Compositions and methods for production of methylxanthines in engineered microbial cells

Abstract

The present disclosure provides engineered microbial cells, compositions, and methods of use thereof for producing methylxanthines including theobromine and caffeine.

Description

[0001] Attorney Docket No. TBN-00125

[0002] COMPOSITIONS AND METHODS FOR PRODUCTION OF METHYLXANTHINES IN

[0003] ENGINEERED MICROBIAL CELLS

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 695,441, filed September 17, 2024, the entire contents of which are incorporated herein by reference.

[0006] BACKGROUND

[0007] Traditional methods for producing chocolate face an expanding list of challenges that stem from their reliance on costly processes including harvesting fastidious crops, processing, fermenting, drying, grinding, refining, tempering, and molding steps. Changing climate patterns, evolving crop diseases, deforestation, and controversial labor practices render traditional chocolate, coffee, and tea production an increasingly difficult endeavor. These factors have led to a global chocolate, coffee, and tea crisis characterized by significant production deficits in these products, leading to exponential increases in the price of each. Accordingly, there is an urgent need for compositions and methods of producing chocolate, coffee, and tea that do not succumb to the challenges facing traditional agricultural production methods.

[0008] SUMMARY

[0009] In some aspects, the disclosure provides an engineered microbial cell comprising at least one of a first heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2, and a third heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 3.

[0010] In some embodiments, the engineered microbial cell comprises the first heterologous polypeptide. In some embodiments, the engineered microbial cell comprises the second heterologous polypeptide. In some embodiments, the engineered microbial cell comprises the third heterologous polypeptide.

[0011] In some embodiments, the engineered microbial cell comprises the first heterologous polypeptide and the second heterologous polypeptide. Attorney Docket No. TBN-00125

[0012] In some embodiments, the engineered microbial cell comprises the first heterologous polypeptide and the third heterologous polypeptide.

[0013] In some embodiments, the engineered microbial cell comprises the second heterologous polypeptide and the third heterologous polypeptide.

[0014] In some embodiments, the engineered microbial cell comprises the first heterologous polypeptide, the second heterologous polypeptide, and the third heterologous polypeptide.

[0015] In some embodiments, the cell comprises a nucleic acid sequence encoding at least one of the first heterologous polypeptide, the second heterologous polypeptide and the third heterologous polypeptide.

[0016] In some embodiments, the nucleic acid sequence is a DNA sequence. In some embodiments, the nucleic acid sequence is an RNA sequence. In some embodiments, the engineered microbial cell produces a methylxanthine at an elevated level compared to a non-engineered microbial cell.

[0017] In some embodiments, the methylxanthine is theobromine. In some embodiments, the methylxanthine is caffeine.

[0018] In some embodiments, the nucleic acid sequence encoding at least one of the first heterologous polypeptide, the second heterologous polypeptide and the third heterologous polypeptide cell comprises a nucleic acid sequence comprising an expression regulator.

[0019] In some embodiments, the expression regulator is an engineered expression regulator. In some embodiments, the expression regulator comprises at least one of a promoter and a terminator.

[0020] In some embodiments, at least one of the first heterologous polypeptide, the second heterologous polypeptide, and the third heterologous polypeptide is encoded in the genome of the engineered microbial cell.

[0021] In some embodiments, at least one of the first heterologous polypeptide, the second heterologous polypeptide, and the third heterologous polypeptide is encoded by a plasmid present in the engineered microbial cell.

[0022] In some embodiments, the cell comprises one or more heterologous polypeptide(s) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. Attorney Docket No. TBN-00125

[0023] In some embodiments, the cell comprises one or more heterologous polypeptide(s) comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0024] In some embodiments, the engineered microbial cell is a bacterial cell. In some embodiments, the bacterial cell is an Escherichia coli, Bacillus subtilis, Bacillus licheniformis , Bacillus megaterium, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Corynebacterium glutamicum, Lactobacillus, Streptomyces, Actinomyces, Salmonella typhimurium, Synechocystis sp., Aquifex aeolicus, Methylacidiphilum infernorum, or Thermophilus cell.

[0025] In some embodiments, the engineered microbial cell is a yeast cell. In some embodiments, the yeast cell is a Saccharomyces cerevisiae, Pichia pastoris, Magnaporthe oryzae, Fusarium graminearum, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Myceliophthora thermophila, Kluyvera lactis, Fusarium oxysporum, or Yarrowia lipolytica cell.

[0026] In some embodiments, the engineered microbial cell is engineered to comprise a genomic mutation in one or more genes. In some embodiments, the engineered microbial cell is engineered to comprise a mutation in one or more of the following genes: Transketolase 1, Transaldolase A, Glucose-6-Phosphate Dehydrogenase, 6-Phosphogluconolactonase, 6-Phosphogluconate Dehydrogenase, Ribose 5-Phosphate Isomerase A, 5-Phosphoribosyl-l -Pyrophosphate (PRPP) Synthase, Amidophosphoribosyltransferase, Phosphoribosylamine-Glycine Ligase, Formatedependent Phosphoribosylglycinamide Formyltransferase, Phosphoribosylformylglycinamide Synthase, Phosphoribosylformylglycinamidine Cyclo-Ligase, N5-Carboxyaminoimidazole Ribonucleotide Synthase, N5 -Carboxy aminoimidazole Ribonucleotide Mutase, Phosphoribosylaminoimidazole-Succinocarboxamide Synthase, Adenylosuccinate Lyase, 5- Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Transformylase, Inosine Monophosphate (IMP) Cyclohydrolase, Inosine Monophosphate (IMP) Dehydrogenase, Methionine Adenosyl Transferase, Adenosine Homocysteinase, Adenosylhomocysteine nucleosidase, S- ribosylhomocysteine Lyase, Methionine Synthase Cobalamin-Depend, Methionine Synthase Cobalamin-Independ, Serine Hydroxymethyltransferase, Methylenetetrahydrofolate Reductase, Phosphoglycerate Dehydrogenase, Phosphoserine Aminotransferase, Phosphoserine Phosphatase, Aspartate Transaminase, Glutamate Dehydrogenase, Glutamine Synthetase, Aspartate Kinase, Aspartate Semialdehyde Dehydrogenase, Homoserine Dehydrogenase, Homoserine O- Succinyltransferase, O-Succinylhomoserine Lyase, Cystathionine b-Lyase, Camellia sinensis Purine Attorney Docket No. TBN-00125

[0027] Permease (CsPUPl), Camellia sinensis Purine Permease (CsPUP3.1), Camellia sinensis Purine Permease (CsPUPlO.l), or Saccharomyces cerevisiae Purine-Cytosine Permease (FCY2).

[0028] In some embodiments, the engineered microbial cell expresses a genetically-modified Transketolase 1 , Transaldolase A, Glucose-6-Phosphate Dehydrogenase, 6- Phosphogluconolactonase, 6-Phosphogluconate Dehydrogenase, Ribose 5-Phosphate Isomerase A, 5 -Phosphoribosyl- 1 -Pyrophosphate (PRPP) Synthase, Amidophosphoribosyltransferase, Phosphoribosylamine-Glycine Ligase, Formate-dependent Phosphoribosylglycinamide Formyltransferase, Phosphoribosylformylglycinamide Synthase, Phosphoribosylformylglycinamidine Cyclo-Ligase, N5-Carboxyaminoimidazole Ribonucleotide Synthase, N5-Carboxyaminoimidazole Ribonucleotide Mutase, Phosphoribosylaminoimidazole- Succinocarboxamide Synthase, Adenylosuccinate Lyase, 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Transformylase, Inosine Monophosphate (IMP) Cyclohydrolase, Inosine Monophosphate (IMP) Dehydrogenase, Methionine Adenosyl Transferase, Adenosine Homocysteinase, Adenosylhomocysteine nucleosidase, S-ribosylhomocysteine Lyase, Methionine Synthase Cobalamin-Depend, Methionine Synthase Cobalamin- Independ, Serine Hydroxymethyltransferase, Methylenetetrahydrofolate Reductase, Phosphoglycerate Dehydrogenase, Phosphoserine Aminotransferase, Phosphoserine Phosphatase, Aspartate Transaminase, Glutamate Dehydrogenase, Glutamine Synthetase, Aspartate Kinase, Aspartate Semialdehyde Dehydrogenase, Homoserine Dehydrogenase, Homoserine O-Succinyltransferase, O- Succinylhomoserine Lyase, Cystathionine b-Lyase, Camellia sinensis Purine Permease (CsPUPl), Camellia sinensis Purine Permease (CsPUP3.1), Camellia sinensis Purine Permease (CsPUPlO.l), or Saccharomyces cerevisiae Purine-Cytosine Permease (FCY2).

[0029] In some aspects, the disclosure provides a plurality of engineered microbial cells, wherein the plurality of engineered microbial cells produces theobromine at a yield of from about 50 mg / L to about 200 g / L. In some embodiments, the composition comprises an engineered microbial cell according to any one disclosed herein.

[0030] In some aspects, the disclosure provides an amino acid sequence at least 90% but less than 100% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. Attorney Docket No. TBN-00125

[0031] In some aspects, the disclosure provides a composition comprising an engineered microbial cell comprising at least one of a first heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2 and a third heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and a cell culture medium.

[0032] In some aspects, the disclosure provides a method of producing a methylxanthine comprising expressing in an engineered microbial cell at least one of a first heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2, and a third heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 3 culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0033] In some embodiments, the culturing comprises providing glucose to the engineered microbial cell. In some embodiments, glucose is provided to a concentration of 20 g / L. In some embodiments, the culturing comprises providing xanthosine to the engineered microbial cell.

[0034] In some aspects, the disclosure provides a nucleic acid encoding at least one of a first heterologous polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 2, and a third heterologous coding polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 3.

[0035] In some embodiments, the nucleic acid is in a plasmid or encapsidated by a virus. In some embodiments, the nucleic acid is in a plasmid. In some embodiments, the plasmid comprises at least one expression regulator. In some embodiments, the nucleic acid is encapsidated in a virus, wherein the virus is a bacteriophage. In some embodiments, the nucleic acid comprises at least one expression regulator.

[0036] In some aspects, the disclosure provides an edible composition comprising a methylxanthine produced using an engineered microbial cell, at least one consumable ingredient, the consumable Attorney Docket No. TBN-00125 ingredient comprising a carbohydrate, a fat, or a protein. In some embodiments, the methylxanthine is theobromine. In some embodiments, the composition is free or substantially free of cocoa.

[0037] In some aspects, the disclosure provides an edible composition prepared by providing a methylxanthine that was produced using an engineered microbial cell, and mixing the methylxanthine with a consumable ingredient, the consumable ingredient comprising a carbohydrate, a fat, or a protein. In some embodiments, the methylxanthine is theobromine. In some embodiments, the composition is a cocoa-free chocolate composition.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 shows molecular structures of xanthine and methylxanthine derivatives.

