Genetically modified Saccharomyces cerevisiae host cell, methods for producing kaurenoic acid, rebaudioside D, rebaudioside ME steviol glycoside, and fermentation composition.

Abstract

Compositions and methods for improved production of steviol glycosides in a host cell are provided herein. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding a kaurenode oxidase from Pisum sativum or its kaurenode oxidase variants. In some embodiments, the host cell further comprises one or more heterologous nucleotide sequences encoding additional enzymes of a pathway capable of producing steviol glycosides in the host cell. The compositions and methods described herein provide an efficient route for the heterologous production of steviol glycosides, including, but not limited to, rebaudioside m.

Description

1 / 105 Genetically modified Saccharomyces cerevisiae host cell, methods for producing kaurenoic acid, rebaudioside D, rebaudioside M and steviol glycoside, and fermentation composition. 1. Cross-reference to related request.

[001] This application claims the benefit of the Provisional Application. US Application No. 62 / 544,718, filed on August 11, 2017, and International Application No. PCT / US2017 / 046637, filed on August 11, 2017, the contents of which are incorporated herein by reference in their entirety. 2. FIELD OF THE INVENTION

[002] The present description refers to certain kaurene oxidases (KOs), compositions comprising the same, host cells comprising the same and methods of using the same to produce rebaudiosides including rebaudioside D and rebaudioside M. 3. FUNDAMENTALS

[003] Zero-calorie sweeteners derived from natural sources are desirable to limit the harmful effects of high sugar consumption (e.g., diabetes and obesity). Rebaudioside M (RebM) is one of the many sweet-tasting compounds produced by the stevia plant (S. rebaudiana Benoni). Of all rebaudiosides, RebM has the highest potency (~200-300x sweeter than sucrose) and is the cleanest-tasting. However, RebM is produced only in small quantities by the Stevia plant and is a small fraction of the total steviol glycoside content (<1.0%). Ohta et al., 2010, J. Appl. Glycosci., 57, 199-209 (2010). As such, it is desirable to produce RebM using biotechnological routes, allowing for production in large quantities and with high purity.

[004] To economically produce a product using biotechnology, each step of the bioconversion of raw material into product Petition 870260074389, dated 07 / 27 / 2026, page 16 / 35 / 105 needs to have a high conversion efficiency (ideally > 90%). In our yeast engineering to produce RebM, we identified a clear limitation in the biosynthetic step at the beginning of the RebM pathway that takes ent-kaurene to kaurenoic acid (FIGS. 1A and 1B).

[005] The KO enzyme is found in all plants and normally acts to produce the plant hormone gibberellin. Gibberellin levels in plant cells are orders of magnitude lower than the RebM levels produced in yeasts for industrial production, and therefore most KO enzymes are not expected to carry the high flux required to produce RebM for commercial manufacturing. Conventionally, the KO enzyme from Stevia rebaudiana (Sr. KO) has been used to convert ent-kaurene to kaurenoic acid in yeasts engineered to produce RebM. The conventional belief is that this plant produces high levels of steviol glycoside, therefore the Sr. KO enzyme should have evolved to have a higher conversion rate or handle a higher flux than most other KO enzymes.

[006] In a yeast strain with high carbon flux to RebM, Sr. KO demonstrated a low conversion efficiency rate to kaurenoic acid (25.6%) and very high levels of upstream intermediate metabolites (ent-kaurenol, kaurenol and kaurenal) were formed (FIG. 1C).

[007] To produce RebM efficiently and with high purity, improved enzymes capable of producing kaurenoic acid with high efficiency are needed. The compositions and methods provided here meet this need and also provide related advantages. 4. SUMMARY OF THE INVENTION

[008] Compositions and methods are provided here for the enhanced conversion of kaurene to kaurenoic acid. These compositions and methods are based in part on the surprising discovery of certain kaurene oxidases (KOs) that are capable of converting kaurene to kaurenoic acid with a Petition 870200018837, dated 07 / 02 / 2020, page 15 / 146 / 105 remarkably high efficiency. Even a modest improvement in strain performance (e.g., ten percent) with new KOs could potentially save more than ten million dollars in production costs in the future, assuming market demand for RebM is 5 billion tons per year.

[009] Certain KOs described herein are also capable of producing kaurenoic acid with little or no residual kaurenol or kaurenal. As such, in certain embodiments, the compositions and methods described herein may reduce downstream processing costs to obtain a high-yield steviol glycoside composition such as RebM.

[0010] In one aspect, genetically modified host cells and methods of their use for the production of industrially useful compounds are provided herein. In one aspect, a genetically modified host cell is provided herein comprising: a heterologous nucleic acid encoding a kaureno oxidase from Pisum sativum. In some embodiments, the genetically modified host cell further comprises one or more enzymatic pathways capable of producing steviol and / or steviol glycosides.

[0011] In certain embodiments, genetically modified host cells are provided herein comprising a heterologous nucleic acid encoding a kaureno oxidase comprising an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to the kaureno oxidase sequence of Pisum sativum (e.g., SEQ ID NO:1). In certain embodiments, the genetically modified host cell is capable of converting kaureno to kaurenoic acid with an efficiency greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98%. In certain embodiments, the genetically modified host cells are yeast cells. In certain modalities, the genetically modified host cells are cells of Petition 870200018837, dated 07 / 02 / 2020, page 16 / 146 / 105 Saccharomyces cerevisiae.

[0012] In another aspect, methods are provided herein for the production of a heterologous steviol glycoside, the method comprising: cultivating a population of genetically modified host cells provided herein, capable of producing the steviol glycoside as described herein, in a medium with a carbon source under conditions suitable for making said steviol glycoside compound; and recovering said steviol glycoside from the medium. In some embodiments, the heterologous steviol glycoside is selected from the group consisting of RebD and RebM.

[0013] In another aspect, methods for the production of RebD are provided herein, the method comprising: cultivating a population of genetically modified host cells provided herein, capable of producing RebD as described herein, in a medium with a carbon source under conditions suitable for making said RebD; and recovering said RebD from the medium.

[0014] In another aspect, methods for the production of RebM are provided herein, the method comprising: cultivating a population of genetically modified host cells provided herein, capable of producing RebM as described herein, in a medium with a carbon source under conditions suitable for making said RebM; and recovering said RebM from the medium.

[0015] In another aspect, methods for the production of kaurenoic acid are provided herein, the method comprising: contacting kaurene with a kaurene oxidase described herein, capable of converting kaurene into kaurenoic acid, under conditions suitable for the formation of kaurenoic acid.

[0016] In some embodiments, the host cell is a yeast cell. In some embodiments, the yeast is Saccharomyces cerevisiae. In some embodiments, the host cell produces RebD or RebM with Petition 870200018837, dated 07 / 02 / 2020, page 17 / 146 / 105 high efficiency. In some embodiments, the host cell produces an increased amount of RebD or RebM compared to a yeast cell that does not understand the kaureno oxidase enzyme of Pisum sativum. 5. BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1A provides a schematic representation of the conversion of farnesyl pyrophosphate to steviol.

[0018] FIG. 1B provides a schematic representation of the conversion of geranylgeranyl pyrophosphate (GGPP) to RebM.

[0019] FIG. 1C provides a schematic representation of the conversion of ent-kaurene to kaurenol to kaurenal to kaurenoic acid.

[0020] FIG. 1D provides a schematic diagram of the mevalonate pathway.

[0021] FIG. 2 provides an exemplary steviol pathway for RebM.

[0022] FIG. 3A provides a schematic diagram of the “landing platform” design used to insert individual KO enzymes for evaluating kaurenoic acid production in yeast.

[0023] FIG. 3B provides a schematic diagram of a KO genetic construct for evaluating the conversion of kaurenoic acid production in yeast.

[0024] FIG. 4 provides a graph illustrating the relative increase in kaurenoic acid produced in vivo with different kaureno oxidases.

[0025] FIG. 5 provides a bar graph illustrating the relative levels of ent-kaurene, caruenol, and caruenal, normalized to the total amount of kaurenoic acid produced in vivo in a yeast strain with high flux to RebM.

[0026] FIG. 6 provides a graph illustrating the relative levels of RebM titrations in high-flux strains containing Sr.KO or Ps.KO. 6. DETAILED DESCRIPTION OF THE MODALITIES 6.1 Terminology Petition 870200018837, dated 07 / 02 / 2020, p. 18 / 146 / 105

[0027] As used herein, the term “heterologous” refers to that which is not normally found in nature. The term “heterologous nucleotide sequence” refers to a nucleotide sequence not normally found in a given cell in nature. As such, a heterologous nucleotide sequence may be: (a) foreign to its host cell (i.e., “exogenous” to the cell); (b) found naturally in the host cell (i.e., “endogenous”), but present in an unnatural amount in the cell (i.e., greater or lesser amount than is naturally found in the host cell); or (c) found naturally in the host cell, but positioned outside its natural locus. The term “heterologous enzyme” refers to an enzyme that is not normally found in a given cell in nature.The term encompasses an enzyme that is: (a) exogenous to a given cell (i.e., encoded by a nucleotide sequence that is not naturally present in the host cell or is not naturally present in a given context in the host cell); and (b) naturally found in the host cell (e.g., the enzyme is encoded by a nucleotide sequence endogenous to the cell), but which is produced in unnatural quantities (e.g., greater or less than that found naturally) in the host cell.

[0028] On the other hand, the term “native” or “endogenous,” as used here with reference to molecules, and in particular enzymes and nucleic acids, indicates molecules that are expressed in the organism in which they originated or are found in nature, regardless of the level of expression, which may be lower, equal to, or higher than the level of expression of the molecule in the native microorganism. It is understood that the expression of native enzymes or polynucleotides may be modified in recombinant microorganisms.

[0029] As used in this document, the term “parental cell” refers to a cell that has an identical genetic background to that of a cell. Petition 870200018837, dated 07 / 02 / 2020, p.19 / 146 / 105 genetically modified host cell disclosed herein, except that it does not comprise one or more specific genetic modifications engineered in the modified host cell, for example, one or more modifications selected from the group consisting of: heterologous expression of an enzyme from a steviol pathway, heterologous expression of an enzyme from a steviol glycoside pathway, heterologous expression of a geranylgeranyl diphosphate synthase, heterologous expression of a copalyl diphosphate synthase, heterologous expression of a kaurene synthase, heterologous expression of a kaurene oxidase (e.g., kaurene oxidase from Pisum sativum), heterologous expression of a steviol synthase (kaurenoic acid hydroxylase), heterologous expression of a cytochrome P450 reductase, heterologous expression of a UGT74, heterologous expression of a UGT76G1, heterologous expression of a UGT85C2, heterologous expression of 91D and heterologous expression of a UGT40087 or its variant.

[0030] As used herein, the term “naturally occurring” refers to what is found in nature. For example, a kaureno oxidase that is present in an organism that can be isolated from a natural source and that has not been intentionally modified by a human in a laboratory is a naturally occurring kaureno oxidase. On the other hand, as used herein, the term “unnaturally occurring” refers to what is not found in nature but is created by human intervention.

[0031] The term “medium” refers to a culture medium and / or fermentation medium.

[0032] The term “fermentation composition” refers to a composition comprising genetically modified host cells and products or metabolites produced by the genetically modified host cells. An example of a fermentation composition is a whole cell broth, which may be the entire contents of a vessel (e.g., flasks, plate, or fermenter), including cells, aqueous phase, and compounds. Petition 870200018837, dated 07 / 02 / 2020, page 20 / 146 / 105 produced from genetically modified host cells.

[0033] As used herein, the term “production” generally refers to an amount of steviol or steviol glycoside produced by a genetically modified host cell provided herein. In some embodiments, production is expressed as a yield of steviol or steviol glycoside by the host cell. In other embodiments, production is expressed as a productivity of the host cell in the production of steviol or steviol glycoside.

[0034] As used herein, the term “productivity” refers to the production of a steviol or steviol glycoside by a host cell, expressed as the amount of steviol or steviol glycoside produced (by weight) per amount of fermentation broth in which the host cell is grown (by volume) over time (per hour).

[0035] As used herein, the term “yield” refers to the production of a steviol or steviol glycoside by a host cell, expressed as the amount of steviol or steviol glycoside produced per amount of carbon source consumed by the host cell, by weight.

[0036] As used herein, the term “an undetectable level” of a compound (e.g., RebM2, steviol glycosides, or other compounds) means a level of a compound that is too low to be measured and / or analyzed by a standard technique for measuring the compound. For example, the term includes the level of a compound that is not detectable by the analytical methods described in Example 6.

[0037] The term “kaurene” refers to the compound kaurene, including any stereoisomer of kaurene. In particular embodiments, the term refers to the enantiomer known in the art as ent-kaurene. In particular embodiments, the term refers to the compound according to the following structure: Petition 870200018837, dated 07 / 02 / 2020, p. 21 / 146 / 105

[0038] The term "kaurenol" refers to the compound kaurenol, including any stereoisomer of kaurenol. In particular embodiments, the term refers to the enantiomer known in the art as ent-kaurenol. In particular embodiments, the term refers to the compound according to the following structure. L XHJ HO~Ί H

[0039] The term “kaurenal” refers to the kaurenal compound, including any stereoisomer of kaurenal. In particular embodiments, the term refers to the enantiomer known in the art as ent-kaurenal. In particular embodiments, the term refers to the compound according to the following structure. [ ThT H

[0040] The term “kaurenoic acid” refers to the compound kaurenoic acid, including any stereoisomer of kaurenoic acid. In particular embodiments, the term refers to the enantiomer known in the art as ent-kaurenoic acid. In particular embodiments, the term refers to the compound according to the following structure. HO

[0041] As used herein, the term “steviol glycoside(s)” refers to Petition 870200018837, dated 07 / 02 / 2020, page 22 / 146 / 105 to a steviol glycoside, including, but not limited to, naturally occurring steviol glycosides, for example steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside L, rebaudioside L, rebaudioside D, rebaudioside N, rebaudioside O, synthetic steviol glycosides, for example enzymatically glycosylated steviol glycosides and combinations thereof.

[0042] As used herein, the term “variant” refers to a polypeptide that differs from a specifically recited “reference” polypeptide (e.g., a wild-type sequence) by amino acid insertions, deletions, mutations, and / or substitutions, but retains activity that is substantially similar to the reference polypeptide. In some embodiments, the variant is created by recombinant DNA techniques such as mutagenesis. In some embodiments, a variant polypeptide differs from its reference polypeptide by the substitution of one basic residue for another (i.e., Arg for Lys), the substitution of one hydrophobic residue for another (i.e., Leu for Ile), or the substitution of one aromatic residue for another (i.e., Phe for Tyr), etc. In some embodiments, variants include analogs in which conservative substitutions are obtained that result in a substantial structural analogy of the reference sequence.Examples of such conservative substitutions, without limitation, include glutamic acid for aspartic acid and vice versa; glutamine for asparagine and vice versa; serine for threonine and vice versa; lysine for arginine and vice versa; or any isoleucine, valine, or leucine for one another.

[0043] As used in this document, the term “sequence identity” or “percent identity,” in the context of two or more nucleic acid or protein sequences, refers to two or more Petition 870200018837, dated 07 / 02 / 2020, page 23 / 146 / 105 sequences or subsequences that are identical or have a specified percentage of amino acid or nucleotide residues that are identical. For example, the sequence may have a percentage of identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or higher identity over a specified region to a reference sequence when compared and aligned for maximum match in a comparison window or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.For example, the percentage of identity is determined by calculating the ratio of the number of identical nucleotides (or amino acid residues) in the sequence divided by the length of the total nucleotides (or amino acid residues) minus the lengths of any gaps.

[0044] For convenience, the extent of identity between two sequences can be determined using computer programs and mathematical algorithms known in the art. Such algorithms that calculate the percentage of sequence identity generally account for gaps and mismatches in the comparison region. Programs that compare and align sequences, such as Clustal W (Thompson et al., (1994) Nucleic Acids Res., 22: 4673-4680), ALIGN (Myers et al., (1988) CABIOS, 4: 11-17), FASTA (Pearson et al., (1988) PNAS, 85: 2444-2448; Pearson (1990), Methods Enzymol., 183: 63-98) and interval BLAST (Altschul et al., (1997) Nucleic Acids Res., 25: 3389-3402) are useful for this purpose. BLAST or BLAST 2.0 (Altschul et al., J. Mol. Biol. 215: 403-10, 1990) is available from various sources, including the National Center for Biotechnology Information (NCBI) and the Internet, for use in conjunction with the BLASTP, BLASTN, and BLASTX sequence analysis programs. Petition 870200018837, dated 07 / 02 / 2020, p. 24 / 146 / 105 TBLASTN and TBLASTX. Additional information can be found on the NCBI website.

[0045] In certain modes, sequence alignments and percentage identity calculations can be determined using the BLAST program using its default, predefined parameters. For nucleotide sequence alignment and sequence identity calculations, the BLASTN program is used with its predefined parameters (opening range penalty = 5, extending range penalty = 2, nucleic match = 2, nucleic mismatch = -3, expectation value = 10.0, word size = 11, Maximum matches in a query range = 0).For polypeptide sequence alignment and sequence identity calculations, the BLASTP program is used with its predefined parameters (Alignment matrix = BLOSUM62; Range costs: Existence = 11, Extension = 1; Composition adjustments = Conditional composition score, matrix adjustment; Expectation value = 10.0; Word size = 6; Maximum matches in a query range = 0). Alternatively, the following programs and parameters are used: Clone Manager Suite Align Plus software, version 5 (Sci-Ed software); DNA comparison: global comparison, standard linear score matrix, Mismatch penalty = 2, Gap opening penalty = 4, Gap extension penalty = 1. Amino acid comparison: global comparison, BLOSUM 62 score matrix.In the modes described in this document, the sequence identity is calculated using the BLASTN or BLASTP programs using their default parameters. In the modes described in this document, the alignment of two or more sequences is performed using Clustal W using the suggested predefined parameters (Misalign input sequences: no; Mbed type clustering guide tree: yes; Mbed type clustering iteration: yes; number of combined iterations: yes). Petition 870200018837, dated 07 / 02 / 2020, page 25 / 146 / 105 predefined (0); Maximum number of iterations of the guide tree: predetermined; Maximum number of HMM iterations: predetermined; Order: input). 6.2 Host cells

[0046] Host cells capable of producing kaurenoic acid (KA) from kaurene are provided here with high efficiency. In certain embodiments, the host cells can produce kaurenoic acid from kaurene as a starting material. In particular embodiments, the host cells can produce kaurenoic acid from a carbon source in a culture medium. In particular embodiments, the host cells can produce kaurenoic acid from a carbon source in a culture medium and can also produce RebA or RebD from kaurenoic acid. In particular embodiments, the host cells can also produce rebaudioside M (RebM) from RebD.

[0047] In particular embodiments, the host cells comprise the kaureno oxidase enzymatic activity of Pisum sativum. A kaurene oxidase enzyme from Pisum sativum is capable of converting kaurene into kaurenoic acid with high efficiency.In certain embodiments, a kaureno oxidase enzyme from Pisum sativum is capable of converting kaureno to kaurenoic acid with an efficiency greater than 30%. In certain embodiments, a kaureno oxidase enzyme from Pisum sativum is capable of converting kaureno to kaurenoic acid with an efficiency greater than 35%. In certain embodiments, a kaureno oxidase enzyme from Pisum sativum is capable of converting kaureno to kaurenoic acid with an efficiency greater than 40%. In certain embodiments, a kaureno oxidase enzyme from Pisum sativum is capable of converting kaureno to kaurenoic acid with an efficiency greater than 95%. In certain embodiments, a kaureno oxidase enzyme from Pisum sativum is capable of converting kaureno to kaurenoic acid with an efficiency greater than 50%. In certain embodiments, a kaureno oxidase enzyme from Pisum sativum is capable of converting kaureno to kaurenoic acid with a... Petition 870200018837, dated 07 / 02 / 2020, p. 26 / 146 / 105 efficiency greater than 55%. In certain embodiments, a kaurene oxidase enzyme from Pisum sativum is capable of converting kaurene to kaurenoic acid with an efficiency greater than 58%. In certain embodiments, a kaurene oxidase enzyme from Pisum sativum is capable of converting kaurene to kaurenoic acid with an efficiency greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0048] In certain embodiments, the host cell is able to convert kaurene to kaurenoic acid with an efficiency greater than 30%. In certain embodiments, the host cell is able to convert kaurene to kaurenoic acid with an efficiency greater than 35%. In certain embodiments, the host cell is able to convert kaurene to kaurenoic acid with an efficiency greater than 40%. In certain embodiments, the host cell is able to convert kaurene to kaurenoic acid with an efficiency greater than 45%. In certain embodiments, the host cell is able to convert kaurene to kaurenoic acid with an efficiency greater than 50%. In certain embodiments, the host cell is able to convert kaurene to kaurenoic acid with an efficiency greater than 55%. In certain embodiments, the host cell is able to convert kaurene to kaurenoic acid with an efficiency of approximately 58%.In certain embodiments, the host cell is able to convert kaurene into kaurenoic acid with an efficiency greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0049] The efficiency of the conversion can be measured by any technique apparent to those skilled in the art. In certain embodiments, the efficiency of the conversion can be measured by contacting kaurene with an enzyme or host cell under conditions suitable for the formation of kaurenoic acid. The efficiency can be measured by comparing the molar amount of kaurene produced compared to the total amount of kaurene and kaureneic acid in the resulting composition. The efficiency also Petition 870200018837, dated 07 / 02 / 2020, p. 27 / 146 / 105 can be measured by comparing the total amount of kaurenoic acid and kaurenoic acid downstream products with the total amount of kaurene, kaurenol, kaurenal, kaurenoic acid and kaurenoic acid downstream products in the resulting composition. For example, the conversion efficiencies of strains comprising Ps.KO shown in FIG. 5 were measured by comparing the total amount of kaurenoic acid and all downstream compounds shown in FIG. 2 to the total amount of kaurene, kaurenol, kaurenal, kaurenoic acid and all downstream compounds shown in FIG. 2 in the resulting composition (i.e., steviol, 1 glucose + steviol, 2 glucose + steviol, 3 glucose + steviol, 4 glucose + steviol, 5 glucose + steviol and 6 glucose + steviol).

[0050] In certain embodiments, host cells comprising a kaureno oxidase comprising the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 65% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:1 are provided herein.In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, they are provided herein. Petition 870200018837, dated 07 / 02 / 2020, p. 28 / 146 / 105 host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1. In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:1 are provided herein.In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:1 are provided herein.In certain embodiments, host cells comprising a kaureno oxidase comprising an amino acid sequence that is at least 60%, at least 99%, or at least any percentage between 60% and 99% identical to the amino acid sequence of SEQ ID NO: 1 are provided herein.

