Biosynthetic production of UDP-rhamnose

Abstract

The present disclosure relates to the biosynthesis of UDP-rhamnose and recombinant polypeptides having enzyme activities of relevant biosynthetic pathways that can be used for the production of UDP-rhamnose. The present invention also provides a method for preparing a steviol glycoside composition comprising at least one rhamnose-containing steviol glycoside.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 825,799, filed on March 29, 2019, the disclosure of which is incorporated herein by reference in its entirety. Field of the invention

[0003] The present disclosure generally relates to the biosynthesis of uridine diphosphate rhamnose (“UDP - rhamnose” or “UDPR” or “UDP - Rh”). More specifically, the present disclosure relates to: biocatalytic methods for preparing UDP - rhamnose, which in turn can be used for the biosynthesis of rhamnose - containing steviol glycosides; and recombinant polypeptides having enzyme activities related to the relevant biosynthetic pathways for producing UDP - rhamnose and rhamnose - containing steviol glycosides. Background art

[0004] Steviol glycosides are a class of compounds found in the leaves of the Stevia rebaudiana Stevia rebaudiana ) plant and can be used as high - intensity, low - calorie sweeteners. These naturally occurring steviol glycosides have the same basic diterpene structure (the steviol backbone), but the number and type of carbohydrate residues (e.g., glucose, rhamnose, and xylose residues) at the C13 and C19 positions of the steviol backbone are different. Interestingly, these variations in the sugar “decoration” of the basic steviol structure often significantly and unpredictably affect the properties of the resulting steviol glycosides. The properties affected can include, but are not limited to, the overall taste profile, the presence and degree of any off - flavors, the crystallization point, “mouthfeel”, solubility, the perceived sweetness, and other differences. Steviol glycosides with known structures include stevioside, rebaudioside A (“Reb A”), rebaudioside B (“Reb B”), rebaudioside C (“Reb C”), rebaudioside D (“Reb D”), rebaudioside E (“Reb E”), rebaudioside F (“Reb F”), rebaudioside M (“Reb M”), rebaudioside J (“Reb J”), rebaudioside N (“Reb N”), and dulcoside A.

[0005] Based on dry weight, steviosin, Reb A, Reb C, and dulcoside A account for approximately 9.1%, 3.8%, 0.6%, and 0.3%, respectively, of the total weight of all steviol glycosides found in wild-type Stevia rebaudiana leaves. Other steviol glycosides such as Reb J and Reb N are present in significantly lower amounts. Extracts from Stevia rebaudiana plants are commercially available. In such extracts, steviosin and Reb A are typically the major components, while other known steviol glycosides are present as minor or trace components. The actual levels of the various steviol glycosides in any given Stevia rebaudiana extract can vary depending on, for example, the climate and soil in which the Stevia rebaudiana plants are grown, the conditions under which the Stevia rebaudiana leaves are harvested, and the method used to extract the desired steviol glycosides. By way of illustration, the amount of Reb A in a commercial preparation can vary between about 20 wt% and over about 90 wt% of the total steviol glycoside content, while the amounts of Reb B, Reb C, and Reb D can be about 1 - 2 wt%, about 7 - 15 wt%, and about 2 wt%, respectively, of the total steviol glycoside content. In such extracts, Reb J and Reb N each typically account for less than 0.5 wt% of the total steviol glycoside content.

[0006] As natural sweeteners, different steviol glycosides have different degrees of sweetness, taste, and aftertaste. The sweetness of steviol glycosides is significantly higher than that of table sugar (i.e., sucrose). For example, steviosin itself has a sweetness that is 100 - 150 times that of sucrose but has a bitter aftertaste, as noted in many taste tests, while Reb A and Reb E have a sweetness that is 250 - 450 times that of sucrose and have much better aftertaste characteristics than steviosin. However, these steviol glycosides still retain an obvious aftertaste themselves. Thus, the overall taste profile of any Stevia rebaudiana extract is profoundly influenced by the relative content of the various steviol glycosides in the extract, which in turn can be affected by the plant source, environmental factors (such as soil content and climate), and the extraction method. In particular, variations in the extraction conditions can result in inconsistent compositions of steviol glycosides in Stevia rebaudiana extracts, such that the taste profiles vary between different batches of extracted products. The taste profile of Stevia rebaudiana extracts can also be affected by plant-derived or environment-derived contaminants (such as pigments, lipids, proteins, phenols, and sugars) that remain in the product after the extraction process. These contaminants typically have off-flavors that are undesirable for using Stevia rebaudiana extracts as sweeteners. In addition, methods for isolating individual or specific combinations of steviol glycosides that are not abundant in Stevia rebaudiana extracts can be cost- and resource-prohibitive.

[0007] In addition, methods for extracting from plants typically employ solid-liquid extraction techniques using solvents such as hexane, chloroform, and ethanol. Solvent extraction is an energy-intensive method and can lead to problems associated with the disposal of toxic waste. Thus, new production methods are needed to reduce the cost of steviol glycoside production and to mitigate the environmental impact of large-scale cultivation and processing.

[0008] Accordingly, there is a need in the art for new methods of preparing steviol glycosides, particularly rhamnose-containing steviol glycosides such as Reb J and Reb N, which can produce products with better and more consistent flavor profiles. Given that the biosynthetic pathways leading to such rhamnose-containing steviol glycosides often use UDP-rhamnose as one of the starting substrates, there is a need in the art for novel and efficient methods for preparing UDP-rhamnose. SUMMARY OF THE INVENTION

[0009] In various embodiments, the present disclosure encompasses biosynthetic methods for preparing UDP-rhamnose. In a preferred embodiment, the present disclosure relates to a biosynthetic method for preparing uridine diphosphate β-L-rhamnose (“UDP-L-rhamnose” or “UDP-L-R” or “UDP-L-Rh”). Generally, the method comprises incubating uridine diphosphate-glucose (“UDP-glucose” or “UDPG”) with one or more recombinant polypeptides in the presence of a source of NAD + and NADPH for a sufficient time to produce UDP-rhamnose, wherein the one or more recombinant polypeptides individually or collectively have UDP-rhamnose synthase activity.

[0010] In certain embodiments, the one or more recombinant polypeptides can be a trifunctional enzyme having UDP-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-glucose 3,5-epimerase, and UDP-4-keto-rhamnose 4-keto-reductase activities. Such a trifunctional polypeptide is also referred to as an RHM enzyme. In such embodiments, the one or more recombinant polypeptides can be selected from those from Ricinus communis ( Ricinus communis )、Ceratopteris thalictroides ( Ceratopteris thalictroides )、Azolla filiculoides ( Azolla filiculoides )、Chlamydomonas reinhardtii ( Ostreococcus lucimarinus )、Nannochloropsis oceanica ( Nannochloropsis oceanica )、Ulva lactuca ( Ulva lactuca )、 Golenkinia longispicula 、Tetraselmis subcordiformis ( Tetraselmis subcordiformis ) or Tetraselmis cordiformis ( Tetraselmis cordiformis) RHM enzymes. In these embodiments, one or more recombinant polypeptides may be selected from recombinant polypeptides comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 or SEQ ID NO: 89. These one or more recombinant polypeptides may be selected from recombinant polypeptides encoded by nucleotides comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48 or SEQ ID NO: 90.

[0011] In certain embodiments, one or more recombinant polypeptides may comprise a first recombinant polypeptide and a second recombinant polypeptide, wherein the first recombinant polypeptide and the second recombinant polypeptide together have UDP - rhamnose synthase activity. Specifically, the first recombinant polypeptide may primarily have UDP - glucose 4,6 - dehydratase activity, and such a recombinant polypeptide is referred to herein as a "DH" (dehydratase) enzyme. The second recombinant polypeptide may be a bifunctional recombinant polypeptide having UDP - 4 - keto - 6 - deoxy - glucose 3,5 - epimerase and UDP - 4 - keto - rhamnose 4 - keto - reductase activity. This bifunctional recombinant polypeptide is referred to herein as an "ER" enzyme (the letter "E" represents epimerase activity and the letter "R" represents reductase activity).

[0012] In such embodiments, the first recombinant polypeptide may be selected from those from Botrytis cinerea ( Botrytis cinerea ), Acrostichum aureum ( Acrostichum aureum ), Neochloris oleoabundans ( Ettlia oleoabundans ), Volvox carteri ( Volvox carteri ), Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii ), Oophila amblystomatis or Dunaliella primolectaDH enzyme. In these embodiments, the first recombinant polypeptide can be selected from recombinant polypeptides comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 7, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO: 37. Such a first recombinant polypeptide can be selected from recombinant polypeptides encoded by nucleotides comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 8, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 or SEQ ID NO: 38.

[0013] Examples of suitable second recombinant polypeptides can include those from Physcomitrella patens Patens subsp.( Physcomitrella patens subsp.Patens ), Magnaporthe oryzae( Pyricularia oryzae ), Nannochloropsis( Nannochloropsis oceanica ), Ulva lactuca( Ulva lactuca ), Tetraselmis cordiformis( Tetraselmis cordiformis ), Tetraselmis subcordiformis( Tetraselmis subcordiformis ), Chlorella sorokiniana( Chlorella sorokiniana ), Chlamydomonas moewusii , Golenkinia longispicula , Chlamydomonas reinhardtii( Chlamydomonas reinhardtii ), Chlorogonium ellipsoideum( Chromochloris zofingiensis ), Dunaliella primolecta , Pavlova lutheri( Pavlova lutheri ), Nitella mirabilis( Nitella mirabilis ), Marchantia polymorpha( Marchantia polymorpha ), Selaginella moellendorffii( Selaginella moellendorffii ), Bryum argenteum argenteum var.( Bryum argenteum var argenteum ), Arabidopsis thaliana( Arabidopsis thaliana ), Magnaporthe oryzae( Pyricularia oryzae ) or Citrus clementina Citrus clementina) ER enzyme. For example, the second recombinant polypeptide may be selected from recombinant polypeptides having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 91, SEQ ID NO: 93 or SEQ ID NO: 95. Such a second recombinant polypeptide may be selected from recombinant polypeptides encoded by nucleotides comprising a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 92, SEQ ID NO: 94 or SEQ ID NO: 96.

