UDP-saccharide bioproduction using microbial hosts

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 of producing UDP-sugars using these engineered cell lines.

Description

[0001] Technical Field

[0002] The present invention relates to materials including engineered cells and cell lines, and methods involving the production of uridine diphosphate (UDP) sugars, including UDP-glucose and UDP-galactose. Background Art

[0003] Nucleotide sugars are key precursors for all glycosylation reactions and are required for both oligosaccharide and polysaccharide synthesis and protein and lipid glycosylation. Of 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 galactitol, which is used in the synthesis of polysaccharides together with sucrose.

[0004] 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. In addition, uridine-5'-diphosphate-glucose (UDP-glucose) is also a very important molecule in the fields of biology, food, biopharmaceuticals and cosmetic chemistry. It is one of the key precursors in the sugar interconversion to form disaccharides and polysaccharides and amino and nucleotide sugar metabolism. In addition, UDP-glucose can also be used as the source of other compounds (such as antibiotics) with industrial value.

[0005] Several chemical methods have been proposed for the synthesis of UDP-glucose, but these methods have problems with reactivity and selectivity, and usually require modification of functional groups to protect residues in the sugar molecule that should not react, while exposing the groups that should react. In addition, these chemical reactions usually require expensive catalysts and organic solvents. Therefore, the chemical synthesis of UDP-glucose is neither cost-effective nor environmentally friendly. Summary of the invention

[0006] In order to get rid of chemical-derived products, the present invention provides engineered cells for the biological production of UDP-sugar. This method provides a feasible approach for the rapid, safe, economical and sustainable production of various important molecules. The present invention provides a feasible approach for the rapid, safe, economical and sustainable production of UDP-sugar (including but not limited to UDP-glucose and UDP-galactose). Aspects of the present invention are achieved by engineered cells, which are used for the biological production of UDP-sugar by engineered host cells comprising one or more genetic modifications. In this article, UDP-sugar is manufactured using improved microbial host organisms. In this article, engineered cells include one or more genetic modifications, which can increase the biological production of UDP-sugar (including UDP-glucose and UDP-galactose).

[0007] Provided herein are engineered cells for the production of UDP-glucose, wherein these engineered cells include one or more genetic modifications, which increase UDP-glucose production by increasing the metabolic flux of UDP-glucose precursors and / or reducing the carbon loss caused by the generation of by-products. As non-limiting examples, genetic modification can be for overexpression or low expression of one or more endogenous genes in this engineered host cell, or can be for expressing one or more non-natural genes in this engineered host cell. Engineered cells as provided herein can include multiple genetic modifications.

[0008] Provided herein are engineered cells for producing UDP-galactose, wherein these engineered cells include one or more genetic modifications, which increase UDP-galactose production by increasing the metabolic flux of UDP-galactose precursors and / or reducing the carbon loss caused by the generation of by-products. As non-limiting examples, genetic modification can be a modification for overexpression or underexpression of one or more endogenous genes in the engineered host cell, or can be a modification for expressing one or more non-natural genes in the engineered host cell. Engineered cells as provided herein can include multiple genetic modifications.

[0009] Provided herein is an engineered cell for producing UDP-glucose by sucrose, wherein these engineered cells include one or more genetic modifications, and these one or more genetic modifications increase UDP-glucose production by increasing the metabolic flux of UDP-glucose precursors and / or reducing the carbon loss caused by the generation of by-products.As non-limiting examples, genetic modification can be for overexpression or low expression of one or more endogenous genes in this engineered host cell, or can be for expressing one or more non-natural genes in this engineered host cell.As provided herein, the engineered cell can include multiple genetic modifications.

[0010] Further provided herein are methods for producing UDP-sugars, which methods include cultivating engineered cells for producing UDP-sugars as provided herein. As non-limiting examples, genetic modification can be modification for overexpression or underexpression of one or more endogenous genes in the engineered host cell, or can be modification for expressing one or more non-natural genes in the engineered host cell. Engineered cells as provided herein can include multiple genetic modifications.

[0011] On the one hand, the present invention provides a kind of engineered host cell, it comprises one or more genetic modifications, and the one or more genetic modifications make the engineered host cell enzymatically convert carbon source into UDP-sugar through a variety of chemical intermediates.In certain embodiments, UDP-sugar is glucose.In certain embodiments, UDP-sugar is UDP-galactose.In certain embodiments, chemical intermediate is UDP-glucose, and UDP-sugar is UDP-galactose.In certain embodiments, the engineered host cell is Escherichia coli (E.coli).In certain embodiments, carbon source is selected from the group consisting of: glycerol, glucose, sucrose, galactose, fructose or any combination thereof.

[0012] In certain embodiments, one or more genetic modifications in the engineered host cell cause increased production of UDP-glucose. In certain embodiments, the one or more genetic modifications that cause increased production of UDP-glucose are overexpression of one or more genes selected from the group consisting of: (i) glucose promoting gene (glf) or a homologue thereof, (ii) glucokinase (glk) or a homologue thereof, (iii) phosphoglucomutase (pgm) or a homologue thereof, (iv) UTP-glucose-1-phosphate-uridyltransferase (galU) or a homologue thereof, (v) fructokinase (cscK) or a homologue thereof, and (vi) any combination thereof.

[0013] 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 uridyltransferase (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 causing the increase in the production of UDP-glucose are downward regulation or disappearance of phosphoglucomutase (pgm). In certain embodiments, the host cell through engineered approaches is supplemented with a culture medium comprising glucose. In certain embodiments, the host cell through engineered approaches is supplemented with a culture medium comprising fructose.

[0014] In certain embodiments, the one or more genetic modifications that cause 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 culture medium comprising sucrose.

[0015] In certain embodiments, one or more genetic modifications in the engineered host cell cause increased production of UDP-glucose. In certain embodiments, the one or more genetic modifications that cause 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 homologue thereof; (ii) 6-phosphogluconate dehydrogenase (gnd) or a homologue thereof; (iii) phosphoribosyl pyrophosphate synthetase (prs) or a homologue thereof; (iv) phosphoribosyltransferase (pyrE) or a homologue thereof; (v) orotidine 5'-phosphate decarboxylase (pyrF) or a homologue thereof; (vi) uridylate kinase (pyrH) or a homologue thereof; (vii) diphosphate nucleoside kinase (ndk) or a homologue thereof and (viii) adenylate kinase (adk) or a homologue thereof, and (ix) any combination thereof. In certain embodiments, the engineered host cell is supplemented with a culture medium comprising orotic acid.

[0016] In certain embodiments, one or more genetic modifications in the engineered host cell result in increased production of UDP-glucose. 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: overexpression of one or more genes selected from the group consisting of: glucose promoting gene (glf) or a homolog thereof, glucokinase (glk) or a homolog thereof, phosphoglucomutase (pgm) or a homolog thereof, UTP-glucose-1-phosphate-uridyltransferase (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, phosphoribosyltransferase (pyrE) or a homolog thereof, orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof. 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 uridyltransferase (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 cells are supplemented with culture medium comprising orotic acid, sucrose, glucose, fructose, or a combination thereof.

[0017] In certain embodiments, one or more genetic modifications in the engineered host cell cause an increase in the production of UDP-galactose. In certain embodiments, one or more genetic modifications in the engineered host cell that cause the enhanced production of UDP-galactose are selected from: (i) overexpression of UDP-glucose 4-epimerase (galE) or its homologue, (ii) overexpression of galactokinase (galK) or its homologue, (iii) overexpression of galactose-1-phosphate uridyl transferase (galT) or its homologue, and (iv) any combination thereof. In certain embodiments, the engineered host cell is supplemented with a culture medium comprising galactose.

[0018] In certain preferred embodiments, the one or more genetic modifications in the engineered host cell for increased production of UDP-galactose include one or more genetic modifications that result in increased production of UDP-glucose. Thus, in certain preferred embodiments, the one or more genetic modifications in the engineered host cell that increases the production of UDP-galactose are selected from: overexpression of one or more genes selected from the group consisting of: glucose promoting gene (glf) or a homolog thereof, glucokinase (glk) or a homolog thereof, phosphoglucomutase (pgm) or a homolog thereof, UTP-glucose-1-phosphate-uridyltransferase (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, phosphoribosyltransferase (pyrE) or a homolog thereof, orotidine 5'-phosphate decarboxylase ( pyrF) or its homologue, uridylate kinase (pyrH) or its homologue, diphosphate nucleoside kinase (ndk) or its homologue and adenylate kinase (adk) or its homologue; overexpression of UDP-glucose 4-epimerase (galE) or its homologue, overexpression of galactokinase (galK) or its homologue, overexpression of galactose-1-phosphate uridyltransferase (galT) or its homologue; 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 uridyltransferase (ugp); phosphoglucomutase (pgm); and / or any combination thereof. In certain embodiments, the engineered host cells are supplemented with culture medium comprising glucose, galactose, orotic acid, sucrose, or any combination thereof.

[0019] On the other hand, the present invention provides a method for increasing the production of UDP-sugars, the method comprising: providing an engineered host cell, the engineered host cell comprising one or more genetic modifications, the one or more genetic modifications causing the engineered host cell to enzymatically convert a carbon source into UDP-sugars through a variety of chemical intermediates. 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 Escherichia coli. In certain embodiments, the carbon source is selected from the group consisting of glycerol, glucose, sucrose, galactose, fructose, or any combination thereof.

[0020] In certain embodiments, the method comprises one or more genetic modifications in the engineered host cell that cause increased production of UDP-glucose. In certain embodiments, the one or more genetic modifications that cause increased production of UDP-glucose are overexpression of one or more genes selected from the group consisting of: (i) glucose promoting gene (glf) or its homologue, (ii) glucokinase (glk) or its homologue, (iii) phosphoglucomutase (pgm) or its homologue, (iv) UTP-glucose-1-phosphate-uridyl transferase (galU) or its homologue, 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 uridyltransferase (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 causing the increase in the production of UDP-glucose are downward regulation or disappearance of phosphoglucomutase (pgm). In certain embodiments, the host cell through engineered approaches is supplemented with a culture medium comprising glucose. In certain embodiments, the host cell through engineered approaches is supplemented with a culture medium comprising fructose.