[0051] In certain forms, host cells are provided here Petition 870200018837, dated 07 / 02 / 2020, p. 29 / 146 / 105 comprising a kaureno oxidase comprising an amino acid sequence described herein and capable of converting kaureno to kaurenoic acid. In certain embodiments, host cells are provided herein comprising a kaureno oxidase comprising an amino acid sequence described herein and capable of oxidizing position 19 of each of kaureno, kaurenol and kaurenal. In certain embodiments, host cells are provided herein comprising a kaureno oxidase capable of converting kaureno to kaurenoic acid with an efficiency greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% or 97%, wherein the kaureno oxidase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0052] In certain embodiments, host cells comprising a nucleic acid encoding a Pisum sativum kaureno oxidase comprising the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 65% identical to the amino acid sequence of SEQ ID NO:1 are provided herein.In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. Petition 870200018837, dated 07 / 02 / 2020, page 30 / 146 / 105 embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1 are provided herein.In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:1 are provided herein.In certain embodiments, host cells comprising an acid are provided here. Petition 870200018837, dated 07 / 02 / 2020, page 31 / 146 / 105 nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:1. In certain embodiments, host cells comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 60%, at least 99%, or any percentage between 60% and 99% identical are provided herein.

[0053] In certain embodiments, host cells comprising a heterologous nucleic acid comprising a nucleotide sequence of SEQ ID NO:14 encoding Pisum sativum kaureno oxidase with the sequence of SEQ ID NO:1 are provided herein. In certain embodiments, host cells comprising a heterologous nucleic acid comprising a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence of SEQ ID NO:14 are provided herein.

[0054] In certain embodiments, the host cell comprises a variant of the Pisum sativum kaureno oxidase polypeptide described above. In certain embodiments, the variant may comprise up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions relative to the Pisum sativum kaureno oxidase polypeptide. In certain embodiments, the variant may comprise up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions conservative relative to the Pisum sativum kaureno oxidase polypeptide. In certain embodiments, any of the nucleic acids described herein may be optimized for the host cell, for example, by codon optimization.

[0055] In embodiments described in this document, any suitable method may be used to determine the corresponding amino acid positions or the corresponding two-loop locations. Petition 870200018837, dated 07 / 02 / 2020, page 32 / 146 / 105 polypeptides. In certain embodiments, the sequences of a kaureno oxidase and the reference sequence SEQ ID NO:1 can be aligned using Clustal (W) using its predefined parameters. In another embodiment, the sequences of a kaureno oxidase and the reference sequence SEQ ID NO:1 can be aligned using structural alignments, such as SWISS-MODEL, which is a protein structure homology modeling server, accessible through the ExPASy web server or the DeepView program (Swiss Pdb-Viewer).

[0056] In certain embodiments, kaurene is as shown in FIG. 1C. In certain embodiments, a Pisum sativum kaurene oxidase or a variant Pisum sativum kaurene oxidase is capable of catalyzing the oxidation of kaurene at C-19 to form kaurenol. In certain embodiments, Pisum sativum kaurene oxidase or a variant Pisum sativum kaurene oxidase is capable of catalyzing the oxidation of kaurenol at C-19 to form kaurenal. In certain embodiments, Pisum sativum kaurene oxidase is capable of catalyzing the oxidation of kaurenal at C-19 to form kaurenoic acid. In particular embodiments, a Pisum sativum kaureno oxidase or a variant Pisum sativum kaureno oxidase is capable of catalyzing the oxidation of kaurenol at C-19 to form kaurenol, the oxidation of kaurenol at C-19 to form kaurenal, and the oxidation of kaurenal at C-19 to form kaurenoic acid.

[0057] In certain embodiments, RebD is as shown in FIG. 2. In certain embodiments, the host cell additionally comprises one or more enzymes capable of converting kaurenoic acid into steviol. In certain embodiments, the host cell additionally comprises one or more enzymes capable of converting steviol into one or more steviol glycosides. In certain embodiments, the host cell additionally comprises one or more enzymes capable of converting RebA into RebD. In certain embodiments, the host cell additionally comprises one or more Petition 870200018837, dated 07 / 02 / 2020, p. 33 / 146 / 105 enzymes capable of converting RebD into RebM.

[0058] While Pisum sativum kaureno oxidase or any variant Pisum sativum kaureno oxidase accepts kaureno as a substrate, the kaureno source can be any source considered suitable by those skilled in the art. In certain embodiments, Pisum sativum kaureno oxidase or any variant Pisum sativum kaureno oxidase can be contacted with kaureno. In certain embodiments, the host cell can be contacted with kaureno. In certain embodiments, Pisum sativum kaureno oxidase or any variant Pisum sativum kaureno oxidase can be contacted with a composition comprising one or more of kaureno, kaurenol, and kaurenal. In certain embodiments, the composition comprises kaureno. In certain embodiments, the composition comprises kaurenol. In certain embodiments, the composition comprises kaurenal. In certain formulations, the composition is derived from natural products isolated from the leaves of Stevia rebaudiana.In certain embodiments, the composition is derived microbially. In certain embodiments, the host cell may be contacted with a composition comprising one or more carbon sources.

[0059] In certain embodiments, any Pisum sativum variant kaurenose suitable for catalyzing a desired reaction can be evaluated by any suitable methods known in the art. For example, a suitable Pisum sativum variant kaurenose can be assayed in vivo by expressing a heterologous nucleic acid encoding a Pisum sativum variant kaurenose and evaluation cells that produce a functional Pisum sativum variant kaurenose capable of catalyzing oxidation at a desired site of a substrate (e.g., C-19 position of kaurenose, kaurenol, and / or kaurenal). Exemplary evaluation methods are described in the Examples below. In another example, a suitable Pisum sativum variant kaurenose can be evaluated in Petition 870200018837, dated 07 / 02 / 2020, p. 34 / 146 / 105 vitro contacting a variant Pisum sativum kaurenoic oxidase with a substrate such as kaureno, kaurenol, and / or kaurenal. In this example, assaying for the presence of kaurenoic acid, steviol, or a steviol glycoside such as RebD can be used as a test to determine if a variant Pisum sativum kaurenoic oxidase is a suitable enzyme. The reaction can be analyzed by LC-MS or other methods known in the art. See, for example, WO 2013 / 022989.

[0060] In certain embodiments, a variant Pisum sativum kaureno oxidase is considered suitable for converting kaureno to kaurenoic acid if it is able to convert kaureno to kaurenoic acid with efficiency greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96% or 97% in vivo.

[0061] In certain embodiments, a variant Pisum sativum kaureno oxidase is considered suitable for converting kaurenol to kaurenol if it is able to convert kaurenol to kaurenol with an efficiency greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96% or 97% in vivo.

[0062] In certain embodiments, a variant Pisum sativum kaureno oxidase is considered suitable for converting kaurenol to kaurenal if it is capable of converting kaurenol to kaurenal with an efficiency greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96% or 97% in vivo.

[0063] In certain embodiments, a variant Pisum sativum kaureno oxidase is considered suitable for converting kaurenolic acid to kaurenoic acid if it is capable of converting kaurenolic acid to kaurenoic acid with an efficiency greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96% or 97% in vivo.

[0064] In certain embodiments, a variant Pisum sativum kaureno oxidase is considered suitable for converting kaureno to kaurenoic acid if the conversion efficiency is greater than 30%, 40%, 50%, 60%, Petition 870200018837, dated 07 / 02 / 2020, p. 35 / 146 / 105 70%, 80%, 90%, 95%, 96% or 97% in vivo, where the conversion efficiency is calculated by the total amount of kaurenoic acid and all downstream compounds shown in FIG. 2 divided by the total amount of kaurenol, kaurenol, kaurenal, kaurenoic acid and all downstream compounds shown in FIG. 2 in the resulting composition (times 100 percent).

[0065] In advantageous embodiments, the host cell may comprise one or more enzymatic pathways capable of making kaurene, said pathways being taken individually or in combination. In certain embodiments, the host cells comprise one or more enzymes capable of converting geranylgeranyl diphosphate into kaurene. Useful enzymes and nucleic acids encoding the enzymes are known to experts. In certain embodiments, the host cells comprise one or more enzymes capable of converting geranylgeranyl diphosphate into kaurene. In other advantageous embodiments, the host cell may comprise one or more enzymatic pathways capable of converting kaurenoic acid into steviol and / or steviol glycosides, said pathways being taken individually or in combination. Useful enzymes and nucleic acids encoding the enzymes are known to experts.Particularly useful enzymes and nucleic acids are described in the sections below and further detailed, for example, in US 2014 / 0329281 A1, US 2014 / 0357588 A1, US 2015 / 0159188, WO 2016 / 038095 A2 and US 2016 / 0198748 A1.

[0066] In additional embodiments, the host cells further comprise one or more enzymes capable of making geranylgeranyl diphosphate from a carbon source. These include enzymes of the DXP pathway and enzymes of the SEM pathway. Useful enzymes and nucleic acids encoding the enzymes are known to those skilled in the art. Exemplary enzymes of each pathway are described below and are further described, for example, in US 2016 / 0177341 A1. The SEM pathway is also shown in FIG. 1D.

[0067] In certain embodiments, the additional enzymes are native. In Petition 870200018837, dated 07 / 02 / 2020, p. 36 / 146 / 105 advantageous embodiments, the additional enzymes are heterologous. In certain embodiments, two enzymes can be combined into a polypeptide. 6.3 Non-naturally occurring kaurene oxidase polypeptides and nucleic acids

[0068] In another aspect, unnaturally occurring variant kaurene oxidases are provided herein, which include modification(s) of amino acid residues compared to a reference sequence (e.g., SEQ ID NO:1) and still retain activity as a kaurene oxidase to convert kaurene to kaurenoic acid, kaurene to kaurenol, kaurenol to kaurenal, and / or kaurenal to kaurenoic acid. In certain embodiments, the unnaturally occurring variant kaurene oxidases may include up to 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, deletions, additions, and / or insertions at certain amino acid positions or locations compared to a reference sequence (e.g., SEQ ID NO:1). In certain embodiments, the non-naturally occurring variant kaurene oxidases comprise any of the variant kaurene oxidases described herein.

[0069] In another aspect, provided herein, are unnaturally occurring variant kaurene oxidases, which include modification(s) of nucleic acid residues compared to a reference sequence (e.g., SEQ ID NO:15) and yet, when translated into a protein, the protein retains activity as a kaurene oxidase to convert kaurene to kaurenoic acid, kaurene to kaurenol, kaurenol to kaurenal, and / or kaurenal to kaurenoic acid. In certain embodiments, the unnaturally occurring variant kaurene oxidase may encode any of the variant kaurene oxidases described herein. 6.4 Cellular strains

[0070] The useful compositions and methods of host cells provided here include archaeal, prokaryotic, or eukaryotic cells. Petition 870200018837, dated 07 / 02 / 2020, p. 37 / 146 / 105

[0071] Suitable prokaryotic hosts include, but are not limited to, any of a variety of Gram-positive, Gram-negative, or Gram-variable bacteria. Examples include, but are not limited to, cells belonging to the genera: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Cromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Mesactylococcus, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, and Zymomonas.Examples of prokaryotic strains include, but are not limited to: Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium beigerinckii, Enterobacter sakazakii, Escherichia coli, Lactococse Pudle, Aerorhizoblasomonas, Mesorhizoblasomonas, Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei and Staphylococcus aureus. In a particular embodiment, the host cell is an Escherichia coli cell.

[0072] Suitable archaeal hosts include, but are not limited to, cells belonging to the genera: Aeropyrum, Archaeglobus, Halobacterium, Methanococcus, Pyrococcus, Sulfolobus, and Thermoplasma. Examples of archaeal strains include, but are not limited to: Archaeoglobus fulgidus, Halobacterium sp., Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Thermoplasma acidophilum, Thermoplasma vulcanium, Pyrococcus horikoshii, Pyrococcus abyssi and Aeropyrum pernix.

[0073] Suitable eukaryotic hosts include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells. In some embodiments, yeasts are useful in the present methods. Petição 870200018837, de 07 / 02 / 2020, pág. 38 / 146 / 105 incluem leveduras que foram depositadas com depositários de microrganismos (por exemplo, IFO, ATCC, etc.) e pertencem aos gêneros Aciculoconidium, Ambrosiozyma, Arthroascus, Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma Brettanomyces, Bullera, Bulleromyces, Candida, Citeromyces, Clavispora, Cryptococcus, Cystofilobasidium, Debaryomyces, Dekkara, Dipodascopsis, Dipodascus, Eeniella, Endomycopsella, Eremascus, Eremotheciumum, Erythrobasidium, Erythrobasidium, Erythrobasidium Holtermannia, Hormoascus, Hyphopichia, Issatchenkia, Kloeckera, Kloeckeraspora, Kluyveromyces, Kondoa, Kuraishia, Kurtzmanomyces, Leucosporidium, Lipomyces, Lodderomyces, Malassezia, Metschnikowia, Mrakia, Myxozymaazson, Nadxiazyma, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Saitoella, Sakaguchia, Saturnospora, Schizoblastosporion, Schizosaccharomyces, Schwanniomyces, Sporidiobolus, Sporobolomyces,Sporopachydermia, Stephanoascus, Sterigmatomyces, Sterigmatosporidium, Symbiotaphrina, Sympodiomyces, Sympodiomycopsis, Torulaspora, Trichosporiella, Trichosporon, Trigonopsis, Tsuchiyaea, Udeniomyces, Waltomyces, Wickerhamia, Wickerhamiella, Williopsis, Yamadazyma, Yarrowia, Zygoascus, Zygosaccharomyces, Zygowilliopsis and Zygozyma, among others.,

[0074] In some embodiments, the host microbe is Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Dekkera bruxellensis, Kluyveromyces lactis (formerly called Saccharomyces lactis), Kluveromyces marxianus, Arxula adeninivorans Phania, or Hansenula polymich (now known as Pichia angusta). In some embodiments, the host microbe is a strain of the genus Candida, such as Candida lipolytica, Candida guilliermondii, Candida krusei, Candida pseudotropicalis, or Candida utilis.

[0075] In one specific embodiment, the host microbe is Petition 870200018837, dated 07 / 02 / 2020, p. 39 / 146 / 105 Saccharomyces cerevisiae. In some embodiments, the host is a strain of Saccharomyces cerevisiae selected from the group consisting of Baker's yeast, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE5, VR-1, BR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1, and AL-1. In some embodiments, the host microbe is a strain of Saccharomyces cerevisiae selected from the group consisting of PE2, CAT-1, VR-1, BG-1, CR-1, and SA-1. In one particular embodiment, the Saccharomyces cerevisiae strain is PE-2. In another particular embodiment, the Saccharomyces cerevisiae strain is CAT-1. In yet another specific embodiment, the Saccharomyces cerevisiae strain is BG-1.

[0076] In some embodiments, the host microbe is a microbe suitable for industrial fermentation. In particular embodiments, the microbe is conditioned to subsist under high solvent concentration, high temperature, expanded substrate utilization, nutrient limitation, osmotic stress due to sugar and salts, acidity, sulfite contamination and bacteria or combinations thereof, which are recognized stress conditions of the industrial fermentation environment. 6.5 Steviol and Steviol Glycoside Biosynthesis Pathways

[0077] In some embodiments, a steviol biosynthesis pathway and / or a steviol glycoside biosynthesis pathway is activated in the genetically modified host cells provided herein, engineering the cells to express polynucleotides and / or polypeptides encoding one or more enzymes of the pathway. FIG. 1B illustrates an exemplary steviol biosynthesis pathway. FIG. 2 illustrates a steviol glycoside biosynthesis pathway from geranylgeranyl pyrophosphate to various steviol glycosides.

[0078] Thus, in some embodiments, the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with geranyl-geranyl activity. Petition 870200018837, dated 07 / 02 / 2020, page 40 / 146 / 105 diphosphate synthase (GGPPS). In some embodiments, the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with copalyl diphosphate synthase or entcopalyl pyrophosphate synthase (CDPS; also referred to as entcopalyl pyrophosphate synthase or CPS activity). In some embodiments, the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with kaureno-synthase (KS; also referred to as ent-kaureno-synthase) activity. In particular embodiments, the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with kaorene oxidase (KO; also referred to as ent-kaorene 19-oxidase) activity, as described herein.In some embodiments, the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with steviol synthase activity (also referred to as ent-kaurenoic acid 13-hydroxylase or KAH). In some embodiments, the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with cytochrome P450 reductase (CPR) activity.

[0079] In some embodiments, the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with UGT74G1 activity. In some embodiments, the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with UGT76G1 activity. In some embodiments, the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with UGT85C2 activity. In some embodiments, the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with UGT91D activity. In some embodiments, Petition 870200018837, dated 07 / 02 / 2020, page 41 / 146 / 105 the genetically modified host cells provided herein comprise a heterologous polynucleotide encoding a polypeptide with UDP glycosyltransferase activity.

[0080] In certain embodiments, the host cell comprises a variant. In certain embodiments, the variant may comprise up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions relative to the relevant polypeptide. In certain embodiments, the variant may comprise up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid substitutions relative to the reference polypeptide. In certain embodiments, any of the nucleic acids described herein may be optimized for the host cell, for example, codon optimized.

[0081] Exemplary nucleic acids and enzymes of a steviol biosynthesis pathway and / or a steviol glycoside biosynthesis pathway are described below. 6.5.1 Geranyl-geranyl diphosphate synthase (GGPPS)

[0082] Geranyl-geranyl diphosphate synthases (EC 2.5.1.29) catalyze the conversion of farnesyl pyrophosphate to geranyl-geranyl diphosphate. Illustrative examples of enzymes include those from Stevia rebaudiana (accession number ABD92926), Gibberella fujikuroi (accession number CAA75568), Mus musculus (accession number AAH69913), Thalassiosira pseudonana (accession number XP_002288339), Slreplomsces clavuligerus (accession number ZP_05004570), Sulfulobus acidocaldarius (accession number BAA43200), Synechococcus sp. (accession number ABC98596), Arabidopsis thaliana (accession number NP_195399), Blakeslea trispora (accession number AFC92798.1) and US 2014 / 0329281 A1. The nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided herein using a nucleic acid with at least 80%, 85%, 90% or 95% sequence identity to at least one of these GGPPS nucleic acids. In certain Petition 870200018837, dated 07 / 02 / 2020, page 42 / 146 / 105 modalities, cells and methods are provided here that utilize a nucleic acid that encodes a polypeptide with at least 80%, 85%, 90%, 95% sequence identity of at least one of these GGPPS enzymes. 6.5.2 Copalyl diphosphate synthase (CDPS)

[0083] Copalyl diphosphate synthases (EC 5.5.1.13) catalyze the conversion of farnesyl pyrophosphate to geranylgeranyl diphosphate. Illustrative examples of enzymes include those from Stevia rebaudiana (accession number AAB87091), Streptomyces clavuligerus (accession number ED Y51667), Bradyrhizobium japonicum (accession number AAC28895.1), Zea mays (accession number AY562490), Arabidopsis thaliana (accession number NM_116512), Oryza sativa (accession number Q5MQ85.1) and US 2014 / 0329281 A1. The nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided here that use a nucleic acid that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these CDPS nucleic acids.In certain embodiments, cells and methods are provided here that use a nucleic acid encoding a polypeptide having at least 80%, 95%, 90%, or 95% sequence identity to at least one of these CDPS enzymes. 6.5.3 Kaurene Synthase (KS)

[0084] Kaurene synthases (EC 4.2.3.19) catalyze the conversion of copalyl diphosphate to kaurene and diphosphate. Illustrative examples of enzymes include those from Bradyrhizobium japonicum (accession number AAC28895.1), Phaeosphaeria sp. (accession number O13284), Arabidopsis thaliana (accession number Q9SAK2), Picea glauca (accession number ADB55711.1), and US 2014 / 0329281 A1. The nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided herein that use a nucleic acid that has at least 80%, 85%, 90%, or 95% identity to Petition 870200018837, dated 07 / 02 / 2020, p. 43 / 146 / 105 sequence to at least one of these KS nucleic acids. In certain embodiments, cells and methods are provided herein that use a nucleic acid encoding a polypeptide having at least 80%, 85%, 85%, 90% or 95% sequence identity to at least one of these KS enzymes. 6.5.4 Bifunctional Copalyl diphosphate synthase (CDPS) and kaurene synthase (KS)

[0085] Bifunctional CDPS-KS enzymes (EC 5.5.1.13 and EC 4.2.3.19) can also be used. Illustrative examples of enzymes include those from *Phomopsis amygdali* (accession number BAG30962), *Physcomitrella patens* (accession number BAF61135), *Gibberella fujikuroi* (accession numbers US 2014 / 0329281 A1, US 2014 / 0357588 A1, US 2015 / 0159188, and WO 2016 / 038095 A2). The nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided herein that use a nucleic acid that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these CDPS-K nucleic acids. In certain embodiments, cells and methods are provided herein that use a nucleic acid encoding a polypeptide having at least 80%, 85%, 90%, or 95% sequence identity to at least one of these CDPS-KS enzymes. 6.5.5 Ent-kaurene oxidase (KO)

[0086] Ent-kaurene oxidases (EC 1.14.13.78; also referred to as kaurene oxidases) are described herein. The nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided herein that use a nucleic acid that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these kaurene oxidase nucleic acids. In certain embodiments, cells and methods are provided herein that use a nucleic acid encoding a polypeptide that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these enzymes. Petition 870200018837, dated 07 / 02 / 2020, p. 44 / 146 / 105 kaurene oxidase. 6.5.6 Steviol Synthase (KAH)

[0087] Steviol synthases or kaurenoic acid hydroxylases (KAH), (EC 1.14.13) catalyze the conversion of kaurenoic acid to steviol. Illustrative examples of enzymes include those from Stevia rebaudiana (accession number ACD93722), Stevia rebaudiana (SEQ ID NO:10), Arabidopsis thaliana (accession number NP_197872), Vitis vinifera (accession number XP_002282091), Medicago trunculata (accession number ABC59076), and US 2014 / 0329281 A1, US 2014 / 0357588 A1, US 2015 / 0159188, and WO 2016 / 038095 A2. The nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided here that use a nucleic acid that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these KAH nucleic acids.In certain embodiments, cells and methods are provided here that use a nucleic acid encoding a polypeptide that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these KAH enzymes. 6.5.7 Cytochrome P450 Reductase (CPR)

[0088] Cytochrome P450 reductases (EC 1.6.2.4) are capable of assisting or facilitating the activity of KO and / or KAH above. Illustrative examples of enzymes include those from Stevia rebaudiana (accession number ABB88839), Arabidopsis thaliana (accession number NP_194183), Gibberella fujikuroi (accession number CAE09055), Artemisia annua (accession number ABC47946.1) and US 2014 / 0329281 A1, US 2014 / 0357588 A1, US 2015 / 0159188 and WO 2016 / 038095 A2. The nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided here that use a nucleic acid that has at least 80%, 85%, 90%, or 95% identity to Petition 870200018837, dated 07 / 02 / 2020, p. 45 / 146 / 105 sequence to at least one of these CPR nucleic acids. In certain embodiments, cells and methods are provided herein that use a nucleic acid encoding a polypeptide that has at least 80%, 85%, 90% or 95% sequence identity to at least one of these CPR enzymes. 6.5.8 UDP 74G1 Glycosyltransferase (UGT74G1)