[0014] In other embodiments, one or more recombinant polypeptides can be fusion enzymes that include a first domain (DH domain) having UDP-glucose 4,6-dehydratase activity and a second domain having bifunctional ER activity (i.e., UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-keto-rhamnose 4-keto-reductase activity). The DH domain can be coupled to the ER domain via a peptide linker. In various embodiments, the peptide linker can include from 2 to 15 amino acids. Exemplary linkers include those that include glycine and serine, such as repeat units of glycine, repeat units of serine, repeat units of certain motifs composed of glycine and serine, and combinations thereof. In a preferred embodiment, the peptide linker can be GSG. Such a fusion enzyme thus includes a DH domain fused to an ER domain that together have UDP-rhamnose synthase activity and the ability to catalyze the conversion of UDP-glucose to UDP-rhamnose.

[0015] In embodiments involving a fusion enzyme, the first domain of the fusion enzyme can include a DH enzyme from Botrytis cinerea, Acrostichum aureum, Neochloris oleoabundans, Volvox carteri, Chlamydomonas reinhardtii, Oophila amblystomatis or Dunaliella primolecta . In these embodiments, the first domain can include a recombinant polypeptide having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37. Such a DH domain can include a recombinant polypeptide encoded by a nucleotide that includes a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. The second domain of the fusion enzyme can include a DH enzyme from Physcomitrium patens Patens subsp., Magnaporthe oryzae, Nannochloropsis sp., Ulva lactuca, Tetraedron cardiacum, Tetraedron subcordiforme, Chlorella sorokiniana, Chlamydomonas moewusii , Golenkinia longispicula , Chlamydomonas reinhardtii, Chlorogonium ellipsoideum, Dunaliella primolecta , Pavlova lutheri, Nitella mirabilis, Marchantia polymorpha, Selaginella moellendorffii, Bryum argenteum argenteumER enzymes of variants, Arabidopsis thaliana, Pyricularia oryzae, or Citrus clementina. For example, the ER domain may comprise a recombinant polypeptide having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 91, SEQ ID NO: 93, or SEQ ID NO: 95. Such an ER domain may comprise a recombinant polypeptide encoded by a nucleotide comprising a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 92, SEQ ID NO: 94, or SEQ ID NO: 96. In certain preferred embodiments, the first domain of the fusion enzyme may comprise an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 7. The second domain may comprise an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 61, or SEQ ID NO: 63.In such a preferred embodiment, the fusion enzyme as a whole may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, SEQ ID NO: 83 or SEQ ID NO: 85. In certain preferred embodiments, the first domain of the fusion enzyme may comprise an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7 or SEQ ID NO: 31, and the second domain of the fusion enzyme may comprise an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 63. The fusion enzyme as a whole may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 87.

[0016] In certain embodiments, the first recombinant polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 83, SEQ ID NO. 85, SEQ ID NO. 87 or SEQ ID NO. 89. In certain embodiments, the first recombinant polypeptide can comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9. In certain embodiments, the first recombinant polypeptide can comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11. In certain embodiments, the first recombinant polypeptide can comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 13. In certain embodiments, the first recombinant polypeptide can comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 83. In certain embodiments, the first recombinant polypeptide can comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 85. In certain embodiments, the first recombinant polypeptide can comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 87. In certain embodiments, the first recombinant polypeptide can comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 89.

[0017] In various embodiments, the biosynthetic methods provided herein can include expressing the first recombinant polypeptide in a transformed cell system. In certain embodiments, the transformed cell system is selected from yeast, plants that do not produce UDP-rhamnose, algae, fungi, and bacteria. In certain embodiments, the bacteria or yeast can be selected from: Escherichia ( Escherichia ); Salmonella ( Salmonella ); Bacillus ( Bacillus ); Acinetobacter ( Acinetobacter ); Streptomyces ( Streptomyces ); Corynebacterium ( Corynebacterium ); Methylosinus ( Methylosinus ); Methylomonas ( Methylomonas ); Rhodococcus ( Rhodococcus ); Pseudomonas ( Pseudomonas ); Rhodobacter (Rhodobacter ); Synechocystis Synechocystis ); Saccharomyces Saccharomyces ); Zygosaccharomyces Zygosaccharomyces ); Kluyveromyces Kluyveromyces ); Candida Candida ); Hansenula Hansenula ); Debaryomyces Debaryomyces ); Mucor Mucor ); Pichia Pichia ); Torulopsis Torulopsis ); Aspergillus Aspergillus ); Arthrobotlys ; Brevibacterium Brevibacteria ); Microbacterium Microbacterium ); Arthrobacter Arthrobacter ); Citrobacter Citrobacter ); Klebsiella Klebsiella ); Pantoea Pantoea ); and Clostridium Clostridium ).

[0018] In certain embodiments, a source of NADPH can be provided after incubating uridine diphosphate - glucose with the first recombinant polypeptide for a sufficient time to produce UDP - 4 - keto - 6 - deoxy - glucose ("UDP4K6G"). In certain embodiments, the source of NADPH can include an oxidation reaction substrate and an NADP + - dependent enzyme. In certain embodiments, the source of NADPH can include malate and malic enzyme. In certain embodiments, the source of NADPH can include formate and formate dehydrogenase. In certain embodiments, the source of NADPH can include phosphite and phosphite dehydrogenase.

[0019] In certain embodiments, the incubation step can be carried out in a transformed cell system. In other embodiments, the incubation step can be carried out in vitro. In certain embodiments, the biosynthetic method disclosed herein can include separating the first recombinant polypeptide from the transformed cell system and carrying out the incubation step in vitro.

[0020] In certain embodiments, the first recombinant polypeptide having rhamnosyl synthase activity and the second recombinant polypeptide having sucrose synthase activity are incubated in a medium containing sucrose and uridine diphosphate ("UDP"). The second recombinant polypeptide can be selected from Arabidopsis thaliana Arabidopsis ) sucrose synthase, mung bean Vigna radiate ) sucrose synthase, and Coffea Coffea)Sucrose synthase. In this embodiment, in the first step of the reaction, sucrose synthase activity produces UDP-glucose, which is then used as a substrate by the first recombinant enzyme to produce UDP-rhamnose. The source of NADPH in this embodiment can include an oxidation reaction substrate and NADP + -dependent enzymes. In certain embodiments, the source of NADPH can include malate and malic enzyme. In certain embodiments, the source of NADPH can include formate and formate dehydrogenase. In certain embodiments, the source of NADPH can include phosphite and phosphite dehydrogenase.

[0021] Also provided herein is a biosynthetic method for preparing a steviol glycoside composition comprising at least one rhamnose-containing steviol glycoside. The method can include incubating UDP-glucose with a first recombinant polypeptide having UDP-rhamnose synthase activity in the presence of a source of NAD + and NADPH to produce UDP-rhamnose; and reacting UDP-rhamnose with a steviol glycoside substrate in the presence of a second recombinant polypeptide having UDP-rhamnosyltransferase activity such that the rhamnose moiety is coupled to the steviol glycoside substrate to produce at least one rhamnose-containing steviol glycoside. In certain embodiments, the steviol glycoside substrate can be Reb A and the resulting steviol glycoside composition can include Reb N, Reb J, or both.

[0022] Aspects of the present disclosure also provide a steviol glycoside composition comprising at least one rhamnose-containing steviol glycoside, the rhamnose-containing steviol glycoside obtainable or producible by any of the biosynthetic methods described herein (including any of the above embodiments).

[0023] Aspects of the present disclosure also provide nucleic acids encoding polypeptides as described herein. In certain embodiments, the nucleic acid comprises a sequence encoding a polypeptide that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 83, SEQ ID NO. 85, SEQ ID NO. 87 or SEQ ID NO. 89. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 2. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 4. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 6. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 10. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 12. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 14. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 40. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 42. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 44. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 46. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 84. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 86. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 88. In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 90. In certain embodiments, the nucleic acid is a plasmid or other vector.

[0024] Aspects of the present disclosure also provide cells that comprise a nucleic acid described herein, including any of the above embodiments.

[0025] Aspects of the present disclosure provide cells comprising at least one polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 83, SEQ ID NO. 85, SEQ ID NO. 87 or SEQ ID NO. 89. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 1. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 3. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 9. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 9. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 11. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 13. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 37. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 41. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 43. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 45. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 47. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 83. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 85. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 87. In certain embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO: 89.In certain embodiments, the cell is a yeast cell, a plant cell that does not produce UDP - rhamnose, an algal cell, a fungal cell, or a bacterial cell. In certain embodiments, the bacterial or yeast cell is selected from: Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotlys ; Brevibacterium; Microbacterium; Arthrobacter; Citrobacter; Klebsiella; Pantoea; and Clostridium. In certain embodiments, the cell further comprises one or more other polypeptides having UDP - rhamnosyltransferase activity, UDP - glucosyltransferase activity, and / or sucrose synthase activity as described herein.

[0026] Regarding the cell system in the embodiments, it can be selected from one or more bacteria, one or more yeasts, and combinations thereof, or any cell system that allows genetic transformation with a selected gene and subsequent biosynthesis production of UDP - rhamnose. In the most preferred microbial system, Escherichia coli is used to produce the desired compound.