[0040] FIG. 2 is a schematic showing a portion of the biosynthetic pathway of 7-methyl-xanthine, theobromine, and caffeine. The pathway involves xanthosine methyltransferase (XMT), theobromine synthase (MXMT), caffeine synthase 1 (DXMT), S-adenosyl-methionine (SAM), and S-adenosyl-homocysteine (SAH).

[0041] FIG. 3 is a schematic showing the methylxanthine biosynthetic pathway. This pathway is engineered for improved methylxanthine production by engineered microbial cells.

[0042] DETAILED DESCRIPTION

[0043] Cacao trees (Theobroma cacao') produce cocoa beans with a unique key ingredient, theobromine, to produce chocolate. This key ingredient alone is bitter in taste, but when combined with sugar, fat, and proteins creates the taste of chocolate. Also, coffee trees (Coffea arabica or Coffea canephora) and tea trees (Camellia sinensis) respectively produce coffee beans and tea leaves both with a common and unique key ingredient, caffeine, to produce coffee and tea beverages. Unfortunately, many factors (such as climate change, diseases, deforestation, and others) have led to a dramatic drop in cocoa bean, coffee bean, and tea leaf production and subsequent price increases in chocolate, coffee, and tea, respectively.

[0044] The present disclosure provides, inter alia, engineered microbial cells, compositions comprising the engineered microbial cells, and methods of use thereof, for the biosynthetic production of methylxanthine (e.g., theobromine and caffeine) compounds. The methylxanthanines produced using the compositions and methods disclosed herein may be used to prepare edible compositions in a sustainable manner, including cocoa-free chocolate, coffee bean-free coffee, and tea leaf-free tea compositions. Attorney Docket No. TBN-00125

[0045] Definitions

[0046] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques and / or substitutions of equivalent techniques that would be apparent to one of skill in the art.

[0047] In this application, unless otherwise clear from context, (i) the terms “a” and “an” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and "including" may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) where ranges are provided, endpoints are included.

[0048] As used herein, the term “substantially free of’ refers to a mass that is at least 95% free of the named ingredient, e.g. 95% free of the ingredient, such as at least 96%, at least 97%, at least 98%, or at least 99% free of the ingredient.

[0049] As used herein, the term “cocoa” also comprises the term “cacao.” The term “cocoa” refers to processed products derived from the Theobroma cacao fruit, in particular the seeds thereof (cacao beans), including fermented, roasted, alkalized, ground, and pressed cacao beans. The term “cacao” refers to the unprocessed products derived from the Theobroma cacao fruit, in particular the seeds (cacao beans) thereof.

[0050] A “cocoa-free chocolate composition” refers to an edible composition produced without any cocoa or any cacao beans, wherein the composition shares one or more organoleptic properties with a chocolate composition produced using cocoa. For example, in some embodiments, a cocoa-free chocolate composition has the same or substantially the same appearance, texture, color, aroma, or flavor as a chocolate composition produced using cocoa.

[0051] A “coffee bean-free coffee composition” refers to an edible composition produced without coffee beans, wherein the composition shares one or more organoleptic properties with a coffee composition produced using coffee beans. For example, in some embodiments, a coffee bean-free coffee composition has the same or substantially the same appearance, texture, color, aroma, or flavor as a coffee composition produced using coffee beans. Attorney Docket No. TBN-00125

[0052] A “tea leaf-free tea composition” refers to an edible composition produced without tea leaves, wherein the composition shares one or more organoleptic properties with a tea composition produced using tea leaves. For example, in some embodiments, a tea leaf-free tea composition has the same or substantially the same appearance, texture, color, aroma, or flavor as a tea composition produced using tea leaves.

[0053] The term “recombinant,” “engineered,” and “non-naturally occurring,” when used with reference to a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (i.e., nonrecombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0054] The term “expression” as used herein includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0055] The term “heterologous” as used herein refers to a polynucleotide or polypeptide that is introduced into a host cell by laboratory techniques, and includes polynucleotides or polypeptides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell. In some embodiments, when “heterologous” is used with reference to a nucleic acid or polypeptide, the term refers to a sequence that is not normally expressed and secreted by an organism (e.g., a “wild-type” organism). In some embodiments, the term encompasses a sequence that comprises two or more subsequences which are not found in the same relationship to each other as normally found in nature, or is recombinantly engineered so that its level of expression, or physical relationship to other nucleic acids or other molecules in a cell, or structure, is not normally found in nature. For instance, a heterologous nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged in a manner not found in nature (e.g., a nucleic acid open reading frame (ORF) operatively linked to a promoter sequence inserted into an expression cassette, such as a vector).

[0056] The term “operably linked” as used herein refers to a configuration in which a control sequence (e.g., an expression regulator) is appropriately placed (i.e., in a functional relationship) at Attorney Docket No. TBN-00125 a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide of interest. Thus, a nucleic acid is “operably linked” to another nucleic acid sequence when it is placed into a functional relationship with the other nucleic acid sequence.

[0057] The term "sequence identity" used herein as relating to an amino acid sequence or a nucleotide sequence represents a percentage (%) determined by aligning two amino acid sequences (or nucleotide sequences) to be compared so as to maximize the number of matching amino acid residues (or nucleotides) and dividing the number of matched amino acid residues (or the number of matched nucleotides) by the total number of amino acid residues (or the total number of nucleotides). Such sequence alignment can be carried out with the use of a well-known program, for example, BLAST, FASTA, or CLUSTAL W (Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A., 87: 2264-2268, 1993; Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997). Unless otherwise indicated, sequence identity is calculated for the purposes of the instant disclosure using the BLAST program available on the world wide web at blast.ncbi.nlm.nih.gov / Blast.cgi.

[0058] The terms “transformed” and “stably transformed” as used herein refer to a cell that has a non-native (i.e., heterologous) polynucleotide sequence integrated into its genome or as an episomal plasmid.

[0059] Engineered cells

[0060] In some aspects, the disclosure provides engineered cells comprising at least one of a first heterologous polypeptide, a second heterologous polypeptide, and a third heterologous polypeptide.

[0061] In some embodiments, the first heterologous polypeptide is XMT (SEQ ID NO: 1). In some embodiments, the second heterologous polypeptide is MXMT (SEQ ID NO: 2). In some embodiments, the third heterologous polypeptide is DXMT (SEQ ID NO: 3).

[0062] In some embodiments, the disclosure provides engineered cells comprising at least one heterologous polypeptide selected from Xanthosine methyltransferase (XMT), theobromine synthase (MXMT), and caffeine synthase 1 (DXMT). In some embodiments, the disclosure provides engineered cells comprising the heterologous polypeptides XMT (SEQ ID NO: 1) and MXMT (SEQ ID NO: 2). In some embodiments, the disclosure provides engineered cells comprising the heterologous polypeptides XMT (SEQ ID NO: 1) and DXMT (SEQ ID NO: 3). In some embodiments, the disclosure provides engineered cells comprising the heterologous polypeptides MXMT (SEQ ID NO: 2) and DXMT (SEQ ID NO: 3). In some embodiments, the Attorney Docket No. TBN-00125 disclosure provides engineered cells comprising the heterologous polypeptides XMT (SEQ ID NO: 1), MXMT (SEQ ID NO: 2), and DXMT (SEQ ID NO: 3).

[0063] In some embodiments, the engineered cells are microbial cells. In some embodiments, the engineered microbial cells are bacterial cells. In some embodiments, the bacterial cells are of a bacterial species such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis , Bacillus megaterium, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Corynebacterium glutamicum, Lactobacillus, Streptomyces, Actinomyces, Salmonella typhimurium, Synechocystis sp., Aquifex aeolicus, Methylacidiphilum infernorum, or Thermophilus.

[0064] In some embodiments, the engineered microbial cells are yeast cells. In some embodiments, the engineered yeast cells are of a species such as Saccharomyces cerevisiae, Pichia pastoris, Magnaporthe oryzae, Fusarium graminearum, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Myceliophthora thermophila, Kluyvera lactis, Fusarium oxysporum, or Yarrowia lipolytica.

[0065] In some embodiments, the engineered cells comprise a first heterologous polypeptide. In some embodiments, the engineered cells comprise a second heterologous polypeptide. In some embodiments, the engineered cells comprise a third heterologous polypeptide. In some embodiments, the engineered cells comprise a first heterologous polypeptide and a second heterologous polypeptide. In some embodiments, the engineered cells comprise a first heterologous polypeptide and a third heterologous polypeptide. In some embodiments, the engineered cells comprise a second heterologous polypeptide and a third heterologous polypeptide. In some embodiments, the engineered cells comprise a first heterologous polypeptide, a second heterologous polypeptide, and a third heterologous polypeptide.

[0066] In some embodiments, the engineered cells comprise a nucleic acid sequence, such as a nucleic acid sequence encoding a heterologous polypeptide. The nucleic acid sequence may be, for example, a DNA sequence or an RNA sequence. Accordingly, in some embodiments, the nucleic acid sequence is a DNA sequence, and in some embodiments, the nucleic acid sequence is an RNA sequence.

[0067] In some embodiments, the engineered cells comprise a nucleic acid sequence encoding at least one of the first heterologous polypeptide, the second heterologous polypeptide and the third heterologous polypeptide. In some embodiments, the engineered cells comprise a nucleic acid sequence encoding the first heterologous polypeptide. In some embodiments, the engineered cells comprise a nucleic acid sequence encoding the second heterologous polypeptide. In some Attorney Docket No. TBN-00125 embodiments, the engineered cells comprise a nucleic acid sequence encoding the third heterologous polypeptide.

[0068] In some embodiments, the engineered cells comprise a first nucleic acid sequence encoding the first heterologous polypeptide and a second nucleic acid sequence encoding the second heterologous polypeptide. In some embodiments, the engineered cells comprise a first nucleic acid sequence encoding the first heterologous polypeptide and a second nucleic acid sequence encoding the third heterologous polypeptide. In some embodiments, the engineered cells comprise a first nucleic acid sequence encoding the second heterologous polypeptide and a second nucleic acid sequence encoding the third heterologous polypeptide. In some embodiments, the engineered cells comprise a first nucleic acid sequence encoding the first heterologous polypeptide, a second nucleic acid sequence encoding the second heterologous polypeptide, and a third nucleic acid sequence encoding the third heterologous polypeptide.