[0089] A UGT74G1 is capable of functioning as a 5'-diphospho glucosyl:steviol 19-COOH transferase and as a uridine 5'-diphospho glucosyl:steviol-13-O-glucoside 19-COOH transferase. As shown in Figure 2, a UGT74G1 is capable of converting steviol to 19-glucoside. A UGT74G1 is also capable of converting steviol monoside to rubusoside. A UGT74G1 may also be capable of converting steviol bioside to stevioside. Illustrative examples of enzymes include those from Stevia rebaudiana (e.g., those from Richman et al., 2005, Plant J. 41: 56-67 and US 2014 / 0329281 and WO 2016 / 038095 A2 and accession number AAR06920.1). The nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided herein that use a nucleic acid that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these nucleic acids of UGT74G1.In certain embodiments, cells and methods are provided here that use a nucleic acid encoding a polypeptide that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these UGT74G1 enzymes. 6.5.9. UDP 76G1 Glycosyltransferase (UGT76G1)

[0090] A UGT76G1 is capable of transferring a glucose moiety to the C-3' of the C-13-O-glucose of the acceptor molecule, a steviol 1,2-glycoside. Thus, a UGT76G1 is capable of functioning as a uridine 5'-diphosphoglucosyl:steviol 13-O-l,2-glycoside C-3'-glycosyltransferase and a uridine 5'-diphosphoglucosyl:steviol-19-O-glucose, 13-O-l,2-bioside C-3'-glycosyltransferase. As shown in Figure 2, a UGT76G1 is capable of Petition 870200018837, dated 07 / 02 / 2020, p. 46 / 146 / 105 convert stevioside to RebB. A UGT76G1 is also capable of converting stevioside to RebA. A UGT76G1 is also capable of converting RebD to RebM. Illustrative examples of enzymes include those from Stevia rebaudiana (e.g., those from Richman et al., 2005, Plant J. 41: 56-67 and US 2014 / 0329281 A1 and WO 2016 / 038095 A2 and accession number AAR06912.1). Nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided here that use a nucleic acid that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these UGT76G1 nucleic acids. In certain embodiments, cells and methods are provided here that use a nucleic acid encoding a polypeptide having at least 80%, 85%, 90%, or 95% sequence identity to at least one of these UGT76G1 enzymes. 6.5.10 UDP glycosyltransferase 85C2 (UGT85C2)

[0091] A UGT85C2 is capable of functioning as a uridine 5'-diphosphoglycosyl:steviol 13-OH transferase, and a uridine 5'-diphosphoglycosyl:steviol-19-O-glucoside 13-OH transferase. Thus, as shown in Figure 2, a UGT85C2 is capable of converting steviol to steviol monoside, and is also capable of converting 19-glucoside to rubusoside. Illustrative examples of enzymes include those from Stevia rebaudiana (e.g., those from Richman et al., 2005, Plant J. 41: 56-67 and US 2014 / 0329281 A1 and WO 2016 / 038095 A2 and accession number AAR06916.1). The nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided here that use a nucleic acid that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these UGT85C2 nucleic acids.In certain embodiments, cells and methods are provided here that use a nucleic acid encoding a polypeptide that has at least 80%, 85%, 90%, or 95% sequence identity. Petition 870200018837, dated 07 / 02 / 2020, p. 47 / 146 / 105 one of these UGT85C2 enzymes. 6.5.11 UDP-glucosyltransferase 91D (UGT91D)

[0092] A UGT91D is capable of functioning as a uridine 5'-diphosphoglucosyl:steviol-13-O-glucoside transferase, transferring a glucose moiety to the C-2' of the 13-O-glucose of the acceptor molecule, steviol-13-O-glucoside (steviolmonoside), to produce steviobioside. A UGT91D is also capable of functioning as a uridine 5'-diphosphoglucosyl:rubusoside transferase, transferring a glucose moiety to the C2' of the 13-O-glucose of the acceptor molecule, rubusoside, to provide stevioside, as shown in Figure 2. A UGT91D is also referred to as UGT91D2, UGT91D2e, or UGT91D-like3. Illustrative examples of UGT91D enzymes include those from Stevia rebauidana (e.g., those with the UGT sequence number ACE87855.1, US 2014 / 0329281 A1, WO 2016 / 038095 A2 and SEQ ID NO:7). The nucleic acids encoding these enzymes are useful in the cells and methods provided herein.In certain embodiments, cells and methods are provided here that use a nucleic acid that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these UGT91D nucleic acids. In certain embodiments, cells and methods are provided here that use a nucleic acid that encodes a polypeptide having at least 80%, 85%, 90%, or 95% sequence identity to at least one of these UGT91D enzymes. 6.5.12 Uridine diphosphate-dependent glycosyltransferase capable of converting RebA to RebD (UGTad)

[0093] A uridine diphosphate-dependent glycosyltransferase (UGTAD) is capable of transferring a glucose moiety to the C-2' position of 19-O-glucose from RebA to produce RebD as shown in Figure 2. A UGTAD is also capable of transferring a glucose moiety to the C2' position of 19-O-glucose from stevioside to RebE. Useful examples of UGTs Petition 870200018837, dated 07 / 02 / 2020, p. 48 / 146 / 105 includes Os_UGT_91C1 of Oryza saliva (also referred to as EUGT11 in Houghton-Larsen et al., WO 2013 / 022989 A2; XP_015629141_1) and S1_UGT_101249881 of Solanum lycopersicum (also referred to as UGTSL2 in Markosyan et al., WO2014 / 193888 A1; XP_004250485.1). Additional useful UGTs include UGT40087 (XP_004982059.1), sr.UGT_9252778 (SEQ ID NO: 16), Bd_UGT10840 (XP_003560669.1), Hv_UGT_V1 (BAJ94055.1), Bd_UGT10850 (XP_010230871.1), and Ob_UGT91B1_like (XP_006650455.1). Any UGT or UGT variant may be used in the compositions and methods described herein. The nucleic acids encoding these enzymes are useful in the cells and methods provided herein. In certain embodiments, cells and methods are provided here that use a nucleic acid that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of the UGTs.In certain embodiments, cells and methods are provided here that use a nucleic acid encoding a polypeptide that has at least 80%, 85%, 90%, or 95% sequence identity to at least one of these UGTs. In certain embodiments, a nucleic acid encoding a UGT variant described here is provided here.

[0094] In certain embodiments, the genetically modified host cells comprise a heterologous nucleic acid encoding a UDP-glycosyltransferase comprising an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to the UGT40087 sequence (e.g., SEQ ID NO:17 or SEQ ID NO:18). In certain embodiments, the genetically modified host cell is capable of converting RebA to RebD at an efficiency greater than 90%, 95%, 96%, or 97%.In certain embodiments, the genetically modified host cell comprises a UDP-glycosyltransferase comprising a sugar acceptor domain, wherein the amino acid sequence of the sugar acceptor domain has at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Petition 870200018837, dated 07 / 02 / 2020, page 49 / 146 / 105 sequence for the amino acid sequence of the sugar acceptor domain of SEQ ID NO:17 or SEQ ID NO:18. In certain embodiments, the genetically modified host cell comprises a UDPglycosyltransferase comprising amino acid sequence loop1, an amino acid sequence variant loop1, an amino acid sequence loop2, an amino acid sequence variant loop2, an amino acid sequence loop3_1, an amino acid sequence variant loop3_1, an amino acid sequence variant loop3_1, an amino acid sequence loop3_2, an amino acid sequence variant loop3_2, an amino acid sequence loop4_1, an amino acid sequence variant loop4_1, an amino acid sequence loop4_2 or any combination thereof.In certain embodiments, the genetically modified host cell comprises a UDP-glycosyltransferase comprising an amino acid sequence having at least 61%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the sugar acceptor domain of SEQ ID NO:17 or SEQ ID NO:18, and further comprises the amino acid sequence loop4_1 of SEQ ID NO:17 or SEQ ID NO:18.

[0095] As used herein, the term “loop1 variant” amino acid sequence refers to an amino acid sequence that differs from the reference loop1 amino acid sequence of SEQ ID NO:17 or 18 (or a modified loop1 sequence of UGT40087 having the sequence of SEQ ID NO:28) by one, two, three, four, five, six, seven, eight, nine, or ten amino acid insertions, deletions, mutations, and / or substitutions, but allows a UDP-glycosyltransferase comprising a loop1 variant amino acid sequence, inserted at a location that corresponds to the location of the amino acid sequence. Petition 870200018837, dated 07 / 02 / 2020, p. 50 / 146 / 105 loopl of SEQ ID NO:17 or 18, respectively, to catalyze the conversion of RebA to RebD and / or stevioside to RebE.

[0096] As used herein, the term “loop2 variant” amino acid sequence refers to an amino acid sequence that differs from the reference loop2 amino acid sequence of SEQ ID NO:17 or 18 by one, two, three, four, five, six, seven, eight, nine, or ten amino acid insertions, deletions, mutations, and / or substitutions, but allows a UDP-glycosyltransferase comprising a loop2 variant amino acid sequence, inserted at a location that corresponds to the location of the loop2 amino acid sequence of SEQ ID NO:17 or 18, respectively, to catalyze the conversion of RebA to RebD and / or stevioside to RebE.

[0097] As used herein, the term “loop3_1 variant” amino acid sequence refers to an amino acid sequence that differs from the reference loop3_1 amino acid sequence of SEQ ID NO:17 or 18 by one, two, three, four, five, six, seven, eight, nine, or ten amino acid insertions, deletions, mutations, and / or substitutions, but allows a UDP-glycosyltransferase comprising a loop3_1 variant amino acid sequence, inserted at a location that corresponds to the location of the loop3_1 amino acid sequence of SEQ ID NO:17 or 18, to catalyze the conversion of RebA to RebD and / or stevioside to RebE.As used herein, the term “loop3_2 variant” amino acid sequence refers to an amino acid sequence that differs from the reference loop3_2 amino acid sequence of SEQ ID NO:17 or 18 by one, two, three, four, five, six, seven, eight, nine, or ten amino acid insertions, deletions, mutations, and / or substitutions, but allows a UDP-glycosyltransferase comprising a variant loop3_2 amino acid sequence, inserted at a location that corresponds to the loop3_2 amino acid sequence location of. Petition 870200018837, dated 07 / 02 / 2020, p. 51 / 146 / 105 SEQ ID NO: 17 or 18, respectively, to catalyze the conversion of RebA to RebD and / or stevioside to RebE. In certain embodiments, the variant amino acid sequence loop3_2 differs from the reference amino acid sequence loop3_2 by one, two, three, four, six, seven, eight, nine, ten, or even thirty amino acid insertions, deletions, mutations, and / or substitutions.

[0098] As used herein, the term “loop4_1 variant” amino acid sequence refers to an amino acid sequence that differs from the reference loop4_1 amino acid sequence of SEQ ID NO:17 or 18 by one, two, three, four, five, six, seven, eight, nine, ten, or up to 30 amino acid insertions, deletions, mutations, and / or substitutions, but allows a UDP-glycosyltransferase comprising a loop4_1 variant sequence, inserted at a location that corresponds to the loop4_1 amino acid location of SEQ ID NO:17 or 18, to catalyze the conversion of RebA to RebD and / or stevioside to RebE.

[0099] In certain embodiments, host cells comprise a functional domain of a UGT40087, wherein the UGT40087 comprises the amino acid sequence of SEQ ID NO:17 or 18. In certain embodiments, host cells comprise a polypeptide comprising the N-terminal sugar acceptor domain of a UGT40087 comprising the amino acid sequence of SEQ ID NO:17 or 18. In certain embodiments, host cells comprise a polypeptide comprising the C-terminal sugar donor domain of a UGT40087 comprising the amino acid sequence of SEQ ID NO:17 or 18. In certain embodiments, the sugar acceptor domain of a UGT40087 comprises approximately amino acid positions 1 to 214 of SEQ ID NO:18 (which correspond to amino acid positions 1 to 215 of SEQ ID NO:17). In certain embodiments, the sugar donor domain of UGT40087 comprises approximately amino acid positions 215 to 435 of SEQ ID NO:18 (which correspond to the positions of Petition 870200018837, dated 07 / 02 / 2020, p. 52 / 146 / 105 amino acids 216 to 436 of SEQ ID NO:17). In certain embodiments, the sugar-accepting domain of UGT40087 comprises approximately amino acid positions 1 to 215 of SEQ ID NO:17. In certain embodiments, the sugar-donating domain comprises approximately amino acid positions 216 to 436 of SEQ ID NO:17. In certain embodiments, the sugar-accepting domain and the sugar-donating domain of UGT40087 comprise a narrower range of amino acid residues than 1 to 214 or 215 to 435, respectively, relative to SEQ ID NO:18. In certain embodiments, the sugar-accepting domain and the sugar-donating domain of UGT40087 comprise a narrower range of amino acid residues than 1 to 215 or 216 to 436, respectively, relative to SEQ ID NO:17.

[00100] In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 65% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18.In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 75% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, such as... Petition 870200018837, dated 07 / 02 / 2020, page. 53 / 146 / 105 host cells comprise a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, host cells comprise a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, host cells comprise a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, host cells comprise a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18.In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18.In certain embodiments, host cells comprising a polypeptide are provided here. Petition 870200018837, dated 07 / 02 / 2020, p. 54 / 146 / 105 comprising an amino acid sequence that is at least 60%, at least 99%, or any percentage between 60% and 99% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18.

[00101] In certain embodiments, the host cells comprise a nucleic acid encoding a UGT40087 comprising the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18.In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 65% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 75% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least... Petition 870200018837, dated 07 / 02 / 2020, p. 55 / 146 / 105 less than 80% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18.In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18.In certain embodiments, the host cells comprise a nucleic acid that encodes a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of the acceptor domain. Petition 870200018837, dated 07 / 02 / 2020, p. 56 / 146 / 105 sugar N-terminal of SEQ ID NO:17 or 18.

[00102] In certain embodiments, when three-dimensional modeled structures of UGT40087 and another UDP-glycosyltransferase were compared and analyzed, they revealed four loops (i.e., loop1, loop2, loop3, and loop4) that possess significant conformational differences in the N-terminal sugar acceptor domain. Experimental results of exchanging the corresponding loop sequences between the two UGTs indicated that loop1, loop2, loop3_l, loop3_2, and loop4_l of UGT40087 can be replaced by their respective corresponding loop sequences from other UDP-glycosyltransferases that are capable of converting RebA to RebD. In these embodiments, two versions of loop3 (i.e., loop3_l and loop3_2) and loop_4 (i.e., loop4_l and loop4_2) were designed to consider two possible loop lengths.

[00103] Thus, in certain embodiments, the host cells comprise a UDP-glycosyltransferase comprising an amino acid sequence that is at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a heterologous nucleic acid encoding a UDP-glycosyltransferase comprising an amino acid sequence that is at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18. In certain embodiments, UDP-glycosyltransferase additionally comprises a loop1 amino acid sequence of UGT40087 (i.e., SEQ ID NO:17 or 18), at a UDP-glycosyltransferase location that corresponds to the loop1 location of SEQ ID NO:17 or 18, respectively.In certain embodiments, the loop1 amino acid sequence of SEQ ID NO:17 or 18 has the sequence of... Petition 870200018837, dated 07 / 02 / 2020, p. 57 / 146 / 105 amino acids of SEQ ID NO:30. In certain embodiments, the loop1 amino acid sequence has the sequence SEQ ID NO:28. In certain embodiments, the UDP-glycosyltransferase additionally comprises a variant loop1 amino acid sequence, at a location of the UDP-glycosyltransferase that corresponds to the loop1 location of SEQ ID NO:17 or 18, respectively. The variant loop1 amino acid sequence refers to an amino acid sequence that differs from the reference loop1 amino acid sequence of SEQ ID NO:17 or 18 or from the loop1 amino acid sequence having SEQ ID NO:28, but allows the UDP-glycosyltransferase comprising the variant loop1 amino acid to retain its activity to convert RebA to RebD and / or to convert stevioside to RebE.

[00104] In certain embodiments, the UDP-glycosyltransferase additionally comprises the loop2 amino acid sequence of UGT40087 (i.e., SEQ ID NO:17 or 18), at a UDP-glycosyltransferase location that corresponds to the loop2 location of SEQ ID NO: 17 or 18, respectively. In certain embodiments, the loop2 amino acid sequence of SEQ ID NO:17 or 18 has the amino acid sequence of SEQ ID NO:24. In certain embodiments, the UDP-glycosyltransferase additionally comprises a variant loop2 amino acid sequence, at a UDP-glycosyltransferase location that corresponds to the loop2 location of SEQ ID NO:17 or 18, respectively. The loop2 variant amino acid sequence refers to an amino acid sequence that differs from the reference loop2 amino acid sequence of SEQ ID NO:17 or 18, but allows the UDP-glycosyltransferase comprising the loop2 variant amino acid to retain its activity to convert RebA to RebD and / or to convert stevioside to RebE.

[00105] In certain embodiments, UDP-glycosyltransferase also comprises loop 3_l amino acid sequence of UGT40087 (i.e., SEQ ID NO:17 or 18), at a location of UDP-glycosyltransferase that Petition 870200018837, dated 07 / 02 / 2020, p. 58 / 146 / 105 corresponds to the loop3_l location of SEQ ID NO: 17 or 18, respectively. In certain embodiments, the loop 3_l amino acid sequence of SEQ ID NO:17 or 18 has the amino acid sequence of SEQ ID NO:25. In certain embodiments, UDP-glycosyltransferase additionally comprises a variant loop3_l amino acid sequence, at a UDP-glycosyltransferase location that corresponds to the loop 3_1 location of SEQ ID NO:17 or 18, respectively. The variant amino acid sequence loop3_l refers to an amino acid sequence that differs from the reference amino acid sequence loop3_l of SEQ ID NO:17 or 18, but allows the UDP-glycosyltransferase comprising the variant amino acid loop3_2 to retain its activity to convert RebA to RebD and / or to convert stevioside to RebE.

[00106] In certain embodiments, the UDP-glycosyltransferase additionally comprises a loop3_2 amino acid sequence of UGT40087 (i.e., SEQ ID NO:17 or 18), at a UDP-glycosyltransferase location that corresponds to the loop3_2 location of SEQ ID NO:17 or 18, respectively. In certain embodiments, the loop3_2 amino acid sequence of SEQ ID NO:17 or 18 has the amino acid sequence of SEQ ID NO:26. In certain embodiments, the UDP-glycosyltransferase additionally comprises a variant loop3_2 amino acid sequence, at a UDP-glycosyltransferase location that corresponds to the loop3_2 location of SEQ ID NO:17 or 18, respectively. The variant amino acid sequence loop3_2 refers to an amino acid sequence that differs from the loop3_2 amino acid sequence of SEQ ID NO:17 or 18, but allows the UDPglycosyltransferase comprising the variant amino acid loop3_2 to retain its activity to convert RebA to RebD and / or to convert stevioside to RebE.

[00107] In certain embodiments, UDP-glycosyltransferase additionally comprises loop4_l amino acid sequence of UGT40087 (i.e., SEQ Petition 870200018837, dated 07 / 02 / 2020, p. 59 / 146 / 105 In certain embodiments, the loop4_l amino acid sequence of SEQ ID NO:17 or 18 has the amino acid sequence of SEQ ID NO:27. In certain embodiments, the UDP-glycosyltransferase additionally comprises a variant loop4_l amino acid sequence, at a UDP-glycosyltransferase location that corresponds to the loop4_l location of SEQ ID NO:17 or 18, respectively. The loop4_l amino acid sequence refers to an amino acid sequence that differs from the loop4_l amino acid sequence of SEQ ID NO:17 or 18, but allows the UDP-glycosyltransferase comprising the variant loop4_l amino acid to retain its activity to convert RebA to RebD and / or to convert stevioside to RebE.

[00108] In certain embodiments, UDP-glycosyltransferase additionally comprises the loop4_2 amino acid sequence of UGT40087 (i.e., SEQ ID NO:17 or 18), at a UDP-glycosyltransferase location that corresponds to the loop4_2 location of SEQ ID NO: 17 or 18, respectively. The loop4_2 amino acid sequence of SEQ ID NO:17 or 18 has the amino acid sequence of SEQ ID NO:28.

[00109] In certain embodiments, the host cells comprise a UDP-glycosyltransferase comprising an amino acid sequence that is at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18 or a heterologous nucleic acid encoding the UDP-glycosyltransferase thereof, and further comprises any combination of the following: (a) the loop1 amino acid sequence of SEQ ID NO:17 or 18, the amino acid sequence of SEQ ID NO:30, or a variant loop1 amino acid sequence, at a UDP-glycosyltransferase location Petition 870200018837, dated 07 / 02 / 2020, p. 60 / 146 / 105, which corresponds to the location of loopl with SEQ ID NO:17 or 18, respectively; (b) the loop2 amino acid sequence of SEQ ID NO:17 or 18 or a variant loop2 amino acid sequence, at a UDP-glycosyltransferase location that corresponds to the loop2 location of SEQ ID NO:17 or 18, respectively; (c) the loop3_l amino acid sequence of SEQ ID NO:17 or 18 or a variant loop3_l amino acid sequence, at a UDP-glycosyltransferase location that corresponds to the loop3_l location of SEQ ID NO:17 or 18, respectively; (d) the loop3_2 amino acid sequence of SEQ ID NO:17 or 18 or a variant loop3_2 amino acid sequence, at a UDP-glycosyltransferase location that corresponds to the loop 3_2 location of SEQ ID NO:17 or 18, respectively; (e) the loop4_1 amino acid sequence of SEQ ID NO:17 or 18 or a variant loop4_1 amino acid sequence, at a UDP-glycosyltransferase location that corresponds to the loop4_1 location of SEQ ID NO:17 or 18, respectively; and (f) the loop4_2 amino acid sequence of SEQ ID NO:17 or 18, at a UDP-glycosyltransferase location that corresponds to the loop4_2 location of SEQ ID NO:17 or 18, respectively.

[00110] In certain embodiments, when the three-dimensional modeled structures of UDP-glycosyltransferases capable of converting RebA to RebD were compared and analyzed, it was discovered that the loop4_1 of UGT40087, when incorporated into the corresponding loop4_1 location of another UDP-glycosyltransferase (and the substitution of its native loop4_1 amino acid sequence) led to superior activity of a variant UDP-glycosyltransferase in terms of its ability to convert RebA to RebD. See Example 12. These results indicate that the sequence of Petition 870200018837, dated 07 / 02 / 2020, page 61 / 146 / 105 amino acids loop4_1 of any suitable UDP-glycosyltransferase can be replaced with the loop4_1 amino acid sequence of SEQ ID NO:17 or 18 to convert RebA to RebD.

[00111] Therefore, in certain embodiments, the host cells comprise a UDP-glycosyltransferase comprising an amino acid sequence that is at least 61%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18, and further comprises the loop 4_l amino acid sequence (i.e., SEQ ID NO:27) of UGT40087 (i.e., SEQ ID NO:17 or 18). In certain embodiments, the host cells comprise a heterologous nucleic acid encoding a UDPglycosyltransferase comprising an amino acid sequence that is at least 61%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18, and further comprises the loop 4_l amino acid sequence (e.g., SEQ ID NO:27) of SEQ ID NO:17 or 18.In certain embodiments, any suitable UDP-glycosyltransferase comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of SEQ ID NO:17 or 18 may be used to integrate the loop4_l amino acid sequence of SEQ ID NO:17 or 18 into its corresponding loop4_l location (replacing its native loop4_l amino acid sequence). For example, Ob_UGT91B_like, Hv_UGT_V1, Sl_UGT_101249881, Sr.UGT_g252778, Os_UGT_91C1, Bd_UGT10840, Bd_UGT10850, or Si91Dlike, can be used as a basis for integrating the loop4_1 amino acid sequence of SEQ ID NO:17 or 18 into its corresponding loop4_1 location. In certain embodiments, UDPglycosyltransferase comprises an amino acid sequence of SEQ ID NO:33.