[0027] Other aspects of the present disclosure provide an in vitro reaction mixture comprising at least one polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 83, SEQ ID NO. 85, SEQ ID NO. 87 or SEQ ID NO. 89. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 1. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 3. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 5. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 9. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 11. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 13. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 37. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 41. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 43. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 45. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 47. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 83. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 85. In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 87.In certain embodiments, the in vitro reaction mixture comprises at least one polypeptide comprising the sequence of SEQ ID NO: 89. In certain embodiments, the in vitro reaction mixture further comprises one or more other recombinant polypeptides having UDP-rhamnosyltransferase activity, UDP-glucosyltransferase activity, and / or sucrose synthase activity as described herein.

[0028] Although the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will be described in detail herein. However, it should be understood that the drawings and detailed description presented herein are not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, are intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0029] Other features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments of the invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The chemical structure of uridine diphosphate β-L-rhamnose is shown.

[0031] Figure 2 The schematic diagram explains the following multi-enzyme synthesis pathway: (a) production of UDP-rhamnose from UDP-glucose; (b) production of Reb N from Reb A and UDP-rhamnose through the intermediate Reb J; (c) regeneration of NADPH from NADP + and malic acid using malic enzyme MaeB; and (d) regeneration of UDP-glucose (UDPG) from UDP and sucrose using sucrose synthase according to the present disclosure.

[0032] Figure 3Shows the UDP-rhamnose biosynthetic pathway involving three different enzymes in plants and fungi. In the first step of this biosynthetic pathway, UDP-glucose 4,6-dehydratase converts UDP-glucose into UDP-4-keto-6-deoxyglucose ("UDP4K6G"). In the second step of this biosynthetic pathway, the enzyme UDP-4-keto-6-deoxy-glucose 3,5-epimerase converts UDP-4-keto-6-deoxyglucose into UDP-4-ketorhamnose. In the third enzymatic step of this biosynthetic pathway, UDP-4-ketorhamnose-4-keto reductase converts UDP-4-ketorhamnose into UDP-rhamnose. A trifunctional polypeptide with all three enzyme activities is called an "RHM" enzyme. A bifunctional polypeptide with UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-ketorhamnose-4-keto reductase activities is called an "ER" enzyme. A polypeptide with only UDP-glucose 4,6-dehydratase activity is called a "DH" enzyme. Additionally, in this embodiment, the NADPH cofactor is regenerated by using NADP + as an oxidant to oxidize malate to pyruvate, and this reaction is catalyzed by NADP + -dependent malic enzyme (MaeB). Furthermore, in this embodiment, UDP-glucose can be converted from UDP and sucrose by sucrose synthase (SUS).

[0033] Figure 4 Shows a one-pot multi-enzyme system for in vitro synthesis of UDP-rhamnose that bioconverts UDP-glucose (UDPG) to UDP-rhamnose using a trifunctional UDP-rhamnose synthase (e.g., NRF1 or NR32) according to the present disclosure. UDP-glucose can be replenished from UDP and sucrose in a reaction catalyzed by sucrose synthase (SUS) as shown. The synthesis of UDP-rhamnose can be coupled with an oxidation reaction to regenerate the NADPH cofactor. In the shown embodiment, the NADPH cofactor is regenerated by using NADP + as an oxidant to oxidize formic acid to carbon dioxide, and this reaction is catalyzed by formate dehydrogenase (FDH).

[0034] Figure 5 Shows a one-pot multi-enzyme system for in vitro synthesis of UDP-rhamnose that bioconverts UDP-glucose to UDP-rhamnose using a trifunctional UDP-rhamnose synthase (e.g., NRF1 or NR32) according to the present disclosure. UDP-glucose can be replenished from UDP and sucrose in a reaction catalyzed by sucrose synthase (SUS) as shown. The synthesis of UDP-rhamnose can be coupled with an oxidation reaction to regenerate the NADPH cofactor. In the shown embodiment, the NADPH cofactor is regenerated by using NADP +It is regenerated by oxidizing phosphorous acid to phosphoric acid as an oxidant, and this reaction is catalyzed by phosphorous acid dehydrogenase (PTDH).

[0035] Figure 6 The results of the enzyme activity analysis of three trifunctional UDP-rhamnose synthase candidates (NR12, NR32, and NR33) for UDP-rhamnose production are shown. The letter "a" after the enzyme indicates a one-step cofactor addition scheme, where NAD + and NADPH are added at the start of the reaction. The letter "b" after the enzyme indicates a two-step cofactor addition scheme, where NAD + is added at the start of the reaction, but NADPH is not added until 3 hours after the start of the reaction. All samples are collected after 3 hours (A), 6 hours (B), and 18 hours (C). The collected samples are extracted with chloroform and analyzed by HPLC. Legend: "UDP-Rh" = UDP-rhamnose; "UDPG" = UDP-glucose; and "UDP4K6G" = UDP-4-keto-6-deoxyglucose.

[0036] Figure 7 Shows how the two-step cofactor addition scheme according to the present disclosure can improve the conversion efficiency of UDP-rhamnose production. In this experiment, the recombinant UDP-rhamnose synthase enzyme NRF1 is used. The collected samples are extracted with chloroform and analyzed by HPLC. All samples are collected after 1 hour, 3 hours, 4 hours, 6 hours, and 18 hours. The letter "a" after the reaction time indicates a one-step cofactor addition scheme, where NAD + and NADPH are added at the start of the reaction. The letter "b" after the reaction time indicates a two-step cofactor addition scheme, where NAD + is added at the start of the reaction, but NADPH is not added until 3 hours after the start of the reaction. Legend: "UDP-Rh" = UDP-rhamnose; "UDPG" = UDP-glucose; and "UDP4K6G" = UDP-4-keto-6-deoxyglucose.

[0037] Figure 8 Compares the production of UDP-glucose (UDPG), UDP-4-keto-6-deoxyglucose (UDP4K6G), and UDP-rhamnose (UDP-Rh) using different one-pot multi-enzyme reaction systems. Figure 8 Subfigure A shows the results after a 6-hour reaction time. Figure 8 Subfigure B shows the results after an 18-hour reaction time. The details of reaction systems 1-6 are summarized in Table 2.

[0038] Figure 9. Enzymatic analysis of DH candidates for UDP-4-keto-6-deoxy-glucose (UDP4K6G) production. The DH candidates included in this experiment were NR55N, NR60N, NR66N, NR67N, NR68N, and NR69N. The following RHM candidates with trifunctional enzyme activity were also included in this experiment: NR53N, NR58N, NR62N, NR64N, and NR65N. All samples were collected at 18 h. The collected samples were extracted with chloroform and analyzed by HPLC. "UDP-Rh": UDP-rhamnose; "UDPG": UDP-glucose; "UDP4K6G": UDP-4-keto-6-deoxy-glucose. "Control": reaction without added enzyme.

[0039] Figure 10 . Enzymatic analysis of ER candidates for the bioconversion of UDP-4-keto-6-deoxy-glucose (UDP4K6G) to UDP-β-L-rhamnose. All samples were collected at 18 h. The collected samples were extracted with chloroform and analyzed by HPLC. "UDP-Rh": UDP-rhamnose; "UDPG": UDP-glucose; "UDP4K6G": UDP-4-keto-6-deoxy-glucose.

[0040] Figure 11 . Enzymatic activity comparison of three fusion enzymes (NRF3, NRF2, and NRF1) with respect to the DH enzyme (NX10) for UDP-rhamnose production. NAD was added at the start of the reaction + , and NADPH was added 3 h after the start of the reaction. All samples were collected at 21 h. The collected samples were extracted with chloroform and analyzed by HPLC. Legend: "UDP-Rh" = UDP-rhamnose; "UDPG" = UDP-glucose; and "UDP4K6G" = UDP-4-keto-6-deoxy-glucose.

[0041] Figure 12 . Enzymatic analysis of the fusion enzyme for UDP-rhamnose production. NAD was added in the initial reaction + , and NADPH was added to the reaction 3 h later. All samples were collected at 21 h. The collected samples were extracted with chloroform and analyzed by HPLC. "UDP-Rh": UDP-rhamnose; "UDPG": UDP-glucose; "UDP4K6G": UDP-4-keto-6-deoxy-glucose.

[0042] Figure 13Shows the production of UDP-4-keto-6-deoxyglucose (UDP4K6G) and UDP-rhamnose (UDP-Rh) using a one-pot multi-enzyme reaction system optimized for the in vitro synthesis of UDP-rhamnose. In this embodiment, NRF1 is used as the RHM enzyme. Using a two-step cofactor addition protocol, NAD + is added at the start of the reaction and NADP + , MaeB, and malic acid are added after 3 hours to regenerate NADPH. The products are analyzed after reaction times of 3 hours and 18 hours.

[0043] Figure 14 Shows HPLC spectra confirming the in vitro production of Reb J and Reb N from Reb A catalyzed by a selected UDP-rhamnosyltransferase (1,2 RhaT) and UDP-glucosyltransferase (UGT) according to the present disclosure. Figure 14 、Panel A shows the Reb J standard. Figure 14 、Panel B shows the Reb N standard. Figure 14 、Panel C shows that when the product is measured at 22 hours, Reb J is enzymatically produced by the EUCP1 enzyme as an exemplary 1,2 RhaT. Fig. Figure 14 、Panel D shows that when the product is measured at 25 hours, Reb N is enzymatically produced from the Reb J product by CP1 as an exemplary UGT. DETAILED DESCRIPTION

[0044] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0045] With respect to the terms "comprising", "having", etc. used in the specification or claims, such terms are intended to be inclusive in a manner similar to the term "including" as interpreted when used as a transitional word in a claim.