[0021] In certain embodiments, the method comprises one or more genetic modifications that cause increased production of UDP-glucose is 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 culture medium comprising sucrose.

[0022] In certain embodiments, the method comprises one or more genetic modifications in the engineered host cell that cause increased production of UDP-glucose. In certain embodiments, the one or more genetic modifications that cause 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) phosphoribosyltransferase (pyrE) or a homolog thereof; (v) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof; (vi) uridylate kinase (pyrH) or a homolog thereof; (vii) diphosphate nucleoside 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 culture medium comprising orotic acid.

[0023] In certain embodiments, the method comprises one or more genetic modifications in the engineered host cell that result in increased production of UDP-glucose. In certain embodiments, the one or more genetic modifications in the engineered host cell that result in enhanced production of UDP-glucose are selected from: overexpression of one or more genes selected from the group consisting of: glucose promoting gene (glf) or a homolog thereof, glucokinase (glk) or a homolog thereof, phosphoglucomutase (pgm) or a homolog thereof, UTP-glucose-1-phosphate-uridyltransferase (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, phosphoribosyltransferase (pyrE) or a homolog thereof, orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof. 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 uridyltransferase (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 cells are supplemented with culture medium comprising orotic acid, sucrose, glucose, fructose, or a combination thereof.

[0024] In certain embodiments, the method comprises one or more genetic modifications in the engineered host cell that cause an increase in the production of UDP-galactose. In certain embodiments, the one or more genetic modifications in the engineered host cell that cause the enhanced production of UDP-galactose are selected from: (i) overexpression of UDP-glucose 4-epimerase (galE) or its homologue, (ii) overexpression of galactokinase (galK) or its homologue, (iii) overexpression of galactose-1-phosphate uridyl transferase (galT) or its homologue, and (iv) any combination thereof. In certain embodiments, the engineered host cell is supplemented with a culture medium comprising galactose.

[0025] In certain preferred embodiments, the method provides that the one or more genetic modifications for increasing the production of UDP-galactose in the engineered host cell include one or more genetic modifications that cause an increase in the production of UDP-glucose. Therefore, in certain preferred embodiments, the one or more genetic modifications in the engineered host cell that increase the production of UDP-galactose are selected from: overexpression of one or more genes selected from the group consisting of: glucose promoting gene (glf) or its homolog, glucokinase (glk) or its homolog, phosphoglucomutase (pgm) or its homolog, UTP-glucose-1-phosphate-uridyltransferase (galU) or its homolog, sucrose transporter (cscB) or its homolog, Sucrose synthase (SuSy) or a homologue thereof, sucrose phosphorylase (SPase) or a homologue thereof, glucose-6-phosphate 1-dehydrogenase (zwf) or a homologue thereof, 6-phosphogluconate dehydrogenase (gnd) or a homologue thereof, phosphoribosyl pyrophosphate synthetase (prs) or a homologue thereof, phosphoribosyltransferase (pyrE) or a homologue thereof, orotidine 5'-phosphate decarboxylase (pyrF) or a homologue thereof, uridylate kinase (pyrH) or a homologue thereof, nucleoside diphosphate kinase (nd k) or its homologues and adenylate kinase (adk) or its homologues; overexpression of UDP-glucose 4-epimerase (galE) or its homologues, overexpression of galactokinase (galK) or its homologues, overexpression of galactose-1-phosphate uridyltransferase (galT) or its homologues, overexpression of fructokinase (cscK) or its homologues; and / or downregulation or deletion of one or more genes selected from the group consisting of glucose-6-phosphate isomerase (pgi), glucose-1-phosphate Acid adenylyltransferase (glgC), UDP-glucose 6-dehydrogenase (ugd), glucan biosynthesis glucosyltransferase (OpgG), UDP-sugar hydrolase (ushA), UTP-glucose-1-phosphate uridyltransferase (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 cell is an engineered host cell supplemented with a culture medium comprising glucose, galactose, orotic acid, sucrose, and any combination thereof.

[0026] On the other hand, the present invention provides an engineered host cell comprising one or more genetic modifications, which allow the engineered host cell to enzymatically convert a carbon source to produce UDP-glucose through a variety of chemical intermediates. In certain embodiments, the one or more genetic modifications are overexpression of a glucose-promoting gene (glf) or a homologue thereof. In certain embodiments, the one or more genetic modifications are overexpression of a glucokinase (glk) or a homologue 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 for expressing one or more non-natural genes in the engineered host cell; and (iv) any combination thereof. In certain embodiments, the engineered host cell is Escherichia 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 promoting gene (glf) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate-uridyltransferase (galU) or a homolog thereof, (v) orotate 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) diphosphate nucleoside 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, the one or more genetic modifications are overexpression of one or more genes selected from the group consisting of: (i) glucose promoting 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-uridyltransferase (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; and (viii) any combination thereof. In certain embodiments, the 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 promoting gene (glf) or a homolog thereof; and (iii) any combination thereof.

[0027] In certain embodiments, one or more genetic modifications are overexpression of phosphoglucomutase (pgm) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of UTP-glucose-1-phosphate-uridyl transferase (galU) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of orotate phosphoribosyl transferase (pyrE) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of orotidine 5' phosphate decarboxylase (pyrF) or its homologue. In certain embodiments, the engineered cells are supplemented with culture medium comprising orotate. In certain embodiments, one or more genetic modifications are overexpression of uridylate kinase (pyrH) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of diphosphate nucleoside kinase (ndk) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of adenylate kinase (adk) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of fructokinase (cscK). In certain embodiments, overexpression of fructokinase (cscK) leads to 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 UDP-glucose from being converted into 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 (OpgG) to prevent the consumption of UDP-glucose and the formation of 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 homologues. In certain embodiments, one or more genetic modifications are overexpression of 6-phosphogluconate dehydrogenase (gnd) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of phosphoribosyl pyrophosphate synthetase (prs) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of sucrose transporter (cscB) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of sucrose synthase (SuSy) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of sucrose phosphorylase (SPase) or its homologue. In certain embodiments, the engineered host cell is supplemented with a culture medium comprising glucose.In certain embodiments, one or more genetic modifications are the deletion or downregulation of UDP-sugar hydrolase (ushA). In certain embodiments, one or more genetic modifications are the deletion or downregulation of UTP-glucose-1-phosphate uridyl transferase (ugp). In certain embodiments, one or more genetic modifications are the 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. Therefore, in certain embodiments, the downregulation of (heptosyl) lipopolysaccharide α-1,3-glucosyltransferase (WaaG) enhances the yield of UDP-glucose. In certain embodiments, one or more genetic modifications are the downregulation or deletion of trehalose-6-phosphate synthase (otsA). Trehalose-6-phosphate synthase (otsA) catalyzes the biosynthesis of trehalose by UDP-glucose. In certain embodiments, the production rate of UDP-glucose is increased by the downward regulation of trehalose-6-phosphate synthase (otsA). In certain embodiments, one or more genetic modifications are the downward regulation or lack of glucose-1-phosphatase (agp). Glucose-1-phosphatase (agp) is a periplasmic protein that hydrolyzes the phosphate in glucose-1-phosphate and other substrates. Therefore, in certain embodiments, the downward regulation or lack of glucose-1-phosphatase (agp) will improve the availability of glucose-1-phosphate for UDP-glucose production, and enhance the production rate of UDP-sugar.

[0028] In certain embodiments, the engineered host cells are supplemented with a culture medium comprising glucose. In certain embodiments, the engineered host cells are supplemented with a culture medium comprising sucrose. In certain embodiments, the engineered host cells are supplemented with a culture medium comprising fructose.

[0029] On the other hand, the present invention provides an engineered host cell comprising one or more genetic modifications, wherein the one or more genetic modifications allow the engineered host cell to enzymatically convert glucose into uridine diphosphate galactose (UDP-galactose) through a variety of chemical intermediates. In certain embodiments, the engineered host cell is Escherichia coli. 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 for expressing one or more non-natural genes in the engineered host cell; and (iv) any combination 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-uridyl transferase (galT) or a homolog thereof, and (iv) any combination thereof. In certain embodiments, one or more genetic modifications are the overexpression of UDP-glucose 4-epimerase (galE) or its homologue. In certain embodiments, one or more genetic modifications are the overexpression of galactokinase (galK) or its homologue. In certain embodiments, one or more genetic modifications are the overexpression of galactose-1-phosphate uridyltransferase (galT) or its homologue. In certain embodiments, the modifications listed herein can be combined with the modifications listed in any other aspects of the present invention. In certain embodiments, the modifications listed herein can be combined with one or more modifications listed for converting chemical intermediates into UDP-glucose. In certain embodiments, the engineered host cell is supplemented with a culture medium comprising galactose. In certain embodiments, the engineered host cell is supplemented with a culture medium comprising fructose.

[0030] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications that allow the engineered host cell to enzymatically convert sucrose to uridine diphosphate glucose (UDP-glucose). 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 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 phosphoribosyltransferase (pyrE) or a homolog thereof, (ii) expression of orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (iii) expression of uridylate kinase (pyrH) or a homolog thereof, (iv) expression of diphosphate nucleoside 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 listed herein may be combined with modifications listed in any other aspect of the invention.