[0069] In some embodiments, at least one of the first heterologous polypeptide, the second heterologous polypeptide, and the third heterologous polypeptide is encoded in the genome of the engineered microbial cell. In some embodiments, the first heterologous polypeptide is encoded in the genome of the engineered microbial cell. In some embodiments, the second heterologous polypeptide is encoded in the genome of the engineered microbial cell. In some embodiments, the third heterologous polypeptide is encoded in the genome of the engineered microbial cell. In some embodiments, the first heterologous polypeptide and the second heterologous polypeptide are encoded in the genome of the engineered microbial cell. In some embodiments, the first heterologous polypeptide and the third heterologous polypeptide are encoded in the genome of the engineered microbial cell. In some embodiments, the second heterologous polypeptide and the third heterologous polypeptide are encoded in the genome of the engineered microbial cell. In some embodiments, the first heterologous polypeptide, the second heterologous polypeptide, and the third heterologous polypeptide are encoded in the genome of the engineered microbial cell.

[0070] In some embodiments, at least one of the first heterologous polypeptide, the second heterologous polypeptide, and the third heterologous polypeptide is encoded by a plasmid present in the engineered microbial cell. In some embodiments, the first heterologous polypeptide is encoded by a plasmid present in the engineered microbial cell. In some embodiments, the second heterologous polypeptide is encoded by a plasmid present in the engineered microbial cell. In some Attorney Docket No. TBN-00125 embodiments, the third heterologous polypeptide is encoded by a plasmid present in the engineered microbial cell.

[0071] In some embodiments, the first heterologous polypeptide and the second heterologous polypeptide are encoded by a plasmid present in the engineered microbial cell. In some embodiments, the first heterologous polypeptide and the third heterologous polypeptide are encoded by a plasmid present in the engineered microbial cell. In some embodiments, the second heterologous polypeptide and the third heterologous polypeptide are encoded by a plasmid present in the engineered microbial cell. In some embodiments, the first heterologous polypeptide, the second heterologous polypeptide, and the third heterologous polypeptide are encoded by a plasmid present in the engineered microbial cell.

[0072] In some embodiments, the engineered microbial cell comprises a first plasmid, a second plasmid, and / or a third plasmid. In some embodiments, the first heterologous polypeptide is encoded by the first plasmid present in the engineered microbial cell. In some embodiments, the second heterologous polypeptide is encoded by a second plasmid present in the engineered microbial cell. In some embodiments, the third heterologous polypeptide is encoded by a third plasmid present in the engineered microbial cell.

[0073] In some embodiments, the first and second plasmids are present in the engineered microbial cell. In some embodiments, the first and third plasmids are present in the engineered microbial cell. In some embodiments, the second and third plasmids are present in the engineered microbial cell. In some embodiments, the first, the second, and the third plasmids are present in the engineered microbial cell.

[0074] In some embodiments, the engineered microbial cell produces a methylxanthine at an elevated level compared to a non-engineered microbial cell. In some embodiments, the methylxanthine is theobromine. In some embodiments, the methylxanthine is caffeine. In some embodiments, the methylxanthine is 7-methylxanthine, 3 -methylxanthine, 1 -methylxanthine, paraxanthine, and theophylline.

[0075] In some embodiments, the engineered microbial cell is engineered to comprise a genomic mutation in one or more genes. In some embodiments, the engineered microbial cell is engineered to comprise a mutation in one or more of the following genes: Transketolase 1, Transaldolase A, Glucose-6-Phosphate Dehydrogenase, 6-Phosphogluconolactonase, 6-Phosphogluconate Dehydrogenase, Ribose 5-Phosphate Isomerase A, 5-Phosphoribosyl-l -Pyrophosphate (PRPP) Attorney Docket No. TBN-00125

[0076] Synthase, Amidophosphoribosyltransferase, Phosphoribosylamine-Glycine Ligase, Formate- dependent Phosphoribosylglycinamide Formyltransferase, Phosphoribosylformylglycinamide Synthase, Phosphoribosylformylglycinamidine Cyclo-Ligase, N5-Carboxyaminoimidazole Ribonucleotide Synthase, N5 -Carboxy aminoimidazole Ribonucleotide Mutase, Phosphoribosylaminoimidazole-Succinocarboxamide Synthase, Adenylosuccinate Lyase, 5- Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Transformylase, Inosine Monophosphate (IMP) Cyclohydrolase, Inosine Monophosphate (IMP) Dehydrogenase, Methionine Adenosyl Transferase, Adenosine Homocysteinase, Adenosylhomocysteine nucleosidase, S- ribosylhomocysteine Lyase, Methionine Synthase Cobalamin-Depend, Methionine Synthase Cobalamin-Independ, Serine Hydroxymethyltransferase, Methylenetetrahydrofolate Reductase, Phosphoglycerate Dehydrogenase, Phosphoserine Aminotransferase, Phosphoserine Phosphatase, Aspartate Transaminase, Glutamate Dehydrogenase, Glutamine Synthetase, Aspartate Kinase, Aspartate Semialdehyde Dehydrogenase, Homoserine Dehydrogenase, Homoserine O- Succinyltransferase, O-Succinylhomoserine Lyase, Cystathionine b-Lyase, Camellia sinensis Purine Permease (CsPUPl), Camellia sinensis Purine Permease (CsPUP3.1), Camellia sinensis Purine Permease (CsPUPlO.l), or Saccharomyces cerevisiae Purine-Cytosine Permease (FCY2).

[0077] In some embodiments, the engineered microbial cell expresses a genetically-modified Transketolase 1 , Transaldolase A, Glucose-6-Phosphate Dehydrogenase, 6- Phosphogluconolactonase, 6-Phosphogluconate Dehydrogenase, Ribose 5-Phosphate Isomerase A, 5 -Phosphoribosyl- 1 -Pyrophosphate (PRPP) Synthase, Amidophosphoribosyltransferase, Phosphoribosylamine-Glycine Ligase, Formate-dependent Phosphoribosylglycinamide Formyltransferase, Phosphoribosylformylglycinamide Synthase, Phosphoribosylformylglycinamidine Cyclo-Ligase, N5-Carboxyaminoimidazole Ribonucleotide Synthase, N5-Carboxyaminoimidazole Ribonucleotide Mutase, Phosphoribosylaminoimidazole- Succinocarboxamide Synthase, Adenylosuccinate Lyase, 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Transformylase, Inosine Monophosphate (IMP) Cyclohydrolase, Inosine Monophosphate (IMP) Dehydrogenase, Methionine Adenosyl Transferase, Adenosine Homocysteinase, Adenosylhomocysteine nucleosidase, S-ribosylhomocysteine Lyase, Methionine Synthase Cobalamin-Depend, Methionine Synthase Cobalamin-Independ, Serine Hydroxymethyltransferase, Methylenetetrahydrofolate Reductase, Phosphoglycerate Dehydrogenase, Phosphoserine Aminotransferase, Phosphoserine Phosphatase, Aspartate Attorney Docket No. TBN-00125

[0078] Transaminase, Glutamate Dehydrogenase, Glutamine Synthetase, Aspartate Kinase, Aspartate Semialdehyde Dehydrogenase, Homoserine Dehydrogenase, Homoserine O-Succinyltransferase, O- Succinylhomoserine Lyase, Cystathionine b-Lyase, Camellia sinensis Purine Permease (CsPUPl), Camellia sinensis Purine Permease (CsPUP3.1), Camellia sinensis Purine Permease (CsPUPlO.l), or Saccharomyces cerevisiae Purine-Cytosine Permease (FCY2).

[0079] Methods, reagents and tools for transforming microbial cells described herein, such as bacteria and yeast, are known in the art. General methods, reagents and tools for transforming, e.g., bacteria can be found, for example, in Sambrook et al (2001) Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Laboratory Press, New York. In some embodiments, introduction of the DNA construct or vector into a host cell is accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation, or other common techniques (See Davis et al., 1986, Basic Methods in Molecular Biology, which is incorporated herein by reference). In some embodiments, a method used to transform E. coli strains is electroporation and reference is made to Dower et al., (1988) NAR 16: 6127-6145. Indeed, any suitable method for transforming host cells may be used to produce the engineered cells described herein. It is not intended that the present disclosure be limited to any particular method for introducing nucleic acids such as constructs into host cells.

[0080] Engineered enzymes

[0081] The heterologous polypeptides described herein may be enzymes in the methylxanthine biosynthetic pathway (see e.g., FIGs. 2 and 3). For example, the heterologous polypeptides may be Xanthosine methyltransferase (XMT), theobromine synthase (MXMT), or caffeine synthase 1 (DXMT). In some embodiments, the enzymes may be 7-Methylxanthosine Synthase 1 (XMT1 COFCA), Coffee Theobromine Synthase 1 (MXMT1_COFAR or CTS1), Coffee Theobromine Synthase 2 (MXMT2_C0FAR or CTS2), Camellia irrawadiensis Caffeine (Theobromine) Synthase (ICS1), Camellia ptilophylla Caffeine (Theobromine) Synthase (PCS1), Coffee Caffeine Synthase 1 (DXMT2_COFAR or CCS1), or Coffee Caffeine Synthase 7 (DXMT1_COFAR or CtCS7). According to some embodiments, the enzymes are wildtype enzymes. In some embodiments, the enzymes are engineered to contain one or more amino acid changes relative to a wildtype version of the enzyme.

[0082] In some embodiments, the first heterologous polypeptide comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the first heterologous polypeptide comprises an amino Attorney Docket No. TBN-00125 acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1 (see Table 1). In some embodiments, the first heterologous polypeptide comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 1.

[0083] In some embodiments, the second heterologous polypeptide comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, the second heterologous polypeptide comprises an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2 (see Table 1). In some embodiments, the second heterologous polypeptide comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 2.

[0084] In some embodiments, the third heterologous polypeptide comprises an amino acid sequence of SEQ ID NO: 3. In some embodiments, the third heterologous polypeptide comprises an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 (see Table 1). In some embodiments, the first heterologous polypeptide comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 3.

[0085] In some embodiments, the cell comprises one or more heterologous polypeptide(s) comprising an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In some embodiments, the cell comprises one or more heterologous polypeptide(s) comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In some embodiments, the cell comprises a heterologous polypeptide comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the cell comprises a heterologous polypeptide comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the cell comprises a heterologous polypeptide comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the cell comprises a heterologous polypeptide comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the cell comprises a Attorney Docket No. TBN-00125 heterologous polypeptide comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the cell comprises a heterologous polypeptide comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the cell comprises a heterologous polypeptide comprising the amino acid sequence of SEQ ID NO: 10.

[0086] In some embodiments, the disclosure provides an engineered enzyme comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 (See Table 1).