[0046] The term "exemplary" is used herein to mean serving as an example, instance, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

[0047] A cell system is any cell that provides heterologous protein expression. It includes bacteria, yeast, plant cells, and animal cells. It includes prokaryotic and eukaryotic cells. It also includes in vitro expression of proteins based on cellular components such as ribosomes.

[0048] A coding sequence will be given its ordinary and customary meaning to one of ordinary skill in the art and is used without limitation to denote a DNA sequence encoding a specific amino acid sequence.

[0049] The term "growing a cell system" refers to providing an appropriate culture medium that allows cells to reproduce and divide. It also includes providing resources such that cells or cell components can translate and produce recombinant proteins.

[0050] Protein expression can occur after gene expression. It consists of the stage after DNA has been transcribed into messenger RNA (mRNA). The mRNA is then translated into a polypeptide chain, which ultimately folds into a protein. DNA is present in cells by transfection, which is a method of deliberately introducing nucleic acids into cells. This term is often used for non-viral methods in eukaryotic cells. It can also denote other methods and cell types, although other terms are preferred: "transformation" is more often used to describe non-viral DNA transfer in bacteria, non-animal eukaryotic cells (including plant cells). In animal cells, transfection is the preferred term because transformation is also used to denote the progression of a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated DNA transfer. Transformation, transduction, and viral infection are included under the definition of transfection in this application.

[0051] According to the present disclosure, the yeast as claimed herein is a eukaryotic single-celled microorganism that is a member of the kingdom Fungi. Yeast are single-celled organisms that evolved from multicellular ancestors, but some of the species useful in the present disclosure are those that have the ability to develop multicellular characteristics by forming chains of budding cells connected by what are called pseudohyphae or false hyphae.

[0052] The names of UGT enzymes used in the present disclosure are consistent with the nomenclature system adopted by the UGT Nomenclature Committee (Mackenzie et al., " The UDP glycosyltransferase gene super family: recommended nomenclature updated based on evolutionary divergence ," Pharmacogenetics, 1997, Vol. 7, pp. 255-269), which classifies UGT genes by a combination of family number, a letter denoting the subfamily, and the number of the individual gene. For example, the name "UGT76Gl" denotes a UGT enzyme encoded by a gene belonging to UGT family number 76 (which is of plant origin), subfamily G, and gene number 1.

[0053] The term "complementary" will be given its ordinary and customary meaning to one of ordinary skill in the art and is used, without limitation, to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the subject technology also includes isolated nucleic acid fragments that are complementary to the complete sequences reported in the accompanying sequence listing and those nucleic acid sequences that are substantially similar.

[0054] The terms "nucleic acid" and "nucleotide" will be given their respective ordinary and customary meanings to those of ordinary skill in the art, and are used non-limitingly to refer to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and their polymers. Unless specifically restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides having binding properties similar to the reference nucleic acid and being metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified or degenerate variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly recited sequence.

[0055] The term "isolated" will be given its ordinary and customary meaning to those of ordinary skill in the art, and when used in the context of an isolated nucleic acid or an isolated polypeptide, is used non-limitingly to refer to a nucleic acid or polypeptide that is present outside of its natural environment by human intervention and is thus not a natural product. An isolated nucleic acid or polypeptide may be in a purified form or may be present in a non-natural environment, such as in a transgenic host cell.

[0056] As used herein, the term "incubate" means the process of mixing two or more chemical or biological entities (such as chemical compounds and enzymes) and allowing them to interact under conditions favorable for the production of one or more chemical or biological entities that are distinct from the original starting entities.

[0057] The term "degenerate variant" refers to a nucleic acid sequence having a residue sequence that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions. Degenerate codon substitutions can be achieved as follows: generating a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. A nucleic acid sequence and all of its degenerate variants will express the same amino acid or polypeptide.

[0058] The terms "polypeptide", "protein" and "peptide" will be given their respective ordinary and customary meanings to those of ordinary skill in the art; these three terms are sometimes used interchangeably and are used non-limitingly to refer to polymers of amino acids or amino acid analogs, regardless of size or function. Although the term "protein" is often used to refer to relatively large polypeptides and the term "peptide" is often used to refer to small polypeptides, the use of these terms in the art overlaps and varies. Unless otherwise indicated, the term "polypeptide" as used herein refers to peptides, polypeptides and proteins. When referring to polynucleotide products, the terms "protein", "polypeptide" and "peptide" may be used interchangeably herein. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments of the foregoing and other equivalents, variants and analogs.

[0059] When the terms "polypeptide fragment" and "fragment" are used in reference to a reference polypeptide, they will be given their ordinary and customary meaning to one of ordinary skill in the art and are used, without limitation, to denote such a polypeptide: in which amino acid residues are missing as compared to the reference polypeptide itself, but in which the remaining amino acid sequence is generally the same as the corresponding positions in the reference polypeptide. Such deletions can occur at the amino terminus or carboxy terminus of the reference polypeptide, or alternatively at both.

[0060] A "functional fragment" of a polypeptide or protein denotes such a peptide fragment that is part of the full-length polypeptide or protein and has substantially the same biological activity as the full-length polypeptide or protein, or performs substantially the same function as the full-length polypeptide or protein (e.g., performs the same enzymatic reaction).

[0061] The terms "variant polypeptide", "modified amino acid sequence", or "modified polypeptide", used interchangeably, denote an amino acid sequence that differs from a reference polypeptide by one or more amino acids (e.g., by one or more amino acid substitutions, deletions, and / or additions). In one aspect, a variant is a "functional variant" that retains some or all of the capabilities of the reference polypeptide.

[0062] The term "functional variant" further includes variants with conservative substitutions. The term "variant with conservative substitutions" denotes such a peptide that has an amino acid sequence that differs from the reference peptide by one or more conservative amino acid substitutions and maintains some or all of the activity of the reference peptide. A "conservative amino acid substitution" is the replacement of an amino acid residue with a residue that is functionally similar. Examples of conservative substitutions include: replacing one nonpolar (hydrophobic) residue such as isoleucine, valine, leucine, or methionine with another; replacing one charged or polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; replacing one basic residue such as lysine or arginine with another; or replacing one acidic residue such as aspartic acid or glutamic acid with another; or replacing one aromatic residue such as phenylalanine, tyrosine, or tryptophan with another. It is expected that such substitutions have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. The phrase "variant with conservative substitutions" also includes peptides in which a residue is replaced with a chemically derivatized residue, provided that the resulting peptide maintains some or all of the activity of the reference peptide as described herein.

[0063] The term "variant" with respect to a polypeptide of the subject technology further includes a functionally active polypeptide having an amino acid sequence that has at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and even 100% identity to the amino acid sequence of a reference polypeptide.

[0064] The term "homologous", in all its grammatical forms and spelling variations, denotes a relationship between polynucleotides or polypeptides having a common evolutionary origin, including polynucleotides or polypeptides from a superfamily and homologous polynucleotides or proteins from different species (Reeck et al., Cell 50:667, 1987). Such polynucleotides or polypeptides have sequence homology as reflected by their sequence similarity, whether in terms of the percentage of identity or the presence of specific amino acids or motifs at conserved positions. For example, two homologous polypeptides can have amino acid sequences with at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and even 100% identity.

[0065] "Suitable regulatory sequences" will be given their ordinary and customary meaning to one of ordinary skill in the art and are used, without limitation, to denote nucleotide sequences that are located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that affect the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translational leader sequences, introns, and polyadenylation recognition sequences.

[0066] "Promoter" will be given its ordinary and customary meaning to one of ordinary skill in the art, and is used without limitation to denote a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' of the promoter sequence. A promoter may be entirely derived from a native gene, or may be composed of different elements from different promoters found in nature, or may even contain synthetic DNA segments. One of ordinary skill in the art will understand that different promoters may direct gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. A promoter that causes a gene to be expressed in most cell types most of the time is commonly referred to as a "constitutive promoter". It is further recognized that, since the precise boundaries of regulatory sequences have not been fully determined in most cases, DNA fragments of different lengths may have the same promoter activity.

[0067] The term "operably linked" denotes the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it can affect the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to the regulatory sequence in a sense or antisense orientation.

[0068] The term "expression" as used herein will be given its ordinary and customary meaning to one of ordinary skill in the art, and is used without limitation to denote the transcription and stable accumulation of sense (mRNA) or antisense RNA from a nucleic acid fragment of the subject technology. "Overexpression" means that the gene product produced in a transgenic or recombinant organism exceeds the production level in a normal or non-transformed organism.

[0069] "Transformation" will be given its ordinary and customary meaning to one of ordinary skill in the art, and is used without limitation to denote the transfer of a polynucleotide into a target cell. The transferred polynucleotide may be incorporated into the genome or chromosomal DNA of the target cell, thereby producing a genetically stable genetic trait, or it may replicate independently of the host chromosome. A host organism containing a transformed nucleic acid fragment is referred to as "transgenic" or "transformed".

[0070] When used in connection with a host cell herein, the terms "transformed," "transgenic," and "recombinant" shall be given their ordinary and customary meanings, respectively, to one of ordinary skill in the art and are used without limitation to indicate a cell of a host organism into which a heterologous nucleic acid molecule has been introduced, such as a plant or microbial cell. The nucleic acid molecule may be stably integrated into the genome of the host cell or the nucleic acid molecule may exist as an extrachromosomal molecule. Such extrachromosomal molecules may be self-replicating. A transformed cell, tissue, or subject is to be understood to include not only the end product of the transformation process, but also its transgenic progeny.