[0031] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications that allow the engineered host cell to enzymatically convert sucrose to uridine diphosphate glucose (UDP-glucose). 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 phosphorylase (SPase) or a homolog thereof, (iii) overexpression of a UTP-glucose-1-phosphate-uridyltransferase (galU) or a homolog thereof, (iv) downregulation or deletion of a UDP-glucose 4-epimerase (galE), (v) downregulation or deletion of a UDP-glucose 6-dehydrogenase (ugd), (vi) downregulation or deletion of a glucan biosynthesis glucosyltransferase (OpgG), (vii) downregulation or deletion of a phosphoglucomutase (pgm), and (viii) any combination thereof. In certain embodiments, the engineered cells comprise overexpression of a heterologous sucrose transporter (cscB) or a homolog thereof, a heterologous sucrose phosphorylase (SPase) or a homolog thereof, a UTP-glucose-1-phosphate uridyltransferase (galU) 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 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 phosphoribosyltransferase (pyrE) or a homolog thereof, (ii) expression of orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (iii) expression of uridylate kinase (pyrH) or a homolog thereof, (iv) expression of diphosphate nucleoside 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 listed herein can be combined with the modifications listed in any other aspect of the invention.

[0032] 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, the engineered host cell comprising one or more genetic modifications, the one or more genetic modifications causing the engineered host cell to enzymatically convert glucose to UDP-glucose through a variety of chemical intermediates. 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 for expressing one or more non-natural genes in the engineered host cell; and (iv) any combination thereof. In certain embodiments, the engineered host cell is Escherichia 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 promoting gene (glf) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate-uridyltransferase (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) diphosphate nucleoside 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, 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 promoting gene (glf) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridyltransferase (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); and (viii) any combination thereof. In certain embodiments, the 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 promoting gene (glf) or a homolog thereof; and (iii) any combination thereof. In certain embodiments, the 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-uridyl transferase (galU) or its homologues. In certain embodiments, one or more genetic modifications are overexpression of fructokinase (cscK) or its homologues. In certain embodiments, one or more genetic modifications are overexpression of orotate phosphoribosyl transferase (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 culture medium comprising orotate. In certain embodiments, one or more genetic modifications are overexpression of uridylate kinase (pyrH). In certain embodiments, one or more genetic modifications are overexpression of diphosphate nucleoside 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 UDP-glucose from being converted to UDP-galactose. In certain embodiments, one or more genetic modifications are down-regulation or deletion of UDP-glucose 6-dehydrogenase (ugd) to prevent UDP-glucose from being converted into UDP-glucuronic acid. In certain embodiments, one or more genetic modifications are down-regulation or deletion of glucose-1-phosphate adenylyltransferase (glgC). In certain embodiments, one or more genetic modifications are down-regulation or deletion of glucan biosynthesis glucosyltransferase (mdoA) to prevent UDP-glucose from being converted into membrane-derived oligosaccharides (MDO). In certain embodiments, one or more genetic modifications are down-regulation or deletion of glucose-6-phosphate isomerase (pgi). In certain embodiments, one or more genetic modifications are down-regulation or deletion of (heptosyl) lipopolysaccharide alpha-1,3-glucosyltransferase (WaaG). (Heptosyl) lipopolysaccharide alpha-1,3-glucosyltransferase (WaaG) uses UDP-glucose as a substrate for lipopolysaccharide synthesis. In certain embodiments, the one or more genetic modifications are the down-regulation or deletion of trehalose-6-phosphate synthase (otsA). Trehalose-6-phosphate synthase (otsA) catalyzes the biosynthesis of trehalose from UDP-glucose. Therefore, in certain embodiments, the down-regulation of trehalose-6-phosphate synthase (otsA) will enhance the yield of UDP-glucose. In certain embodiments, the one or more genetic modifications are the down-regulation or deletion of glucose-1-phosphatase (agp). Glucose-1-phosphatase (agp) is a periplasmic protein that hydrolyzes phosphates in glucose-1-phosphate and other substrates.In certain embodiments, one or more genetic modifications are the overexpression of glucose-6-phosphate 1-dehydrogenase (zwf) or its homologue. In certain embodiments, one or more genetic modifications are the overexpression of 6-phosphogluconate dehydrogenase (gnd) or its homologue. In certain embodiments, one or more genetic modifications are the overexpression of phosphoribosyl pyrophosphate synthetase (prs) or its homologue. In certain embodiments, the engineered host cell comprises overexpression of (i) glucokinase (glk) or a homolog thereof, (ii) glucose promoting gene (glf) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate-uridyltransferase (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) diphosphate nucleoside 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, the engineered host cell comprises overexpression of (i) glucokinase (glk) or a homolog thereof, (ii) glucose promoting gene (glf) or a homolog thereof, (iii) phosphoglucomutase (pgm) or a homolog thereof, (iv) UTP-glucose-1-phosphate uridyltransferase (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 UDP-galactose, the method comprising: providing an engineered host cell, the engineered host cell comprising one or more genetic modifications, the one or more genetic modifications causing the engineered host cell to enzymatically convert glucose and / or galactose to UDP-galactose via a variety of chemical intermediates. In certain embodiments, the engineered host cell is E. coli.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 for expressing one or more non-native genes in the engineered host cell; and (iv) any combination 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 its homolog, (ii) overexpression of galactokinase (galK) or its homolog, (iii) overexpression of galactose-1-phosphate uridyltransferase (galT) or its homolog, and (iv) any combination thereof. In certain embodiments, the one or more genetic modifications are overexpression of UDP-glucose 4-epimerase (galE) or its homolog. In certain embodiments, the one or more genetic modifications are overexpression of galactokinase (galK) or its homolog. In certain embodiments, the one or more genetic modifications are overexpression of hydroxyproline O-galactosyltransferase (galT) or a homologue thereof. In certain embodiments, the genetic modification is selected from the group consisting of: (i) expression of phosphoribosyltransferase (pyrE) or a homologue thereof, (ii) expression of orotidine 5'-phosphate decarboxylase (pyrF) or a homologue thereof, (iii) expression of uridylate kinase (pyrH) or a homologue thereof, and (iv) any combination thereof. In certain embodiments, the modifications listed herein may be combined with the modifications listed in any other aspect of the invention. In certain embodiments, the engineered host cell is supplemented with a culture medium comprising galactose.

[0033] In another aspect, the present invention provides a method for increasing the production of UDP-glucose, the method comprising: providing an engineered host cell, the engineered host cell comprising one or more genetic modifications, the one or more genetic modifications causing the engineered host cell to enzymatically convert sucrose to uridine diphosphate glucose (UDP-glucose). 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 phosphorylase (SPase) or a homolog thereof, (iii) overexpression of a UTP-glucose-1-phosphate-uridyltransferase (galU) or a homolog thereof, (iv) downregulation or deletion of a UDP-glucose 4-epimerase (galE), (v) downregulation or deletion of a UDP-glucose 6-dehydrogenase (ugd), (vi) downregulation or deletion of a glucan biosynthesis glucosyltransferase (OpgG), (vii) downregulation or deletion of a phosphoglucomutase (pgm), and (viii) any combination thereof. In certain embodiments, the engineered cells comprise overexpression of a heterologous sucrose transporter (cscB) or a homolog thereof, a heterologous sucrose phosphorylase (SPase) or a homolog thereof, a UTP-glucose-1-phosphate uridyltransferase (galU) 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 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 phosphoribosyltransferase (pyrE) or a homolog thereof, (ii) expression of orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (iii) expression of uridylate kinase (pyrH) or a homolog thereof, (iv) expression of diphosphate nucleoside 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 listed herein can be combined with the modifications listed in any other aspect of the invention.

[0034] In another aspect, the present invention provides a method for increasing the production of UDP-glucose from sucrose, the method comprising: providing an engineered host cell comprising one or more genetic modifications that allow the engineered host cell to enzymatically convert sucrose to UDP-glucose. 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 a UDP-glucose 4-epimerase (galE), (iv) downregulation or deletion of a 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 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 phosphoribosyltransferase (pyrE) or a homolog thereof, (ii) expression of orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof, (iii) expression of uridylate kinase (pyrH) or a homolog thereof, and (iv) any combination thereof. In certain embodiments, the engineered cells are supplemented with culture medium comprising orotic acid, sucrose, fructose, and / or glucose. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Shown are pathways for the bioproduction of UDP-glucose and / or UDP-galactose in engineered cells and methods for making UDP-glucose and / or UDP-galactose described herein.

[0036] Figure 2 Genetic modifications for increasing the production of UDP-glucose and / or UDP-galactose in engineered host cells are provided. DETAILED DESCRIPTION

[0037] The application provides engineered cells for producing UDP-sugars (including but not limited to UDP-glucose and / or UDP-galactose), cultures comprising 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' may refer to any intermediate present in a biosynthetic pathway that results in the production of UDP-glucose and / or UDP-galactose. UDP-sugar precursors may 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), 5'-phosphoorotidine (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.

[0038] Cells engineered to produce UDP-sugars can have one or more modifications including, but not limited to, down-regulation, disruption or deletion of endogenous genes, up-regulation of endogenous genes, and introduction of exogenous genes.

[0039] The term "non-naturally occurring", when used to refer to an enzyme, is intended to mean that a nucleic acid or polypeptide includes at least one genetic alteration 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 that includes at least one genetic modification resulting from human intervention may also be referred to as "non-naturally occurring", "engineered", "genetically engineered", or "recombinant".

[0040] The host cell, organism or microorganism that is engineered to express or overexpress a gene or nucleic acid sequence, or overexpress an enzyme or polypeptide has been genetically engineered by recombinant DNA technology to include genes or nucleic acid sequences that do not naturally encode enzymes or polypeptides, or to express endogenous genes at a level that exceeds its expression level in unmodified cells. As a non-limiting example, the host cell, organism or microorganism that is engineered to express or overexpress a gene or nucleic acid sequence, or overexpress an enzyme or polypeptide can have any modification of the coding sequence of the gene, the position of the gene on the chromosome, or the regulatory elements associated with the gene that affect the gene. The overexpression of a gene can also be achieved by increasing the copy number of the gene in the cell or organism. Similarly, the host cell, organism or microorganism that is engineered to have insufficient expression of a gene, nucleic acid sequence, or reduced expression or insufficient expression of an enzyme or polypeptide can have any modification of the coding sequence of the gene, the position of the gene on the chromosome, or the regulatory elements associated with the gene that affect the gene. Specifically, gene disruptions are included, which include any insertion, deletion, or sequence mutation of a gene or a part of a gene that affects its expression or the activity of the encoded polypeptide. Gene disruption includes "knockout" mutations that eliminate the expression of a gene. Modifications to under-express a gene also include modifications to the regulatory regions of a gene that can reduce its expression.