[0087] In some embodiments, the engineered enzyme comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 1. In some embodiments, the engineered enzyme comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 2. In some embodiments, the engineered enzyme comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 3. In some embodiments, the engineered enzyme comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 4. In some embodiments, the engineered enzyme comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 5. In some embodiments, the engineered enzyme comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 6. In some embodiments, the engineered enzyme comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 7. In some embodiments, the engineered enzyme comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, Attorney Docket No. TBN-00125

[0088] 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 8. In some embodiments, the engineered enzyme comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 9. In some embodiments, the engineered enzyme comprises an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid(s) mutations relative to SEQ ID NO: 10.

[0089] In some embodiments, the engineered enzymes comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4 are homologs. In some embodiments, the engineered enzymes comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4 catalyze one N- methylation, producing 7-methylxanthine, which may be the first step to produce all subsequent methylxanthine products in an engineered microbial cell.

[0090] In some embodiments, the engineered enzymes comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 are homologs. In some embodiments, the engineered enzymes comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 catalyze one N-methylation, producing theobromine (3,7 -dimethylxanthine).

[0091] In some embodiments, the engineered enzymes comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 10, are homologs. In some embodiments, the engineered enzymes comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 10, catalyze two N-methylations, the first producing theobromine and the second producing caffeine (1,3 ,7-trimethylxanthine) .

[0092] In some embodiments, to produce theobromine, the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 1 or a homolog thereof (e.g., the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 4) and the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 2, or a homolog thereof (e.g., the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8) are co-expressed in an engineered microbial cell.

[0093] In some embodiments, to produce caffeine, the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 1 or a homolog thereof (e.g., the engineered enzyme comprising the Attorney Docket No. TBN-00125 amino acid sequence of SEQ ID NO: 4) and the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 3, or a homolog thereof (e.g., the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10) are co-expressed in an engineered microbial cell. In some embodiments, to produce theobromine, a nucleic acid encoding the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 1 or a homolog thereof (e.g., the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 4) and a nucleic acid encoding the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 2, or a homolog thereof (e.g., the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8) are present in an engineered microbial cell.

[0094] In some embodiments, to produce caffeine, a nucleic acid encoding the engineered enzyme, comprising the amino acid sequence of SEQ ID NO: 1 or a homolog thereof (e.g., the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 4) and a nucleic acid encoding the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 3, or a homolog thereof (e.g., the engineered enzyme comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO:

[0095] 10) are present in an engineered microbial cell.

[0096] Table 1. Heterologous polypeptide sequences Attorney Docket No. TBN-00125 Attorney Docket No. TBN-00125

[0097] Compositions

[0098] In some embodiments, the disclosure provides a composition comprising a plurality of engineered microbial cells. In some embodiments, a plurality of engineered microbial cells comprises from 2 to about 1012engineered microbial cells. In some embodiments, a plurality of engineered microbial cells comprises about 10, about 100, about 1,000, about 10,000, about 100,000, about 106, about 107, about 108, about 109, about 1010, about 1011, or about 1012engineered microbial cells. In some embodiments, a plurality of engineered microbial cells comprises from about IxlO6to about IxlO12(e.g., about IxlO6, about 2xl06, about 3xl06, about 4xl06, about 5xl06, about 6xl06, about 7xl06, about 8xl06, about 9xl06, about IxlO7, about 2xl07, about 3xl07, about 4xl07, about 5xl07, about 6xl07, about 7xl07, about 8xl07, about 9xl07, about IxlO8, about 2xl08, about 3xl08, about 4xl08, about 5xl08, about 6xl08, about 7xl08, about 8xl08, about 9xl08, about IxlO9, about 2xl09, about 3xl09, about 4xl09, about 5xl09, about 6xl09, about Attorney Docket No. TBN-00125

[0099] 7xl09, about 8xl09, about 9xl09, about IxlO10, about 2xlO10, about 3xlO10, about 4xlO10, about 5xlO10, about 6xlO10, about 7xlO10, about 8xlO10, about 9xlO10, about IxlO11, about 2xlOn, about 3xl0n, about 4xlOn, about 5xl0u, about 6xlOn, about 7xlOn, about 8xl0n, about 9xlOn, or about IxlO12) engineered microbial cells. In some embodiments, the composition comprises an engineered microbial cell as disclosed herein.

[0100] In some embodiments, the plurality of engineered microbial cells is in a cell culture composition comprising from about 2 engineered microbial cells / mL to about 1012engineered microbial cells / mL. In some embodiments, the cell culture composition comprises about 10, about 100, about 1,000, about 10,000, about 100,000, about 106, about 107, about 108, about 109, about 1010, about 1011, or about 1012engineered microbial cells / mL. In some embodiments, a plurality of engineered microbial cells comprises from about IxlO6engineered microbial cells / mL to about IxlO12engineered microbial cells / mL (e.g., about IxlO6, about 2xl06, about 3xl06, about 4xl06, about 5xl06, about 6xl06, about 7xl06, about 8xl06, about 9xl06, about IxlO7, about 2xl07, about 3xl07, about 4xl07, about 5xl07, about 6xl07, about 7xl07, about 8xl07, about 9xl07, about IxlO8, about 2xl08, about 3xl08, about 4xl08, about 5xl08, about 6xl08, about 7xl08, about 8xl08, about 9xl08, about IxlO9, about 2xl09, about 3xl09, about 4xl09, about 5xl09, about 6xl09, about 7xl09, about 8xl09, about 9xl09, about IxlO10, about 2xlO10, about 3xl010, about 4xlO10, about 5xl010, about 6xlO10, about 7xlO10, about 8xl010, about 9xlO10, about IxlO11, about 2xlOn, about 3xl0n, about 4xlOn, about 5xl0n, about 6xlOn, about 7xlOn, about 8xl0n, about 9xlOn, or about IxlO12) engineered microbial cells / mL.

[0101] In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 50 mg / L to about 200 g / L (e.g., from about 100 mg / L to about 200 g / L, from about 200 mg / L to about 200 g / L, from about 500 mg / L to about 200 g / L, from about 1 g / L to about 200 g / L, from about 2 g / L to about 200 g / L, from about 3 g / L to about 200 g / L, from about 4 g / L to about 200 g / L, from about 5 g / L to about 200 g / L, from about 6 g / L to about 200 g / L, from about 7 g / L to about 200 g / L, from about 8 g / L to about 200 g / L, from about 9 g / L to about 200 g / L, from about 10 g / L to about 200 g / L, from about 15 g / L to about 200 g / L, from about 20 g / L to about 200 g / L, from about 30 g / L to about 200 g / L, from about 40 g / L to about 200 g / L, from about 50 g / L to about 200 g / L, from about 60 g / L to about 200 g / L, from about 70 g / L to about 200 g / L, from about 80 g / L to about 200 g / L, from about 90 g / L to about 200 g / L, from about 100 g / L to about 200 g / L, from about 110 g / L to about 200 g / L, from about 120 g / L to about 200 g / L, from about 130 g / L to Attorney Docket No. TBN-00125 about 200 g / L, from about 140 g / L to about 200 g / L, from about 150 g / L to about 200 g / L, from about 160 g / L to about 200 g / L, from about 170 g / L to about 200 g / L, from about 180 g / L to about 200 g / L, from about 190 g / L to about 200 g / L, from about 195 g / L to about 200 g / L, from about 50 mg / L to about 150 g / L, from about 50 mg / L to about 100 g / L, from about 50 mg / L to about 50 g / L, from about 50 mg / L to about 25 g / L, from about 50 mg / L to about 10 g / L, from about 50 mg / L to about 5 g / L, from about 50 mg / L to about 1 g / L, from about 50 mg / L to about 900 mg / L, from about 50 mg / L to about 800 mg / L, from about 50 mg / L to about 700 mg / L, from about 50 mg / L to about 600 mg / L, from about 50 mg / L to about 500 mg / L, from about 50 mg / L to about 400 mg / L, from about 50 mg / L to about 300 mg / L, from about 50 mg / L to about 200 mg / L, from about 50 mg / L to about 100 mg / L, from about 1 g / L to about 190 g / L, from about 10 g / L to about 180 g / L, from about 20 g / L to about 170 g / L, from about 30 g / L to about 160 g / L, from about 40 g / L to about 150 g / L, from about 50 g / L to about 140 g / L, from about 60 g / L to about 130 g / L, from about 70 g / L to about 120 g / L, from about 80 g / L to about 110 g / L, or from about 90 g / L to about 100 g / L). In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 1 g / L to about 10 g / L. In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 10 g / L to about 20 g / L. In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 20 g / L to about 30 g / L. In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 30 g / L to about 40 g / L. In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 40 g / L to about 50 g / L. In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 50 g / L to about 75 g / L. In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 75 g / L to about 100 g / L. In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 100 g / L to about 125 g / L. In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 125 g / L to about 150 g / L. In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 150 g / L to about 175 g / L. In some embodiments, the plurality of engineered microbial cells produces theobromine at a yield of from about 175 g / L to about 200 g / L.

[0102] In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 50 mg / L to about 200 g / L (e.g., from about 100 mg / L to about 200 g / L, from Attorney Docket No. TBN-00125 about 200 mg / L to about 200 g / L, from about 500 mg / L to about 200 g / L, from about 1 g / L to about 200 g / L, from about 2 g / L to about 200 g / L, from about 3 g / L to about 200 g / L, from about 4 g / L to about 200 g / L, from about 5 g / L to about 200 g / L, from about 6 g / L to about 200 g / L, from about 7 g / L to about 200 g / L, from about 8 g / L to about 200 g / L, from about 9 g / L to about 200 g / L, from about 10 g / L to about 200 g / L, from about 15 g / L to about 200 g / L, from about 20 g / L to about 200 g / L, from about 30 g / L to about 200 g / L, from about 40 g / L to about 200 g / L, from about 50 g / L to about 200 g / L, from about 60 g / L to about 200 g / L, from about 70 g / L to about 200 g / L, from about 80 g / L to about 200 g / L, from about 90 g / L to about 200 g / L, from about 100 g / L to about 200 g / L, from about 110 g / L to about 200 g / L, from about 120 g / L to about 200 g / L, from about 130 g / L to about 200 g / L, from about 140 g / L to about 200 g / L, from about 150 g / L to about 200 g / L, from about 160 g / L to about 200 g / L, from about 170 g / L to about 200 g / L, from about 180 g / L to about 200 g / L, from about 190 g / L to about 200 g / L, from about 195 g / L to about 200 g / L, from about 50 mg / L to about 150 g / L, from about 50 mg / L to about 100 g / L, from about 50 mg / L to about 50 g / L, from about 50 mg / L to about 25 g / L, from about 50 mg / L to about 10 g / L, from about 50 mg / L to about 5 g / L, from about 50 mg / L to about 1 g / L, from about 50 mg / L to about 900 mg / L, from about 50 mg / L to about 800 mg / L, from about 50 mg / L to about 700 mg / L, from about 50 mg / L to about 600 mg / L, from about 50 mg / L to about 500 mg / L, from about 50 mg / L to about 400 mg / L, from about 50 mg / L to about 300 mg / L, from about 50 mg / L to about 200 mg / L, from about 50 mg / L to about 100 mg / L, from about 1 g / L to about 190 g / L, from about 10 g / L to about 180 g / L, from about 20 g / L to about 170 g / L, from about 30 g / L to about 160 g / L, from about 40 g / L to about 150 g / L, from about 50 g / L to about 140 g / L, from about 60 g / L to about 130 g / L, from about 70 g / L to about 120 g / L, from about 80 g / L to about 110 g / L, or from about 90 g / L to about 100 g / L). In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 1 g / L to about 10 g / L. In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 10 g / L to about 20 g / L. In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 20 g / L to about 30 g / L. In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 30 g / L to about 40 g / L. In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 40 g / L to about 50 g / L. In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 50 g / L to about 75 g / L. In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield Attorney Docket No. TBN-00125 of from about 75 g / L to about 100 g / L. In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 100 g / L to about 125 g / L. In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 125 g / L to about 150 g / L. In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 150 g / L to about 175 g / L. In some embodiments, the plurality of engineered microbial cells produces caffeine at a yield of from about 175 g / L to about 200 g / L.