[00112] In certain forms, the host cells comprise Petition 870200018837, dated 07 / 02 / 2020, p. 62 / 146 / 105 a polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 65% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18.In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 75% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18.In certain forms, the host cells comprise a polypeptide comprising an amino acid sequence that is... Petition 870200018837, dated 07 / 02 / 2020, p. 63 / 146 / 105 less than 95% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18.In certain embodiments, the host cells comprise a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18.

[00113] In certain embodiments, the host cells comprise a nucleic acid encoding a UGT40087 comprising the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is by Petition 870200018837, dated 07 / 02 / 2020, p. 64 / 146 / 105 less 65% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 75% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18.In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18.In certain embodiments, the host cells comprise a nucleic acid that encodes a polypeptide comprising an amino acid sequence that is at least 96% identical to the amino acid sequence of the donor domain. Petition 870200018837, dated 07 / 02 / 2020, p. 65 / 146 / 105 C-terminal sugar of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 97% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18.

[00114] Thus, in certain embodiments, the host cells comprise a UDP-glycosyltransferase comprising an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the host cells comprise a heterologous nucleic acid encoding a UDP-glycosyltransferase comprising an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of the C-terminal sugar donor domain of SEQ ID NO:17 or 18. In certain embodiments, the UDP-glycosyltransferase further comprises a C-terminal sugar donor domain. from another UDP-glycosyltransferase. Examples of other UDP-glycosyltransferases with suitable C-terminal sugar donor domains include Ob_UGT91B_like, Hv_UGT_V1, SI_UGT_101249881, Sr.UGT_g252778, Os_UGT_91C1, Bd_UGT10840, Bd_UGT10850 or Si91Dlike. Petition 870200018837, dated 07 / 02 / 2020, p. 66 / 146 / 105

[00115] In certain embodiments, it has been discovered that certain amino acid residues in the N-terminal sugar acceptor domain can restore the catalytic activity of a non-functional UDP-glycosyltransferase to an active UDP-glycosyltransferase. Therefore, host cells comprise a UDP-glycosyltransferase comprising an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the N-terminal sugar acceptor domain of SEQ ID NO:17 or 18, and further comprises one or more of the following amino acid residues: (a) valine at an amino acid position of UDPglycosyltransferase that corresponds to amino acid position 11 of SEQ ID NO:18; (b) isoleucine at an amino acid position of UDPglycosyltransferase that corresponds to amino acid position 12 of SEQ ID NO:18; (c) proline at an amino acid position of UDPglycosyltransferase that corresponds to amino acid position 55 of SEQ ID NO:18; (d) glutamic acid at an amino acid position of UDPglycosyltransferase that corresponds to amino acid position 90 of SEQ ID NO:18; (e) serine at an amino acid position of UDPglycosyltransferase that corresponds to amino acid position 203 of SEQ ID NO:18; (f) glutamic acid at an amino acid position of UDPglycosyltransferase that corresponds to amino acid position 223 of SEQ ID NO:18; or (g) valine at an amino acid position of UDP Petition 870200018837, dated 07 / 02 / 2020, p. 67 / 146 / 105 glycosyltransferase that corresponds to amino acid position 413 of SEQ ID NO:18, wherein the amino acid positions of UDP-glycosyltransferase that correspond to the amino acid positions of SEQ ID NO:18 are determined by sequence alignment.

[00116] In certain embodiments, the host cells comprise a UDP-glycosyltransferase comprising an amino acid sequence of SEQ ID NO:32.

[00117] In certain embodiments, the host cell comprises a variant of the UGT40087 polypeptide described above. In certain embodiments, the variant may comprise up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions relative to the UGT40087 polypeptide. In certain embodiments, the variant may comprise up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid substitutions relative to the UGT40087 polypeptide. In certain embodiments, any of the nucleic acids described herein may be optimized for the host cell, for example, codon optimized. Useful nucleic acids include SEQ ID NO:35 and 36. 6.6 Production of FPP and / or GGPP via SEM

[00118] In some embodiments, a genetically modified host cell provided herein comprises one or more heterologous enzymes of the SEM pathway, useful for the formation of FPP and / or GGPP. See Figure 1D. In some embodiments, one or more enzymes of the SEM pathway comprise an enzyme that condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA. In some embodiments, one or more enzymes of the SEM pathway comprise an enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA. In some embodiments, one or more enzymes of the SEM pathway comprise an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA. In some embodiments, one or more enzymes of the SEM pathway comprise a Petition 870200018837, dated 07 / 02 / 2020, page 68 / 146 / 105 enzyme that converts HMG-CoA into mevalonate. In some embodiments, one or more enzymes of the SEM pathway comprise an enzyme that phosphorylates mevalonate into mevalonate 5-phosphate. In some embodiments, one or more enzymes of the SEM pathway comprise an enzyme that converts mevalonate 5-phosphate into mevalonate 5-pyrophosphate. In some embodiments, one or more enzymes of the SEM pathway comprise an enzyme that converts mevalonate 5-pyrophosphate into isopentyl pyrophosphate.

[00119] In some embodiments, one or more enzymes of the SEM pathway are selected from the group consisting of acetyl-CoA thiolase, acetoacetyl-CoA synthetase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate pyrophosphate decarboxylase. In some embodiments, with respect to the SEM pathway enzyme capable of catalyzing the formation of acetoacetyl-CoA, the genetically modified host cell comprises an enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA, for example, acetyl-CoA thiolase; or an enzyme that condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA, for example, acetoacetyl-CoA synthase.In some embodiments, the genetically modified host cell comprises both an enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA, for example, acetyl-CoA thiolase; and an enzyme that condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA, for example, acetoacetyl-CoA synthase.

[00120] In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding more than one enzyme of the SEM pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding two enzymes of the SEM pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding an enzyme that can convert HMG-CoA to mevalonate and Petition 870200018837, dated 07 / 02 / 2020, page 69 / 146 / 105 an enzyme that can convert mevalonate to mevalonate 5-phosphate. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding three enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding four enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding five enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding six enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences that encode seven enzymes of the SEM pathway.In some embodiments, the host cell comprises a plurality of heterologous nucleic acids that encode all the enzymes of the SEM pathway.

[00121] In some embodiments, the genetically modified host cell further comprises a heterologous nucleic acid encoding an enzyme that can convert isopentenyl pyrophosphate (IPP) into dimethylallyl pyrophosphate (DMAPP). In some embodiments, the genetically modified host cell further comprises a heterologous nucleic acid encoding an enzyme that can condense IPP and / or DMAPP molecules to form a polyprenyl compound. In some embodiments, the genetically modified host cell further comprises a heterologous nucleic acid encoding an enzyme that can modify IPP or a polyprenyl to form an isoprenoid compound such as FPP. 6.6.1 Conversion of Acetyl-CoA to Acetoacetyl-CoA

[00122] In some embodiments, the genetically modified host cell comprises a heterologous nucleotide sequence encoding an enzyme that can condense two acetyl-coenzyme molecules. Petition 870200018837, dated 07 / 02 / 2020, p. 70 / 146 / 105 A to form acetoacetyl-CoA, for example, an acetyl-CoA thiolase. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (REGION NC_000913: 2324131.2325315; Escherichia coli), (D49362; Paracoccus denitrificans), and (L20428; Saccharomyces cerevisiae).

[00123] Acetyl-CoA thiolase catalyzes the reversible condensation of two acetyl-CoA molecules to yield acetoacetyl-CoA, but this reaction is thermodynamically unfavorable; acetoacetyl-CoA thiolysis is favored over acetoacetyl-CoA synthesis. Acetoacetyl-CoA synthase (AACS) (alternatively referred to as acetyl-CoA:malonyl-CoA acyltransferase; EC 2.3.1.194) condenses acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA. In contrast to acetyl-CoA thiolase, AACS-catalyzed acetoacetyl-CoA synthesis is essentially an energy-favored reaction due to the associated decarboxylation of malonyl-CoA. Furthermore, AACS does not exhibit thiolysis activity against acetoacetyl-CoA, and thus the reaction is irreversible.

[00124] In host cells comprising acetyl-CoA thiolase and a heterologous ADA and / or phosphotransacetylase (PTA), the reversible reaction catalyzed by acetyl-CoA thiolase, which favors acetacetyl-CoA thiolysis, can result in a large pool of acetyl-CoA. Given the reversible activity of ADA, this acetyl-CoA group can, in turn, drive ADA toward the reverse reaction of converting acetyl-CoA to acetaldehyde, thus diminishing the benefits provided by ADA in relation to acetyl-CoA production. Similarly, the activity of PTA is reversible, and thus, a large pool of acetyl-CoA can drive PTA toward the reverse reaction of converting acetyl-CoA to acetyl phosphate. Therefore, in some embodiments, in order to provide a strong pull on acetyl-CoA to drive the advanced ADA and PTA reaction, the SEM pathway of the genetically modified host cell provided here utilizes an acetoacetyl-CoA synthase to form acetoacetyl-CoA. Petition 870200018837, dated 07 / 02 / 2020, p. 71 / 146 / 105 of Acetyl-CoA and malonyl-CoA.

[00125] In some embodiments, AACS is from the CL190 strain of Streptomyces sp. (Okamura et al., Proc Natl Acad Sci USA 107(25):11265-70 (2010). Representative AACS nucleotide sequences from the CL190 strain of Streptomyces sp. include accession number AB540131.1. Representative AACS protein sequences from the CL190 strain of Streptomyces sp. include accession numbers D7URV0, BAJ10048. Other acetoacetyl-CoA synthases useful for the compositions and methods provided herein include, but are not limited to, Streptomyces sp. (AB183750; KO-3988 BAD86806); Actinoplanes sp. A40644 (AB113568; BAD07381); Streptomyces sp. C (NZ_ACEW010000640; ZP_05511702); Nocardiopsis dassonvillei DSM 43111 (NZ_ABUI01000023; ZP_04335288); Mycobacterium ulcerans Agy99 (NC_008611; YP_907152); Mycobacterium marinum M (NC_010612; YP_001851502); Streptomyces sp. Mg1 (NZ_DS570501; ZP_05002626); Streptomyces sp. AA4 (NZ_ACEV01000037; ZP_05478992); S. roseosporus NRRL 15998 (NZ_ABYB01000295; ZP_04696763); Streptomyces sp. ACT (NZ_ADFD01000030; ZP_06275834); S. viridochromogenes DSM 40736 (NZ_ACEZ01000031; ZP_05529691); Frankia sp. CcI3 (NC_007777; YP_480101); Nocardia brasiliensis (NC_018681; YP_006812440.1); and Austwickia chelonae (NZ_BAGZ01000005; ZP_10950493.1). Additional suitable acetoacetylCoA synthases include those described in U.S. Patent Application Publications Nos. 2010 / 0285549 and 2011 / 0281315, the contents of which are incorporated by reference in their entirety.

[00126] The acetoacetyl-CoA synthases also useful in the compositions and methods provided herein include those molecules which are referred to as being “derivatives” of any of the acetoacetyl-CoA synthases described herein. Such a “derivative” has the following characteristics: (1) it shares Petition 870200018837, dated 07 / 02 / 2020, p. 72 / 146 / 105 substantial homology with any of the acetoacetyl-CoA synthases described herein; and (2) is capable of catalyzing the irreversible condensation of acetyl-CoA with malonyl-CoA to form acetoacetyl-CoA. An acetoacetyl-CoA synthase derivative is said to share “substantial homology” with acetoacetyl-CoA synthase if the amino acid sequences of the derivative are at least 80%, and more preferably at least 90%, and most preferably at least 95%, the same as that of acetoacetyl-CoA synthase. 6.6.2 Conversion of Acetoacetyl-CoA to HMG-CoA

[00127] In some embodiments, the host cell comprises a heterologous nucleotide sequence that encodes an enzyme that can condense acetoacetyl-CoA with another acetyl-CoA molecule to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), for example, an HMG-CoA synthase. Illustrative examples of nucleotide sequences that encode such an enzyme include, but are not limited to: (NC_001145. complement 19061.20536; Saccharomyces cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907; Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_002758, Locus tag SAV2546, GeneID 1122571; Staphylococcus aureus). 6.6.3 Conversion of HMG-CoA to Mevalonate

[00128] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can convert HMG-CoA to mevalonate, for example, an HMG-CoA reductase. In some embodiments, the HMG-CoA reductase is an NADH-using hydroxymethylglutaryl-CoA reductase. HMG-CoA reductases (EC 1.1.1.34; EC 1.1.1.88) catalyze the reductive deacylation of (S)-HMGCoA to (R)-mevalonate, and can be categorized into two classes, class I and class II HMGs. Class I includes the enzymes of eukaryotes and most archaea, and class II includes the HMG-CoA reductases of certain prokaryotes and archaea. Aside from the divergence in sequences, the enzymes of the two classes Petition 870200018837, dated 07 / 02 / 2020, page 73 / 146 / 105, also differs in relation to its cofactor specificity. Unlike class I enzymes, which use NADPH exclusively, class II HMG-CoA reductases vary in their ability to discriminate between NADPH and NADH. See, for example, Hedl et al. Journal of Bacteriology 186 (7): 1927-1932 (2004). The cofactor specificities for selecting class II HMG-CoA reductases are provided below. Cofactor specificities for the selectivity of HMG-CoA class II reductases Source Coenzyme Specificity Km nadph (pM) Km nadh (pM) P. mevalonii NADH 80 A. fulgidus NAD(P)H 500 160 S. aureus NAD(P)H 70 100 E. faecalis NADPH 30

[00129] The HMG-CoA reductases useful for the compositions and methods provided herein include HMG-CoA reductases that are capable of using NADH as a cofactor, for example, HMG-CoA reductase from P. mevalonii, A. fulgidus, or S. aureus. In particular embodiments, the HMG-CoA reductase is capable of using only NADH as a cofactor, for example, HMG-CoA reductase from P. mevalonii, S. pomeroyi, or D. acidovorans.

[00130] In some embodiments, the NADH-using HMG-CoA reductase is from Pseudomonas mevalonii. The wild-type mvA gene sequence of Pseudomonas mevalonii, which encodes HMG-CoA reductase (EC 1.1.1.88), has been previously described. See Beach and Rowell, J. Bacteriol. 171: 2994-3001 (1989). Representative mvA nucleotide sequences from Pseudomonas mevalonii include accession number M24015. Representative HMG-CoA reductase protein sequences from Pseudomonas mevalonii include accessions AAA25837, P13702, MVAA_PSEMV.

[00131] In some embodiments, the NADH-using HMG-CoA reductase is from Silicibacter pomeroyi. Representative HMG-CoA reductase nucleotide sequences from Silicibacter pomeroyi include accession number NC_006569.1. The HMG-CoA reductase protein sequences Petition 870200018837, dated 07 / 02 / 2020, page 74 / 146 / 105 representative of Silicibacter pomeroyi include accession number YP_164994.

[00132] In some embodiments, the HMG-CoA using NADH reductase is from Delftia acidovorans. The nucleotide sequences of HMG-CoA reductase representative of Delftia acidovorans include NC_010002 REGION: complement (319980..321269). The protein sequences of HMG-CoA reductase representative of Delftia acidovorans include accession number YP 001561318.

[00133] In some embodiments, the HMG-CoA using NADH reductases are from Solanum tuberosum (Crane et al., J. Plant Physiol. 159:1301-1307 (2002)).

[00134] HMG-CoA using NADH reductases also useful in the compositions and methods provided herein include those molecules that are referred to as being “derivatives” of any of the HMG-CoA using NADH reductases described herein, for example, from P. mevalonii, S. pomeroyi, and D. acidovorans. Such a “derivative” has the following characteristics: (1) it shares substantial homology with any of the HMG-CoA reductases described herein; and (2) it is capable of catalyzing the reductive deacylation of (S)-HMG-CoA to (R)-mevalonate while preferentially using NADH as a cofactor. A derivative of HMG-CoA using NADH reductase is said to share "substantial homology" with HMG-CoA using NADH reductase if the amino acid sequences of the derivative are at least 80%, and more preferably at least 90%, and most preferably at least 95%, the same as that of HMG-CoA using NADH reductase.

[00135] As used herein, the expression “using NADH” means that HMG-CoA using NADH reductase is selective for NADH over NADPH as a cofactor, for example, demonstrating greater specific activity for NADH than for NADPH. In some embodiments, the selectivity for NADH as a cofactor is expressed as a kcat(NADH) / ratio. Petition 870200018837, dated 07 / 02 / 2020, page 75 / 146 / 105 kcat(NADPH). In some embodiments, HMG-CoA using NADH reductase has a kcat(NADH) / kcat(NADPH) ratio of at least 5, 10, 15, 20, 25 or greater than 25. In some embodiments, HMG-CoA using NADH reductase uses NADH exclusively. For example, an HMG-CoA using NADH reductase that uses NADH exclusively exhibits some activity with NADH supplied as the sole cofactor in vitro, and exhibits no detectable activity when NADPH is supplied as the sole cofactor. Any method for determining cofactor specificity known in the art may be used to identify HMG-CoA reductases having a preference for NADH as a cofactor, including those described by Kim et al., Protein Science 9: 1226-1234 (2000); and Wilding et al., J Bacteriol. 182 (18): 5147-52 (2000), the contents of which are incorporated herein in their entirety.

[00136] In some embodiments, HMG-CoA using NADH reductase is engineered to be selective for NADH over NAPDH, for example, through mutagenesis directed at the cofactor binding cavity site. Methods for engineering NADH selectivity are described in Watanabe et al. Microbiology 153:3044-3054 (2007), and methods for determining the cofactor specificity of HMG-CoA reductases are described in Kim et al. Protein Sci. 9:1226-1234 (2000), the contents of which are incorporated herein by reference in their entirety.

[00137] In some embodiments, NADH-using HMG-CoA reductase is derived from a host species that natively comprises a mevalonate-degrading pathway, for example, a host species that catabolizes mevalonate as its sole carbon source. Within these embodiments, NADH-using HMG-CoA reductase, which normally catalyzes the oxidative acylation of internalized (R)-mevalonate to (S)-HMG-CoA within its native host cell, is used to catalyze the reverse reaction, i.e., the reductive deacylation of (S)-HMG-CoA to (R)-mevalonate, in a genetically modified host cell. Petition 870200018837, dated 07 / 02 / 2020, page 76 / 146 / 105, comprising a mevalonate biosynthetic pathway. Prokaryotes capable of growing on mevalonate as their sole carbon source have been described by: Anderson et al., J. Bacterial, 171(12):6468-6472 (1989); Beach et al., J. Bacterial. 171:2994-3001 (1989); Bensch et al., J. Bial. Chem. 245:3755-3762; Fimongnari et al., Biochemistry 4:2086-2090 (1965); Siddiqi et al., Biachem. Biaphys. Res. Cammun. 8:110-113 (1962); Siddiqi et al., J. Bacterial. 93:207-214 (1967); and Takatsuji et al., Biachem. Biaphys. Res. Cammun. 110:187-193 (1983), whose contents are incorporated herein by reference in their entirety.

[00138] In some embodiments of the compositions and methods provided herein, the host cell comprises both an HMGr using NADH and an HMH-CoA using NADPH reductase. Illustrative examples of nucleotide sequences encoding an HMG-CoA reductase using NADPH include, but are not limited to: (NM_206548; Drasaphila melanagaster), (NC_002758, Locus tag SAV2545, GeneID 1122570; Staphylococcus aureus), (AB015627; Streptamyces sp. KO 3988), (AX128213, providing the sequence encoding a truncated HMG-CoA reductase; Saccharamyces cerevisiae), and (NC_001145: complement (115734.118898; Saccharamyces cerevisiae). 6.6.4 Conversion of Mevalonate to Mevalonate-5-Phosphate

[00139] In some embodiments, the host cell comprises a heterologous nucleotide sequence that encodes an enzyme that can convert mevalonate to mevalonate 5-phosphate, for example, a mevalonate kinase. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (L77688; Arabidapsis thaliana), and (X55875; Saccharamyces cerevisiae). 6.6.6 Conversion of Mevalonate-5-Phosphate to Mevalonate-5-Pyrophosphate

[00140] In some embodiments, the host cell comprises a heterologous nucleotide sequence that encodes an enzyme that can Petition 870200018837, dated 07 / 02 / 2020, page 77 / 146 / 105 convert mevalonate 5-pyrophosphate into mevalonate 5-pyrophosphate, for example, a phosphomevalonate kinase. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (AF429385; Hevea brasiliensis), (NM_006556; Homo sapiens), and (NC_001145. complement 712315.713670; Saccharomyces cerevisiae). 6.6.6 Conversion of Mevalonate-5-Pyrophosphate to IPP

[00141] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can convert mevalonate 5-pyrophosphate to isopentyl diphosphate (IPP), for example, a mevalonate pyrophosphate decarboxylase. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (X97557; Saccharomyces cerevisiae), (AF290095; Enterococcus faecium), and (U49260; Homo sapiens). 6.6.7 Conversion of IPP to DMAPP

[00142] In some embodiments, the host cell additionally comprises a heterologous nucleotide sequence encoding an enzyme that can convert IPP generated via the SEM pathway into dimethylallyl pyrophosphate (DMAPP), for example, an IPP isomerase. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (NC_000913, 3031087.3031635; Escherichia coli) and (AF082326; Haematococcus pluvialis). 6.6.8 Polyprenyl Synthases

[00143] In some embodiments, the host cell additionally comprises a heterologous nucleotide sequence encoding a polyprenyl synthase that can condense IPP and / or DMAPP molecules to form polyprenyl compounds containing more than five carbons.

[00144] In some embodiments, the host cell comprises a heterologous nucleotide sequence that encodes an enzyme that can condense an IPP molecule with a DMAPP molecule to form Petition 870200018837, dated 07 / 02 / 2020, page 78 / 146 / 105 a geranyl pyrophosphate (“GPP”) molecule, for example, a GPP synthase. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (AF513111; Abies grandis), (AF513112; Abies grandis), (AF513113; Abies grandis), (AY534686; Antirrhinum majus), (AY534687; Antirrhinum majus), (Y17376; Arabidopsis thaliana), (AE016877, Locus AP11092; Bacillus cereus; ATCC 14579), (AJ243739; Citrus sinensis), (AY534745; Clarkia breweri), (AY953508; Ips pini), (DQ286930; Lycopersicon esculentum), (AF182828; Mentha x piperita), (AF182827; Mentha x piperita), (MPI249453; Mentha x piperita), (PZE431697, Locus CAD24425; Paracoccus zeaxanthinifaciens), (AY866498; Picrorhiza kurrooa), (AY351862; Vitis vinifera) and (AF203881, Locus AAF12843; Zymomonas mobilis).