[0041] The term "exogenous" or "heterologous" is intended to mean that a reference molecule or reference activity is introduced into a host microorganism. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the host genetic material, such as by integration into a host chromosome or as non-chromosomal genetic material that can be introduced onto a medium such as a plasmid. Thus, the term "endogenous" refers to a reference molecule or activity that is naturally present in the host.

[0042] Gene or nucleotide sequence can be stably or transiently introduced into host cell using technology well known in the art, and these technologies include but are not limited to conjugation, electroporation, chemical transformation, transduction and transfection.Optionally, for exogenous expression in Escherichia coli or other prokaryotic cells, some nucleotide sequences in the gene or cDNA of eukaryotic nucleic acid can encode targeting signals, such as N-terminal mitochondria or other targeting signals, which can be removed before being converted into prokaryotic host cells if desired. In addition, gene can be subjected to codon optimization to realize optimized protein expression with technology well known in the art.

[0043] The identity percentage (identity %) between two sequences is determined when the sequences are aligned to obtain maximum homology. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal Ω etc. compare and determine the similarity or identity of the original sequence, and also determine the presence or significance of the gap in the sequence, which can be assigned weights or scores. Such algorithms are also known in the art, and are equally applicable to determining nucleotide or amino acid sequence similarity or identity, and can be used to identify the orthologs of the gene being paid attention to. Other sequences added to the polypeptide sequence, such as but not limited to immune detection markers, purification markers, localization sequences (presence or absence), etc., will not affect identity %.

[0044] Homologs are one or more genes that have the same or identical function in different organisms. Orthologous genes can encode proteins with sequence similarity of about 45% to 100% amino acid sequence identity, and more preferably about 60% to 100% amino acid sequence identity. If genes share a three-dimensional structure (but not necessarily sequence similarity) sufficient to indicate that they evolved from a common ancestor, but the amount of primary sequence similarity cannot be identified, then these genes can also be considered as orthologs. Paralogs are genes related by duplication within the genome, and new functions can be evolved, even if these functions are related to the original function.

[0045] The engineered cells for producing UDP-glucose may include an exogenous nucleic acid sequence encoding glucose facilitator (glf) activity or a homolog thereof, an exogenous nucleic acid sequence encoding glucokinase (glk) activity or a homolog thereof, an exogenous nucleic acid sequence encoding phosphoglucomutase (pgm) activity or a homolog thereof, an exogenous nucleic acid sequence encoding UTP-glucose-1-phosphate uridyltransferase (galU) activity or a homolog thereof, and an exogenous nucleic acid sequence encoding phosphoribosyltransferase (pyrE) activity, orotidine 5'-phosphate decarboxylase (pyrF) activity or a homolog thereof, an exogenous nucleic acid sequence encoding uridylate kinase (pyrH) activity or a homolog thereof, an exogenous nucleic acid sequence encoding diphosphate kinase (nPK) activity or a homolog thereof. The invention relates to an exogenous nucleic acid sequence encoding an activity of adenylate kinase (adk) or a homolog thereof, an exogenous nucleic acid sequence encoding an activity of glucose-6-phosphate 1-dehydrogenase (zwf) or a homolog thereof, an exogenous nucleic acid sequence encoding an activity of 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof, an exogenous nucleic acid sequence encoding an activity of phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof, an exogenous nucleic acid sequence encoding an activity of a sucrose transporter (cscB) or a homolog thereof, an exogenously expressed nucleic acid sequence encoding an activity of sucrose synthase (SuSy) or a homolog thereof, and an exogenously expressed nucleic acid sequence encoding an activity of sucrose phosphorylase (SPase) or a homolog thereof. Optionally, the engineered cells may further include 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 uridyltransferase (ugp) activity, and downregulation or deletion of phosphoglucomutase (pgm) activity.

[0046] For example, a glucose promoting gene or glucose transporter gene (glf) can be a member of the major promoting gene superfamily or sugar transporter (TC 2.A.1.1) that promotes cellular uptake of glucose. An exemplary glucose promoting gene can be glf from Zymomonas mobilis (NCBI Accession ID: AAA27691.1). Also contemplated for use in the engineered cells provided herein are glf homologues 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, which have the activity of transporting glucose into the cell.

[0047] For example, glucokinase (glk) can be a member of the hexokinase family of proteins that phosphorylates glucose to produce glucose-6-phosphate. An exemplary glucokinase (glk) is Zymomonas mobilis (EC: 2.7.1.2, NCBI reference: AAA27694.1). Also contemplated for use in the engineered cells provided herein are 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, which have the activity of a glucokinase that produces glucose-6-phosphate from glucose.

[0048] Phosphoglucomutase (pgm) catalyzes the conversion of 6-phosphogluconate into glucose 1-phosphate. Exemplary phosphogluconate mutase (pgm) can be pgm from Escherichia coli (EC: 5.4.2.2, NCBI accession AAC73782.1). Also contemplated for use in the engineered cells provided herein are pgm with SEQ ID NOs: 4-22, pgm homologues and variants with 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 with SEQ ID NOs: 3-22, which have the activity of catalyzing the conversion of 6-phosphogluconate into glucose 1-phosphate.

[0049] UTP-glucose-1-phosphate-uridyl transferase (galU) is an enzyme that synthesizes UDP-glucose from glucose-1-phosphate and UTP. Exemplary UTP-glucose-1-phosphate-uridyl transferase (galU) can be galU from Escherichia coli (EC: 2.7.7.9, NCBI accession EEW2752841.1). Also contemplated for use in the engineered cells provided herein are galUs with SEQ ID NOs: 23-39 or 72-74. Also contemplated for use in the engineered cells provided herein are ugpA from Bifidobacterium bifidum (Bifidobacterium bifidum, NCBI accession WP_021648042.1). Also contemplated for use in the engineered cells provided herein are ugpA with SEQ ID NOs: 41-47. Also contemplated for use in the engineered cells provided herein are 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, which have activity in synthesizing UDP-glucose from glucose-1-phosphate.

[0050] Orotate phosphoribosyltransferase (pyrE) is an enzyme that catalyzes the transfer of a ribosyl phosphate group from 5-phosphoribosyl 1-bisphosphate to orotate, resulting in the formation of orotidine monophosphate (OMP). An exemplary orotate phosphoribosyltransferase can be pyrE from Enterobacteriaceae (NCBI accession WP_000806177.1). Also contemplated for use in the engineered cells provided herein are pyrE with SEQ ID NO: 49, pyrE homologs and variants with 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: 48-49, which have the activity of catalyzing the conversion of phosphoribosyl 1-bisphosphate (PRPP) into orotidine monophosphate (OMP).

[0051] 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 Escherichia 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, 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 NO: 50-51, which have the activity of catalyzing the conversion of orotidine monophosphate (OMP) to uridine monophosphate (UMP).

[0052] Uridylate kinase (pyrH) is an enzyme that catalyzes the conversion of uridine monophosphate (UMP) to uridine diphosphate (UDP). An exemplary uridine kinase (pyrH) can be pyrH from Escherichia coli (EC: 2.7.4.22, NCBI accession WP_000224573.1). Also contemplated for use in the engineered cells provided herein are pyrH with SEQ ID NO: 53, 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 NO: 52-53, which have an activity of catalyzing the conversion of uridine monophosphate (UMP) to uridine diphosphate (UDP).

[0053] Nucleoside diphosphate kinase (NDK) is an enzyme that catalyzes the conversion of uridine diphosphate (UDP) into uridine triphosphate (UTP). Exemplary nucleoside diphosphate kinase (NDK) can be an NDK from Enterobacteriaceae (EC: 2.7.4.6, NCBI accession WP_000963837.1). Also contemplated for use in the engineered cells provided herein are NDKs with SEQ ID NO: 55, NDK homologs and variants with 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: 54-55, which have an activity of catalyzing the conversion of uridine diphosphate (UDP) into uridine triphosphate (UTP).

[0054] Adenylate kinase (ADK) is an enzyme that catalyzes the interconversion of various adenylic acids (ATP, ADP and AMP). An exemplary adenylate kinase (ADK) can be an ADK from Escherichia coli (EC: 2.7.4.3, NCBI accession AAC73576.1). Also contemplated for use in the engineered cells provided herein are ADKs with SEQ ID NO: 57, ADK homologs and variants with 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: 56-57, which have the activity of catalyzing the interconversion of various adenosine phosphates (ATP, ADP and AMP).

[0055] Glucose-6-phosphate 1-dehydrogenase (zwf) is an enzyme that catalyzes the oxidation of 6-phosphate glucose to 6-phosphogluconolactone. Exemplary glucose-6-phosphate 1-dehydrogenase (zwf) can be zwf from Escherichia coli (EC: 1.1.1.49, NCBI accession UGE34297.1). Also contemplated for use in the engineered cells provided herein are zwf homologues and variants with SEQ ID NO:58 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, which have the activity of catalyzing the oxidation of 6-phosphate glucose to 6-phosphogluconolactone.

[0056] 6-phosphogluconate dehydrogenase (gnd) is an enzyme that catalyzes the oxidative decarboxylation of 6-phosphogluconate to ribose 5-phosphate and CO2, while reducing NADP to NADPH. An exemplary 6-phosphogluconate dehydrogenase (gnd) can be a 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, which have the activity of catalyzing the oxidative decarboxylation of 6-phosphogluconate to ribose-5-phosphate and CO2, while reducing NADP to NADPH.

[0057] PRPP synthase (prs) is an enzyme that catalyzes the conversion of ribose-5-phosphate to PRPP by transferring a pyrophosphoryl group from ATP to PRPP. An exemplary PRPP synthase (prs) can be a prs from the family 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, which have an activity of catalyzing the conversion of ribose-5-phosphate to PRPP by transferring a pyrophosphoryl group from ATP to PRPP.