[0071] When used in connection with a polynucleotide herein, the terms "recombinant," "heterologous," and "exogenous" shall be given their ordinary and customary meanings to one of ordinary skill in the art and are used without limitation to indicate such a polynucleotide (e.g., a DNA sequence or gene): which is derived from a source that is exogenous to a particular host cell, or which has been modified relative to its original form if derived from the same source. Thus, a heterologous gene in a host cell includes a gene that is endogenous to a particular host cell but has been modified, for example, by using site-directed mutagenesis or other recombinant techniques. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term indicates a DNA segment that is exogenous or heterologous to the cell, or homologous to the cell but in a position or form in which the element is not normally found within the host cell.

[0072] Similarly, when used in connection with a polypeptide or amino acid sequence herein, the terms "recombinant," "heterologous," and "exogenous" refer to such a polypeptide or amino acid sequence: which is derived from a source that is exogenous to a particular host cell, or which has been modified relative to its original form if derived from the same source. Thus, a recombinant DNA segment may be expressed in a host cell to produce a recombinant polypeptide.

[0073] The terms "plasmid", "vector" and "cassette" will be given their respective ordinary and customary meanings to one of ordinary skill in the art, and are used without limitation to denote extrachromosomal elements that often carry genes that are not part of the central metabolism of the cell and are usually in the form of circular double-stranded DNA molecules. Such elements can be self-replicating sequences, genomic integration sequences, phages or nucleotide sequences of single-stranded or double-stranded DNA or RNA, linear or circular, from any source, many of which have been ligated or recombined into unique constructs that are capable of introducing a promoter fragment and DNA sequence of a selected gene product, along with appropriate 3' untranslated sequences, into a cell. A "transformation cassette" denotes a particular vector that contains a foreign gene and, in addition to the foreign gene, has elements that facilitate the transformation of a particular host cell. An "expression cassette" denotes a particular vector that contains a foreign gene and, in addition to the foreign gene, has elements that permit enhanced expression of the gene in a foreign host.

[0074] In certain embodiments, the present disclosure relates to the biosynthetic production of UDP-rhamnose. In a preferred embodiment, the invention relates to the production of UDP-L-rhamnose, the chemical structure of which is shown in Figure 1 FIG. Because UDP-rhamnose can be used as a rhamnose donor moiety in the biosynthetic production of rhamnose-containing steviol glycosides such as Reb J and Reb N, the present disclosure also relates in part to biosynthetic pathways for preparing rhamnose-containing steviol glycosides, which include, for example, the preparation of UDP-rhamnose from UDP-glucose.

[0075] Reference Figure 2 , aspects of the present disclosure relate to reaction systems that at least include a first recombinant polypeptide having UDP-rhamnose synthase activity that catalyzes the bioconversion of UDP-glucose to UDP-rhamnose via the intermediate UDP-4-keto-6-deoxyglucose ("UDP4K6G"). In the Figure 2 illustrated embodiment, the first recombinant polypeptide is a trifunctional enzyme that catalyzes the bioconversion of UDP-glucose to UDP4K6G and the bioconversion of UDP4K6G to UDP-rhamnose. In certain embodiments, the first polypeptide can include two different enzymes, each responsible for a different step in the bioconversion. The reaction system can also include a second polypeptide that catalyzes the regeneration reaction of NADPH, which is a cofactor for the bioconversion of UDP-glucose to UDP-rhamnose. The reaction system can further include a third recombinant polypeptide that converts UDP and sucrose into UDP-glucose. In embodiments in which UDP-rhamnose is used as a rhamnose donor moiety in the biosynthetic production of rhamnose-containing steviol glycosides such as Reb J and Reb N, the reaction system can include additional enzymes having rhamnosyltransferase and glucosyltransferase activity.

[0076] The UDP-rhamnose biosynthetic pathway in plants and fungi involves three different enzymes. In the first step of this biosynthetic pathway, UDP-glucose 4,6-dehydratase ("DH") converts UDP-glucose into UDP-4-keto-6-deoxyglucose (UDP4K6G). In the second step of this biosynthetic pathway, the enzyme UDP-4-keto-6-deoxy-glucose 3,5-epimerase converts UDP-4-keto-6-deoxyglucose into UDP-4-ketorhamnose. In the third enzymatic step of this biosynthetic pathway, UDP-4-ketorhamnose-4-keto reductase converts UDP-4-ketorhamnose into UDP-rhamnose. In various embodiments, the present invention provides a trifunctional recombinant polypeptide having UDP-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-glucose 3,5-epimerase, and UDP-4-ketorhamnose 4-keto-reductase activities. Such a trifunctional polypeptide is also referred to as an RHM enzyme. Since the trifunctional recombinant polypeptide exhibits three different enzyme functions, this trifunctional recombinant protein is also referred to as a multi-enzyme protein.

[0077] In certain embodiments, the present invention provides a recombinant polypeptide having only UDP-glucose 4,6-dehydratase activity, and this recombinant polypeptide is referred to herein as a "DH" (dehydratase) polypeptide. In another embodiment, the present invention provides a bifunctional recombinant polypeptide having UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-ketorhamnose 4-keto-reductase activities. This bifunctional recombinant polypeptide is referred to herein as an "ER" (the letter "E" represents epimerase activity and the letter "R" represents reductase activity). In another embodiment, the present invention provides a recombinant fusion polypeptide in which an enzyme having UDP-glucose 4,6-dehydratase activity (DH polypeptide) is fused with a bifunctional ER polypeptide having UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-ketorhamnose 4-keto-reductase activities. It has been found that such a fusion polypeptide has the ability to catalyze the conversion of UDP-glucose to UDP-rhamnose.

[0078] The cofactor NAD is required in the step catalyzed by DH + , and the cofactor NADPH is required in the second step of the step catalyzed by ER.

[0079] Referring to Table 1, the inventors have identified various trifunctional UDP-rhamnose synthases for the bioconversion of UDP-glucose to UDP-rhamnose. As described below Figure 6As shown, NR12 [SEQ ID NO: 1] from Ricinus communis, NR32 [SEQ ID NO: 3] from Ceratopteris thalictroides, and NR33 [SEQ ID NO: 5] from Azolla filiculoides have been shown to be capable of catalyzing the conversion of UDP-glucose to UDP-rhamnose. Accordingly, in certain embodiments, the present disclosure relates to a biosynthetic method for preparing UDP-rhamnose, which comprises incubating a recombinant polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 with a substrate such as UDP-glucose in the presence of cofactors NAD + and NADPH.

[0080] In certain embodiments, the present disclosure relates to a biosynthetic method for preparing UDP-rhamnose, which comprises incubating a substrate such as UDP-glucose with an artificial fusion enzyme obtained by fusing a highly active DH enzyme and a highly active ER enzyme. The DH and ER enzymes can be obtained from a variety of sources shown in the following examples, and their activities can be determined using biochemical assays. Using recombinant techniques well known to those skilled in the art, a nucleic acid sequence encoding a selected DH enzyme can be fused with a nucleic acid encoding a selected ER enzyme to produce a recombinant fusion peptide that catalyzes the synthesis of UDP-rhamnose from UDP-glucose. The DH enzyme and the ER enzyme can be coupled by a peptide linker. In different embodiments, the peptide linker can comprise 2-15 amino acids. Exemplary linkers include those comprising glycine and serine. In a preferred embodiment, the DH enzyme and the ER enzyme can be coupled by a GSG linker (Table 3).

[0081] In different embodiments, UDP-glucose can be prepared in situ from UDP and sucrose in the presence of sucrose synthase (SUS). For example, SUS can have an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 15.

[0082] As shown in Figures 3 - 5 the reaction system can include an NADP + -dependent enzyme for regenerating the cofactor NADPH and an oxidation reaction substrate. Referring to Figure 3 a cofactor NAD is required in the reaction catalyzed by DH +, in which UDP-glucose is converted into UDP-4-keto-6-deoxy-glucose. UDP-4-keto-6-deoxy-glucose is then converted into UDP-4-keto-rhamnose by UDP-4-keto-6-deoxy-glucose 3,5-epimerase. The last step of catalyzing the conversion of UDP-4-keto-rhamnose into UDP-rhamnose by UDP-4-keto-rhamnose 4-keto-reductase requires the cofactor NADPH. Therefore, it is beneficial to integrate side reactions that can help regenerate the NADPH cofactor to ensure the continuous conversion of UDP-rhamnose.

[0083] Continuing to refer to Figure 3 , it can include malic acid and NADP + -dependent malic enzyme (“MaeB”) to optimize this pathway. As shown, in the presence of NADP + , MaeB oxidizes malic acid into pyruvic acid, during which the NADP + factor is reduced back to NADPH, thus regenerating NADPH for the bioconversion of UDP-rhamnose.

[0084] Figure 4 An alternative embodiment is shown, in which another NADP + -dependent enzyme formate dehydrogenase (“FDH”) and formic acid are used. Similar to malic acid and MaeB, formic acid is oxidized into CO 2 by the FDH enzyme, which uses NADP + as a cofactor. The electrons removed from formic acid are transferred to NADP + , thus reducing NADP + back to NADPH.

[0085] Figure 5 Another alternative embodiment for regenerating NADPH is shown. Phosphite dehydrogenase (“PTDH”), another exemplary NADP + -dependent enzyme, is added together with phosphite. Similar to malic acid and MaeB, phosphite is oxidized into phosphoric acid by the PTDH enzyme, and the PTDH enzyme uses NADP + as a cofactor. The electrons removed from phosphite are transferred to NADP + , thus reducing NADP + back to NADPH.