[0103] In some embodiments, the composition comprises an engineered microbial cell comprising a first heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1 and a cell culture medium. In some embodiments, the composition comprises an engineered microbial cell comprising a second heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2 and a cell culture medium. In some embodiments, the composition comprises an engineered microbial cell comprising a third heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 and a cell culture medium.

[0104] In some embodiments, the composition comprises an engineered microbial cell comprising a first heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 and a cell culture medium. In some embodiments, the composition comprises an engineered microbial cell comprising a second heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 and a cell culture medium. In some embodiments, the composition comprises an engineered microbial cell comprising a third heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 3 and a cell culture medium.

[0105] In some embodiments, the composition comprises an engineered microbial cell comprising a first heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2, and a cell culture medium. In some embodiments, the composition comprises an engineered microbial cell comprising a first heterologous polypeptide comprising an Attorney Docket No. TBN-00125 amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, and a cell culture medium. In some embodiments, the composition comprises an engineered microbial cell comprising a first heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2, a second heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, and a cell culture medium.

[0106] In some embodiments, the composition comprises an engineered microbial cell comprising a first heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2, and a third heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, and a cell culture medium.

[0107] In some embodiments, the composition comprises an engineered microbial cell comprising a first heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, and a cell culture medium. In some embodiments, the composition comprises an engineered microbial cell comprising a first heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 3, and a cell culture medium. In some embodiments, the composition comprises an engineered microbial cell comprising a first heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, a second heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 3, and a cell culture medium.

[0108] In some embodiments, the composition comprises an engineered microbial cell comprising a first heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, Attorney Docket No. TBN-00125 a third heterologous polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 3, and a cell culture medium.

[0109] Methods of producing methylxanthines

[0110] Also provided herein are methods for producing methylxanthines. In some embodiments, the methods comprise expressing one or more heterologous polypeptides in an engineered microbial cell. In some embodiments, the methods comprise culturing an engineered microbial cell described herein. In some embodiments, the methods comprise culturing the engineered microbial cell under conditions in which the methylxanthine is produced and collecting the methylxanthine. The methylxanthine produced using these methods may be, for example, theobromine or caffeine.

[0111] In some embodiments, the method comprises expressing in an engineered microbial cell at least one of a first heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2, and a third heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0112] In some embodiments, the method comprises expressing in an engineered microbial cell at least one of a first heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 2, and a third heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 3, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0113] In some embodiments, the method comprises expressing in an engineered microbial cell a first heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0114] In some embodiments, the method comprises expressing in an engineered microbial cell a first heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 1, culturing the Attorney Docket No. TBN-00125 engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0115] In some embodiments, the method comprises expressing in an engineered microbial cell a second heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0116] In some embodiments, the method comprises expressing in an engineered microbial cell a second heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 2, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0117] In some embodiments, the method comprises expressing in an engineered microbial cell a third heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0118] In some embodiments, the method comprises expressing in an engineered microbial cell a third heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 3, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0119] In some embodiments, the method comprises expressing in an engineered microbial cell a first heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, and a second heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0120] In some embodiments, the method comprises expressing in an engineered microbial cell a first heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 1, and a second heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 2, culturing the Attorney Docket No. TBN-00125 engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0121] In some embodiments, the method comprises expressing in an engineered microbial cell a first heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, and a third heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0122] In some embodiments, the method comprises expressing in an engineered microbial cell a first heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 1, and a third heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 3, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0123] In some embodiments, the method comprises expressing in an engineered microbial cell a second heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2, and a third heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0124] In some embodiments, the method comprises expressing in an engineered microbial cell a second heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 2, and a third heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 3, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0125] In some embodiments, the method comprises expressing in an engineered microbial cell a first heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino Attorney Docket No. TBN-00125 acid sequence of SEQ ID NO: 2, and a third heterologous polypeptide comprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0126] In some embodiments, the method comprises expressing in an engineered microbial cell a first heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 2, and a third heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 3, culturing the engineered microbial cell under conditions in which the methylxanthine is produced, and collecting the methylxanthine.

[0127] Conditions under which the methylxanthine is produced will be known to those of ordinary skill in the art. In some embodiments, the engineered microbial cells can be grown under conditions that are selected, for example based upon cell culture parameters associated with optimized growth of specific microbial cells used in an embodiment of the disclosure. In some embodiments, the engineered microbial cells are grown at a temperature between 30-40° C, for a period of time from about 4 hours to about 48 hours. In some embodiments, the engineered microbial cells are grown at a pH from about 6.5 to about 8.5. In some embodiments, the pH of the cell culture media can be about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, or about 8.1. In some embodiments, the engineered microbial cells are grown in a cell culture media comprising lysogeny broth (LB), terrific broth (TB), yeast extract peptone dextrose (YPD), or yeast nitrogen base (YNB) media.

[0128] In some embodiments, the conditions for culturing the engineered microbial cells include supplementing the cell culture media with one or more nutrients which support methylxanthine production.

[0129] In some embodiments, the method comprises providing glucose to the engineered microbial cell. In some embodiments, the culturing comprises providing glucose to the engineered microbial cell to a final concentration of from about 1 g / L to about 150 g / L (e.g., about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 11 g / L, about 12 g / L, about 13 g / L, about 14 g / L, about 15 g / L, about 16 g / L, about 17 g / L, about 18 g / L, about 19 g / L, about 20 g / L, about 21 g / L, about 22 g / L, about 23 g / L, about 24 g / L, about 25 g / L, about 26 g / L, about 27 g / L, about 28 g / L, about 29 g / L, about 30 g / L, about 31 Attorney Docket No. TBN-00125 g / L, about 32 g / L, about 33 g / L, about 34 g / L, about 35 g / L, about 36 g / L, about 37 g / L, about 38 g / L, about 39 g / L, about 40 g / L, about 41 g / L, about 42 g / L, about 43 g / L, about 44 g / L, about 45 g / L, about 46 g / L, about 47 g / L, about 48 g / L, about 49 g / L, about 50 g / L, about 51 g / L, about 52 g / L, about 53 g / L, about 54 g / L, about 55 g / L, about 56 g / L, about 57 g / L, about 58 g / L, about 59 g / L, about 60 g / L, about 61 g / L, about 62 g / L, about 63 g / L, about 64 g / L, about 65 g / L, about 66 g / L, about 67 g / L, about 68 g / L, about 69 g / L, about 70 g / L, about 71 g / L, about 72 g / L, about 73 g / L, about 74 g / L, about 75 g / L, about 76 g / L, about 77 g / L, about 78 g / L, about 79 g / L, about 80 g / L, about 81 g / L, about 82 g / L, about 83 g / L, about 84 g / L, about 85 g / L, about 86 g / L, about 87 g / L, about 88 g / L, about 89 g / L, about 90 g / L, about 91 g / L, about 92 g / L, about 93 g / L, about 94 g / L, about 95 g / L, about 96 g / L, about 97 g / L, about 98 g / L, about 99 g / L, about 100 g / L, about 101 g / L, about 102 g / L, about 103 g / L, about 104 g / L, about 105 g / L, about 106 g / L, about 107 g / L, about 108 g / L, about 109 g / L, about 110 g / L, about 111 g / L, about 112 g / L, about 113 g / L, about 114 g / L, about 115 g / L, about 116 g / L, about 117 g / L, about 118 g / L, about 119 g / L, about 120 g / L, about 121 g / L, about 122 g / L, about 123 g / L, about 124 g / L, about 125 g / L, about 126 g / L, about 127 g / L, about 128 g / L, about 129 g / L, about 130 g / L, about 131 g / L, about 132 g / L, about 133 g / L, about 134 g / L, about 135 g / L, about 136 g / L, about 137 g / L, about 138 g / L, about 139 g / L, about 140 g / L, about 141 g / L, about 142 g / L, about 143 g / L, about 144 g / L, about 145 g / L, about 146 g / L, about 147 g / L, about 148 g / L, about 149 g / L, or about 150 g / L). In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 10 g / L. In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 20 g / L. In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 30 g / L. In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 40 g / L. In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 50 g / L. In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 60 g / L. In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 70 g / L. In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 80 g / L. In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 90 g / L. In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 100 g / L. In some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 110 g / L. In Attorney Docket No. TBN-00125 some embodiments, the method comprises providing glucose to the microbial cell to a final concentration of 120 g / L.

[0130] In some embodiments, the culturing comprises providing xanthosine to the engineered microbial cell. In some embodiments, the culturing comprises providing xanthosine to the engineered microbial cell to a final concentration of from about 0.1 g / L to about 200 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L).

[0131] In some embodiments, the culturing comprises providing xanthine to the engineered microbial cell. In some embodiments, the culturing comprises providing xanthine to the engineered microbial cell to a final concentration of from about 0.1 g / L to about 200 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L).

[0132] In some embodiments, the culturing comprises providing hypoxanthine to the engineered microbial cell. In some embodiments, the culturing comprises providing hypoxanthine to the engineered microbial cell to a final concentration of from about 0.1 g / L to about 200 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L).

[0133] In some embodiments, the culturing comprises providing adenine to the engineered microbial cell. In some embodiments, the culturing comprises providing adenine to the engineered Attorney Docket No. TBN-00125 microbial cell to a final concentration of from about 0.1 g / L to about 200 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L).

[0134] In some embodiments, the culturing comprises providing guanine to the engineered microbial cell. In some embodiments, the culturing comprises providing guanine to the engineered microbial cell to a final concentration of from about 0.1 g / L to about 200 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L).