[0058] Sucrose permease (cscB) is responsible for transporting sucrose into the cell. Exemplary sucrose permease (cscB) can be cscB from Enterobacteriaceae (EC: 2.7.1.69, NCBI accession WP_001197025.1). Also contemplated for use in the engineered cells provided herein are cscB homologues and variants with SEQ ID NO:61 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, which have the activity of transporting sucrose in the cell.

[0059] Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and diphosphate nucleosides into fructose and nucleotide (NDP)-glucose. An exemplary sucrose synthase (SuSy) can be SuSy from Acidithiobacillus caldus (EC 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, which have the activity of catalyzing the reversible conversion of sucrose and diphosphate nucleosides into fructose and nucleotide (NDP)-glucose.

[0060] Sucrose phosphorylase (SPase) catalyzes the reversible phosphorolysis of sucrose to α-D-glucose 1-phosphate (Glc1P) and D-fructose. An exemplary sucrose phosphorylase can be sucP from Bifidobacterium adolescentis (EC 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, which have activity of catalyzing the reversible phosphorolysis of sucrose to α-D-glucose 1-phosphate (Glc1P) and D-fructose.

[0061] UDP-glucose 4-epimerase (galE) is an enzyme that catalyzes the reversible conversion of UDP-galactose to UDP-glucose by a mechanism that participates in the transient reduction of NAD. An exemplary UDP-glucose 4-epimerase (galE) can be galE from Escherichia coli (EC: 5.1.3.2, NCBI accession AAV80748.1). It is also contemplated that the overexpression, downregulation or deletion in the engineered cells provided herein is 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% homologs and variants with SEQ ID NO: 64, which have the activity of catalyzing the reversible conversion of UDP-galactose to UDP-glucose by a mechanism that participates in the transient reduction of NAD.

[0062] UDP-glucose 6-dehydrogenase (ugd) is an enzyme that catalyzes the conversion of UDP-glucose into UDP-glucuronic acid. Exemplary UDP-glucose 6-dehydrogenase (ugd) can be ugd from Escherichia coli (EC: 1.1.1.22, NCBI login WP_089615770.1). It is also contemplated that the downregulation or deletion in the engineered cell provided herein is an ugd homologue and variant with 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 with SEQ ID NO:65, which has the activity of catalyzing the reversible conversion of UDP-glucose into UDP-glucuronic acid.

[0063] Glucose-1-phosphate adenylyltransferase (glgC) is an enzyme that catalyzes the conversion of glucose-1-phosphate into ADP-α-D-glucose. An exemplary glucose-1-phosphate adenylyltransferase (glgC) can be glgC from Escherichia coli (EC: 2.7.7.27, NCBI accession WP_097472330.1). It is also contemplated that the downregulation or deletion of the engineered cells provided herein is 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% identical to SEQ ID NO: 66, and has the activity of catalyzing the reversible conversion of glucose-1-phosphate into ADP-α-D-glucose.

[0064] Glucan biosynthesis glucosyltransferase (OpgG) is an enzyme involved in converting UDP-glucose into MDO. An exemplary glucan biosynthesis glucosyltransferase (OpgG) can be an 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, which have an activity of catalyzing the reversible conversion of UDP-glucose into MDO.

[0065] Glucose-6-phosphate isomerase (pgi) catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. An exemplary glucose-6-phosphate isomerase (pgi) can be pgi from Escherichia coli (EC: 5.3.1.9, NCBI accession AAC76995.1). It is also contemplated that the downregulation or deletion in the engineered cells provided herein is 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% homologs and variants with SEQ ID NO: 68, which have the activity of catalyzing the reversible conversion of glucose-6-phosphate to fructose-6-phosphate.

[0066] UDP-sugar hydrolase (ushA) is an enzyme with UDP-sugar hydrolase activity. Exemplary UDP-sugar hydrolase (ushA) can be ushA from Escherichia coli (EC: 3.1.3.5, NCBI accession WP_000771748.1). It is also contemplated that the downregulation or deletion in the engineered cells provided herein is 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% homologs and variants with SEQ ID NO: 69, which have the activity of hydrolyzing UDP-sugar.

[0067] In addition to the modifications in the engineered cell lines discussed above, the present invention further includes engineered cells that produce 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).

[0068] Galactokinase (galK) catalyzes the formation of galactose-6-phosphate from galactose. Exemplary galactokinase (galK) can be galK from Escherichia coli (EC: 2.7.1.6, NCBI accession WP_000053415.1). Also contemplated for use in the engineered cells provided 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, which have the activity of catalyzing the formation of galactose-6-phosphate from galactose.

[0069] Galactose-1-phosphate uridyl transferase (galT) catalyzes the formation of UDP-galactose from galactose-1-phosphate. An exemplary galactose-1-phosphate uridyl transferase (galT) can be galT from Escherichia 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, which have the activity of catalyzing the formation of UDP-galactose from galactose-1-phosphate.

[0070] Fructose kinase (cscK) phosphorylates fructose to produce fructose-6-phosphate. Exemplary fructokinase (cscK) can be cscK from Escherichia 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, which have an activity of catalyzing the formation of fructose-6-phosphate from fructose.

[0071] (Heptosyl) lipopolysaccharide alpha-1,3-glucosyltransferase (WaaG) uses UDP-glucose as a substrate for lipopolysaccharide synthesis. An exemplary (heptosyl) lipopolysaccharide alpha-1,3-glucosyltransferase (WaaG) can be WaaG from Escherichia 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, which have the activity of catalyzing lipopolysaccharide synthesis performed by UDP-glucose.

[0072] Trehalose-6-phosphate synthase (otsA) catalyzes the biosynthesis of trehalose from UDP-glucose. Exemplary trehalose-6-phosphate synthase (otsA) can be otsA from Escherichia coli (EC 2.4.1.15, AAC74966.1). Also contemplated for use in the engineered cells provided herein are otsA homologues and variants with 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, which have the activity of catalyzing the synthesis of lipopolysaccharide by UDP-glucose.

[0073] Glucose-1-phosphatase (agp) is a periplasmic protein that hydrolyzes phosphates in glucose-1-phosphate and other substrates. Exemplary glucose-1-phosphatase (agp) can be agp from Escherichia coli (EC 3.1.3.10, accession AAC74087.1). Also contemplated for use in engineered cells provided herein are agp homologues and variants with 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, which have the activity of hydrolyzing phosphate from glucose-1-phosphate.

[0074] The present invention further provides an engineered host cell comprising one or more genetic modifications, wherein the one or more genetic modifications enable the engineered host cell to enzymatically convert sucrose into uridine diphosphate glucose (UDP-glucose). The engineered host cell includes an exogenous nucleic acid sequence encoding a sucrose transporter (cscB) activity or its homologue, and an exogenously expressed nucleic acid sequence encoding a sucrose synthase (SuSy) activity or its homologue. The engineered host cell optionally further includes genetic modifications, including but not limited to the down-regulation or lack of UDP-glucose 4-epimerase (galE), the down-regulation or lack of UDP-glucose 6-dehydrogenase (ugd), the down-regulation or lack of glucan biosynthesis glucosyltransferase (mdoA), the down-regulation or lack of glucose-1-phosphate adenylyltransferase (glgC), and the down-regulation or lack of glucose-6-phosphate isomerase (pgi). In certain embodiments, the engineered cell comprises a heterologous sucrose transporter (cscB) or a homologue thereof, a heterologous sucrose synthase (SuSy) or a homologue thereof, and a genetic modification selected from the group consisting of: (i) downregulation or deletion of UDP-glucose 4-epimerase (galE) or a homologue thereof, (ii) downregulation or deletion of UDP-glucose 6-dehydrogenase (ugd) or a homologue thereof, (iii) downregulation or deletion of glucan biosynthetic glucosyltransferase (mdoA) or a homologue thereof, and (iv) any combination thereof. In certain embodiments, the modifications listed herein can be combined with the modifications listed in any other aspect of the invention. In certain embodiments, the modifications listed herein can be combined with one or modifications listed for converting a chemical intermediate to UDP-glucose.

[0075] The present invention Figure 2 An overview of genetic modifications of engineered host cells for increased production of UDP-sugars, including UDP-glucose and UDP-galactose, is provided.

[0076] As described herein, the invention provides an engineered host cell comprising one or more genetic modifications that cause the engineered host cell to enzymatically convert a carbon source into a UDP-sugar through a variety of chemical intermediates. 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 Escherichia coli. In certain embodiments, the carbon source is selected from the group consisting of glycerol, glucose, sucrose, galactose, fructose, or any combination thereof.

[0077] like Figure 2 As described, one or more genetic modifications in the engineered host cell result in increased production of UDP-glucose. Figure 2 The one or more genetic modifications in the engineered host cell that cause enhanced production of UDP-galactose are selected from: overexpression of one or more genes selected from the group consisting of: glucose promoting gene (glf) or a homolog thereof, glucokinase (glk) or a homolog thereof, phosphoglucomutase (pgm) or a homolog thereof, UTP-glucose-1-phosphate-uridyltransferase (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 pyrophosphate synthetase (prs) or a homolog thereof, Transferase (pyrE) or its homologue, orotidine 5'-phosphate decarboxylase (pyrF) or its homologue, uridylate kinase (pyrH) or its homologue, diphosphate nucleoside kinase (ndk) or its homologue and adenylate kinase (adk) or its homologue; 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 uridyltransferase (ugp), phosphoglucomutase (pgm); and / or any combination thereof. Figure 2 As described, the engineered host cells are supplemented with a culture medium comprising orotic acid, sucrose, glucose, or a combination thereof.

[0078] like Figure 2As described, one or more genetic modifications in the engineered host cell result in increased production of UDP-galactose. Figure 2 The one or more genetic modifications in the engineered host cell that result in enhanced production of UDP-galactose are selected from: (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 uridyltransferase (galT) or a homolog thereof, and (iv) any combination thereof. Figure 2 As described, the engineered host cells are supplemented with culture medium containing galactose.