[00145] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can condense two IPP molecules with a DMAPP molecule or add an IPP molecule to a GPP molecule to form a farnesyl pyrophosphate (“FPP”) molecule, for example, an FPP synthase. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (ATU80605; Arabidopsis thaliana), (ATHFPS2R; Arabidopsis thaliana), (AAU36376; ​​Artemisia annua), (AF461050; Bos taurus), (D00694; Escherichia coli K-12), (AE009951, Locus AAL95523; Fusobacterium nucleatum subsp. AAW60034; Gluconobacter oxydans 621H), (AF019892; Helianthus annuus), (HUMFAPS; Homo sapiens), (KLPFPSQCR; Kluyveromyces lactis), (LAU15777; Lupinus albus), (LAU20771; Lupinus albus), (AF309508; Mus musculus), (NCFPPSGEN; Neurospora crassa), (PAFPS1; Parthenium argentatum), (PAFPS2; Parthenium argentatum), (RATFAPS; Rattus norvegicus), (YSCFPP; Saccharomyces cerevisiae), (D89104; Petition 870200018837, of 07 / 02 / 2020, p. 79 / 146 / 105 Schizosaccharomyces pombe), (CP000003, Locus AAT87386; Streptococcus pyogenes), (CP000017, Locus AAZ51849; Streptococcus pyogenes), (NC_008022, Locus YP_598856; Streptococcus pyogenes MGAS10270), (NC_008023, Locus YP_600845; Streptococcus pyogenes MGAS2096), (NC_008024, Locus YP_602832; Streptococcus pyogenes MGAS10750), (MZEFPS; Zea mays), (AE000657, Locus AAC06913; Aquifex aeolicus VF5), (NM_202836; Arabidopsis thaliana), (D84432, Locus BAA12575; Bacillus subtilis), (U12678, Location AAC28894; Bradyrhizobium japonicum USDA 110), (BACFDPS; Geobacillus stearothermophilus), (NC_002940, Locus NP_873754; Haemophilus ducreyi 35000HP), (L42023, Locus AAC23087; Haemophilus influenzae Rd KW20), (J05262; Homo sapiens), (YP_395294; Lactobacillus sakei subsp. sakei 23K), (NC_005823, Locus YP_000273; Leptospira interrogans serovar Copenhageni str. Fiocruz L1130), (AB003187; Micrococcus luteus), (NC_002946, Locus YP_208768; Neisseria gonorrhoeae FA 1090), (U00090, Locus AAB91752; Rhizobium sp.NGR234), (J05091; Saccharomyces cerevisae), (CP000031, Locus. AAV93568; Silicibacter pomeroyi DSS-3), (AE008481, Locus AAK99890; Streptococcus pneumoniae R6) and (NC_004556, Locus NP 779706; Xylella fastidiosa Temecula1).

[00146] In some embodiments, the host cell additionally comprises a heterologous nucleotide sequence encoding an enzyme that can combine IPP and DMAPP or IPP and FPP to form geranylgeranyl pyrophosphate (“GGPP”). Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (ATHGERPYRS; Arabidopsis thaliana), (BT005328; Arabidopsis thaliana), (NM_119845; Arabidopsis thaliana), (NZ_AAJM01000380, Locus ZP_00743052; Bacillus thuringiensis serovar israelensis, ATCC 35646 sq1563), (CRGGPPS; Catharanthus roseus), (NZ_AABF02000074, Locus ZP_00144509; Fusobacterium nucleatum subsp. vincentii, ATCC 49256), Petition 870200018837, of 07 / 02 / 2020, p. 80 / 146 / 105 (GFGGPPSGN; Gibberella fujikuroi), (AY371321; Ginkgo biloba), (AB055496; Hevea brasiliensis), (AB017971; Homo sapiens), (MCI276129; Mucor circinelloides f. lusitanicus), (AB016044; Mus musculus), (AABX01000298, Locus NCU01427; Neurospora crassa), (NCU20940; Neurospora crassa), (NZ_AAKL01000008, Locus ZP_00943566; Ralstonia solanacearum UW551), (AB118238; Rattus norvegicus), (SCU31632; Saccharomyces cerevisiae), (AB016095; Synechococcus elongates), (SAGGPS; Sinapis alba), (SSOGDS; Sulfolobus acidocaldarius), (NC_007759, Locus YP_461832; Syntrophus aciditrophicus SB), (NC_006840, Locus YP_204095; Vibrio fischeri ES114), (NM_112315; Arabidopsis thaliana), (ERWCRTE; Pantoea agglomerans), (D90087, Locus BAA14124; Pantoea ananatis), (X52291, Locus CAA36538; Rhodobacter capsulatus), (AF195122, Locus AAF24294; Rhodobacter sphaeroides), and (NC_004350, Locus NP_721015; Streptococcus mutans UA159).

[00147] Although examples of mevalonate pathway enzymes are described above, in certain embodiments, DXP ​​pathway enzymes can be used as an alternative or additional pathway to produce DMAPP and IPP in host cells, compositions and methods described herein. Enzymes and nucleic acids encoding DXP pathway enzymes are well known and distinguished in the art. WO 2012 / 135591 A2. 6.7 Methods for Producing Steviol Glycosides

[00148] In another aspect, a method is provided here for the production of a steviol glycoside, the method comprising the steps of: (a) cultivating a population of any of the genetically modified host cells described herein that are capable of producing a steviol glycoside in a medium with a carbon source under conditions suitable for making the steviol glycoside compound; and (b) recovering said steviol glycoside compound from the medium.

[00149] In some forms, the genetically modified host cell Petition 870200018837, dated 07 / 02 / 2020, page 81 / 146 / 105 modified produces an increased amount of steviol glycoside compared to a parent cell not comprising one or more modifications or a parent cell comprising only a subset of one or more modifications of the genetically modified host cell, but otherwise genetically identical. In some embodiments, the increased quantity is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more than 100%), as measured, for example, in yield, production, productivity, in grams per liter of cell culture, milligrams per gram of dry cell weight, on a volume per unit basis of cell culture, on a basis of dry cell weight per unit, on a basis of unit volume of cell culture per unit time or on a basis of dry cell weight per unit time.

[00150] In some embodiments, the host cell produces a high level of a steviol glycoside that is greater than about 10 grams per liter of fermentation medium. In some of these embodiments, the steviol glycoside is produced in an amount of about 10 to about 50 grams, more than about 15 grams, more than about 20 grams, more than about 25 grams, or more than about 30 grams per liter of cell culture.

[00151] In some embodiments, the host cell produces a high level of a steviol glycoside that is greater than about 50 milligrams per gram of dry cell weight. In some of these embodiments, the steviol glycoside is produced in an amount of about 50 to about 1500 milligrams, more than about 100 milligrams, more than about 150 milligrams, more than about 200 milligrams, more than about 250 milligrams, more than about 500 milligrams, more than about 750 milligrams, or more than about 1000 milligrams per gram of dry cell weight. Petition 870200018837, dated 07 / 02 / 2020, page 82 / 146 / 105

[00152] In some embodiments, the host cell produces a high level of steviol glycoside that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1.000 times or more superior to the level of steviol glycoside produced by a parent cell, in a volume per unit of cell culture basis.

[00153] In some embodiments, the host cell produces a high level of a steviol glycoside that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1.000 times or more superior to the level of steviol glycoside produced by the parent cell, on a dry cell weight basis. Petition 870200018837, dated 07 / 02 / 2020, page 83 / 146 / 105 unit.

[00154] In some embodiments, the host cell produces a high level of steviol glycoside that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1.000 times or more superior to the level of steviol glycoside produced by the parent cell, in a unit volume of cell culture per unit time.

[00155] In some embodiments, the host cell produces a high level of steviol glycoside that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1,000 times or more, higher than the steviol glycoside level. Petition 870200018837, dated 07 / 02 / 2020, page 84 / 146 / 105 produced by the parent cell, based on dry unit per unit per unit of time.

[00156] In most embodiments, the production of high levels of steviol glycoside by the host cell is inducible by an inducing compound. Such a host cell can be easily manipulated in the absence of the inducing compound. The inducing compound is then added to induce the production of high levels of steviol glycoside by the host cell. In other embodiments, the production of high levels of steviol glycoside by the host cell is inducible by changing culture conditions, such as, for example, growth temperature, media constituents, and the like. 6.8 Culture Media and Conditions

[00157] Materials and methods for the maintenance and growth of microbial cultures are well known to those versed in the art of microbiology or fermentation science (see, for example, Bailey et al. Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986). Consideration should be given to the appropriate culture medium, pH, temperature, and requirements for aerobic, microaerobic, or anaerobic conditions, depending on the specific needs of the host cell, the fermentation, and the process.

[00158] The methods for producing steviol glycosides provided herein can be carried out in a suitable culture medium (e.g., with or without pantothenate supplementation) in a suitable container, including but not limited to a cell culture plate, a flask, or a fermenter. Furthermore, the methods can be carried out at any fermentation scale known in the art to support the industrial production of microbial products. Any suitable fermenter can be used, including a stirred tank fermenter, an air transport fermenter, a bubble fermenter, or any combination thereof. Petition 870200018837, dated 07 / 02 / 2020, pp. 85 / 146 / 105 same. In particular embodiments that utilize Saccharomyces cerevisiae as the host cell, the strains can be grown in a fermenter as described in detail by Kosaric, et al. in Ullmann's Encyclopedia of Industrial Chemistry, sixth edition, Volume 12, pages 398-473, WileyVCH Verlag GmbH & Ampt Co KDaA, Weinheim, Germany.

[00159] In some embodiments, the culture medium is any culture medium in which a genetically modified microorganism capable of producing a steviol glycoside can subsist, that is, maintain growth and viability. In some embodiments, the culture medium is an aqueous medium comprising assimilable sources of carbon, nitrogen, and phosphate. Such a medium may also include salts, minerals, metals, and other appropriate nutrients. In some embodiments, the carbon source and each of the essential cell nutrients are added incrementally or continuously to the fermentation medium, and each required nutrient is maintained essentially at the minimum level necessary for efficient assimilation by growing cells, for example, according to a predetermined cell growth curve based on the metabolic or respiratory function of the cells that convert the carbon source into biomass.

[00160] The appropriate conditions and media for the culture of microorganisms are well known in the art. In some embodiments, the appropriate medium is supplemented with one or more additional agents, such as, for example, an inducer (e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressor (e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter) or a selection agent (e.g., an antibiotic to select microorganisms comprising the genetic modifications).

[00161] In some forms, the carbon source is a Petition 870200018837, dated 07 / 02 / 2020, page 86 / 146 / 105 a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or one or more combinations thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, xylose, ribose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof. Non-limiting examples of suitable non-fermentable carbon sources include acetate and glycerol.

[00162] The concentration of a carbon source, such as glucose, in the culture medium should promote cell growth, but should not be so high as to suppress the growth of the microorganism used. Typically, cultures are carried out with a carbon source, such as glucose, being added at levels to achieve the desired level of growth and biomass, but at undetectable levels (with detection limits being around 0.1 g / L). In other embodiments, the concentration of a carbon source, such as glucose, in the culture medium is greater than about 1 g / L, preferably greater than about 2 g / L, and more preferably greater than about 5 g / L. Furthermore, the concentration of a carbon source, such as glucose, in the culture medium is typically less than about 100 g / L, preferably less than about 50 g / L, and more preferably less than about 20 g / L.It should be noted that references to culture component concentrations may refer to initial and / or ongoing component concentrations. In some cases, it may be desirable to allow the culture medium to become depleted of a carbon source during the culture process.

[00163] Sources of assimilable nitrogen that can be used in a suitable culture medium include, but are not limited to, simple nitrogen sources, organic nitrogen sources, and complex nitrogen sources. Petition 870200018837, dated 07 / 02 / 2020, page 87 / 146 / 105 nitrogen. Such nitrogen sources include anhydrous ammonia, ammonium salts, and substances of animal, vegetable, and / or microbial origin. Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptone, yeast extract, ammonium sulfate, urea, and amino acids. Typically, the concentration of nitrogen sources in the culture medium is greater than about 0.1 g / L, preferably greater than about 0.25 g / L, and most preferably greater than about 1.0 g / L. Beyond certain concentrations, however, the addition of a nitrogen source to the culture medium is not advantageous for the growth of microorganisms.As a result, the concentration of nitrogen sources in the culture medium is less than about 20 g / L, preferably less than about 10 g / L, and most preferably less than about 5 g / L; in some cases, it may be desirable to allow the culture medium to become depleted of nitrogen sources during cultivation.

[00164] The effective culture medium may contain other compounds such as inorganic salts, vitamins, trace metals or growth promoters. Such other compounds may also be present as carbon, nitrogen or mineral sources in the effective medium or may be specifically added to the medium.

[00165] The culture medium may also contain a suitable phosphate source. Such phosphate sources include both inorganic and organic phosphate sources. Preferred phosphate sources include, but are not limited to, phosphate salts such as mono- or dibasic sodium and potassium phosphates, ammonium phosphate, and mixtures thereof. Typically, the phosphate concentration in the culture medium is greater than about 1.0 g / L, preferably greater than about 2.0 g / L, and most preferably greater than about 5.0 g / L. Beyond certain concentrations, however, the addition of phosphate to the culture medium is not advantageous for the growth of microorganisms. Consequently, the phosphate concentration in the medium of Petition 870200018837, dated 07 / 02 / 2020, page 88 / 146 / 105 culture is typically less than about 20 g / L, preferably less than about 15 g / L and, more preferably, less than about 10 g / L.

[00166] A suitable culture medium may also include a source of magnesium, preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium may be used. Typically, the magnesium concentration in the culture medium is greater than about 0.5 g / L, preferably greater than about 1.0 g / L, and more preferably greater than about 2.0 g / L. Beyond certain concentrations, however, the addition of magnesium to the culture medium is not advantageous for the growth of microorganisms. Consequently, the magnesium concentration in the culture medium is typically less than about 10 g / L, preferably less than about 5 g / L, and more preferably less than about 3 g / L. Furthermore, in some cases it may be desirable to allow the culture medium to become depleted of a magnesium source during culture.

[00167] In some embodiments, the culture medium may also include a biologically acceptable chelating agent, such as trisodium citrate dihydrate. In such a case, the concentration of a chelating agent in the culture medium is greater than about 0.2 g / L, preferably greater than about 0.5 g / L, and more preferably greater than about 1 g / L. Beyond certain concentrations, however, the addition of a chelating agent to the culture medium is not advantageous for the growth of microorganisms. Consequently, the concentration of a chelating agent in the culture medium is typically less than about 10 g / L, preferably less than about 5 g / L, and more preferably less than about 2 g / L.

[00168] The culture medium may also initially include a biologically acceptable acid or base to maintain the desired pH of the culture medium. Biologically acceptable acids include, but are not limited to Petition 870200018837, dated 07 / 02 / 2020, page 89 / 146 / 105 a, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and mixtures thereof. Biologically acceptable bases include, without limitation, ammonium hydroxide, sodium hydroxide, potassium hydroxide and mixtures thereof. In some embodiments, the base used is ammonium hydroxide.

[00169] The culture medium may also include a biologically acceptable calcium source, including, but not limited to, calcium chloride. Typically, the concentration of the calcium source, such as calcium chloride dihydrate, in the culture medium is in the range of about 5 mg / L to about 2,000 mg / L, preferably in the range of about 20 mg / L to about 1,000 mg / L, and more preferably in the range of about 50 mg / L to about 500 mg / L.

[00170] The culture medium may also include sodium chloride. Typically, the concentration of sodium chloride in the culture medium is within the range of about 0.1 g / L to about 5 g / L, preferably within the range of about 1 g / L to about 4 g / L, and more preferably in the range of about 2 g / L to about 4 g / L.

[00171] In some embodiments, the culture medium may also include trace metals. Such trace metals may be added to the culture medium as a stock solution which, for convenience, may be prepared separately from the rest of the culture medium. Typically, the amount of such a trace metal solution added to the culture medium is greater than about 1 mL / L, preferably greater than about 5 mL / L, and more preferably greater than about 10 mL / L. Beyond certain concentrations, however, the addition of trace metals to the culture medium is not advantageous for the growth of microorganisms. Consequently, the amount of such a trace metal solution added to the culture medium is typically less than about 100 mL / L, preferably less than about 50 mL / L, and more preferably less than about 30 mL / L. It should be noted that, in addition to the addition of trace metals in a stock solution, the Petition 870200018837, dated 07 / 02 / 2020, page 90 / 146 / 105: Individual components can be added separately, each within the corresponding ranges independently of the component quantities dictated by the ranges above the trace metal solution.

[00172] Culture media may include other vitamins, such as pantothenate, biotin, calcium, pantothenate, inositol, pyridoxine-HCl, and thiamine-HCl. Such vitamins may be added to the culture medium as a stock solution which, for convenience, may be prepared separately from the rest of the culture medium. Beyond certain concentrations, however, the addition of vitamins to the culture medium is not advantageous for the growth of microorganisms.

[00173] The fermentation methods described herein can be carried out in conventional culture modes, which include, but are not limited to, batch, fed-batch, cell recycling, continuous, and semi-continuous. In some embodiments, fermentation is carried out in fed-batch mode. In such a case, some of the medium components are depleted during the culture, including pantothenate during the production stage of fermentation. In some embodiments, the culture may be supplemented with relatively high concentrations of such components at the beginning, for example, of the production stage, so that growth and / or steviol glycoside production is supported for a period of time before additions are necessary. Preferred ranges of these components are maintained throughout the culture, with additions being made as levels are depleted by the culture.The levels of components in the culture medium can be monitored, for example, by sampling the culture medium periodically and assaying for concentrations. Alternatively, once a standard culture procedure has been developed, additions can be made at timed intervals that correspond to known levels at particular times throughout the culture. As will be recognized by those skilled in the art, the rate of nutrient consumption increases during the... Petition 870200018837, dated 07 / 02 / 2020, page 91 / 146 / 105 culture as the cell density of the medium increases. Furthermore, to avoid the introduction of foreign microorganisms into the culture medium, the addition is carried out using aseptic addition methods, as is known in the art. Additionally, a small amount of antifoaming agent may be added during the culture.

[00174] The temperature of the culture medium can be any temperature suitable for the growth of the genetically modified cells and / or the production of steviol glycoside. For example, before inoculation of the culture medium with an inoculum, the culture medium can be brought to a temperature in the range of about 20°C to about 45°C, preferably in the range of about 25°C to about 40°C, and more preferably in the range of about 28°C to about 32°C.

[00175] The pH of the culture medium can be controlled by adding acid or base to the culture medium. In such cases, when ammonia is used to control the pH, it also conveniently serves as a nitrogen source in the culture medium. Preferably, the pH is maintained from about 3.0 to about 8.0, more preferably from about 3.5 to about 7.0, and most preferably from about 4.0 to about 6.5.

[00176] In some embodiments, the concentration of the carbon source, such as the glucose concentration, in the culture medium is monitored during culture. The glucose concentration of the culture medium can be monitored using known techniques, such as, for example, the use of the glucose oxidase enzyme assay or high-performance liquid chromatography, which can be used to monitor the glucose concentration in the supernatant, for example, a cell-free component of the culture medium. As previously established, the concentration of the carbon source must be maintained below the level at which cell growth inhibition occurs. Although such a concentration may vary from organism to organism, for glucose as a carbon source, cell growth inhibition occurs at concentrations Petition 870200018837, dated 07 / 02 / 2020, p. 92 / 146 / 105 of glucose greater than about 60 g / L, and can be determined immediately by assay. Consequently, when glucose is used as a carbon source, glucose is preferably fed to the fermenter and kept below detection limits. Alternatively, the glucose concentration in the culture medium is maintained in the range of about 1 g / L to about 100 g / L, more preferably in the range of about 2 g / L to about 50 g / L, and even more preferably in the range of about 5 g / L to about 20 g / L. Although the carbon source concentration can be maintained within desired levels by adding, for example, a substantially pure glucose solution, it is acceptable, and may be preferred, to maintain the carbon source concentration of the culture medium by adding aliquots of the original culture medium.The use of aliquots of the original culture medium may be desirable because the concentrations of other nutrients in the medium (e.g., nitrogen and phosphate sources) can be maintained simultaneously. Similarly, trace metal concentrations can be maintained in the culture medium by adding aliquots of the trace metal solution.

[00177] Other suitable fermentation means and methods are described, for example, in WO 2016 / 196321. 6.9 Fermentation Compositions

[00178] In another aspect, fermentation compositions comprising a genetically modified host cell described herein and steviol glycosides produced from genetically modified host cells are provided herein. The fermentation compositions may further comprise a medium. In certain embodiments, the fermentation compositions comprise a genetically modified host cell and further comprise RebA, RebD, and RebM. In certain embodiments, the fermentation compositions provided herein comprise RebM as a major component of the Petition 870200018837, dated 07 / 02 / 2020, page 93 / 146 / 105 steviol glycosides produced from the genetically modified host cell. In certain embodiments, the fermentation compositions comprise RebA, RebD, and RebM in a ratio of at least 1:7:50. In certain embodiments, the fermentation compositions comprise RebA, RebD, and RebM in a ratio of at least 1:7:50 to 1:100:1000. In certain embodiments, the fermentation compositions comprise a ratio of at least 1:7:50 to 1:200:2000. In certain embodiments, the ratio of RebA, RebD, and RebM is based on the total content of steviol glycosides that are associated with the genetically modified host cell and the medium. In certain embodiments, the ratio of RebA, RebD, and RebM is based on the total steviol glycoside content in the medium. In certain embodiments, the ratio of RebA, RebD, and RebM is based on the total steviol glycoside content that is associated with the genetically modified host cell.

[00180] In certain embodiments, the fermentation compositions presented here contain RebM2 at an undetectable level. In certain embodiments, the fermentation compositions presented here contain non-naturally occurring steviol glycosides at an undetectable level. In certain embodiments, the fermentation compositions provided here, when subjected to GC chromatography, do not produce a “steviol + 2 glucose” peak between a RebA peak and a RebB peak at a detectable level. 6.10 Recovery of Steviol Glycosides

[00181] Since steviol glycoside is produced by the host cell, it can be recovered or isolated for subsequent use using any suitable separation and purification methods known in the art. In some embodiments, an organic phase comprising steviol glycoside is separated from the fermentation by centrifugation. In other embodiments, an organic phase comprising steviol glycoside separates from the fermentation spontaneously. In other embodiments, an organic phase comprising steviol glycoside is separated from the fermentation. Petition 870200018837, dated 07 / 02 / 2020, pp. 94 / 146 / 105, regarding the addition of a demulsifier and / or a nucleating agent in the fermentation reaction. Illustrative examples of demulsifiers include flocculants and coagulants. Illustrative examples of nucleating agents include droplets of the steviol glycoside itself and organic solvents such as dodecane, isopropyl myristate, and methyl oleate.

[00182] The steviol glycoside produced in these cells may be present in the culture supernatant and / or associated with the host cells. In embodiments where the steviol glycoside is associated with the host cell, recovery of the steviol glycoside may comprise a method of permeabilization or lysis of the cells. Alternatively or simultaneously, the steviol glycoside in the culture medium may be recovered using a recovery process including, but not limited to, chromatography, extraction, solvent extraction, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization, and crystallization.