[0135] In some embodiments, the culturing comprises providing inosine to the engineered microbial cell. In some embodiments, the culturing comprises providing inosine to the engineered microbial cell to a final concentration of from about 0.1 g / L to about 200 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L).

[0136] In some embodiments, the culturing comprises providing adenosine to the engineered microbial cell. In some embodiments, the culturing comprises providing adenosine to the engineered microbial cell to a final concentration of from about 0.1 g / L to about 200 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, Attorney Docket No. TBN-00125 about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L).

[0137] In some embodiments, the culturing comprises providing guanosine to the engineered microbial cell. In some embodiments, the culturing comprises providing guanosine to the engineered microbial cell to a final concentration of from about 0.1 g / L to about 200 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L).

[0138] In some embodiments, the culturing comprises providing xanthosine-5 -phosphate to the engineered microbial cell. In some embodiments, the culturing comprises providing xanthosine-5- phosphate to the engineered microbial cell to a final concentration of from about 0.1 g / L to about 200 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L).

[0139] In some embodiments, the culturing comprises providing inosine-5-phosphate to the engineered microbial cell. In some embodiments, the culturing comprises providing inosine-5- phosphate to the engineered microbial cell to a final concentration of from about 0.1 g / L to about 200 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L). Attorney Docket No. TBN-00125

[0140] In some embodiments, the culturing comprises providing two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more compounds selected from glucose, xanthosine, xanthine, hypoxanthine, adenine, guanine, inosine, adenosine, guanosine, xanthosine-5 -phosphate, and inosine-5-phosphate to the engineered microbial cell. In some embodiments, the culturing comprises providing glucose, xanthosine, xanthine, hypoxanthine, adenine, guanine, inosine, adenosine, guanosine, xanthosine-5 -phosphate, and inosine-5 -phosphate to the engineered microbial cell.

[0141] In some embodiments, the methylxanthines are collected using standard methods known in the art, e.g., separating the engineered microbial cells from the cell culture medium using centrifugation and withdrawal of methylxanthine-containing supernatant after centrifugation. In some embodiments, the methylxanthines are collected using methods described in Li, M., et al., (2017) RSC Advances 7, 56382-56389; and Jin, L., et al., (2014) PLoS One 9(8), el05368; incorporated by reference herein in their entireties. Purification and quantification of methylxanthines produced using the methods herein disclosed is conducted using standard methods and equipment known in the art, e.g., analysis of methylxanthines using high-performance liquid chromatography (HPLC) and identification of methylxanthines collected using mass spectrometry (e.g., triple-quadrupole tandem mass spectrometry).

[0142] Nucleic acids

[0143] In some embodiments, the disclosure provides a nucleic acid encoding at least one of the heterologous polypeptides herein disclosed. In some embodiments, the nucleic acid is in a plasmid (e.g., an expression vector). In some embodiments, the nucleic acid is encapsidated by a virus.

[0144] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 1.

[0145] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 1.

[0146] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 2. Attorney Docket No. TBN-00125

[0147] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 2.

[0148] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 3.

[0149] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 3.

[0150] In some embodiments, the nucleic acid encodes a first heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 1 and a second heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 2.

[0151] In some embodiments, the nucleic acid encodes a first heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 1 and a second heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 2.

[0152] In some embodiments, the nucleic acid encodes a first heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 1 and a third heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 3.

[0153] In some embodiments, the nucleic acid encodes a first heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 1 and a third heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 3.

[0154] In some embodiments, the nucleic acid encodes a second heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) Attorney Docket No. TBN-00125 identical to the amino acid sequence of SEQ ID NO: 2 and a third heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 3.

[0155] In some embodiments, the nucleic acid encodes a second heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 2 and a third heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 3.

[0156] In some embodiments, the nucleic acid encodes a first heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 1, a second heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 2, and a third heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 3.

[0157] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 1, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 2, and the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 3.

[0158] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 4.

[0159] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 4.

[0160] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 5. Attorney Docket No. TBN-00125

[0161] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 5.

[0162] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 6.

[0163] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 6.

[0164] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 7.

[0165] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 7.

[0166] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 8.

[0167] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 8.

[0168] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 9.

[0169] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 9.

[0170] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence at least 90% (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 100%) identical to the amino acid sequence of SEQ ID NO: 10. Attorney Docket No. TBN-00125

[0171] In some embodiments, the nucleic acid encodes a heterologous polypeptide comprising an amino acid sequence of SEQ ID NO: 10.

[0172] In some embodiments, the nucleic acid sequence encoding at least one of the first heterologous polypeptide, the second heterologous polypeptide and the third heterologous polypeptide cell comprises a nucleic acid sequence comprising an expression regulator.

[0173] In some embodiments, the nucleic acid sequence encoding the first heterologous polypeptide comprises a nucleic acid sequence comprising an expression regulator. In some embodiments, the nucleic acid sequence encoding the second heterologous polypeptide comprises a nucleic acid sequence comprising an expression regulator. In some embodiments, the nucleic acid sequence encoding the third heterologous polypeptide comprises a nucleic acid sequence comprising an expression regulator. The term “expression regulator,” as used herein refers to any nucleotide sequence which modulates (e.g., increases or decreases) expression of a second nucleic acid sequence operably linked thereto. In some embodiments, an expression regulator is a promoter. In some embodiments, an expression regulator is a terminator.

[0174] In some embodiments, the nucleic acid sequence encoding the first heterologous polypeptide and the second heterologous polypeptide comprises a nucleic acid sequence comprising an expression regulator.

[0175] In some embodiments, the nucleic acid sequence encoding the first heterologous polypeptide and the third heterologous polypeptide comprises a nucleic acid sequence comprising an expression regulator.

[0176] In some embodiments, the nucleic acid sequence encoding the second heterologous polypeptide and the third heterologous polypeptide comprises a nucleic acid sequence comprising an expression regulator.

[0177] In some embodiments, the nucleic acid sequence encoding any of the heterologous polypeptides herein disclosed comprises a nucleic acid sequence comprising an expression regulator.

[0178] In some embodiments, the nucleic acid comprises at least one expression regulator. In some embodiments, the expression regulator is an engineered expression regulator. In some embodiments, the expression regulator comprises at least one of a promoter and a terminator.

[0179] In some embodiments, the promoter is an inducible or constitutive promoter, such as but not limited to, pT7, pT5, pTrc, pTrp, pTac, Lac, pLacUV5, pTet, pPrp, TEF1, GALI, GPD, and ADH1 Attorney Docket No. TBN-00125 as well as promoters with one or more additional repressor binding sites such as LacO for tighter control. Known promoters in the art such as those found in the following Addgene database, available on the world wide web at blog.addgene.org / plasmids- 101-the-promoter-region, herein incorporated by reference in its entirety, may be used as described herein. In some embodiments, the promoter is a eukaryotic promoter. In some embodiments, the promoter is CMV, EFla, SV40, PGK1 (human or mouse), Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GALI, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, U6. In some embodiments, the promoter is a prokaryotic promoter. In some embodiments, the promoter is T7, T71ac, Sp6, araBAD, trp, lac, Ptac, pL, or T3. Known promoters in the art such as those found in the following iGEM catalog, available on the world wide web at parts.igem.org / Promoters / Catalog, herein incorporated by reference in its entirety, may be used as described herein. Suitable promoters include, but are not limited to constitutive promoters, regulated promoters, and inducible promoters. Appropriate promoter sequences can be obtained from genes encoding extracellular or intracellular polypeptides which are either endogenous or heterologous to the host cell. Methods for the isolation, identification and manipulation of promoters of varying strengths are available in or readily adapted from the art. See e.g., Nevoigt et al. (2006) Appl. Environ. Microbiol. 72:5266-5273, the disclosure of which is herein incorporated by reference in its entirety.

[0180] In some embodiments, the terminator is a rho-independent terminator. In some embodiments, the terminator is a rho-dependent terminator. Known terminators in the art such as those found in the following Addgene database, available on the world wide web at blog.addgene.org / plasmids-101-terminators-and-polya-signals, herein incorporated by reference in its entirety, may be used as described herein. In some embodiments, the terminator comprises T7, rrnB, TO, SV40, hGH, BGH, and rbGlob. In some embodiments, the terminator comprises the sequence motif AAUAAA (SEQ ID NO: 11). In some embodiments, a rho-independent terminator comprises an intrinsic terminator that relies on the formation of a GC-rich hairpin in the RNA transcript followed by a weakly bound poly -uracil tract. Known terminators in the art such as those found in the following iGEM catalog, parts.igem.org / Terminators / Catalog, herein incorporated by reference in its entirety, may be used in the compositions and methods disclosed herein. In some embodiments, the nucleic acid comprises double terminators. In some embodiments, the terminator is a high affinity terminator. Attorney Docket No. TBN-00125

[0181] In some embodiments, the plasmid comprises at least one codon-optimized nucleic acid sequence encoding a heterologous polypeptide. In some embodiments, this disclosure provides plasmids, cosmids, or artificial chromosomes (e.g., bacterial artificial chromosome, yeast artificial chromosomes, etc.) comprising a nucleic acid encoding at least one of the heterologous polypeptides as disclosed herein.

[0182] In some embodiments, the plasmid comprises an origin of replication sequence, a selectable marker, and one or more suitable sites for one or more nucleic acid sequences encoding heterologous polypeptides. In some embodiments, the plasmid comprises at least one expression regulator. In some embodiments, when two or more plasmids are present in an engineered microbial cell, each plasmid comprises a different antibiotic selection marker, and a different origin of replication as the other plasmids present in the engineered microbial cell.

[0183] In some embodiments, a single plasmid comprises one or more nucleic acid sequences encoding two or more heterologous polypeptides herein disclosed. In some embodiments, when a plasmid comprises two or more nucleic acid sequences encoding two or more heterologous polypeptides, the nucleic acid sequences encoding the heterologous polypeptides are designed in tandem as in a bi-cistronic (or poly-cistronic) construct as described in, for example, Vickers, C. E., et al., (December 2013) Microbial Cell Factories, 12 (96): 96; Tan, S., et al., Protein Expr Purif. 2005; 40:385-395; and Tan, S., Protein Expr Purif. 2001; 21:224-234, herein incorporated by reference in their entireties. Suitable multi-gene expression plasmids for use in embodiments of this disclosure will be apparent to those of skill in the art.

[0184] In some embodiments, the plasmid comprises elements necessary for gene expression. In some embodiments, the plasmid comprises elements necessary for gene expression in prokaryotic cells. In some embodiments, the plasmid comprises elements necessary for gene expression in eukaryotic cells.