[0086] Part of this disclosure relates to the production of rhamnose-containing steviol glycosides using UDP-rhamnose as the rhamnose donor moiety. Referring back to Figure 2, rhamnosyl steviol glycosides such as Reb J and Reb N can be produced from Reb A. In certain embodiments, using rhamnosyltransferase (RhaT) such as EU11 [SEQ ID No. 97], EUCP1 [SEQ ID No. 23], HV1 [SEQ ID No. 99], UGT2E-B [SEQ ID No. 101] or NX114 [SEQ ID No. 103] and a rhamnose donor moiety such as UDP-rhamnose, Reb A can be converted to Reb J. Subsequently, using UDP-glycosyltransferase (UGT) such as UGT76G1 [SEQ ID No. 107], CP1 [SEQ ID No. 25], CP2 [SEQ ID No. 105] or a fusion enzyme of UGT76G1 and SUS [SEQ ID No. 109], Reb J can be converted to Reb N. Example

[0087] Example 1

[0088] Enzyme Activity Screening of UDP-Rhamnose Synthase

[0089] Phylogenetic, gene cluster, and protein BLAST analyses were used to identify candidate UDP-rhamnose synthase ("RHM") genes for the production of UDP-rhamnose from UDP-glucose. Based on the codon preference of Escherichia coli (Gene Universal, DE), full-length DNA fragments of all candidate RHM genes were optimized and synthesized. The synthesized DNA fragments were cloned into the bacterial expression vector pETite N-His SUMO Kan Vector (Lucigen).

[0090] Each expression construct was transformed into Escherichia coli BL21 (DE3), and then grown at 37 °C in LB medium containing 50 μg / mL kanamycin until an OD of 0.8 - 1.0 was reached. 600 . Protein expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture was further incubated at 16 °C for 22 hours. Cells were harvested by centrifugation (3,000 x g; 10 min; 4 °C). The cell pellet was collected and used immediately or stored at -80 °C.

[0091] The cell pellet is usually resuspended in lysis buffer (50 mM potassium phosphate buffer, pH 7.2, 25 µg / ml lysozyme, 5 µg / ml DNase I, 20 mM imidazole, 500 mM NaCl, 10% glycerol, and 0.4% Triton X-100). The cells are disrupted by sonication at 4 °C and cell debris is clarified by centrifugation (18,000 x g; 30 min). The supernatant is loaded onto a pre-equilibrated (equilibration buffer: 50 mM potassium phosphate buffer, pH 7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol) Ni-NTA (Qiagen) affinity column. After loading the protein sample, the column is washed with equilibration buffer to remove unbound contaminant proteins. The His-tagged RHM recombinant polypeptide is eluted with equilibration buffer containing 250 mM imidazole.

[0092] The UDP-rhamnose synthase activity of the purified candidate RHM recombinant polypeptide is assayed by using UDP-glucose as a substrate. Typically, the recombinant polypeptide (20 - 50 µg) is assayed in a 200 µl in vitro reaction system. The reaction system contains 50 mM potassium phosphate buffer pH 8.0, 3 mM MgCl 2 , 3 - 6 mM UDP-glucose, 1 - 3 mM NAD + , 1 mM DTT and 1 - 3 mM NADPH. The reaction is carried out at 30 - 37 °C and terminated by adding 200 µL chloroform. The sample is extracted by vortexing with an equal volume of chloroform for 10 minutes. After centrifugation for 10 minutes, the supernatant is collected for high performance liquid chromatography (HPLC) analysis.

[0093] Then HPLC analysis is performed using an Agilent 1200 system (Agilent Technologies, CA), which includes a quaternary pump, a temperature-controlled column compartment, an autosampler, and a UV absorbance detector. Chromatographic separation is carried out using a Dionex Carbo PA10 column (4 x 120 mm, Thermo Scientific) with the mobile phase delivered at a flow rate of 1 ml / min. The mobile phase is H 2 O (MPA) and 700 mM ammonium acetate (pH 5.2) (MPB). The gradient concentration of MPB is programmed for sample analysis. The detection wavelength used in the HPLC analysis is 261 nm. After activity screening, three RHM enzymes (NR12, NR32, and NR33) are identified as candidates for the bioconversion of UDP-glucose to UDP-rhamnose (Table 1).

[0094] The activities of three different RHM enzymes, namely NR12, NR32, and NR33, were studied at three different time intervals (3 hours, 6 hours, and 18 hours). The enzyme activities at the end of 3 hours are shown in the top inset (A) of Figure 6 . The enzyme activities at the end of 6 hours and 18 hours are shown in the middle inset (B) and bottom inset (C) of Figure 6 respectively. In addition, in these experiments, an attempt was also made to understand the effect of NADPH on the reduction of NAD + during the UDP-glucose 4,6-dehydratase component of these three RHM enzymes. Under one experimental condition, the cofactors NAD + and NADPH were added at the start of the experiment. This process variant is referred to as "one-step cofactor addition" and is labeled with the letter "a" after the enzyme name in Figure 6 (NR12-a, NR32-a, and NR33-a). In a second set of experiments, NAD + was added at the start of the experiment, and NADPH was added only 3 hours after the reaction had started. This process variant is referred to as "two-step cofactor addition" and is labeled with the letter "b" after the enzyme name in Figure 6 (NR12-b, NR32-b, and NR33-b).

[0095] Continuing to refer to Figure 6 , it can be seen that in the presence of both factors, all three candidate enzymes started producing UDP-rhamnose as early as the 3-hour mark (inset A). When the reaction time was extended for a longer period, more UDP-rhamnose was produced ([[]] Figure 6 insets B and C). For the one-step cofactor addition protocol, NR32-a exhibited the highest UDP-rhamnose production activity among the three candidate enzymes (0.57 g / L UDP-Rh at 18 hours). In this first set of experiments (a), it was observed that NR12-a had high UDP-glucose 4,6-dehydratase (DH) activity but very low UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-keto-rhamnose 4-keto-reductase (ER) activities, as demonstrated by the high-level (almost complete) conversion from UDP-glucose (UDPG) to UDP-4-keto-6-deoxy-glucose (UDP4K6G). These results indicate that all three enzymes are trifunctional UDP-rhamnose synthases for the bioconversion of UDP-glucose to UDP-rhamnose.

[0096] In addition, the inventors also found that the two-step cofactor addition protocol could improve the conversion efficiency, indicating that late addition of NADPH could avoid the negative feedback regulation of UDP-rhamnose on the DH enzyme. During the two-step cofactor addition process, NAD +, and NADPH was added to the reaction 3 hours later. As shown in Figure 6 , both NR32 (NR32-b) and NR33 (NR33-b) have higher UDP-rhamnose production than the one-step reaction (NR32-a and NR33-a). NR32-b has the highest activity for producing UDP-Rh, reaching 1.1 g / L UDP-Rh at 18 hours (Figure C). Consistent with the results of the first set of experiments, NR12-b showed high DH activity but very low ER activity, as demonstrated by the high-level conversion from UDP-glucose to UDP4K6G, but very little UDP-rhamnose was produced.

[0097] These results indicate that a two-step cofactor addition scheme can be used to improve the conversion efficiency from UDP-glucose to UDP-rhamnose.

[0098] Example 2

[0099] Two-step addition of cofactors

[0100] Figure 7 Shows how the two-step cofactor addition scheme according to the present disclosure can improve the conversion efficiency of UDP-rhamnose production in a reaction involving the trifunctional enzyme NRF1. In the two-step reaction (b-1 hour, b-3 hours, b-4 hours, b-6 hours, b-18 hours), NAD + was added to the initial reaction. The UDPG substrate was completely converted to UDP-4-keto-6-deoxyglucose by DH activity at 3 hours (b-3 hours). Then NADPH was added to the reaction, and it was confirmed that UDP-4-keto-6-deoxyglucose had been completely converted to UDP-rhamnose at 18 hours (b-18 hours). In the one-step reaction (a-1 hour, a-3 hours, a-4 hours, a-6 hours, a-18 hours), NAD+ and NADPH were added to the initial reaction, and UDPG was not completely converted to UDP-rhamnose, thus supporting the reported negative feedback effect of UDP-rhamnose on DH activity. Under both schemes ("a" represents the one-step scheme and "b" represents the two-step scheme), the levels of UDP-glucose (UDPG), UDP-4-keto-6-deoxyglucose (UDP4K6G), and UDP-rhamnose (UDP-Rh) were measured after 1 hour, 3 hours, 4 hours, 6 hours, and 18 hours.

[0101] Example 3

[0102] Optimization of a one-pot multi-enzyme system for in vitro synthesis of UDP-rhamnose

[0103] Sucrose synthase (SUS) can break down one molecule of sucrose to produce one molecule of fructose and one molecule of glucose. In addition, SUS can transfer a glucose to UDP to form UDP-glucose. Thus, by including sucrose, UDP, and SUS in the starting materials, the UDP-glucose component required in the UDP-rhamnose synthesis pathway disclosed herein can be supplemented in the presence of sucrose synthase.

[0104] In addition, NADPH is a key cofactor for ER activity. During the reaction catalyzed by ER, NADPH is oxidized to NADP + . By incorporating the NADP + -dependent oxidation reaction as part of the UDP-rhamnose synthesis disclosed herein, NADPH can be regenerated. Exemplary NADP + -dependent oxidation reactions include the oxidation of malate to pyruvate, the oxidation of formate to CO 2 , and the oxidation of phosphite to phosphate. By including malate, formate, or phosphite in the starting materials and the corresponding enzymes (MaeB, FDH, and PTDH, respectively) that can catalyze each of these oxidation reactions, NADPH is continuously regenerated, thereby further optimizing the total UDP-rhamnose production. Table 1 provides information on the sequences of various enzymes.

[0105] In this example, six different experiments were conducted using different combinations of starting materials in a one-pot multi-enzyme reaction system using a two-step cofactor addition scheme. Table 2 provides the compositions of the six different reaction systems tested in this experiment.