[00183] In some embodiments, steviol glycoside is separated from other products that may be present in the organic phase. In some embodiments, the separation is achieved using adsorption, distillation, gas-liquid extraction (extraction), liquid-liquid extraction (solvent extraction), vacuum extraction, evaporation, ultrafiltration, and standard chromatographic techniques. Other suitable fermentation media and methods are described in, for example, US 2016 / 0185813. 6.11 Methods for Making Genetically Modified Cells

[00184] Methods are also provided herein for producing a host cell that is genetically engineered to comprise one or more of the modifications described above, for example, one or more nucleic acids encoding kaureno oxidase from Pisum sativum, and / or enzymes of biosynthetic pathways, for example, for a steviol glycoside compound. The expression of a heterologous enzyme in a host cell can be achieved by introducing into the host cells Petition 870200018837, dated 07 / 02 / 2020, pp. 95 / 146 / 105: a nucleic acid comprising a nucleotide sequence that encodes the enzyme under the control of regulatory elements that allow expression in the host cell. In some embodiments, the nucleic acid is an extrachromosomal plasmid. In other embodiments, the nucleic acid is the chromosomal integration vector that can integrate the nucleotide sequence into the host cell chromosome.

[00185] The nucleic acids that encode these proteins can be introduced into the host cell by any method known to those skilled in the art without limitation (see, for example, Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1292-3; Cregg et al. (1985) Mol. Cell. Biol. 5:3376-3385; Goeddel et al. eds, 1990, Methods in Enzymology, vol. 185, Academic Press, Inc., CA; Krieger, 1990, Gene Transfer and Expression -- A Laboratory Manual, Stockton Press, NY; Sambrook et al., 1989, Molecular Cloning -- A Laboratory Manual, Cold Spring Harbor Laboratory, NY; Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY). Exemplary techniques include, but are not limited to, spheroplast transformation, electroporation, PEG-1000 mediated transformation, and lithium acetate or lithium chloride mediated transformation.

[00186] The number of copies of an enzyme in a host cell can be altered by modifying the transcription of the gene that encodes the enzyme. This can be achieved, for example, by modifying the copy number of the nucleotide sequence that encodes the enzyme (e.g., by using a higher or lower copy number expression vector comprising the nucleotide sequence, or by introducing additional copies of the nucleotide sequence into the host cell genome, or by deletion or breakage of the nucleotide sequence in the host cell genome), by changing the order of coding sequences in a polycistronic mRNA of an operon, or by breaking an operon in genes. Petition 870200018837, dated 07 / 02 / 2020, p. 96 / 146 / 105 individuals, each with its own control elements, either by increasing the resistance of the promoter or operator to which the nucleotide sequence is operably linked. Alternatively or additionally, the number of copies of an enzyme in a host cell can be altered by modifying the translation level of an mRNA encoding the enzyme.This can be achieved, for example, by modifying mRNA stability, modifying the ribosome binding site sequence, modifying the distance or sequence between the ribosome binding site and the start codon of the enzyme coding sequence, modifying the entire intercistronic region located “upstream of” or adjacent to the 5' side of the start codon of the enzyme coding region, stabilizing the 3' end of the mRNA transcript using pins and specialized sequences, modifying enzyme codon utilization, altering the expression of rare codon tRNAs used in enzyme biosynthesis, and / or increasing enzyme stability, such as via mutation of its coding sequence.

[00187] The activity of an enzyme in a host cell can be altered in several ways, including, but not limited to, expressing a modified form of the enzyme that exhibits increased or decreased solubility in the host cell, expressing an altered form of the enzyme that lacks a domain through which the enzyme's activity is inhibited, expressing a modified form of the enzyme that has a higher or lower Kcat or a lower or higher Km for the substrate, or expressing an altered form of the enzyme that is more or less affected by feedback or forward feed regulation by another molecule in the pathway.

[00188] In some embodiments, a nucleic acid used to genetically modify a host cell comprises one or more selectable markers useful for selecting transformed host cells and for placing selective pressure on the host cell. Petition 870200018837, dated 07 / 02 / 2020, pp. 97 / 146 / 105 to retain the foreign DNA.

[00189] In some embodiments, the selectable marker is an antibiotic resistance marker. Illustrative examples of antibiotic resistance markers include, but are not limited to, the BLAA, NAT1, PAT, AUR1-C, PDR4, SMR1, CAT, mouse dhfr, HPH, DSDA, KANR, and SH BLE genes. The E. coli BLA gene product confers resistance to beta-lactam antibiotics (e.g., cephalosporins, cephamycins and carbapenems (ertapenem), cefamandole, and narrow-spectrum cefoperazone) and to all anti-Gram-negative bacterial penicillins except temoccilin; the S. noursei NAT1 gene product confers resistance to nurseotricin; the S. PAT gene product confers resistance to phlebotomine penicillin.The viridochromogenes Tu94 gene confers resistance to bialophos; the Saccharomyces cerevisiae AUR1-C gene product confers resistance to Auerobasidine A (AbA); the PDR4 gene product confers resistance to cericulin; the SMR1 gene product confers resistance to sulfometuron methyl; the CAT gene product of transposon Tn9 confers resistance to chloramphenicol; the mouse dhfr gene product confers resistance to methotrexate; the Klebsiella pneumoniae HPH gene product confers resistance to hygromycin B; the E. coli DSDA gene product allows cells to grow on plates with Dserine as the sole nitrogen source; the KANR gene of transposon Tn903 confers resistance to G418; and the Streptoalloteichus hindustanus SH BLE gene product confers resistance to zeocin (bleomycin). In some embodiments, the antibiotic resistance marker is suppressed after the genetically modified host cell described here is isolated.

[00190] In some embodiments, the selectable marker evokes an auxotrophy (e.g., a nutritional auxotrophy) in the genetically modified microorganism. In such embodiments, a parent microorganism comprises a functional disruption in one or more gene products. Petition 870200018837, dated 07 / 02 / 2020, pp. 98 / 146 / 105, which function in an amino acid or nucleotide biosynthetic pathway and which, when non-functional, render a parent cell incapable of growing in media without supplementation with one or more nutrients. Such gene products include, but are not limited to, the genes HIS3, LEU2, LYS1, LYS2, MET15, TRP1, ADE2, and URA3 in yeast. The auxotrophic phenotype can then be restored by transforming the parent cell with an expression vector or a chromosomal integration construct that encodes a functional copy of the disrupted gene product, and the resulting genetically modified host cell can be selected based on the loss of the parent cell's auxotrophic phenotype. The use of the URA3, TRP1, and LYS2 genes as selectable markers has a marked advantage because both positive and negative selection are possible.Positive selection is achieved through auxotrophic complementation of URA3, TRP1, and LYS2 mutations, while negative selection is based on specific inhibitors, namely 5-fluoroortic acid (FOA), 5-fluoroanthranilic acid, and aminoadipic acid (aA), respectively, which prevent the growth of prototrophic strains but allow the growth of URA3, TRP1, and LYS2 mutants, respectively. In other modalities, the selectable marker may represent other non-lethal deficiencies or phenotypes that can be identified by a known selection method.

[00191] Described here are specific genes and proteins useful in the methods, compositions, and organisms of the description; however, it will be recognized that absolute identity to such genes is not required. For example, changes in a given gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be made and examined for activity. Typically, such changes comprise conservative mutations and silent mutations. Such modified or mutated polynucleotides and polypeptides can be selected for the expression of a functional enzyme using methods Petition 870200018837, dated 07 / 02 / 2020, pp. 99 / 146 / 105 known in the technical field.

[00192] Due to the inherent degeneracy of the genetic code, other polynucleotides that encode substantially the same functionally equivalent polypeptides can also be used to clone and express the polynucleotides that encode these enzymes.

[00193] As will be understood by those skilled in the art, it can be advantageous to modify a coding sequence to improve its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically utilize a subset of these codons. The codons that are used most frequently in a species are called optimal codons, and those not used very frequently are classified as rare or low-consumption codons. Codons can be substituted to reflect the host's preferred codon usage, in a process sometimes called "codon optimization" or "species codon drift control". Codon optimization for other host cells can be readily determined using codon usage tables or can be performed using commercially available software, such as CodonOp (www.idtdna.com / CodonOptfrom) from Integrated DNA Technologies.

[00194] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et al., 1989, Nucl Acids Res. 17: 477-508) can be prepared, for example, to increase the translation rate or produce recombinant RNA transcripts having desirable properties, such as longer half-life, compared to transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, while Petition 870200018837, dated 07 / 02 / 2020, p. 100 / 146 / 105 Insects and E. coli commonly use UAA as the stop codon (Dalphin et al., 1996, Nucl Acids Res. 24: 216-8).

[00195] Those skilled in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA molecules differing in their nucleotide sequences can be used to encode a particular enzyme of the description. The native DNA sequence encoding the biosynthetic enzymes described above is referred to herein merely to illustrate one embodiment of the description, and the description includes DNA molecules of any sequence encoding the amino acid sequences of the polypeptides and proteins of the enzymes used in the methods of the invention. Similarly, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant impairment of a desired activity.The description includes such polypeptides with different amino acid sequences than the specific proteins described herein, provided that the modified or variant polypeptides have the enzymatic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the description.

[00196] In addition, enzyme homologs useful for the compositions and methods provided herein are covered by the description. In some embodiments, two proteins (or a region of proteins) are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%), 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be inserted into one or both of a first and a second sequence). Petition 870200018837, dated 07 / 02 / 2020, page 101 / 146 / 105 amino acids or nucleic acid for optimal alignment and non-homologous sequences may be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30% >, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid or nucleotide residues in corresponding amino acid positions or nucleotide positions are then compared when a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position (as here used amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each interval, which needs to be introduced for optimal alignment of the two sequences.

[00197] When “homologous” is used with reference to proteins or peptides, it is recognized that the positions of residues that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology can be adjusted upwards to correct for the conservative nature of the substitution. Means of doing this Petition 870200018837, dated 07 / 02 / 2020, p. 102 / 146 / 105 adjustments are well known to those versed in the technique (see, for example, Pearson WR, 1994, Methods in Mol Biol 25: 365-89).

[00198] The following six groups each contain amino acids that are conservative substitutions for each other: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Alanine (A), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[00199] Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. A typical algorithm used to compare a molecule sequence with a database containing a large number of sequences from different organisms is the BLAST computer program. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences.

[00200] Furthermore, any of the genes encoding the above enzymes (or any others mentioned herein (or any of the regulatory elements that control or modulate their expression)) can be optimized by genetic / protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those skilled in the art. Such action allows those skilled in the art to optimize the enzymes for expression and activity in yeast.

[00201] Furthermore, genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed to modulate this pathway. A variety of organisms could serve as sources for these enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Petition 870200018837, dated 07 / 02 / 2020, pp. 103 / 146 / 105 Sources of genes from anaerobic fungi include, but are not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp. Sources of useful prokaryotic enzymes include, but are not limited to, Escherichia. Yamadazyma spp., including Y. spp. stipitis, Torulaspora pretoriensis, Issatchenkia orientalis, Schizosaccharomyces spp., including S. pombe, Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustilago spp. coli, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., and Salmonella spp.

[00202] Techniques known to those skilled in the art may be suitable for identifying additional homologous genes and homologous enzymes. Generally, analogous genes and / or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable for identifying analogous genes and analogous enzymes. For example, to identify homologous or analogous UDP glycosyltransferases, PTA, or any genes of biosynthetic pathways, proteins, or enzymes, techniques may include, but are not limited to, cloning a gene by PCR using primers based on a published sequence of a gene / enzyme of interest or by degenerate PCR using degenerate primers designed to amplify a conserved region between a gene of interest.Furthermore, someone skilled in the technique can use techniques to identify homologous or analogous genes, proteins, or enzymes with homology or functional similarity. Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity (e.g., as described here or in Kiritani, K., Branched-Chain Amino Acids Methods Enzymology, 1970), then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as degradation. Petition 870200018837, dated 07 / 02 / 2020, pp. 104 / 146 / 105 Edman, design of PCR primers for a probable nucleic acid sequence, amplification of said DNA sequence via PCR, and cloning of said nucleic acid sequence. To identify homologous or similar genes and / or homologous or similar enzymes, analog genes and / or analog enzymes or proteins, techniques also include comparing data relating to a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC. The candidate gene or enzyme can be identified within the aforementioned databases according to the teachings herein. 7. EXAMPLES Example 1: Generation of a base yeast strain capable of high flux for farnesyl pyrophosphate (FPP) and the isoprenoid farnesene.

[00203] The farnesene-producing strain was created from a wild-type Saccharomyces cerevisiae strain (CEN.PK2) by expressing mevalonate pathway genes (Figure 1D) under the control of GAL1 or GAL10 promoters. This strain comprised the following mevalonate pathway genes integrated from S. cerevisiae: acetylCoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, and IPP:DMAPP isomerase. All genes described here were codon-tagged using publicly available or other appropriate algorithms. In addition, the strain contained six copies of farnesene synthase from Artemisinin annua, also under the control of either GAL1 or GAL10 promoters.The strain also contained a deletion of the GAL80 gene and an additional copy of GAL4 under the GAL4oc promoter, wherein the coding sequence of the Saccharomyces cerevisiae GAL4 gene is under the regulatory control of an “operational constitutive” version of its native promoter (PGAL4oc; see, for example, Griggs & Johnston (1991) PNAS 88(19):8597-8601). Finally, the ERG9 gene, which encodes squalene synthase, is downregulated by... Petition 870200018837, dated 07 / 02 / 2020, page 105 / 146 / 105 replacement of the native promoter with the yeast MET3 gene promoter (Westfall et al. PNAS 2012). Example 2. Generation of a base yeast strain capable of high flux for Rebaudioside A (RebA).

[00204] Figure 1B shows an exemplary biosynthetic pathway from FPP to steviol. Figure 2 shows an exemplary biosynthetic pathway from steviol to the glycoside RebM. To convert the farnesene base strain described above to have high flux to the C-20 isoprenoid kaurene, six copies of a geranylgeranylpyrophosphate synthase (GGPPs) were integrated into the genome, followed by four copies each of a copalyl diphosphate synthase and kaurene synthase. Table 1 lists all the genes and promoters used to convert FPP to RebA. At this point, the six copies of farnesene synthase were removed from the strain. Once the new strain was confirmed to make ent-kaurene, the remaining genes for the conversion of ent-kaurene to RebA were inserted into the genome. Each gene was integrated with a single copy, except for the Sr.KAH enzyme which had two copies (Table 1). The strain containing all the genes described in Table 1 produced primarily RebA.The enzyme UGT91D_like3 has some low activity to convert RebA to Rebaudioside D (RebD). It was measured that a single copy of 91D_like3 is capable of converting approximately (3%) of RebA in the RebD strain in vivo in the yeast strain described above (Figure 3 and Table 2). UGT76G1 can then convert RebD to the final product Rebaudioside M (RebM). Example 3. Screening of kaorene oxidase (KO) enzymes to convert kaorene to kaurenoic acid more efficiently.

[00205] To generate a strain with high flux for RebM, the strain described in Example 2 was transformed with a single copy of the UGT40087 gene (as described in Example 8 and in the tables and figures in PCT Application AM-7400 PCT, which is attached as an appendix here) under the GAL1 promoter. This strain primarily produces RebM. To screen alleles of Petition 870200018837, dated 07 / 02 / 2020, pages 106 / 146 / 105 Due to different KO factors for the conversion of kaurene to kaurenoic acid in vivo, the Stevia rebuaudiana KO gene in this RebM strain was removed and replaced with a resting block containing only the GAL1 promoter and terminator, with an F-CphI restriction sequence between the promoter and the terminator (Figure 3). This screening strain now lacks any KO enzyme and only makes ent-kaurene.

[00206] Thirteen KO enzymes (Table 1) obtained from the literature were optimized at the codon for optimal expression in S. cerevisiae and synthesized with 60 bp of sequence homologous to the PGAL1 promoter and yeast flanking the F-CphI sequences in the landing block described in Figure 3A. Each synthesized KO gene was individually tested, with a single copy, for the ability to convert ent-kaurenic acid to kaurenoic acid in vivo in the yeast strain described above. Yeast was transformed with KO donor DNA and a plasmid containing the F-Cphl endonuclease to cut the DNA at the landing block. Correct integrations were verified by colony PCR using a reverse primer internal to the KO gene specific in each transformation and a universal forward primer at the GAL1 promoter end. Figure 3B shows the final genetic construct after correct F-Cphl cleavage and homologous recombination with KO DNA. Table 1. Kaurene oxidase enzymes tested in yeast for higher conversion of kaurene to kaurenoic acid. Species Abbreviation Uniprot Number SEQ ID NO Arabidopsis thaliana At Q93ZB2 SEQ ID NO:3 Cucurbita maxima Cm Q9FQY5 SEQ ID NO:4 Cucumis sativus Cs J7I3T1 SEQ ID NO:5 Gibberella fujikuroi Gf O94142 SEQ ID NO:6 Gibberella moniliformis Gm B6HY18 SEQ ID NO:7 Lactuca sativa Ls_1 B5MEX5 SEQ ID NO:8 Lactuca sativa Ls_2 B5MEX6 SEQ ID NO:9 Oryza sativa subsp. japonica Os Q5Z5R4 SEQ ID NO:10 Physcomitrella patens subsp. patens Pp A9TVB8 SEQ ID NO:11 Pisum Sativum Ps Q6XAF4 SEQ ID NO:1 Sphaceloma manihoticola Sm B5DBY4 SEQ ID NO:12 Stevia rebaudiana Sr Q4VCL5 SEQ ID NO:2 Zea mays Zm B4FYL7 SEQ ID NO:13 Petition 870200018837, dated 07 / 02 / 2020, p. 107 / 146 / 105

[00207] Figure 4 shows the results of the KO screening. It was observed that a KO enzyme (Ps.KO) from the plant Pisum sativum (garden peas) has an increased capacity (improved approximately 3.5x) to convert ent-kaurene to kaurenoic acid, compared to the KO enzyme from Stevia rebaudiana (Sr.KO) in this baseline strain. The optimized nucleic acid sequence of the Pisum sativum KO enzyme codon used for expression in yeast cells is shown as SEQ ID NO:15. Example 4. Generation of a high-flux RebM strain with improved conversion of ent-kaurene to kaurenoic acid.

[00208] The activity of Ps.KO was then tested against Sr.KO in a strain with very high flux for RebM. KO enzymes normally act in most plants to produce the plant hormone gibberellin. Gibberellin levels in plant cells are orders of magnitude lower than the RebM levels produced in yeast for industrial production and therefore KO enzymes are not expected to drive the high flux required to produce RebM for commercial manufacturing. Table 3 lists all genes and promoters contained in a strain with higher RebM flux than the strain used to initially select the KO enzymes (i.e., the “base strain” of KO). All genes in Table 3 were inserted into the yeast genome. The KO enzyme drives ent-kaurene through three rounds of subsequent oxidation to produce kaurenoic acid.The order of reactions and intermediates is: the first oxidation yields ent-kaurene to kaurenol (K-OL), the second oxidation yields kaurenol to kaurenal (K-AL), and the third oxidation yields kaurenal to kaurenoic acid (-acid) (Figure 1C). To obtain the maximum flux of entkaurene to RebM, the KO enzyme must completely oxidize ent-kaurene to K-acid. Incomplete conversion will waste carbon, reduce total RebM titrations, and produce intermediate compounds. Petition 870200018837, dated 07 / 02 / 2020, p. 108 / 146 / 105 potentially toxic. The data in Figure 5 show that in a strain with high carbon flux to RebM, the Sr.KO allele accumulates significant amounts of the upstream intermediates ent-kaurene, kaurenol (K-OL), kaurenal (K-AL), while the Ps.KO enzyme shows a significantly reduced accumulation of these intermediates.

[00209] Figure 6 shows that Ps.KO increases the amount of RebM made in the cell, due to the greater amount of kaurenoic acid produced with Ps.KO compared to Sr.KO. In the high-flux ResbM strain, there is a 16% increase in RebM titers in a strain with Ps.KO compared to an identical strain with Sr.KO. This higher ResbM titer is due to more kaurenoic acid being produced in the Ps.KO strain. Example 5. Yeast culture conditions

[00210] Yeast colonies verified to contain the expected kaurene oxidase gene were harvested in 96-well microtiter plates containing Bird Seed Media (BSM, originally described by van Hoek et al., Biotechnology and Bioengineering 68 (5), 2000, pp. 517-523) with 20 g / L sucrose and 37.5 g / L ammonium sulfate. Cells were cultured at 30°C in a high-capacity microtiter plate incubator, shaking at 1000 RPM and 80% humidity for 3 days until the cultures reached carbon exhaustion. Saturated growth cultures were subcultured onto fresh plates containing BSM with 40 g / L sucrose and 150 g / L ammonium sulfate, taking 14.4 μL of the saturated cultures and diluting them in 360 μL of fresh medium. The cells in the production medium were cultured at 30°C in a high-capacity microtiter plate shaker at 1000 RPM and 80% humidity for an additional 3 days before extraction and analysis.After completion, the complete cell broth is diluted with 360 μL of 100% ethanol, sealed with a blade seal, and stirred at 1250 rpm for 30 minutes to extract the rebaudiosides. 490 μL of. Petition 870200018837, dated 07 / 02 / 2020, page 109 / 146 / 105 water:ethanol 50:50 is added to a new 1.1 mL test plate and 10 μE of the culture / ethanol mixture is added to the test plate. The mixture is centrifuged to subject any solids to granulation, and 400 μg of solution is transferred to a new 1.1 mL plate and analyzed by LC-MS. Example 6. Analytical Methods Steviol and Steviol Glycoside Mass Spectrometer Detection:

[00211] Samples are analyzed by LCMS mass spectrometer (AB QTrap 4000) using a Sigma Ascentis Express ES-C18 peptide (5 cm, 2.1 mm, 2.7 μm; part #53301-U) with the following gradient: Time (min.) %B 1 0 25 2 2.50 25 3 10.00 60 4 10.50 100 5 12.50 100 6 12.51 25 Mobile Phase A: Water + Formic acid 0.1% Mobile Phase B: Acetonitrile + Formic acid 0.1% Flow rate: 250uL / min.