[0185] In some embodiments, the elements of the plasmid include a promoter, a translation initiation sequence (e.g., a ribosomal binding site), a start codon, a termination codon, and a transcription termination sequence. Suitable configuration of plasmid elements for use in embodiments of this disclosure will be apparent to those of skill in the art.

[0186] In some embodiments, the plasmid comprises a Shine-Dalgarno sequence at the translation initiation site. In some embodiments, the plasmid comprises a Kozak consensus sequence. Attorney Docket No. TBN-00125

[0187] In some embodiments, the plasmid comprises a nucleic acid sequence encoding a gene product conferring resistance to an antibiotic to a bacterial host cell. In some embodiments, the antibiotic is chloramphenicol, kanamycin, tetracycline, tetracycline, spectinomycin or apramycin, or ampicillin. Additional suitable origins of replication and antibiotics for use in embodiments of this disclosure will be apparent to those of skill in the art. The disclosure is not limited in this respect.

[0188] In some embodiments, the plasmid is a low, medium, or high copy number plasmid with an origin of replication such as, but not limited to, pMBl, ColEl, R6K, pUC, CDF, pBR322, pl5A, SC101, 2-micron, or CEN. In some embodiments, the plasmid is one of the plasmids described in del Solar, G., et al., Microbiol Mol Biol Rev. 1998 Jun; 62(2): 434-464; Sikorski, R.S., et al., Genetics. 1989 May; 122(1): 19-27; or Christianson, T.W., et al., Gene. 1992 Jan 2; 110(1): 119-22, herein incorporated by reference in their entireties.

[0189] In some embodiments, the nucleic acid is encapsidated in a virus. The virus may be, for example, a bacteriophage.

[0190] Edible compositions

[0191] In some embodiments, the disclosure provides an edible composition. In some embodiments, the edible composition comprises a methylxanthine produced using an engineered microbial cell herein disclosed, and at least one consumable ingredient. The consumable ingredient may be, for example, a carbohydrate, a fat, or a protein.

[0192] In some embodiments, the methylxanthine in the edible composition is theobromine. In some embodiments, the methylxanthine in the edible composition is caffeine. In some embodiments, the edible composition is free or substantially free of cocoa. In some embodiments, the edible composition is a cocoa-free chocolate composition. In some embodiments, the cocoa-free chocolate composition shares one or more organoleptic properties with a chocolate composition produced using cocoa. In some embodiments, the cocoa-free chocolate composition has the same or substantially the same appearance, texture, color, or flavor as a chocolate composition produced using cocoa. In some embodiments, the cocoa-free chocolate composition comprises theobromine produced using a method of producing methylxanthines disclosed herein.

[0193] In some embodiments, the edible composition is free or substantially free of coffee beans. In some embodiments, the edible composition is a coffee bean-free coffee composition. In some embodiments, the coffee bean-free coffee composition shares one or more organoleptic properties Attorney Docket No. TBN-00125 with a coffee composition produced using coffee beans. In some embodiments, the coffee bean-free coffee composition has the same or substantially the same appearance, texture, color, or flavor as a coffee composition produced using coffee beans. In some embodiments, the coffee bean-free coffee composition comprises caffeine produced using a method of producing methylxanthines disclosed herein.

[0194] In some embodiments, the edible composition is free or substantially free of tea leaves. In some embodiments, the edible composition is a tea leaf-free composition. In some embodiments, the leaf-free tea composition shares one or more organoleptic properties with a tea composition produced using tea leaves. In some embodiments, the leaf-free tea composition has the same or substantially the same appearance, texture, color, or flavor as a tea composition produced using tea leaves. In some embodiments, the tea leaf-free tea composition comprises caffeine produced using a method of producing methylxanthines disclosed herein.

[0195] In some embodiments, edible composition comprises a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, fructose, galactose), a disaccharide (e.g., sucrose, lactose, maltose), or a complex carbohydrate (i.e., a carbohydrate comprising three or more saccharide molecules).

[0196] In some embodiments, edible composition comprises a fat. In some embodiments, the fat is a consumable lipid comprising polar and nonpolar compounds, such as triglycerides (TGs), diglycerides, monoglycerides, fatty acids, phospholipids, or sterols.

[0197] In some embodiments, the edible compositions comprises a protein. In some embodiments, the protein is a complete protein comprising essential amino acids. In some embodiments, the protein ingredient is from an animal-based or plant-based source.

[0198] In some embodiments, the disclosure provides an edible composition prepared by providing a methylxanthine that was produced using an engineered microbial cell disclosed herein and mixing the methylxanthine with one or more consumable ingredients (e.g., a carbohydrate, a fat, or a protein)

[0199] EXAMPLES

[0200] The following examples, which are included herein for illustration purposes only, are not intended to be limiting. Attorney Docket No. TBN-00125

[0201] Example 1: Engineering of microbial cell for production of methylxanthines

[0202] Electrocompetent cells of Escherichia coli (E. coli) are engineered to express heterologous enzymes for the production of theobromine and / or caffeine. A culture of E. coli cells is grown in LB media to an ODeoo of ~3 and concentrated 100-fold by centrifugation. The cells are washed three times in ice-cold water, and then washed once with ice-cold water 10% glycerol. The cells are transformed with a first plasmid encoding a heterologous xanthosine methyltransferase (e.g., the xanthosine methyltransferase having the amino acid sequence set forth in SEQ ID NO: 1), a second plasmid encoding a heterologous theobromine synthase (e.g., the theobromine synthase having the amino acid sequence set forth in SEQ ID NO: 2), and / or a third plasmid encoding a heterologous caffeine synthase (e.g., the caffeine synthase having the amino acid sequence set forth in SEQ ID NO: 3) using standard molecular biology methods (Dower et al., 1988 NAR 16:6127-6145).

[0203] The plasmids comprise nucleic acid sequences that encode xanthosine methyltransferase (SEQ ID NO: 1), theobromine synthase (SEQ ID NO: 2), and caffeine synthase (SEQ ID NO: 3), each of which is operably linked to one or more control sequences (e.g., promoters) useful for production of xanthosine methyltransferase, theobromine synthase, and caffeine synthase. The plasmid comprises a T7 ribosomal binding site (e.g., T7gl0 RBS) and a selection marker (e.g., an antibiotic resistance gene).

[0204] The cells are treated with an antibiotic to eliminate any untransformed cells. The cells are then aliquoted and frozen in liquid nitrogen for future use.

[0205] Example 2: Theobromine production

[0206] Recombinant E. coli cells, produced as described in Example 1, expressing a heterologous theobromine synthase (SEQ ID NO: 2) are cultivated in 100 ml LB medium containing 2% glucose and the specified antibiotic at 30 °C with shaking at 200 rpm for 96 h. The cell cultures are supplemented with additional glucose (to a 20 g-L’1final concentration). Optionally, the cell cultures are supplemented with one or more of xanthosine, xanthine, hypoxanthine, adenine, guanine, inosine, adenosine, guanosine, xanthosine-5-phosphate, and inosine-5-phosphate.

[0207] Theobromine is extracted by subjecting the cell culture to centrifugation at 12,000 x g for 10 minutes at various times, and collecting the supernatants. The culture supernatants are analyzed using high-performance liquid chromatography (HPLC). Attorney Docket No. TBN-00125

[0208] To determine the extracellular theobromine content, 1 ml of supernatant is diluted 10-fold with distilled water. After being mixed by vortexing, the diluted sample is filtered through a Millipore filter (0.22 pm) and stored at -20 °C. For the identification and quantification of the theobromine content, an HPLC system (Waters; America) equipped with a reverse-phase Cis column and ultraviolet detector at a flow rate of 1 mL min-1is used. In addition, a triplequadrupole tandem mass spectrometer (QQQ-MS / MS) system is used to further identify the intermediates and theobromine products.

[0209] Recombinant E. coli cells harboring the heterologous theobromine synthase produce a high yield of theobromine (approximately 50 mg / L1) from 20 g-L1glucose in flask batch culture, which is approximately 100-fold higher than theobromine yields from non-engineered E. coli cells.

[0210] Example 3: Preparation of an edible theobromine composition

[0211] A cocoa-free chocolate composition is prepared using theobromine produced with engineered E. coli cells described in Examples 1 and 2. The edible cocoa-free chocolate composition is prepared by mixing theobromine with food-grade ingredients including a carbohydrate, a fat, and a protein. The cocoa- free chocolate composition shares one or more organoleptic properties with a chocolate composition produced using cocoa.

[0212] Example 4: Caffeine production

[0213] Recombinant E. coli cells, produced as described in Example 1, expressing a heterologous caffeine synthase (SEQ ID NO: 3) are cultivated in 100 ml LB medium containing 2% glucose and the specified antibiotic at 30 °C with shaking at 200 rpm for 96 h. The cell cultures are supplemented with additional glucose (to a 20 g-L’1final concentration). Optionally, the cell cultures are supplemented with one or more of xanthosine, xanthine, hypoxanthine, adenine, guanine, inosine, adenosine, guanosine, xanthosine-5-phosphate, and inosine-5-phosphate.

[0214] Caffeine is extracted by subjecting the cell culture to centrifugation at 12,000 x g for 10 minutes at various times, and collecting the supernatants. The culture supernatants are analyzed using high-performance liquid chromatography (HPLC).

[0215] To determine the extracellular caffeine content, 1 ml of supernatant is diluted 10-fold with distilled water. After being mixed by vortexing, the diluted sample is filtered through a Millipore filter (0.22 pm) and stored at -20 °C. For the identification and quantification of the caffeine content, an HPLC system (Waters; America) equipped with a reverse-phase Cis column and Attorney Docket No. TBN-00125 ultraviolet detector at a flow rate of 1 mL min-1is used. In addition, a triple-quadrupole tandem mass spectrometer (QQQ-MS / MS) system is used to further identify the intermediates and caffeine products.

[0216] Recombinant E. coli cells harboring the heterologous caffeine synthase produce a high yield of caffeine (approximately 50 mg-L1) from 20 g-L1glucose in flask batch culture, which is approximately 100-fold higher than caffeine yields from non-engineered E. coli cells.

[0217] Example 5: Preparation of an edible caffeine composition

[0218] A coffee bean-free coffee composition is prepared using caffeine produced with engineered E. coli cells described in Examples 1 and 4. The edible coffee bean-free coffee composition is prepared by mixing caffeine with food-grade ingredients. The coffee bean-free coffee composition shares one or more organoleptic properties with a coffee composition produced using coffee bean.

[0219] INCORPORATION BY REFERENCE

[0220] All publications, websites, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, website, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0221] Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.

[0222] EQUIVALENTS

[0223] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

Attorney Docket No. TBN-00125CLAIMSWhat is claimed is:

1. An engineered microbial cell comprising at least one of: a) a first heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1; b) a second heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2; and c) a third heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 3.