[0106] In each of the six systems, UDP-glucose was not included. Instead, UDP, sucrose, and SUS were provided to produce the required UDP-glucose. Referring Figure 8 , the results from system 1 indicated that UDP-glucose was produced, thus confirming that SUS can completely convert UDP into UDP-glucose. By providing sucrose synthase (SUS) together with the RHM enzyme (e.g., NRF1), UDP-rhamnose can be produced using UDP as a substrate (system 2).

[0107] The experiments also confirmed the role of NADPH regeneration in UDP-rhamnose production. Continuing to refer Figure 8 , by adding the MaeB enzyme and malate to a reaction system (system 4) containing a small amount of NADPH, a high level of UDP-rhamnose could still be obtained, thus confirming the regeneration of NADPH. In contrast, in system 3 containing the same amount of NADPH but in the absence of the MaeB enzyme, a much lower amount of UDP-Rh was produced. Similarly, in reaction systems (systems 5 and 6) containing a small amount of NADP + if the MaeB enzyme is present, the added NADP+ can be converted into NADPH by MaeB and continuously regenerated for UDP - rhamnose production (System 6). Only 1 mM NADP was used in System 6. + The amount of UDP - rhamnose obtained was comparable to that obtained with 3 mM NADPH in System 2. In contrast, in System 5 which does not include NADPH and does not include MaeB, almost no UDP4K6G was converted into UDP - rhamnose. As described above, the malate / MaeB system can be replaced by other NADP + - dependent oxidation systems such as formate / FDH and phosphite / PTDH.

[0108] Example 4

[0109] Enzyme Activity Screening of UDP - glucose 4,6 - dehydratase

[0110] UDP - glucose 4,6 - dehydratase (DH) can catalyze the enzymatic reaction of the bioconversion of UDP - glucose (UDPG) to UDP - 4 - keto - 6 - deoxy - glucose (UDP4K6G). To identify a specific DH enzyme, enzyme candidates were selected based on multi - gene genetic analysis and Blast analysis.

[0111] Full - length DNA fragments of all candidate DH genes were commercially synthesized. Almost all codons of the cDNA were changed to codons preferred by Escherichia coli (Gene Universal, DE). The synthesized DNA was cloned into the bacterial expression vector pETite N - His SUMO Kan Vector (Lucigen).

[0112] Each expression construct was transformed into Escherichia coli BL21 (DE3) and then grown in LB medium containing 50 μg / mL kanamycin at 37 °C until an OD600 of 0.8 - 1.0 was reached. Protein expression was induced by adding 1 mM isopropyl β - D - 1 - thiogalactopyranoside (IPTG), and the culture was further incubated at 16 °C for 22 hours. Cells were harvested by centrifugation (3,000 x g; 10 min; 4 °C). The cell pellet was collected and used immediately or stored at - 80 °C.

[0113] The cell pellet is usually resuspended in lysis buffer (50 mM potassium phosphate buffer, pH 7.2, 25 μg / ml lysozyme, 5 μg / ml DNase I, 20 mM imidazole, 500 mM NaCl, 10% glycerol, and 0.4% Triton X-100). The cells are disrupted by sonication at 4 °C and cell debris is clarified by centrifugation (18,000 x g; 30 min). The supernatant is loaded onto a pre-equilibrated (equilibration buffer: 50 mM potassium phosphate buffer, pH 7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol) Ni-NTA (Qiagen) affinity column. After loading the protein sample, the column is washed with equilibration buffer to remove unbound contaminant proteins. The His-tagged DH recombinant polypeptide is eluted with equilibration buffer containing 250 mM imidazole.

[0114] The UDP-4-keto-6-deoxy-glucose synthesis of the purified candidate DH recombinant polypeptide is assayed by using UDPG as a substrate. Typically, the recombinant polypeptide (20 μg) is assayed in a 200 μl in vitro reaction system. The reaction system contains 50 mM potassium phosphate buffer pH 8.0, 3 mM MgCl 2 , 3 mM UDPG, 3 mM NAD + and 1 mM DTT. The reaction is carried out at 30 - 37 °C and terminated by adding 200 μL chloroform. The samples are extracted by vortexing with an equal volume of chloroform for 10 minutes. After centrifugation for 10 minutes, the supernatant is collected for high performance liquid chromatography (HPLC) analysis.

[0115] Then HPLC analysis is performed using an Agilent 1200 system (Agilent Technologies, CA), which includes a quaternary pump, a temperature-controlled column compartment, an autosampler, and a UV absorbance detector. Chromatographic separation is carried out using a Dionex Carbo PA10 column (4 x 120 mm, Thermo Scientific), and the mobile phase is delivered at a flow rate of 1 ml / min. The mobile phase is H 2 O (MPA) and 700 mM ammonium acetate (pH 5.2) (MPB). The gradient concentration of MPB is programmed for sample analysis. The detection wavelength used in the HPLC analysis is 261 nm.

[0116] After activity screening, 12 novel DH enzymes were identified for the bioconversion of UDPG into UDP4K6G (Table 1). As in Figure 9As shown, the DH enzyme showed different levels of enzyme activity for UDP4K6G production. In addition, six candidates (NR15N, NR53N, NR58N, NR62N, NR64N, and NR65N) also showed low enzyme activity for UDP-rhamnose production, indicating that these enzymes may have trifunctional activity (RHM) for synthesizing UDP-L-rhamnose from UDPG.

[0117] Example 5

[0118] Enzyme Activity Screening of Bifunctional UDP-4-Keto-6-Deoxy-Glucose 3,5-Epimerase / UDP-4-Keto-Rhamnose 4-Keto Reductase

[0119] The bifunctional UDP-4-keto-6-deoxy-glucose 3,5-epimerase / UDP-4-keto-rhamnose 4-keto reductase (ER) enzyme can convert UDP-4-keto-6-deoxy-glucose into UDP-β-L-rhamnose. To identify specific ER enzymes, certain enzyme candidates were selected based on multi-gene genetic analysis and Blast analysis.

[0120] Full-length DNA fragments of all candidate ER genes were commercially synthesized. Almost all codons of the cDNA were changed to codons preferred by Escherichia coli (Gene Universal, DE). The synthesized DNA was cloned into the bacterial expression vector pETite N-His SUMO Kan Vector (Lucigen).

[0121] Each expression construct was transformed into Escherichia coli BL21 (DE3) and subsequently grown in LB medium containing 50 μg / mL kanamycin at 37 °C until an OD600 of 0.8 - 1.0 was reached. Protein expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture was further incubated at 16 °C for 22 hours. Cells were harvested by centrifugation (3,000 x g; 10 min; 4 °C). The cell pellet was collected and used immediately or stored at -80 °C.

[0122] The cell pellet is typically resuspended in lysis buffer (50 mM potassium phosphate buffer, pH 7.2, 25 μg / ml lysozyme, 5 μg / ml DNase I, 20 mM imidazole, 500 mM NaCl, 10% glycerol, and 0.4% Triton X-100). The cells are disrupted by sonication at 4 °C and the cell debris is clarified by centrifugation (18,000 x g; 30 min). The supernatant is loaded onto a pre-equilibrated (equilibration buffer: 50 mM potassium phosphate buffer, pH 7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol) Ni-NTA (Qiagen) affinity column. After loading the protein sample, the column is washed with equilibration buffer to remove unbound contaminant proteins. The His-tagged ER recombinant polypeptide is eluted with equilibration buffer containing 250 mM imidazole.

[0123] The UDP-rhamnose synthesis of the purified candidate ER recombinant polypeptide was assayed by using UDP-4-keto-6-deoxy-glucose (UDP4K6G) as a substrate. Typically, the recombinant polypeptide (20 μg) was assayed in a 200 μl in vitro reaction system. The reaction system contained 50 mM potassium phosphate buffer pH 8.0, 3 mM MgCl 2 , 3 mM UDP-4-keto-6-deoxy-glucose, 3 mM NADPH, and 1 mM DTT. The reaction was carried out at 30 - 37 °C and terminated by adding 200 μL chloroform. The samples were extracted by vortexing with an equal volume of chloroform for 10 minutes. After centrifugation for 10 minutes, the supernatant was collected for high performance liquid chromatography (HPLC) analysis.

[0124] Then HPLC analysis was performed using an Agilent 1200 system (Agilent Technologies, CA), which included a quaternary pump, a temperature-controlled column compartment, an autosampler, and a UV absorbance detector. Chromatographic separation was carried out using a Dionex Carbo PA10 column (4 x 120 mm, Thermo Scientific) with the mobile phase delivered at a flow rate of 1 ml / min. The mobile phase was H 2 2O (MPA) and 700 mM ammonium acetate (pH 5.2) (MPB). The gradient concentration of MPB was programmed for sample analysis. The detection wavelength used in the HPLC analysis was 261 nm.

[0125] After activity screening, 17 novel ER enzymes were identified for the bioconversion of UDP-4-keto-6-deoxy-glucose into UDP-L-rhamnose (Table 1). As in Figure 10As shown, 17 candidates showed different levels of enzyme activity for UDP-L-rhamnose production. Among the 17 enzyme candidates, the following enzymes showed high ER activity: NR21C, NR37C, NR40C, NR41C, and NR46C.

[0126] Example 6

[0127] Identification of novel fusion enzymes for UDP-rhamnose production

[0128] The construction of fusion enzymes by recombinant DNA technology can be used to obtain novel trifunctional enzymes with UDP-rhamnose synthase activity. However, the fusion of two functional enzymes does not necessarily provide an active fusion enzyme with the activities of both enzyme components. In addition, suitable linkers often can only be identified empirically.