[00212] The mass spectrometer was operated in negative ion multiple reaction monitoring mode. Each rebaudioside isomer was identified by retention time, determined from an authentic standard, and MRM transition: RT (min.) Compound Q1 Mass (Da) Q3 Mass (Da) 10.5 Steviol 317,328 317,300 8.2 Steviolmonoside 479,354 317,200 7.9 19-glucoside 479,369 317,100 7.4 Steviolbioside 641,451 479,300 6.9 Rubusoside 641,491 479,400 7.3 RebB 803,612 641,500 6.2 Stevioside 803,550 641,400 3.3 RebE 965,441 479,400 6.2 RebA 965,441 803,700 3.8 RebD 1127.140 803.500 4.5 RebM 1289.540 803.400 2.4 RebM2 1289.540 641.400 Kaorene quantification:

[00213] The titration of ent-kaurene in culture broth is measured using a gas chromatograph equipped with a thermal mass oven. Petition 870200018837, dated 07 / 02 / 2020, page 110 / 146 / 105 limited and a flame ionization detector. Broth samples are extracted using equal parts broth and methanol and stirred in a sealed container for 30 minutes to recover ent-kaurene from the cells. A 240 µL aliquot of the broth:methanol solution is then diluted with 1 mL of ethyl acetate, sealed, and stirred for an additional 30 minutes to extract entkaurene in the organic phase. The organic phase is diluted as appropriate to fall within the linear range of the assay and aliquoted into a sample vial. Samples are injected at the appropriate split ratio to fall within the linear range.Sample separation occurs on an Agilent DB-1MS LTM II column, with hydrogen as the carrier gas in constant pressure mode, using the temperature gradient: (1) initial temperature 150°C for 0 minutes, (2) temperature increase of 25°C / minute to a temperature of 230°C, (3) temperature increase of 1800°C / minute to a temperature of 320°C and held for 1 minute. External calibration, using an authentic ent-kaurene standard, is used to determine the amount of entkaurene. Kaurenoic acid, Kaurenol and Kaurenol Quantification:

[00214] The titration of kaurenoic acid, kaurenol, and kaurenal in culture broth is determined using a high-performance liquid chromatograph equipped with a variable wavelength detector. A broth sample (100 μL) is diluted in 300 μL of ethanol and stirred in a sealed container for 30 min. 200 μL of water is added to the broth:ethanol mixture, mixed, and centrifuged. An aliquot of the resulting solution (avoiding the cell bead) is transferred to a sample vial and analyzed using HPLC. Sample separation occurs in an Aglient Eclipse Plus C18 USP L1 (4.6 mm x 50 mm x 1.8 μm) with the following solvents: • Mobile Phase A: 0.1% Formic Acid in water (v / v) • Mobile Phase B: 0.1% Formic Acid in acetonitrile (v / v) with the following solvent gradient: Petition 870200018837, dated 07 / 02 / 2020, pages 111 / 146 / 105 Time (min.) Channel A (%) Channel B (%) 0.00 50 50 2.50 50 50 5.75 0 100 8.00 0 100 8.10 50 50 9.0 50 50

[00215] The analytes are detected using UV absorbance at 200 nm, and quantified with external calibration with response factors relative to a Steviol standard. Method for quantifying rebaudioside M used for the data shown in Figure 6:

[00216] Rebaudioside M titrations in broth are determined using a high-performance liquid chromatograph equipped with a triple quadrupole mass spectrometer. A broth sample is divided into an Eppendorf tube diluted between 200 and 800 times in ethanol:water 50:50, mixed for 20 minutes, centrifuged to subject cells and debris to granule formation, and an aliquot of the supernatant is transferred to a sample vial for analysis. Samples are run in flow injection mode where analytes are quantified based on the signal intensity of MRM transitions. The mobile phase is 40% water + 0.1% formic acid and 60% acetonitrile + 0.1% formic acid with a flow rate of 1.1 mL / min. The concentration of Rebaudioside M is determined by its normalized response to that of an internal standard (Rebaudioside N). Table 2. Genes, promoters, and amino acid sequences of the enzymes used to convert FPP to RebA.__________________________ Enzyme Name Accession Number or Sequence ID Promoter Btrispora.GGPPS AFC92798.1 PGAL1 ent-CDPS_Os Q5MQ85.11 PGAL1 KS_Pg ADB55711.1 PGAL1 Sr.KO AAQ63464.1 PGAL1 Sr.KAH SEQ ID:10 PGAL1 Aa.CPR ABC47946.1 PGAL3 UGT85C2 AAR06916.1 PGAL1 UGT74G1 AAR06920.1 PGAL10 UGT91D_like3 SEQ ID NO:7 PGAL1 UGT76G1 AAR06912.1 PGAL10 The first 65 amino acids were removed and replaced with methionine. Table 3. Genes, promoters, copy number and sequences of Petition 870200018837, dated 07 / 02 / 2020, pp. 112 / 146 100 / 105 amino acids of enzymes in a RebM-producing strain. Enzyme Name Gene Copy Number Accession Number or Sequence ID Promoter Btrispora.GGPPS 6 AFC92798.1 PGAL1 ent- CDPS_Os 4 Q5MQ85.11 PGAL1 KS_Pg 4 ADB55711.1 PGAL1 Sr.KO 1 AAQ63464.1 PGAL1 Sr.KAH 3 SEQ ID:10 PGAL1 ATR2 1 NP_194750.1 PGAL3 UGT85C2 2 AAR06916.1 PGAL1 or PGAL10 UGT74G1 2 AAR06920.1 PGAL1 or PGAL10 UGT91D_like3 2 SEQ ID NO:7 PGAL1 or PGAL10 UGT76G1 4 AAR06912. 1 PGAL1 or PGAL10 The first 65 amino acids were removed and replaced with methionine.

[00217] All publications, patents and patent applications cited in this descriptive report are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the invention above has been described in some detail by way of illustration and example for the sake of clarity of understanding, it will be readily apparent to those ordinarily skilled in the art in the light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. Petition 870200018837, dated 07 / 02 / 2020, pp. 113 / 146 101 / 105 SEQUENCE LISTING >Seq_ID_1 MDTLTLSLGFLSLFLFLFLLKRSTHKHSKLSHVPVVPGLPVIGNLLQLKEKKPHKTFTKMAQKYGPIFSIKAG SSKIIVLNTAHLAKEAMVTRYSSISKRKLSTALTILTSDKCMVAMSDYNDFHKMVKKHILASVLGANAQKRLR FHREVMMENMSSKFNEHVKTLSDSAVDFRKIFVSELFGLALKQALGSDIESIYVEGLTATLSREDLYNTLVVD FMEGAIEVDWRDFFPYLKWIPNKSFEKKIRRVDRQRKIIMKALINEQKKRLTSGKELDCYYDYLVSEAKEVTE EQMIMLLWEPIIETSDTTLVTTEWAMYELAKDKNRQDRLYEELLNVCGHEKVTDEELSKLPYLGAVFHETLRK HSPVPIVPLRYVDEDTELGGYHIPAGSEIAINIYGCNMDSNLWENPDQWIPERFLDEKYAQADLYKTMAFGGG KRVCAGSLQAMLIACTAIGRLVQEFEWELGHGEEENVDTMGLTTHRLHPLQVKLKPRNRIY >Seq_ID_2 MDAVTGLLTVPATAITIGGTAVALAVALIFWYLKSYTSARRSQSNHLPRVPEVPGVPLLGNLLQLKEKKPYMT FTRWAATYGPIYSIKTGATSMVVVSSNEIAKEALVTRFQSISTRNLSKALKVLTADKTMVAMSDYDDYHKTVK RHILTAVLGPNAQKKHRIHRDIMMDNISTQLHEFVKNNPEQEEVDLRKIFQSELFGLAMRQALGKDVESLYVE DLKITMNRDEIFQVLVVDPMMGAIDVDWRDFFPYLKWVPNKKFENTIQQMYIRREAVMKSLIKENKKRIASGE KLNSYIDYLLSEAQTLTDQQLLMSLWEPIIESSDTTMVTTEWAMYELAKNPKLQDRLYRDIKSVCGSEKITEE HLSQLPYITAIFHETLRRHSPVPIIPLRHVHEDTVLGGYHVPAGTELAVNIYGCNMDKNVWENPEEWNPERFM KENETIDFQKTMAFGGGKRVCAGSLQALLTASIGIGRMVQEFEWKLKDMTQEEVNTIGLTTQMLRPLRAIIKP RI >Seq_ID_3 MAFFSMISILLGFVISSFIFIFFFKKLLSFSRKNMSEVSTLPSVPVVPGFPVIGNLLQLKEKKPHKTFTRWSE IYGPIYSIKMGSSSLIVLNSTETAKEAMVTRFSSISTRKLSNALTVLTCDKSMVATSDYDDFHKLVKRCLLNG LLGANAQKRKRHYRDALIENVSSKLHAHARDHPQEPVNFRAIFEHELFGVALKQAFGKDVESIYVKELGVTLS KDEIFKVLVHDMMEGAIDVDWRDFFPYLKWIPNKSFEARIQQKHKRRLAVMNALIQDRLKQNGSESDDDCYLN FLMSEAKTLTKEQIAILVWETIIETADTTLVTTEWAIYELAKHPSVQDRLCKEIQNVCGGEKFKEEQLSQVPY LNGVFHETLRKYSPAPLVPIRYAHEDTQIGGYHVPAGSEIAINIYGCNMDKKRWERPEDWWPERFLDDGKYET SDLHKTMAFGAGKRVCAGALQASLMAGIAIGRLVQEFEWKLRDGEEENVDTYGLTSQKLYPLMAIINPRRS >Seq_ID_4 MAVATDPLGCMQKLVQMLQAPPYVAAAVQSSALLLTFFIGDWRKRRRSPLPLLPAIPGIPVLGNLLQLKEKKP HKTFAQWSETYGPIYSIKAGASTVIVLNSSDLAKEAMVTRYSSISSRKLSKALTILTADKCMVAMSDYNDFHK LVKRYILANVLGANAQKRLRQRRDTMIDNISRELFACVKDSSSESVNFRKIFESELFGLALKETFGRDMESLY VDGLGTTLLREDLFRTLVIDPMEGAIEVDWRDFFPYLRWIPNKGVEDRIRKMDFRRRVTMKSLMEEKKKQIAA GEDLNCYSEFLLSEAKSLTEEQISMLLWEIIIETSDTTLVVTEWAMYELAQNPKRQERLYQHIQSVCGSAKIT EENLSQLPYLTAVFHETLRKYSPVSIVPLRYAHEDTQLGGYFIPAGSEVAVNIYACNMDKKQWESPEEWKPER FLDESYDPMDLYKTMAFGGGKRVCAGAPKAMLIACTTLGRLVQGFTWKLREGEEDKVDTLGLTARKLQPLHIV AKPRIN >Seq_ID_5 MAVVTDPLASMQLLANTIPAPPYAAAAVLGGVSLVLSVFFVADCRKKKRRNFLPPPVPAVPGVPVLGNLLQLKEK KPHKTFARWAETYGAVYSIRTGASTVIVLNTTEVAKEAMVTRYGISSRKLSKALTITADKCMVAMSDYNEF HKMVKRYILANVLGANAQKKHRQRRDAMIENISRELFAHVKEFPLDTVNFRKIFEAELFRLALKETLGKDIES IYVDGLGTTLPREDLFRILVIDPMEGAIEVDWRDFFPYLRWIPNKRVENKIRNMDFRRRRMTMKKLMEEPKKRI AAGEETYCYADFLLSEAKTLTEDQISMLLWETIIETSDTTLVVTEWAMYELSKDPRRQDYLYQQIQSVCGSAT LTEENLSQLPYLTAIFHETLRKHSPPVVPLRYAHEDTQLGGYFVPAGSEIAVNIYACNMDKDHWESPEEWKP ERFLDDKYDPMLHKTMAFGGGKRVCAGALKAMLIACTTIGRMVQEFEWKLREGEEEKVDTLGLTARKLQPLH VVIKPRNN >Seq_ID_6 MSKSNSMNSTSHETLFQQLVLGLDRMPLMDVHWLIYVAFGAWLCSYVIHVLSSSSTVKVPVVGYRSVFEPTWL LRLRFVWEGGSIIGQGYNKFKDSIFQVRKLGTDIVIIPPNYIDEVRKLSQDKTRSVEPFINDFAGQYTRGMVF LQSDLQNRVIQQRLTPKLVSLTKVMKEELDYALTKEMPDMKNDEWVEVDISSIMVRLISRISARVFLGPEHCR NQEWLTTTAEYSESLFITGFILRVVPHILRPFIAPLLPSYRTLLRNVSSGRRVIGDIIRSQQGDGNEDILSWM RDAATGEEKQIDNIAQRMLILSLASIHTTAMTMTHAMYDLCACPEYIEPLRDEVKSVVGASGWDKTALNRFHK LDSFLKESQRFNPVFLLTFNRIYHQSMTLSDGTNIPSGTRIAVPSHAMLQDSAHVPGPTPPTEFDGFRYSKIR SDSNYAQKYLFSMTDSSNMAFGYGKYACPGRFYASNEMKLTLAILLLQFEFKLPDGKGRPRNITIDSDMIPDP RARLCVRKRSLRDE >Seq_ID_7 MNKFNSMNNTINETLLRQLVSGLDEIPLMDIHWLIYVAFGAWLCSYVIHLLSSPSTVNVPVGYRSVFEPTWF LRLRFVWEGGSIISQGYSKFKDSIFQVRKLGTDIVIIPPNYIDEVRKLSQDKTRSVEPFAGDYTRGMVF LQSDLQNRVIQQRLTPKLVSLTKVMKEELDYALTKGMPDMKDDEWVEDIASIMVRLISARVFLGPEHCR NQEWLTTTAEYSESLFMTGFILRVVPHILRPFVAPLLPSYRTLLRSVSSGRKVIGDIIRSQQGSENEDILSWM VEAATGEEKQVDNIAQRMLILSLASIHTTAMTMTHAMYDLCARPEYTKPLREEVKGVVGASGWDKTALNRLHK LDSFLKESQRFNPVFLLTFNRIYHQPMTLSDGTNLPSGTRIAVPSHAMLQDSAHVPGPAPPTDFDGFRYSKIR SDSNYAQKYLFSMTDSSNMAFGYGKYACPGRFYASNEMKLTLAILLLQFEFKLPDGKGRPRNITIDSDMVPDP Petition 870200018837, de 07 / 02 / 2020, pág. 114 / 146 102 / 105 RARLCVRKRSLREE >Seq_ID_8 MDLQTMAPMGSAAIAIGGPAVAVAGGISLLFLKSFLSQQPGNPNHLPSVPAVPGVPLLGNLLELKEKKPYKTF TKWAETYGPIYSIKTGATSMVVVNSNQLAKEAMVTRFDSISTRKLSKALQILTADKTMVAMSDYDDYHKTVKR NLLTSILGPAAQKRHRAHRDAMGDNLSRQLHALALNSPQEAINFRQIFQSELFTLAFKQTFGRDIESIFVGDL GTTMTREEMFQILVVDPMMGAIDVDWRDFFPYLKWIPNAKLEEKIEQMYIRRKAVMKAVIQEHRKRIDSGENL DSYIDFLLAEAQPLTEKQLLMSLWEPIIETSDTTMVTTEWAMYELSKHPNKQQRLYNEIRNICGSEKITEEKL CKMPYLSAVFHETLRVHSPVSIIPLRYVHENTELGGYHVPAGTELAVNIYGCNMEREIWENPEEWSPERFLAE NEPVNLQKTMAFGAGKRVCAGAMQAMLLACVGIGRMVQEFEWRLKDDVEEDVNTLGLTTQRLNPMLAVIKPRN >Seq_ID_9 MDGVIDMQTIPLRTAIAIGGTAVALVVALYFWFLRSYASPSHHSNHLPPVPEVPGVPVLGNLLQLKEKKPYMT FTKWAEMYGPIYSIRTGATSMVVVSSNEIAKEVVVTRFPSISTRKLSYALKVLTEDKSMVAMSDYHDYHKTVK RHILTAVLGPNAQKKFRAHRDTMMENVSNELHAFFEKNPNQEVNLRKIFQSQLFGLAMKQALGKDVESIYVKD LETTMKREEIFEVLVVDPMMGAIEVDWRDFFPYLKWVPNKSFENIIHRMYTRREAVMKALIQEHKKRIASGEN LNSYIDYLLSEAQTLTDKQLLMSLWEPIIESSDTTMVTTEWAMYELAKNPNMQDRLYEEIQSVCGSEKITEEN LSQLPYLYAVFQETLRKHCPVPIMPLRYVHENTVLGGYHVPAGTEVAINIYGCNMDKKVWENPEEWNPERFLS EKESMDLYKTMAFGGGKRVCAGSLQAMVISCIGIGRLVQDFEWKLKDDAEEDVNTLGLTTQKLHPLLALINPR KS >Seq_ID_10 MEAFVPGGAGAAAAAVGGFVAAAALAERAGVIAPRKRPNAPPAVPGLPIIGNLHQLKEKKPHQTFAKWAEIYG PIYTIRTGASSVVLNSTEVAKEAMVAKFSSISTRKLSKALTVLTRDKSMVATSDYCDFHKMVKRYVMSMLG TSAQKQFRDIRDMMIHNMLSTFHKLVKDDPHAPLIFRDVFKDELFRLSMIQSLGEDVSSVYVDEFGRDISKEE IYNATVTDMMMCAIEVDWRDFFPYLSWVPNKSFETRVFTTETRRTAVMRALIKQQKERIVRGEAKTCYLDFLL AENTLTDEQLMMLVWEALIEAADTTLVTTEWAMYELAKNPDKQERLYQEIREVCGDETVTEEHLPRLPYLNAV FHETLRRHSPVPLIPPRFVHEDTKLAGYDVPAGTEMVINLYGCNMNRKEWESPEEWVPERFAGGRLEVADMYK TMAFGAGRRACAGSLQATHIACAAVARFVQEFGWRLREGDEEKVDTVQLTAYKLHPLHVHLTRRGRM >Seq_ID_11 MLETKVIAHHVSHSPCAAIPGGLPVLGNLLQLTEKKPHRTFTAWSKEHGPIFTIKVGSVPQAVVNNSEIAKEV LVTKFASISKRQMPMALRVLTRDKTMVAMSDYGEEHRMLKKLVMTNLGPTTQNKNRSLRDDALIGMIEGVLA ELKASPTSPKVVNVRDYVQRSLFPFALQQVFGYIPDQVEVLELGTCVSTWDMFDALVVAPLSAVINVDWRDFF PALRWIPNRSVEDLVRTVDFKRNSIMKALIRAQRMRLANLKEPPRCYADIALTEATHLTEKQLEMSLWEPIIE SADTTLVTSEWAMYEIAKNPDCQDRLYREIVSVAGTERMVTEDDLPNMPYLGAIIKETLRKYTPVPLIPSRFV EEDITLGGYDIPKGYQILVNLFAIANDPAVWSNPEKWDPERMLANKKVDMGFRDFSLMPFGAGKRMCAGITQA MFIIPMNVAALVQHCEWRLSPQEISNINNKIEDVVYLTTHKLSPLSCEATPRISHRLP >Seq_ID_12 MMDDTTSPYSTYHSVRSIRNQSAWALAPIAVFICYVVLRHNRKSVPAASAGSHSILEPLWLARLRFIRDSRFI IGQGYSKFKDTIFKVTKVGADIIVVAPKYVEEIRRLSRDTGRSVEPFIHDFAGELLGGLNFLESDLQTRVVQQ KLTPNLKTIVPVMEDEMHYALVSELDSCLDGSEHWTRVDMIHMLSRIVSRISARIFLGPKYCRNDLWLKTTAE YTENLFLTGTLLRFVPRMLQKWIAPLLPSFRQLQENRQAARKIISEILTDHQPEKHDETSDNGDPYPDILTLM FQAARGKEKDIEDIAQHTLLLSLSSIHTTALTMTQALYDLCAYPQYLDPVKHEIADTLQSEGSWSKAMLDKLH MMDSLLRESQRLSPVFLLTFNRILHTPLTLSNGIHLPKGTRIAAPSDAILNDPSLVPGPQPADTFDPFRYINH STGDAKKTKTNFQTTSLQNMAFGYGKYACPGRFYVANEIKLVLGHLLMHYEFKFPPGMGRPVNSTVDTDMYPD LGARLLVRKRKMEE >Seq_ID_13 MESLVAALPAGGAAAAAAFGGLVAAAALAGKVGLVGSKKHLNAPPAVSGLPLIGNLHQLKEKKPHQTFTKWAE IYGPIYTIRTGSSTVVLNSAQVAKEAMIAKFSSISTRKLSKALSALTRDK™VATSDYGDFHKMIKRIIMTF MLGTSGQKQFRDTRNMMVDNMLNTFHTLLMDDPNSPLNFREVFKNELFRLSLVQALGEDVSSIYVEEYGKVIS KEEIYKATVVDMMMCAIEVDWRDFFPYPNRTFETRVLTTEARRTTVMQALIKQQKERIARGETRISYLD FLLAENTLTDEQLLMLVWEAVIEAADTTLVTTEWAMYEIAKHPEKQEYLYQEIQKVCGNKTVTEDHLPELPYL NAVFHETMRRHSPVPLVPRLVHENTNLAGYEVPAGTEIIINLYGCNMNKNDWAEPEEWKPERFLDGRFEAVD MHKTMAFGAGRRACAGSMQAMNISCTAIGRFVQEFAWRLEEGDKVDTIQLTTNRLYPLHVYLAPRGRK >SEQ ID NO:14 mRNA for ent-caureno oxidase AY245442.1 [PIsum sativum] GTGGTGAAGCAACTAGCAGTAGCCATCAGTTCAGTTCAGTTCTGTTCTTA TCTCTCTTTTTGTTCCTCTTCTTACTAAAGATCTACTCACAAAACATTCCAAGCTTTCC CATGTACCAGTGGTTCCAGGTTTGCCAGTGATTGGGAATCTGCTGCAATTGAAAGAGAAG AAAC CT CACAAGACAT T CACAAAGAT GGCT CAGAAATAT GGAC CCATTTTTTCCAT CAAA GCTGGTTCTTCCAAAATCAATTCTCAACACTGCTCATCCTTGTCTAAGGCAGTGATTGTCATTCATG CAAAAAGGAAGC TAT CAAC TGCACT GAC GATTC TAAC TTCG GATAAAT GCATGGTTGCTAT GAGC GAC TACAATGATTTT CACAAAAT GGTTAAAAAACAT Petition 870200018837, of 07 / 02 / 2020, p. 115 / 146 103 / 105 ATTCTTGCAAGTGTTCTTGGAGCCAATGCACAGAAGCGACTCCGTTTTCACAGAGAGGTT ATGATGGAAAATATGTCTAGTTTAATGAACATGTGAAGACCCTCTCAGATTCTGCT GTTGATTTTAGGAAAATTTGTCTGAACTTTTCGGATTAGCACTAAAGCAAGCTCTGTTG GGAAGTGATATTGAATCCATTTATGTGGAGGGTTTGACGGCTACATTATCAAGAGAGGAC TTATATAACACTCTAGTGGTTGATTTTATGGAGGGTGCAATTGAGGTGGATTGGAGAGAT TTCTTCCCGTACCTGAAATGGATTCCAAATAAGCTTCGAGAAGAAAGAAATCAATCAATCAATCATCATT GAAGGCAC TAATTAAT GAGCAAAAGAAGC GGTT GACA T CAGGAAAAGAAT TAGAT TGTTATTATGATTACCTAGTAT CAGAAGC TAAAGAAGTGAC T GAAGAACAAATGATCATGCTGCTCTGGGAGCCAATTATTGAGACATCCGATACTACCTTA GTCACGACAGACTATCAGCAGAGAGGAGGATT >Seq_ID_15 ATGGATACCTTAACTTTGTCTTTAGGTTTCTTATCTTTGTTCTTATTTTTATTCTTGTTAAAGAGATCTACTC ACAAGCACTCCAAGTTATCCCACGTTCCAGTTGTTCCAGGTTTGCCTGTCATTGGTAACTTATTGCAATTGAA AGAAAAGAAGC CACACAAGAC TTTCAC CAAGAT GGCT CAAAAGTAC GGTC CAAT TTTCTCCAT CAAAGC CGGT TCTTCTAAAATCATTGTTTTAAACACTGCCCACTTGGCTAAAGAAGCTATGGTTACTAGATATTCTTCCATCT CCAAGAGAAAGTTGTCTACTGCTTTGACCATCTTGACTTCTGATAAGTGCATGGTTGCTATGTCCGATTATAA CGACTTCCACAAGATGGTTAAGAAGCACATCTTGGCTTCTGTTTTGGGTGCCAACGCCCAAAAGAGATTGCGT TTCCACAGAGAAGTCATGATGGAAAACATGTCTTCCAAATTCAATGAACATGTCAAGACTTTGTCTGATTCTG CTGTTGACTTCAGAAAGATTTTCGTTTCTGAATTATTTGGTTTGGCTTTGAAGCAAGCTTTGGGTTCCGATAT CGAATCTATCTACGTTGAAGGTTTGACTGCTACTTTATCTAGAGAAGATTTGTATAACACCTTGGTCGTCGAC TTCATGGAAGGTGCTATCGAAGTTGATTGGAGAGACTTTTTCCCTTATTTGAAGTGGATTCCAAACAAATCCT TCGAAAAGAAGATCAGAAGAGTTGATAGACAAAGAAAAATTATCATGAAAGCTTTGATCAACGAACAAAAGAA AAGAT T GAC CTCT GGTAAGGAAT T GGAC TGTTACTACGATTACTTAGTTTCT GAAGC TAAGGAAGTCAC C GAA GAACAAATGATCATGTTGTTGTGGGAACCAATTATTGAGACTTCTGATACTACTTTAGTTACCACCGAATGGGCTATGTATGAGTTGGCTAAGGACAAGAACCGTCAAGACAGATTGTACGAAGAATTGTTGAACGTTTGTGGTCA CGAAAAGGTTACTGATGAAGAATTGTCCAAGTTGCCATACTTAGGTGCTGTCTTTCACGAAACCTTGCGTAAA CACTCTCCAGTTCCAATCGTCCCATTGAGATACGTTGATGAAGATACCGAATTGGGTGGTTATCATATTCCTG CCGGTTCCGAAATCGCTATCAACATTTACGGTTGTAATATGGATTCCAACTTGTGGAGAAACCCAGATCAATG GATCCCTGAAAGATTTTTAGATGAAAAATACGCCCAAGCTGATTTGTATAAGACTATGGCTTTCGGTGGTGGT AAAGAGTCTGTGCTGGTTCCTTACAAGCTATGTTGATTGCCTGTACTGCTATTGGTAGATTGGTCAAGAAT TTGAATGGGAATTGGGTCACGGTGAAGAAGAAGAAAACGTTGACACCATGGGTTTAACTACCCATAGATTACACCC ATTGCAAGTCAAATTAAAGCCAAGAAACAGAATTTACTAA >SEQ ID NO:16 (sr.UGT_g252778) MATNDDDRKQLHVAMFPWLAFGHILPFLELSKLIAQNGHKVSFLSTTRNIQRLPSHLTPLINLVKLTLRVQE LPEDAEATTDIKHDDQDHLLNASDGLQPEVTRFLEEESPDWIIFDYSYYWLPPVAAELGISRAFFMTFPTWTM ALTRLPSDQLTAEDLMTLSKISFKKHEIVNLMYGTSTQGDLYRLTMACNGSDCILIRCCYEFEPQWLTLLEKL LPVPVVPVGLLPPEIHGDEKDDTTWVSVKEWLDGQHKGHVVYVALGSEAMVSKDELGELALGLELSGLPFFWA LRKPPGSTESDSVELPDGFMERTRNRGVWTSWAPQLRILSHESVCGFLTHCGVSSIVEGLMFGHPLIMLPIF GDQIMNAQVLADKQVGIEIPRNEEDGWFTKESVAKSLRSVVDDEGEIYKANARELSKIFSDTDLGKKYISHF IDFLMMEIVKT* SEQ ID NO:17 (UGT40087 version 1) MDASDSSPLHIVIFPWLAFGHMLASLELAERLAARGHRVSFVSTPRNISRLRPPVPAPLIDFVALPLP RVDGLPDGAEATSDIPPGKTELHLKALDGLAAPFAFLDAACADGSTNKVDWLFLDNFQYWAAAAAADHK IPCALNLTFAASTSAEYGVPRVEPPVDGSTASILQRFVLTLEKCQFVIQRACFELEPEPLPLLSDIFGKP VIPYGLVPPCPPAEGHKREHGNAALSWLDKQQPESVLFIALGSEPPVTVEQLHEIALGLELAGTTFLWAL KKPNGLLLEADGDILPPGFEERTRDRGLVAMGWVPQPIILAHSSVGAFLTHGGWASTIEGVMSGHPMFLL TFLDEQRINAQLIERKKAGLRVPRREKDGSYDRQGIAGAIRAVMCEEESKSVFAANAKKMQEIVSDRNCQ EKYIDELIQRLGSFEK SEQ ID NO:18 (UGT40087 version 2) MDASSSPLHIVIFPWLAFGHMLASLELAERLAARGHRVSFVSTPRNISRLRPVPPALAPLIDFVALPLP RVDGLPDGAEATSDIPPGKTELHLKALDGLAAPFAAFLDAACADGSTNKVDWLFLDNFQYWAAAAAADHK IPCALNLTFAASTSAEYGVPRVEPPVDGSTASILQRFVLTLEKCQFVIQRACFELEPEPLPLLSDIFGKP VIPYGLVPPCPPAEGHKREHGNAALSWLDKQQPESVLFIALGSEPPVTVEQLHEIALGLELAGTTFLWAL KKPNGLLLEADGDILPPGFEERTRDRGLVAMGWVPQPIILAHSSVGAFLTHGGWASTIEGVMSGHPMLFL TFLDEQRINAQLIERKKAGLRVPRREKDGSYDRQGIAGAIRAVMCEEESKSVFAANAKKMQEIVSDRNCQ EKYIDELIQRLGSFEK SEQ ID NO:19 (loop2 of Os_UGT_91C1) EGLPDGAESTNDVPHDRPDMV Petition 870200018837, dated 07 / 02 / 2020, pages 116 / 146 104 / 105 SEQ ID NO:20 (loop3_1 of Os_UGT_91C1) SEFLGTACAD SEQ ID NO:21 (loop3_2 of Os_UGT_91C1) SEFLGTACADWVIVDVFHH SEQ ID NO:22 (loop4_1 of Os_UGT_91C1) ADRRLERAETESSPAAAGQGRPAAAPTFEVARMKLIRTKGSSGM SEQ ID NO:23 (loop4_2 of Os_UGT_91C1) MMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGM SEQ ID NO:24 (loop2 to UGT40087) DGLPDGAEATSDIPPGKT SEQ ID NO:25 (loop3_1 to UGT40087) AAFLDAACADGSTNKVD SEQ ID NO:26 (loop3_2 to UGT40087) AAFLDAACADGSTNKVDWLFLDNFQY SEQ ID NO:27 (loop4_1 to UGT40087) GVPRVEPPVDGSTA SEQ ID NO:28 (loop4_2 to UGT40087) LNLTFAASTSAEYGVPRVEPPVDGSTA SEQ ID NO:29 (loop1 modified from Os_UGT_91C1 present in UGT40087_loop1) TPRNISRLPPVPPALAP SEQ ID NO:30 (loop1 modified by UGT40087 present at Os_UGT_91C1_loop1) TPRNISRLRPVRPALAP SEQ ID NO:31 (loop1 to Os_UGT_91C1 tendo SEQ ID NO:8) TPRNISRLPPVRPALAP SEQ ID NO:32 (loop1 to UGT40087 tendo SEQ ID NO:11) TPRNISRLRPVPPALAP SEQ ID NO:33 (UGT40087 / Si91Dlike chimera) MDASSSPLHIVIFPWLAFGHMLASLELAERLAARGHRVSFVSTPRNISRLRPVPPALAPLIDFVALPLPRVDG LPDGAEATSDIPPGKTELHLKALDGLAAPFAAFLDAACADGSTNKVDWLFLDNFQYWAAAAAADHKIPCALNL TFAASTSAEYGVPRVEPPVDGSTASILQRFVLTLEKCQFVIQRACFELEPEPLPLLSDIFGKPVIPYGLVPPC PPAQGHIEHDNAALSWLDKQQPESVLFIALGSEPPVTVEQLHEIALGLELAGTTFLWALKKPNGLLLEADGDI LPPGFEERTRDRGLVAMGWVPQLSILAHSSVGAFLTHGGWSSTIEGAMSGHPMVFLTFLDEQRINAQLIERKK AGLRVPRCEKDGSYDRQGIAGAIRAVMCEEESKSVFAANAKKMQEIINDRKCQERYIDELIQRLRSFEK SEQ ID NO:34 (Os_UGT_91C1_loop4_1) MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVAL PLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCA MMLLGSAHMIASIGVPRVEPPVDGSTASLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLM PPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLL PAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNA GLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKD SEQ ID NO:35 (non-optimized nucleic acid sequences of UGT40087 having SEQ ID NO:17) tcgtgacgca acagagcaac tctcgccggc accggtcgcc ccttccgcag gcaggcagca ggctcgcgcg catggacgcc tccgactcct ccccgctgca catcgtcatc ttcccgtggc tcgcgttcgg ccacatgctc gccagcctgg agctcgccga gcgcctggcc gcgcgaggcc Petition 870200018837, dated 07 / 02 / 2020, pp. 117 / 146 105 / 105 accgcgtgtc cgctggcgcc acggggcgga tagacggcct ccaacaaggt ccgaccataa acggtgtgcc ttgtgctaac cggagcccct tcccgccgtg ggctcgacaa tgaccgtcga tctgggctct caggtttcga ccatcatact ccattgaagg ggataaacgc aggacggctc aagaaagtaa ggaattgcca gaataaggt tggccattcg tgacttactg tcgcgccgg t cttcgtctcc gctcatcgac ggccaccagc cgccgcgccc ggactggctc gataccctgc acgcgttgag cttggagaaa gcctctcctg tccccccgca gcagcagccc acagctgcac ggaagcct ggagcggac ggctcacagc ggttatgtcc gcaactgatc gtacgatcgc gagcgtcttc gggaagtac gaaatatcct cgcctctgat aatttcctaa attagcgata accccgcgca ttcgtggcgc gacatcccgc ttcgcagctt ttcctcgaca gcgctgaacc ccgccggtgg tgccagtttg tcagacatct gaaggtcaca gagtctgtcc gagatcgcgc aacggcctcc cgtgaccgtg tccgtgggcg gggcatccca gagagagaa caaggcatcg gcggctaatg atcgacgagc aataaccg ctccatgttc tatgtatgcc atgtcaccgc acatcagccg tgccgctgcc ccggcaagac tcctcgacgc acttccaata tgacattcgc atggctcaac tcatccaacg tcggcaagcc aaagagagca tgttcattgc ttgggctgga tcctcgaggc ggctcgtggc cgttcctgac tgctcttcct aggccgggttccggagcgat ccaagaagat ttatccagcg cctgttgatg cggcaataaa caaacacatg agtcgccagc cctccgcccg gcgcgtcgac cgagctccac cgcctgcgcc ctgggccgcc agcgtcgacg agcctcaata cgcctgcttc ggtgatcccg cggcaacgca tctgggaagc gctcgccggg ggacggcgac catgggctgg gcacggcgga gacgttctta gcgagtgcca ccgggctgtc gcaggagatt tctgggatcc gcttgatgca tccaccatat cataggttgc acaggtgtag gtcccgcccg ggcctccccg ctcaaggccc gacgggagca gccgccgctg tcagcggagt ctccagcgat gagctggagc tacggcctag gctctgtcat gagcctccgg acgacattcc atcctgcccc gttcctcagc tgggcctcca gatgaacaga aggcgtgaga atgtgcgagg gtgagcgaca ttcgagaagt acgatgtagg gttatggctc tagttgcccc caatttgaca SEQ ID NO:36 (UGT40087-1 optimized nucleic acid sequence at codon) ATGGATGCTTCCAGTAGTCCTTTACACATCGTTATCTTTCCATGGTTAGCTTTCGGTCATATGTTGGCTTCCT TGGAATTGGCTGAGAGATTGGCTGCTCGTGGTCACAGAGTCTCCTTCGTTTCCACCCCTAGAAACATCTCTAG ATTACGTCCAGTTCCACCAGCTTTAGCTCCATTGATTGATTTTGTCGCTTTGCCATTGCCTAGAGTCGATGGT TTAC CAGAT GGTGCC GAAGC TACCTCT GACAT TCCAC CAGGTAAGAC C GAAT TACACTT GAAGGC TTT GGAC G GTTTGGCTGCTCCATTCGCCGCTTTTTTGGACGCTGCCTGTGCTGATGGTTCCACCAACAAGGTTGATTGGTT GTTTTTGGACAACTTCCAATACTGGGCTGCCGCTGCCGCTGCTGATCACAAAATTCCTTGCGCCTTAAACTTG ACTTTTGCCGCTTCCACCTCCGCTGAATACGGTGTTCCACGTGTTGAACCACCAGTTGACGGTTCCACTGCCT CCATCTTACAAAGATTTGTCTTAACCTTAGAAAAATGTCAATTCGTTATCCAAAGAGCTTGTTTCGAATTGGA ACCTGAACCATTGCCATTGTTGTCCGACATTTTCGGTAAGCCAGTCATCCCATACGGTTTAGTTCCTCCATGT CCACCAGCTGAAGGTCACAAAAGAGAACACGGTAACGCTGCTTTGTCCTGGTTGGATAAGCAACAACCAGAAT CTGTTTTGTTCATCGCTTTGGGTTCTGAACCACCTGTTACCGTCGAACAATTGCACGAAATCGCTTTGGGTTT AGAATTGGCCGGTACCACCTTCTTGTGGGCCTTGAAAAAGCCAAACGGTTTGTTGTTAGAAGCCGATGGTGAT ATTTTGCCACCAGGTTTCGAAGAAAGAACTAGAGATAGAGGTTTAGTCGCTATGGGTTGGGTTCCACAACCAATTATCTTGGCCCATTCCTCTGTTGGTGCCTTTTTGACTCACGGTGGTTGGGCCTCCACTATTGAAGGTGTCAT GTCCGGTCACCCTATGTTGTTCTTAACCTTCTTGGACGAACAACGTATCAACGCCCAATTGATCGAAAGAAAA AAGGCTGGTTTAAGAGTCCCAAGAAGAGAAAAGGATGGTTCCTACGACAGACAAGGTATTGCTGGTGCTATTA GAGCCGTCATGTGTGAAGAAGAATCTAAGTCTGTCTTCGCTGCTAACGCTAAGAAAATGCAAGAGATCGTTTC TGACAGAAACTGTCAAGAAAAGTACATCGACGAATTGATTCAAAGATTGGGTTCTTTCGAAAAGTAA Petição 870200018837, the 07 / 02 / 2020, pág. 118 / 146