2. The engineered microbial cell of claim 1, comprising the first heterologous polypeptide.

3. The engineered microbial cell of claim 1, comprising the second heterologous polypeptide.

4. The engineered microbial cell of claim 1, comprising the third heterologous polypeptide.

5. The engineered microbial cell of claim 1, comprising: a) the first heterologous polypeptide; and b) the second heterologous polypeptide.

6. The engineered microbial cell of claim 1, comprising: a) the first heterologous polypeptide; and b) the third heterologous polypeptide.

7. The engineered microbial cell of claim 1, comprising: a) the second heterologous polypeptide; and b) the third heterologous polypeptide.

8. The engineered microbial cell of claim 1, comprising:Attorney Docket No. TBN-00125 a) the first heterologous polypeptide; b) the second heterologous polypeptide; and c) the third heterologous polypeptide.

9. The engineered microbial cell of claim 1, wherein the cell comprises a nucleic acid sequence encoding at least one of the first heterologous polypeptide, the second heterologous polypeptide and the third heterologous polypeptide.

10. The engineered microbial cell of claim 9, wherein the nucleic acid sequence is a DNA sequence.

11. The engineered microbial cell of claim 9, wherein the nucleic acid sequence is an RNA sequence.

12. The engineered microbial cell of any one of claims 1-11, wherein the engineered microbial cell produces a methylxanthine at an elevated level compared to a non-engineered microbial cell.

13. The engineered microbial cell of claim 12, wherein the methylxanthine is theobromine.

14. The engineered microbial cell of claim 12, wherein the methylxanthine is caffeine.

15. The engineered microbial cell of any one of claims 9-14, wherein the nucleic acid sequence encoding at least one of the first heterologous polypeptide, the second heterologous polypeptide and the third heterologous polypeptide cell comprises a nucleic acid sequence comprising an expression regulator.

16. The engineered microbial cell of claim 15, wherein the expression regulator is an engineered expression regulator.

17. The engineered microbial cell of claim 15 or 16, wherein the expression regulator comprises at least one of a promoter and a terminator.Attorney Docket No. TBN-0012518. The engineered microbial cell of any one of claims 1-17, wherein at least one of the first heterologous polypeptide, the second heterologous polypeptide, and the third heterologous polypeptide is encoded in the genome of the engineered microbial cell.

19. The engineered microbial cell of any one of claims 1-17, wherein at least one of the first heterologous polypeptide, the second heterologous polypeptide, and the third heterologous polypeptide is encoded by a plasmid present in the engineered microbial cell.

20. The engineered microbial cell of any one of claims 1-19, wherein the cell comprises one or more heterologous polypeptide(s) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

21. The engineered microbial cell of any one of claims 1-20, wherein the cell comprises one or more heterologous polypeptide(s) comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

22. The engineered microbial cell of any one of claims 1-21, wherein the engineered microbial cell is a bacterial cell.

23. The engineered microbial cell of claim 22, wherein the bacterial cell is an Escherichia coli, Bacillus subtilis, Bacillus licheniformis , Bacillus megaterium, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Corynebacterium glutamicum, Lactobacillus, Streptomyces, Actinomyces, Salmonella typhimurium, Synechocystis sp., Aquifex aeolicus, Methylacidiphilum infernorum, or Thermophilus cell.

24. The engineered microbial cell of any one of claims 1-21, wherein the engineered microbial cell is a yeast cell.Attorney Docket No. TBN-0012525. The engineered microbial cell of claim 24, wherein the yeast cell is a Saccharomyces cerevisiae, Pichia pastoris, Magnaporthe oryzae, Fusarium graminearum, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Myceliophthora thermophila, Kluyvera lactis, Fusarium oxysporum, or Yarrowia lipolytica cell.

26. The engineered microbial cell of any one of claims 1-25, wherein the engineered microbial cell is engineered to comprise a genomic mutation in one or more genes.

27. The engineered microbial cell of claim 26, wherein the engineered microbial cell is engineered to comprise a mutation in one or more of the following genes: Transketolase 1, Transaldolase A, Glucose-6-Phosphate Dehydrogenase, 6-Phosphogluconolactonase, 6- Phosphogluconate Dehydrogenase, Ribose 5-Phosphate Isomerase A, 5 -Phosphoribosyl- 1- Pyrophosphate (PRPP) Synthase, Amidophosphoribosyltransferase, Phosphoribosylamine-Glycine Ligase, Formate-dependent Phosphoribosylglycinamide Formyltransferase, Phosphoribosylformylglycinamide Synthase, Phosphoribosylformylglycinamidine Cyclo-Ligase, N5-Carboxyaminoimidazole Ribonucleotide Synthase, N5-Carboxyaminoimidazole Ribonucleotide Mutase, Phosphoribosylaminoimidazole-Succinocarboxamide Synthase, Adenylosuccinate Lyase, 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Transformylase, Inosine Monophosphate (IMP) Cyclohydrolase, Inosine Monophosphate (IMP) Dehydrogenase, Methionine Adenosyl Transferase, Adenosine Homocysteinase, Adenosylhomocysteine nucleosidase, S- ribosylhomocysteine Lyase, Methionine Synthase Cobalamin-Depend, Methionine Synthase Cobalamin-Independ, Serine Hydroxymethyltransferase, Methylenetetrahydrofolate Reductase, Phosphoglycerate Dehydrogenase, Phosphoserine Aminotransferase, Phosphoserine Phosphatase, Aspartate Transaminase, Glutamate Dehydrogenase, Glutamine Synthetase, Aspartate Kinase, Aspartate Semialdehyde Dehydrogenase, Homoserine Dehydrogenase, Homoserine O- Succinyltransferase, O-Succinylhomoserine Lyase, Cystathionine b-Lyase, Camellia sinensis Purine Permease (CsPUPl), Camellia sinensis Purine Permease (CsPUP3.1), Camellia sinensis Purine Permease (CsPUPlO.l), or Saccharomyces cerevisiae Purine-Cytosine Permease (FCY2).

28. The engineered microbial cell of claim 26, wherein the engineered microbial cell expresses a genetically-modified Transketolase 1 , Transaldolase A, Glucose-6-Phosphate Dehydrogenase, 6-Attorney Docket No. TBN-00125Phosphogluconolactonase, 6-Phosphogluconate Dehydrogenase, Ribose 5-Phosphate Isomerase A, 5 -Phosphoribosyl- 1 -Pyrophosphate (PRPP) Synthase, Amidophosphoribosyltransferase, Phosphoribosylamine-Glycine Ligase, Formate-dependent Phosphoribosylglycinamide Formyltransferase, Phosphoribosylformylglycinamide Synthase, Phosphoribosylformylglycinamidine Cyclo-Ligase, N5-Carboxyaminoimidazole Ribonucleotide Synthase, N5-Carboxyaminoimidazole Ribonucleotide Mutase, Phosphoribosylaminoimidazole- Succinocarboxamide Synthase, Adenylosuccinate Lyase, 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Transformylase, Inosine Monophosphate (IMP) Cyclohydrolase, Inosine Monophosphate (IMP) Dehydrogenase, Methionine Adenosyl Transferase, Adenosine Homocysteinase, Adenosylhomocysteine nucleosidase, S-ribosylhomocysteine Lyase, Methionine Synthase Cobalamin-Depend, Methionine Synthase Cobalamin- Independ, Serine Hydroxymethyltransferase, Methylenetetrahydrofolate Reductase, Phosphoglycerate Dehydrogenase, Phosphoserine Aminotransferase, Phosphoserine Phosphatase, Aspartate Transaminase, Glutamate Dehydrogenase, Glutamine Synthetase, Aspartate Kinase, Aspartate Semialdehyde Dehydrogenase, Homoserine Dehydrogenase, Homoserine O-Succinyltransferase, O- Succinylhomoserine Lyase, Cystathionine b-Lyase, Camellia sinensis Purine Permease (CsPUPl), Camellia sinensis Purine Permease (CsPUP3.1), Camellia sinensis Purine Permease (CsPUPlO.l), or Saccharomyces cerevisiae Purine-Cytosine Permease (FCY2).

29. A composition comprising a plurality of engineered microbial cells, wherein the plurality of engineered microbial cells produces theobromine at a yield of from about 50 mg / L to about 200 mg / L.

30. The composition of claim 29, wherein the composition comprises an engineered microbial cell according to any one of claims 1-28.

31. An engineered enzyme comprising an amino acid sequence at least 90% but less than 100% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.Attorney Docket No. TBN-0012532. A composition comprising: a) an engineered microbial cell comprising at least one of: i) a first heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1 ; ii) a second heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2; and iii) a third heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and b) a cell culture medium.

33. A method of producing a methylxanthine comprising: a) expressing in an engineered microbial cell at least one of: i) a first heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1 ; ii) a second heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2; and iii) a third heterologous polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 3, b) culturing the engineered microbial cell under conditions in which the methylxanthine is produced; and c) collecting the methylxanthine.

34. The method of claim 33, wherein the culturing comprises providing glucose to the engineered microbial cell.

35. The method of claim 34, wherein glucose is provided to a concentration of 20 g / L.

36. The method of claim 33, wherein the culturing comprises providing xanthosine to the engineered microbial cell.Attorney Docket No. TBN-0012537. A nucleic acid encoding at least one of: a) a first heterologous polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1; b) a second heterologous polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 2; and c) a third heterologous coding polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 3.

38. The nucleic acid of claim 37, wherein the nucleic acid is in a plasmid or encapsidated by a virus.

39. The nucleic acid of claim 37, wherein the nucleic acid is in a plasmid.

40. The nucleic acid of claim 39, wherein the plasmid comprises at least one expression regulator.

41. The nucleic acid of claim 37, wherein the nucleic acid is encapsidated in a virus, wherein the virus is a bacteriophage.

42. The nucleic acid of claim 41 , wherein the nucleic acid comprises at least one expression regulator.

43. An edible composition comprising: a) a methylxanthine produced using an engineered microbial cell, b) at least one consumable ingredient, the consumable ingredient comprising: i) a carbohydrate, ii) a fat, or iii) a protein.

44. The edible composition of claim 43, wherein the methylxanthine is theobromine.Attorney Docket No. TBN-0012545. The edible composition of claim 43 or 44, wherein the composition is free or substantially free of cocoa.

46. An edible composition prepared by: a) providing a methylxanthine that was produced using an engineered microbial cell; and b) mixing the methylxanthine with a consumable ingredient, the consumable ingredient comprising: i) a carbohydrate; ii) a fat; or iii) a protein.

47. The edible composition of claim 46, wherein the methylxanthine is theobromine.

48. The edible composition of claim 46 or 47, wherein the composition is a cocoa-free chocolate composition.