[0079] like Figure 2 As described, the one or more genetic modifications for increasing the production of UDP-galactose in the engineered host cell include one or more genetic modifications that cause increased production of UDP-glucose. Figure 2 The one or more genetic modifications in the engineered host cell that increase the production of UDP-galactose are selected from: overexpression of one or more genes selected from the group consisting of: glucose promoting gene (glf) or a homolog thereof, glucokinase (glk) or a homolog thereof, phosphoglucomutase (pgm) or a homolog thereof, UTP-glucose-1-phosphate-uridyltransferase (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, phosphoribosyltransferase (pyrE) or a homolog thereof, orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof. or its homologues, uridylate kinase (pyrH) or its homologues, diphosphate nucleoside kinase (ndk) or its homologues and adenylate kinase (adk) or its homologues; overexpression of UDP-glucose 4-epimerase (galE) or its homologues, overexpression of galactokinase (galK) or its homologues, overexpression of galactose-1-phosphate uridyltransferase (galT) or its homologues; 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 uridyltransferase (ugp); phosphoglucomutase (pgm); and / or any combination thereof. Such as Figure 2As described, the engineered host cells are supplemented with a culture medium comprising glucose, galactose, orotic acid, sucrose, or any combination thereof.

[0080] This article Figure 1 Exemplary embodiments are provided illustrating combinations of modifications to the E. coli host genome, including deletion and overexpression of enzymes from other organisms, to recapitulate the biological production pathways depicted in the figure.

[0081] The present invention provides engineered host cells (e.g. Figure 1 As shown in Example 1 and described above in this application), the one or more genetic modifications allow the engineered host cell to enzymatically convert glucose to UDP-glucose via a variety of chemical intermediates. Figure 1 As shown, in certain embodiments, one or more of the genetic modifications result in an increase in metabolic flux to a UDP-glucose precursor. Figure 1 As shown, in certain embodiments, one or more of the genetic modifications results in reduced formation of byproducts. Figure 1 As shown, in certain embodiments, the genetic modification is selected from the group consisting of: (i) glucose promoting gene (glf) or its homolog, (ii) glucokinase (glk) or its homolog, (iii) phosphoglucomutase (pgm) or its homolog, (iv) UTP-glucose-1-phosphate-uridyltransferase (galU) or its homolog, (v) phosphoribosyltransferase (pyrE) or its homolog, (vi) expression of orotidine 5'-phosphate decarboxylase (pyrF) or its homolog, (vii) uridylate kinase (pyrH) or its homolog, (viii) diphosphate nucleoside kinase (ndk) or its homolog, (ix) adenylate kinase (adk) or its homolog; and (x) any combination thereof. In certain embodiments, the one or more genetic modifications are overexpression of fructokinase (cscK) or its homolog.

[0082] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are overexpression of one or more genes selected from the group consisting of: (i) glucose promoting 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-uridyltransferase (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; and (viii) any combination thereof.

[0083] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) glucose promoting gene (glf) or its homologue, (ii) glucokinase (glk) or its homologue; and (iii) any combination thereof. In certain embodiments, the one or more genetic modifications are overexpression of phosphoglucomutase (pgm) or its homologue. In certain embodiments, the one or more genetic modifications are overexpression of UTP-glucose-1-phosphate-uridyltransferase (galU) or its homologue.

[0084] like Figure 1 In certain embodiments, one or more genetic modifications are overexpression of orotate phosphoribosyltransferase (pyrE) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of orotidine 5' phosphate decarboxylase (pyrF) or its homologue. In certain embodiments, a culture medium comprising orotate is also provided. In certain embodiments, one or more genetic modifications are overexpression of uridylate kinase (pyrH) or its homologue.

[0085] like Figure 1 As shown, in certain embodiments, one or more genetic modifications are overexpression of diphosphate nucleoside kinase (NDK) or its homologue. In certain embodiments, one or more genetic modifications are overexpression of adenylate kinase (ADK) or its homologue.

[0086] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are downregulation or deletion of UDP-glucose 4-epimerase (galE). Figure 1 As shown, in certain embodiments, the 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.

[0087] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are downregulation or deletion of glucose-1-phosphate adenylyltransferase (glgC).

[0088] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are downregulation or deletion of a glucan biosynthetic glucosyltransferase (OpgG) to prevent conversion of UDP-glucose to membrane-derived oligosaccharides (MDO).

[0089] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are downregulation or deletion of glucose-6-phosphate isomerase (pgi).

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

[0091] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are overexpression of 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof.

[0092] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are overexpression of phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof.

[0093] like Figure 1 As shown, in certain embodiments, the engineered host cell includes overexpression of: (i) glucose promoting 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-uridyltransferase (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) diphosphate nucleoside kinase (ndk) or a homolog thereof, and (ix) adenylate kinase (adk) or a homolog thereof.

[0094] like Figure 1 As shown, in certain embodiments, the engineered host cell includes overexpression of: (i) glucose promoting 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-uridyltransferase (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.

[0095] Reference now Figure 1 Various aspects of the invention are described.

[0096] Step 1: Glucose is converted into glucose-6-phosphate. The glucose-promoting gene (glf) or its homologue and glucokinase (glk) or its homologue are overexpressed. In addition, phosphoribosyltransferase or its homologue may be overexpressed.

[0097] Step 2: To convert glucose-6-phosphate into glucose-1-phosphate, phosphoglucomutase (pgm) or its homolog is overexpressed.

[0098] Step 3: Conversion of glucose-1-phosphatase and UTP into UDP-glucose UTP-glucose-1-phosphate-uridyltransferase (galU) or its homologue is overexpressed.

[0099] Step 4: Convert 1-pyrophosphate 5-phosphoribosyl (PRPP) to uridine monophosphate (UMP). Orotate phosphoribosyltransferase (pyrE) or a homologue thereof and orotidine 5'-phosphate decarboxylase (pyrF) or a homologue thereof are overexpressed. In certain embodiments, this step includes supplementing orotic acid.

[0100] Step 5: Conversion of uridine monophosphate (UMP) to uridine diphosphate (UDP) Uridylate kinase (pyrH) or its homologue is overexpressed.

[0101] Step 6: Converting uridine diphosphate (UDP) to uridine triphosphate (UTP) Nucleoside diphosphate kinase (NDK) or its homologue and / or adenylate kinase (ADK) or its homologue is overexpressed.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] Step 10: Conversion of UDP-glucose into membrane-derived oligosaccharides (MDO). Glucan biosynthesis glucosyltransferase (OpgG) is downregulated or deleted to prevent the conversion of UDP-glucose into membrane-derived oligosaccharides (MDO), including the conversion into ADP-α-D-glucose.

[0106] 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 through the glycolytic pathway.

[0107] Step 12: Conversion of glucose-6-phosphate into 6-phosphogluconic acid Glucose-6-phosphate 1-dehydrogenase (zwf) or a homologue thereof may be overexpressed.

[0108] Step 13: Conversion of 6-phosphogluconate into ribose-5-phosphate (R5P) 6-phosphogluconate dehydrogenase (gnd) or its homologue can be overexpressed.

[0109] Step 14: Conversion of ribose-5-phosphate (R5P) into 5-phosphoribosyl-1-pyrophosphate (PRPP) Phosphoribosyl pyrophosphate synthetase (prs) or a homologue thereof may be overexpressed.

[0110] In certain preferred embodiments, the engineered host cell comprises one or more of the genetic modifications listed in steps 1-3 and 4-6 above. In these preferred embodiments, the engineered host cell may include overexpression of: (i) glucose promoting 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-uridyltransferase (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) diphosphate nucleoside kinase (ndk) or a homolog thereof, and (ix) adenylate kinase (adk) or a homolog thereof.

[0111] In certain preferred embodiments, the engineered cell line comprises one or more of the genetic modifications listed in steps 1-3 and 12-14 above. In these preferred embodiments, the engineered host cell comprises overexpression of: (i) glucose promoting 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-uridyltransferase (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.

[0112] In certain preferred embodiments, the engineered cell line comprises one or more of the genetic modifications listed in steps 1-3, 4-6 and 12-14 above. In these preferred embodiments, the engineered host cell may include overexpression of: (i) glucose promoting gene (glf) or its homolog, (ii) glucokinase (glk) or its homolog, (iii) phosphoglucomutase (pgm) or its homolog, (iv) UTP-glucose-1-phosphate-uridyltransferase (galU) or its homolog, (v) phosphoribosyltransferase (pyrE) or its homolog, (vi) orotidine 5'-phosphotransferase (PPT) or its homolog. acid decarboxylase (pyrF) or a homologue thereof, (vii) uridylate kinase (pyrH) or a homologue thereof, (viii) diphosphate nucleoside kinase (ndk) or a homologue thereof, (ix) adenylate kinase (adk) or a homologue thereof, (x) glucose-6-phosphate 1-dehydrogenase (zwf) or a homologue thereof, (xi) 6-phosphogluconate dehydrogenase (gnd) or a homologue thereof, and (xii) phosphoribosyl pyrophosphate synthetase (prs) or a homologue thereof.

[0113] In another aspect, the present invention provides a method for increasing the production of UDP-glucose by utilizing the engineered host cells described in this example.

[0114] In another aspect, the invention provides engineered host cells (e.g. Figure 1 The one or more genetic modifications allow the engineered host cell to enzymatically convert glucose and / or galactose to UDP-galactose via a variety of chemical intermediates.

[0115] like Figure 1 In another aspect, the present invention provides an engineered host cell (e.g. Figure 1 As shown in Example 1 and described above in this application), the one or more genetic modifications allow the engineered host cell to enzymatically convert glucose and / or galactose to UDP-galactose via a variety of intermediates. Figure 1 As shown, in certain embodiments, in addition to all of the genetic modifications listed above, one or more of the genetic modifications result in an increased metabolic flux to a UDP-galactose precursor. Figure 1 As shown, in certain embodiments, one or more of the genetic modifications result in reduced formation of by-products.