Claims

CLAIMS 1. A genetically modified Saccharomyces cerevisiae host cell, characterized in that it is capable of producing a steviol glycoside comprising a heterologous kaureno oxidase having an amino acid sequence of SEQ ID NO:1, wherein the genetically modified Saccharomyces cerevisiae host cell further comprises a polypeptide having geranyl-geranyl diphosphate synthase (GGPPS) activity, a polypeptide having copalyl diphosphate synthase (CDPS) or enicopalyl pyrophosphate synthase (CPS) activity, a polypeptide having enicopalyl pyrophosphate synthase activity, a polypeptide having steviol synthase activity, and a polypeptide having uridine-5'-diphosphate (UDP)-glycosyltransferase (UGT) activity.

2. Genetically modified Saccharomyces cerevisiae host cell according to claim 1, characterized in that it is capable of converting kaurene into kaurenoic acid with an efficiency greater than 30%, 35%, 40%, 45%, 50% or 55%.

3. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that the kaurenol oxidase is capable of oxidizing the C19 position of kaurenol, kaurenol and / or kaurenal.

4. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that the kaureno oxidase is encoded by a heterologous nucleic acid with the sequence SEQ ID NO:

15.

5. Genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized by being able to convert kaurene into kaurenoic acid with an efficiency greater than 30%, 35%, 40%, 45%, 50% or 55%. Petition 870260074389, dated 07 / 27 / 2026, page 17 / 35 2 / 7 6. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized by being able to produce kaurenoic acid.

7. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized by being able to produce steviol.

8. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized by being capable of producing RebD.

9. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized by being able to produce RebM.

10. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that it is capable of producing RebM and RebM2 at a ratio of at least 10:1, 100:1 or 1000:

1.

11. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that the genetically modified host cell produces an undetectable level of RebM2.

12. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that it additionally comprises enzymes of a pathway for making steviol.

13. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that it additionally comprises enzymes of a pathway for making a steviol glycoside.

14. Host cell of Saccharomyces cerevisiae Petition 870260074389, dated 07 / 27 / 2026, page 18 / 35 2>!Ί genetically modified according to any of the preceding claims, characterized in that it additionally comprises more enzymes of a pathway to make a RebA.

15. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that it additionally comprises enzymes of a pathway for making RebM.

16. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that it additionally comprises enzymes of a pathway for making RebE.

17. A genetically modified Saccharamyces cerevisiae host cell according to any of the preceding claims, characterized in that it additionally comprises enzymes of a pathway for the production of steviol, a steviol glycoside, RebA, RebM, RebD and / or RebE.

18. A genetically modified Saccharamyces cerevisiae host cell according to any of the preceding claims, characterized in that the polypeptide having CDPS activity is a copalyl diphosphate synthase.

19. A genetically modified Saccharamyces cerevisiae host cell according to any of the preceding claims, characterized in that the polypeptide having enikaure synthase activity is an enikaure synthase.

20. A genetically modified Saccharamyces cerevisiae host cell according to any of the preceding claims, characterized in that the polypeptide having kaurenoic acid hydroxylase activity is a kaurenoic acid hydroxylase.

21. Genetically modified Saccharomyces cerevisiae host cell Petition 870260074389, dated 07 / 27 / 2026, page 19 / 35 according to any of the preceding claims, characterized in that the genetically modified host cell further comprises a polypeptide having cytochrome P450 reductase (CPR) activity.

22. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that the polypeptide having UGT activity is a uridine 5'-bisphosphate-dependent glycosyltransferase.

23. Genetically modified Saccharomyces cerevisiae host cell according to claim 22, characterized in that it is capable of converting kaurene into kaurenoic acid with an efficiency greater than 55%.

24. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that the polypeptide having UGT activity is selected from an Os_UGT_91Cl (SEQ ID NO: 37), a Sl_UGT_101249881 (SEQ ID NO: 38), a UGT40087 (SEQ ID NO: 39, SEQ ID NO: 17, 18 or 33), a sr.UGT_9252778 (SEQ ID NO: 16), a Bd_UGT10840 (SEQ ID NO: 40), an Hv_UGT_Vl (SEQ ID NO: 41), a Bd_UGT10850 (SEQ ID NO: 42), or an Ob_UGT91Bl_like (SEQ ID NO: 43).

25. Genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that the polypeptide having UGT activity is selected from among a UGT74G1, a UGT76G1, a UGT85C2, a UGT91D or a UGT40087 (SEQ ID NO: 39, SEQ ID NO: 17, 18, or 33).

26. Genetically modified Saccharomyces cerevisiae host cell in accordance with any of the claims Petition 870260074389, dated 07 / 27 / 2026, p. 20 / 35 5 / 7 above, characterized in that the polypeptide having steviol synthase activity is a kaurenoic acid hydroxylase, and the polypeptide having UGT activity comprises a polypeptide selected from an Os_UGT_91Cl (SEQ ID NO: 37), a Sl_UGT_101249881 (SEQ ID NO: 38), a UGT40087 (SEQ ID NO: 39, SEQ ID NO: 17, 18 or 33), a sr.UGT_9252778 (SEQ ID NO: 16), a Bd_UGT10840 (SEQ ID NO: 40), an Hv_UGT_Vl (SEQ ID NO: 41), a Bd_UGT10850 (SEQ ID NO: 42) or an Ob_UGT91Bl_like (SEQ ID NO: 43), a UGT74G1, UGT76G1, UGT85C2 and UGT91D.

27. Genetically modified Saccharomyces cerevisiae host cell according to claim 26, characterized in that the genetically modified Saccharomyces cerevisiae host cell comprises a cytochrome P450 reductase, and the polypeptide having UGT activity is selected from among a UGT40087 (SEQ ID NO: 39, SEQ ID NO: 17, 18, or 33), a UGT74G1, a UGT76G1, a UGT85C2, and a UGT91D.

28. Genetically modified Saccharomyces cerevisiae host cell according to any one of claims 24 to 27, characterized in that the genetically modified Saccharomyces cerevisiae host cell comprises UGT40087 having an amino acid sequence of SEQ ID NO: 17, 18 or 33.

29. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that the polypeptide having CDPS activity is a bifunctional copalyl diphosphate synthase and a kaurene synthase.

30. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that each polypeptide is encoded together by nucleic acids under the control of a single transcriptional regulator. Petition 870260074389, dated 07 / 27 / 2026, p. 21 / 35 6 / 7 31. A genetically modified Saccharomyces cerevisiae host cell according to any of the preceding claims, characterized in that each polypeptide is encoded together by nucleic acids under the control of multiple heterologous transcriptional regulators.

32. Method for producing kaurenoic acid, characterized in that it comprises: (a) cultivating a population of genetically modified Saccharomyces cerevisiae host cells as defined in any of the preceding claims in a medium with a carbon source under conditions suitable for making kaurenoic acid; and (b) recovering said kaurenoic acid compound from the medium.

33. Method for producing Rebaudioside D (RebD), characterized in that it comprises: (a) cultivating a population of the genetically modified host cells as defined in any one of claims 1 to 31 in a medium with a carbon source under conditions suitable for making RebD; and (b) recovering said RebD compound from the medium.

34. Method for producing Rebaudioside M (RebM), characterized in that it comprises: (a) cultivating a population of the genetically modified host cells as defined in any one of claims 1 to 31 in a medium with a carbon source under conditions suitable for making RebM; and (b) recovering said RebM compound from the medium.

35. Fermentation composition, characterized in that it comprises: (a) a genetically modified Saccharomyces cerevisiae host cell comprising: i. a heterologous kaurene oxidase having an amino acid sequence of SEQ ID NO: 1, capable of converting kaurene into kaurenoic acid; and (b) steviol glycosides produced from the genetically modified Saccharomyces cerevisiae host cell.

36. Fermentation composition according to claim 35, characterized in that the steviol glycosides comprise RebA, RebD and ReM at a RebA:RebD:RebM ratio of 1:7:50 to 1:100:1000.

37. Method for producing a steviol glycoside, characterized in that it comprises: (a) cultivating a population of the genetically modified host cells as defined in any one of claims 1 to 31 in a medium with a carbon source under conditions suitable for making a steviol glycoside. Petition 870260074389, dated 27 / 07 / 2026, p. 23 / 35

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