolog, nucleoside diphosphate kinase (ndk) or its homolog, and (viii) adenylate kinase (adk) or its homolog; overexpression of UDP-glucose 4-epimerase (galE) or its homolog, overexpression of galactokinase (galK) or its homolog, overexpression of galactose-1-phosphate uridylyltransferase (galT) or its homolog, overexpression of fructokinase (cscK) or its homolog; and / or Downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi); glucose-1-phosphate adenylyltransferase (glgC); UDP-glucose 6-dehydrogenase (ugd); glucan biosynthesis glucosyltransferase (OpgG); UDP-glucose 4-epimerase (galE); UDP-sugar hydrolase (ushA); UTP-glucose-1-phosphate uridylyltransferase (ugp); (heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG); trehalose-6-phosphate synthase (otsA), glucose-1-phosphatase (agp); and / or Any combination thereof The method according to claim 21, 22 or 32, selected from **Claim 35** The method according to claim 21, 22, 32 or 34, wherein the manipulated host cell is supplemented with a medium containing galactose, orotic acid, sucrose, fructose and / or glucose. **Claim 36** The method according to claim 19, wherein the carbon source is selected from the group consisting of (i) glycerol, (ii) glucose, (iii) sucrose, (iv) galactose, (v) fructose, and (v) any combination thereof.

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