[0116] like Figure 1As shown, 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 uridyltransferase (galT) or a homolog thereof, and (iv) any combination thereof.

[0117] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are overexpression of UDP-glucose 4-epimerase (galE) or a homolog thereof.

[0118] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are overexpression of galactokinase (galK) or a homologue thereof. In certain embodiments, a supplement comprising galactose is also provided.

[0119] like Figure 1 As shown, in certain embodiments, the one or more genetic modifications are overexpression of galactose-1-phosphate uridyltransferase (galT) or a homolog thereof.

[0120] like Figure 1 In another aspect, the present invention provides an engineered host cell (e.g. Figure 1 As shown in Example 1 and described herein above in this application), the one or more genetic modifications allow the engineered host cell to enzymatically convert glucose to UDP-glucose via a variety of intermediates.

[0121] Reference now Figure 1 Various aspects of the invention are described.

[0122] Step 1: Glucose is converted to glucose-6-phosphate. Glucose stimulating gene (glf) or its homologue and / or glucokinase (glk) or its homologue are overexpressed. In addition, phosphoribosyltransferase or its homologue may be overexpressed.

[0123] Step 2: To convert glucose-6-phosphate into glucose-1-phosphate, phosphoglucomutase (pgm) or its homolog is overexpressed.

[0124] Step 3: Conversion of glucose-1-phosphatase and UTP into UDP-glucose UTP-glucose-1-phosphate-uridyltransferase (galU) or its homologue is overexpressed.

[0125] Step 4: Converting 1-pyrophosphate 5-phosphoribosyl (PRPP) to uridine monophosphate (UMP). Orotate phosphoribosyltransferase (pyrE) or a homologue thereof and / or orotidine 5'-phosphate decarboxylase (pyrF) or a homologue thereof is overexpressed. In certain embodiments, a supplement comprising orotic acid is also provided.

[0126] Step 5: Conversion of uridine monophosphate (UMP) to uridine diphosphate (UDP) Uridylate kinase (pyrH) or its homologue is overexpressed.

[0127] Step 6: Converting uridine diphosphate (UDP) to uridine triphosphate (UTP) Nucleoside diphosphate kinase (NDK) or its homologue and / or adenylate kinase (ADK) or its homologue is overexpressed.

[0128] Step 7: Conversion of UDP-glucose to UDP-galactose UDP-glucose 4-epimerase (galE) or its homologue 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 into membrane-derived oligosaccharides (MDO). Glucan biosynthesis glucosyltransferase (OpgG) is downregulated or deleted to prevent the formation of membrane-derived oligosaccharides (MDO), including 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 through the glycolytic pathway.

[0133] Step 12: Conversion of glucose-6-phosphate into 6-phosphogluconic acid Glucose-6-phosphate 1-dehydrogenase (zwf) or a homologue thereof may be overexpressed.

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

[0135] Step 14: Conversion of ribose-5-phosphate (R5P) into 5-phosphoribosyl-1-pyrophosphate (PRPP) Phosphoribosyl pyrophosphate synthetase (prs) or a homologue thereof may be overexpressed.

[0136] Step 15: Convert galactose to galactose-1-phosphate. Galactokinase (galK) or its homologue is overexpressed. In certain embodiments, a supplement comprising galactose is also provided.

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

[0138] In certain preferred embodiments, the engineered host cell comprises one or more of the genetic modifications listed in steps 1-3, 4-7 and 15-16 above. In these preferred embodiments, the engineered host cell may include overexpression of: (i) glucose promoting gene (glf) or its homolog, (ii) glucokinase (glk) or its homolog, (iii) phosphoglucomutase (pgm) or its homolog, (iv) UTP-glucose-1-phosphate-uridyltransferase (galU) or its homolog, (v) orotate phosphoribosyltransferase (pyrE) or its homolog, (vi) orotidine 5'-phosphate decarboxylase (pyrE) or its homolog. rF) or its homologue is overexpressed, (vii) uridylate kinase (pyrH) or its homologue, (viii) diphosphate nucleoside kinase (ndk) or its homologue, (ix) adenylate kinase (adk) or its homologue, (x) overexpression of UDP-glucose 4-epimerase (galE) or its homologue, (xi) overexpression of galactokinase (galK) or its homologue, and (xii) overexpression of galactose-1-phosphate uridyltransferase (galT) or its homologue.

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

[0140] In certain preferred embodiments, the engineered cell line comprises one or more of the genetic modifications listed in steps 7, 15, and 16 above. 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 uridyltransferase (galT) or a homolog thereof. In certain embodiments, a supplement comprising galactose is also provided.

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

[0142] In another aspect, the invention provides engineered host cells (e.g. Figure 1 The one or more genetic modifications allow the engineered host cell to enzymatically convert sucrose to UDP-glucose via a variety of chemical intermediates.

[0143] In certain embodiments, the genetic modification in the engineered host cell 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 a UDP-glucose 4-epimerase (galE), (iv) downregulation or deletion of a 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 the genetic modification is 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.

[0145] like Figure 1 In another aspect, the present invention provides an engineered host cell (e.g. Figure 1 As shown in Example 1 and described above in this application), the one or more genetic modifications allow the engineered host cell to enzymatically convert sucrose to UDP-glucose. In addition, Figure 1 As described, the present invention provides engineered host cells that allow enzymatic conversion of sucrose to UDP-glucose by sucrose phosphorylase (SPase) or a homologue thereof and / or sucrose synthase (SuSy) or a homologue thereof. Thus, the present invention provides overexpression of sucrose phosphorylase (SPase) or a homologue thereof and / or sucrose synthase (SuSy) or a homologue thereof to increase the conversion of sucrose to UDP-glucose.

[0146] Reference now Figure 1 Various aspects of the invention are described.

[0147] Step 1: Glucose is converted to glucose-6-phosphate. The glucose stimulating gene (glf) or its homologue and / or the glucokinase gene (glk) or its homologue are overexpressed. In addition, a phosphotransferase or its homologue may be overexpressed.

[0148] Step 2: To convert glucose-6-phosphate into glucose-1-phosphate, phosphoglucomutase (pgm) or its homolog is overexpressed.

[0149] Step 3: Conversion of glucose-1-phosphatase and UTP into UDP-glucose UTP-glucose-1-phosphate-uridyltransferase (galU) or its homologue is overexpressed.

[0150] Step 4: Converting 1-pyrophosphate 5-phosphoribosyl (PRPP) to uridine monophosphate (UMP). Orotate phosphoribosyltransferase (pyrE) or a homologue thereof and / or orotidine 5'-phosphate decarboxylase (pyrF) are overexpressed. In certain embodiments, a supplement comprising orotic acid is also provided.

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

[0152] Step 6: Converting uridine diphosphate (UDP) to uridine triphosphate (UTP) Nucleoside diphosphate kinase (NDK) or its homologue and / or adenylate kinase (ADK) or its homologue is overexpressed.

[0153] 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.

[0154] 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.

[0155] 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 UDP-glucuronic acid.

[0156] Step 10: Conversion of UDP-glucose into membrane-derived oligosaccharides (MDO). Glucan biosynthesis glucosyltransferase (OpgG) is downregulated or deleted to prevent the conversion of UDP-glucose into membrane-derived oligosaccharides (MDO), including the conversion into ADP-α-D-glucose.

[0157] 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 through the glycolytic pathway.

[0158] Step 12: Conversion of glucose-6-phosphate into 6-phosphogluconic acid Glucose-6-phosphate 1-dehydrogenase (zwf) or a homologue thereof may be overexpressed.

[0159] Step 13: Conversion of 6-phosphogluconate into ribose-5-phosphate (R5P) 6-phosphogluconate dehydrogenase (gnd) or its homologue can be overexpressed.

[0160] Step 14: Conversion of ribose-5-phosphate (R5P) into 5-phosphoribosyl-1-pyrophosphate (PRPP) Phosphoribosyl pyrophosphate synthetase (prs) or a homologue thereof may be overexpressed.

[0161] In another aspect, the present invention provides a method for increasing the production of UDP-glucose by utilizing the engineered host cells described in this example.

[0162] Table 1 below provides a list of sequences of exemplary enzymes of the invention.

[0163] Table 1: Exemplary enzyme sequences of the invention

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] By reference Incorporation

[0183] Other documents, such as patents, patent applications, patent publications, journals, books, papers, web page content, publicly accessible databases, have been referenced and cited throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0184] Equivalent form

[0185] Various modifications to the invention and many other embodiments of the invention in addition to the embodiments shown and described herein will become apparent to those skilled in the art based on the entire content of this document (including references to the scientific literature and patent literature cited herein). The subject matter of this article contains important information, illustrations, and guidance that can be adapted to practice the invention in its various embodiments and equivalent forms.

Claims

1. An engineered host cell comprising one or more genetic modifications that allow the engineered host cell to enzymatically convert a carbon source into UDP-sugars via a variety of chemical intermediates.

2. The engineered host cell of claim 1, wherein the UDP-sugar is UDP-glucose.

3. The engineered host cell of claim 1, wherein the UDP-sugar is UDP-galactose.

4. The engineered host cell of claim 1, wherein the chemical intermediate is UDP-glucose and the UDP-sugar is UDP-galactose.

5. The engineered host cell of claim 1, wherein the engineered host cell is Escherichia coli (E. coli).

6. The engineered host cell of claim 2, wherein the one or more genetic modifications are overexpression of one or more genes selected from the group consisting of: (i) glucose promoting 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-uridyltransferase (galU) or a homolog thereof, (v) fructokinase (cscK) or a homolog thereof, and (vi) any combination thereof.

7. The engineered host cell of claim 2, wherein the one or more genetic modifications 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.