[0129] Based on a wide screening of various DH and ER enzyme candidates as well as the N-terminal and C-terminal domains of the trifunctional RHM enzyme, a series of fusion enzymes with specific DH and ER domains were identified and screened.

[0130] After such further screening, six fusion enzymes were found to have trifunctional activity for bioconverting UDP-glucose into UDP-rhamnose (Table 3).

[0131] Specifically, five of these fusion enzymes are based on the high-activity DH enzyme NX10 fused with different ER enzymes (NX5C, NX13, NR5C, NR40C, and NR41C), namely NRF1 (NX10-NX5C), NRF2 (NX10-NX13), NRF3 (NX10-NR5C), NRF4 (NX10-NR40C), and NRF5 (NX10-NR41C). Another fusion enzyme with trifunctional activity, NRF7 (NR66N-NR41C), is based on the high-activity DH enzyme NR66N fused with the high-activity ER enzyme NR41C. As shown in Figure 11 NX10 signal enzyme can completely convert UDP-glucose into UDP-4-keto-6-deoxyglucose (UDP4K6G). Meanwhile, Figure 11 and 12 showed that the fusion enzymes NRF1, NRF2, NRF3, NRF4, NRF5, and NRF7 all had trifunctional activity for UDP-rhamnose synthesis in a two-step cofactor addition reaction (where NADPH was added after a 3-hour reaction). Notably, the NRF1, NRF2, NRF4, NRF5, and NRF7 fusion enzymes had higher enzyme activity than NRF3.

[0132] Example 7

[0133] Combination of UDP-rhamnose and steviol glycoside production

[0134] As described in co-owned International Application No. PCT / US2019 / 021876 (now published as WO2019 / 178116A1), the inventors have identified various UDP-rhamnosyltransferases (1,2 RhaT) for the biosynthesis of rhamnose-containing steviol glycosides such as Reb J and Reb N. Specifically, Reb J and Reb N can be synthesized from Reb A and UDP-rhamnose.

[0135] Reference Figure 2 , by coupling the biosynthetic pathway for the production of UDP-rhamnose disclosed herein with the biosynthetic pathway for the production of Reb J / N from Reb A disclosed in International Application No. PCT / US2019 / 021876, a one-pot multi-enzyme reaction system is provided for the in vitro bioconversion of Reb J / N from Reb A and UDP-glucose.

[0136] In the first step, UDP-glucose is converted to UDP-rhamnose by an RHM enzyme such as NRF1 (SEQ ID NO: 9) through a two-step cofactor addition process. UDP-glucose (6 mM) is completely converted to UDP-4-keto-6-deoxyglucose ( Figure 13 ) at 3 hours. Subsequently, 0.5 mM NADP + and an NADPH-regeneration system (e.g., MaeB enzyme and malic acid) are added to the reaction to convert UDP-4-keto-6-deoxyglucose to UDP-rhamnose. Reference Figure 13 , and almost 3 g / L of UDP-Rh is obtained after 18 hours.

[0137] In the second step, Reb A and a UDP-rhamnosyltransferase such as EUCP1 (SEQ ID NO: 23) are added to the reaction system. The UDP-rhamnosyltransferase transfers one rhamnose moiety from UDP-rhamnose to the C-2’ of the 19-O-glucose of the Reb A substrate, thereby converting Reb A to Reb J. The level of Reb J is measured at 22 hours. The activity of EUCP1 is confirmed by HPLC, which indicates the presence of Reb J ( Figure 14 , inset C). UDP is released as a byproduct.

[0138] In the third step, UDP-glycosyltransferase enzymes such as CP1 (SEQ ID NO: 25), sucrose synthase enzymes such as SUS (SEQ ID NO: 15), and sucrose are added to the reaction mixture. The SUS enzyme catalyzes the reaction to produce UDP-glucose and fructose from UDP and sucrose. The CP1 enzyme catalyzes the conversion of Reb J to Reb N, specifically, by transferring a glucose moiety from UDP-glucose to the C-3' of the 19-O-glucose of Reb J to produce Reb N and UDP. In the presence of sucrose, the UDP produced is converted back to UDP-glucose by the SUS enzyme for UDP-rhamnose and Reb N production. HPLC analysis confirmed that Reb N was produced from Reb J at 25 hours ( Figure 14 , panel D).

[0139] Based on these results and referring again to Figure 2 , the one-pot multi-enzyme reaction of the present invention can be summarized as follows. In the reaction, UDP-glucose can be converted to UDP-rhamnose by UDP-rhamnose synthase (e.g., NRF1) through a two-step cofactor addition process. UDP-rhamnosyltransferase (e.g., EUCP1) can be used to transfer a rhamnose moiety from UDP-rhamnose to the C-2' of the 19-O-glucose of Reb A to produce Reb J and UDP. The UDP produced can be converted to UDP-glucose by the SUS enzyme, using sucrose as the glucose source. UDP-glycosyltransferase enzyme (e.g., CP1) can be used to transfer a glucose moiety from UDPG to the C-3' of the 19-O-glucose of Reb J to produce Reb N and UDP. The UDP produced can be converted back to UDP-glucose by the SUS enzyme for UDP-rhamnose and Reb N production.

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

Claims

1. A biosynthetic method for preparing uridine diphosphate - rhamnose (UDP - rhamnose) from uridine diphosphate - glucose (UDP - glucose), the method comprising incubating UDP - glucose with one or more recombinant polypeptides having UDP - rhamnose synthase activity in the presence of a source of NAD + and NADPH for a sufficient time to produce UDP - rhamnose, wherein the one or more recombinant polypeptides comprise a first recombinant polypeptide, the first recombinant polypeptide being a fusion enzyme comprising a first domain having UDP - glucose 4,6 - dehydratase activity and a second domain having UDP - 4 - keto - 6 - deoxy - glucose 3,5 - epimerase and UDP - 4 - keto - rhamnose 4 - keto - reductase activity, wherein the first domain is coupled to the second domain via a GSG linker, and Wherein: i) The first domain of the fusion enzyme is the amino acid sequence of SEQ ID NO:7 and the second domain of the fusion enzyme is the amino acid sequence of SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:61 or SEQ ID NO:63; or ii) The first domain of the fusion enzyme is the amino acid sequence of SEQ ID NO:31, and the second domain of the fusion enzyme is the amino acid sequence of SEQ ID NO:

63.

2. The method according to claim 1, wherein the fusion enzyme is an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13, SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:

87.

3. The method according to claim 2, wherein the first recombinant polypeptide is an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:

87.

4. The method according to claim 1, wherein the one or more recombinant polypeptides comprise a first recombinant polypeptide, which is a fusion polypeptide encoded by a nucleotide produced by the fusion between a first nucleotide encoding UDP-glucose 4,6-dehydratase and a second nucleotide encoding a bifunctional enzyme having UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-keto-rhamnose 4-keto-reductase activities.

5. The method according to claim 4, wherein the first nucleotide is the nucleotide sequence of SEQ ID NO:

8.

6. The method according to claim 4, wherein the first nucleotide is the nucleotide sequence of SEQ ID NO:

32.

7. The method according to any one of claims 4-5, wherein the second nucleotide is the nucleotide sequence of SEQ ID NO:

62.

8. The method according to any one of claims 4-6, wherein the second nucleotide is the nucleotide sequence of SEQ ID NO:

64.

9. The method according to any one of claims 1-3, the method comprising expressing the one or more recombinant polypeptides in a transformed cell system.

10. The method according to claim 9, wherein the transformed cell system is selected from plants, algae, fungi and bacteria that do not produce UDP-rhamnose.

11. The method according to claim 10, wherein the transformed cell system is a bacterium or yeast selected from the following: Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Aspergillus; Arthrobotrys; Brevibacterium; Microbacterium; Arthrobacter; Citrobacter; Klebsiella; Pantoea; and Clostridium.

12. The method according to any one of claims 1-3, wherein UDP-rhamnose is produced from UDP-glucose via UDP-4-keto-6-deoxy-glucose as an intermediate.

13. The method according to claim 12, wherein a source of NADPH is provided after UDP-glucose has been incubated with one or more recombinant polypeptides for a sufficient time to produce UDP-4-keto-6-deoxy-glucose.

14. The method according to claim 13, wherein the source of NADPH comprises an oxidation reaction substrate and an NADP + -dependent enzyme.

15. The method according to claim 13, wherein the source of NADPH comprises malate and malic enzyme.

16. The method according to claim 13, wherein the source of NADPH comprises formate and formate dehydrogenase.

17. The method according to claim 13, wherein the source of NADPH comprises phosphite and phosphite dehydrogenase.

18. The method according to any one of claims 1-3, wherein the uridine diphosphate-glucose and the one or more recombinant polypeptides are incubated with sucrose and a third recombinant polypeptide having sucrose synthase activity.

19. The method according to claim 18, wherein the third recombinant polypeptide is selected from Arabidopsis thaliana sucrose synthase, mung bean sucrose synthase, and Coffea sucrose synthase.

20. A nucleic acid comprising a sequence encoding a polypeptide having an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO.13, SEQ ID NO.83, SEQ ID NO.85, or SEQ ID NO.

87.

21. A cell comprising the nucleic acid of claim 20, wherein the cell is not a plant cell.

22. A composition comprising at least one polypeptide having an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO.13, SEQ ID NO.83, SEQ ID NO.85, or SEQ ID NO.

87.

23. A cell comprising at least one polypeptide having an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO.13, SEQ ID NO.83, SEQ ID NO.85, or SEQ ID NO.87, wherein the cell is not a plant cell.

24. The cell according to claim 21 or 23, wherein the cell is a fungal cell or a bacterial cell that does not produce UDP-rhamnose.

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