8. An engineered host cell according to claim 2, 6 or 7, wherein the 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 (OpgG), (v) UDP-glucose 4-epimerase (galE), (vi) UDP-glycohydrolase (ushA), (vii) UTP-glucose-1-phosphate uridyltransferase (ugp), (viii) phosphoglucomutase (pgm), (ix) (heptosyl) lipopolysaccharide alpha-1,3-glucosyltransferase (WaaG), (x) trehalose-6-phosphate synthase (otsA), (xi) glucose-1-phosphatase (agp), and (xii) any combination thereof.

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

10. An engineered host cell according to claims 2 to 4, wherein the 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 a homolog thereof; (ii) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof; (iii) phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof; (iv) phosphoribosyltransferase (pyrE) or a homolog thereof; (v) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof; (vi) uridylate kinase (pyrH) or a homolog thereof; (vii) diphosphate nucleoside kinase (ndk) or a homolog thereof, and (viii) adenylate kinase (adk) or a homolog thereof, and (ix) any combination thereof.

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

12. The engineered host cell of claim 2, wherein the one or more genetic modifications are selected from: Overexpression of one or more genes selected from the group consisting of: glucose facilitating gene (glf) or homologs thereof, glucokinase (glk) or homologs thereof, phosphoglucomutase (pgm) or homologs thereof, UTP-glucose-1-phosphate-uridyltransferase (galU) or homologs thereof, sucrose transporter (cscB) or homologs thereof, sucrose synthase (SuSy) or homologs thereof, sucrose phosphorylase (SPase) or homologs thereof, glucose-6-phosphate 1-dehydrogenase (zwf ) or a homologue thereof; 6-phosphogluconate dehydrogenase (gnd) or a homologue thereof; phosphoribosyl pyrophosphate synthetase (prs) or a homologue thereof; phosphoribosyltransferase (pyrE) or a homologue thereof; orotidine 5'-phosphate decarboxylase (pyrF) or a homologue thereof, uridylate kinase (pyrH) or a homologue thereof, nucleoside diphosphate kinase (ndk) or a homologue thereof, (viii) adenylate kinase (adk) or a homologue thereof, and (ix) fructokinase (cscK) or a homologue 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 (OpgG), UDP-glucose 4-epimerase (galE), UDP-glycohydrolase (ushA), UTP-glucose-1-phosphate uridyltransferase (ugp), phosphoglucomutase (pgm), (heptosyl) lipopolysaccharide alpha-1,3-glucosyltransferase (WaaG), trehalose-6-phosphate synthase (otsA), glucose-1-phosphatase (agp); and / or Any combination thereof.

13. The engineered cell of claim 2, wherein the engineered cell is an engineered host cell supplemented with a culture medium comprising orotic acid, fructose, glucose and / or sucrose.

14. An engineered host cell according to claim 3 or 4, 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 uridyltransferase (galT) or a homolog thereof, and (iv) any combination thereof.

15. The engineered host cell of claim 3, 4 or 14, wherein the engineered host cell is supplemented with culture medium comprising galactose.

16. The engineered host cell of claim 3, 4 or 14, wherein the one or more genetic modifications are selected from: Overexpression of one or more genes selected from the group consisting of: glucose promoting gene (glf) or homologs thereof, glucokinase (glk) or homologs thereof, phosphoglucomutase (pgm) or homologs thereof, UTP-glucose-1-phosphate-uridyltransferase (galU) or homologs thereof, sucrose transporter (cscB) or homologs thereof, sucrose synthase (SuSy) or homologs thereof, sucrose phosphorylase (SPase) or homologs thereof, glucose-6-phosphate 1-dehydrogenase (zwf) or homologs thereof; 6-phosphogluconate dehydrogenase (gnd) or homologs thereof; phosphoribosyl pyrophosphate synthase (RPS) or homologs thereof. enzyme (prs) or its homologue; phosphoribosyltransferase (pyrE) or its homologue; orotidine 5'-phosphate decarboxylase (pyrF) or its homologue, uridylate kinase (pyrH) or its homologue, diphosphate nucleoside kinase (ndk) or its homologue, and (viii) adenylate kinase (adk) or its homologue; overexpression of UDP-glucose 4-epimerase (galE) or its homologue, overexpression of galactokinase (galK) or its homologue, overexpression of galactose-1-phosphate uridyltransferase (galT) or its homologue, overexpression of fructokinase (cscK) or its homologue; 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-glycohydrolase (ushA), UTP-glucose-1-phosphate uridyltransferase (ugp), phosphoglucomutase (pgm), (heptosyl) lipopolysaccharide alpha-1,3-glucosyltransferase (WaaG), trehalose-6-phosphate synthase (otsA), glucose-1-phosphatase (agp); and / or Any combination thereof.

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

18. The engineered host cell of 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-sugars, the method comprising: providing an engineered host cell, the engineered host cell comprising one or more genetic modifications, the one or more genetic modifications allowing the engineered host cell to enzymatically convert a carbon source into UDP-sugars via a variety of chemical intermediates.

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

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

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

23. The method of claim 19, wherein the engineered host cell is Escherichia coli.

24. The method of claim 20, wherein the one or more genetic modifications are overexpression of one or more genes selected from the group consisting of: (i) glucose promoting 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-uridyltransferase (galU) or a homolog thereof, (v) fructokinase (cscK) or a homolog thereof, and (v) any combination thereof.

25. The method of claim 20, wherein the one or more genetic modifications 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), and (iv) any combination thereof.

26. The method of claim 20, 24 or 25, wherein the 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 (OpgG); (v) UDP-glucose 4-epimerase (galE); (vi) UDP-glycohydrolase (ushA); (vii) UTP-glucose-1-phosphate uridyltransferase (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 of claims 24 to 26, wherein the cells are supplemented with glucose, fructose and / or sucrose.

28. The method of claim 20 to 22, wherein the 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 a homolog thereof; (ii) 6-phosphogluconate dehydrogenase (gnd) or a homolog thereof; (iii) phosphoribosyl pyrophosphate synthetase (prs) or a homolog thereof; (iv) phosphoribosyltransferase (pyrE) or a homolog thereof; (v) orotidine 5'-phosphate decarboxylase (pyrF) or a homolog thereof; (vi) uridylate kinase (pyrH) or a homolog thereof; (vii) diphosphate nucleoside kinase (ndk) or a homolog thereof, and (viii) adenylate kinase (adk) or a homolog thereof, and (ix) any combination thereof.

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

30. The method of claim 20, wherein the one or more genetic modifications are selected from: Overexpression of one or more genes selected from the group consisting of: glucose facilitating gene (glf) or homologs thereof, glucokinase (glk) or homologs thereof, phosphoglucomutase (pgm) or homologs thereof, UTP-glucose-1-phosphate-uridyltransferase (galU) or homologs thereof, sucrose transporter (cscB) or homologs thereof, sucrose synthase (SuSy) or homologs thereof, sucrose phosphorylase (SPase) or homologs thereof, glucose-6-phosphate 1-dehydrogenase (zw f) or a homologue thereof; 6-phosphogluconate dehydrogenase (gnd) or a homologue thereof; phosphoribosyl pyrophosphate synthetase (prs) or a homologue thereof; phosphoribosyltransferase (pyrE) or a homologue thereof; orotidine 5'-phosphate decarboxylase (pyrF) or a homologue thereof, uridylate kinase (pyrH) or a homologue thereof, nucleoside diphosphate kinase (ndk) or a homologue thereof, and (viii) adenylate kinase (adk) or a homologue thereof, and fructokinase (cscK) or a homologue 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 (OpgG); UDP-glucose 4-epimerase (galE); UDP-sugar hydrolase (ushA); UTP-glucose-1-phosphate uridyltransferase (ugp); (heptosyl) lipopolysaccharide alpha-1,3-glucosyltransferase (WaaG); trehalose-6-phosphate synthase (otsA); glucose-1-phosphatase (agp); and / or Any combination thereof.

31. The method of claim 20, wherein the engineered cell is an engineered host cell supplemented with a culture medium comprising orotic acid, glucose, fructose, and / or sucrose.

32. The method of 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 uridyltransferase (galT) or a homolog thereof, and (iv) any combination thereof.

33. The method of claim 21, 22 or 32, wherein the engineered host cell is supplemented with culture medium comprising galactose.

34. The method of claim 21, 22 or 32, wherein the one or more genetic modifications are selected from: Overexpression of one or more genes selected from the group consisting of: glucose promoting gene (glf) or homologs thereof, glucokinase (glk) or homologs thereof, phosphoglucomutase (pgm) or homologs thereof, UTP-glucose-1-phosphate-uridyltransferase (galU) or homologs thereof, sucrose transporter (cscB) or homologs thereof, sucrose synthase (SuSy) or homologs thereof, sucrose phosphorylase (SPase) or homologs thereof, glucose-6-phosphate 1-dehydrogenase (zwf) or homologs thereof; 6-phosphogluconate dehydrogenase (gnd) or homologs thereof; phosphoribosyl pyrophosphate synthase (RPS) or homologs thereof. enzyme (prs) or its homologue; phosphoribosyltransferase (pyrE) or its homologue; orotidine 5'-phosphate decarboxylase (pyrF) or its homologue, uridylate kinase (pyrH) or its homologue, diphosphate nucleoside kinase (ndk) or its homologue, and (viii) adenylate kinase (adk) or its homologue; overexpression of UDP-glucose 4-epimerase (galE) or its homologue, overexpression of galactokinase (galK) or its homologue, overexpression of galactose-1-phosphate uridyltransferase (galT) or its homologue, overexpression of fructokinase (cscK) or its homologue; 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 uridyltransferase (ugp); (heptosyl) lipopolysaccharide alpha-1,3-glucosyltransferase (WaaG); trehalose-6-phosphate synthase (otsA); glucose-1-phosphatase (agp); and / or Any combination thereof.

35. The method of claim 21, 22, 32 or 34, wherein the engineered cell is an engineered host cell supplemented with a culture medium comprising galactose, orotic acid, sucrose, fructose and / or glucose.

36. The method of 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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