Genetically modified bacteria capable of producing cytokinins with isoprenoid side chains

Abstract

The present invention relates generally to biotechnology engineering, and specifically to genetically modified bacteria capable of producing cytokinins with isoprenoid side chains (isoprenoid cytokinins), and their production and applications.

Description

[Technical Field]

[0001] This invention generally relates to biotechnology engineering, and more specifically to genetically modified bacteria capable of producing cytokinins having isoprenoid side chains (isoprenoid cytokinins), as well as their fabrication and application.

[0002] Background of the Invention Cytokinins are essential plant hormones that control many plant growth and developmental processes, from seed germination to plant and leaf senescence, and are characterized by their ability to induce cell division (Mok, Martin, and Mok 2000). Naturally occurring cytokinins are either aromatic side chains (aromatic cytokinins) or N of adenine. 6 It is an adenine derivative having one of the terminal isoprene-derived side chains (isoprenoid cytokinin). 6 The structure and arrangement of the side chains determine both the type and activity of cytokinins. Isoprenoid cytokinins are widely produced naturally in plants and algae, as well as in many species of bacteria, fungi, nematodes, and parasitic insects (Stirk and van Staden 2010). Natural isoprenoid cytokinins are N 6 These are (D2-isopentenyl)adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin (DZ) (Figure 1).

[0003] Isoprenoid cytokinins are derivatives of adenine or adenosine, in which the extracyclic amino group at position 6 is modified by the addition of a dimethylallyl side chain to the parent compound N 6 -(D2-isopentenyl)adenine (iP) and its riboside N 6-(D2-isopentenyl)adenosine (iPR) is produced. Hydroxylation of the dimethylallyl side chain at the 9' position yields trans-zeatin (tZ) or its riboside, trans-ribosylzeatin (tZR). Furthermore, reduction of the extraring double bond yields dihydrozeatin (DZ) or its riboside, ribosyldihydrozeatin (DZR) (Figure 1). All six compounds, iP, iPR, tZ, tZR, DZ, and DZR, are biologically active and occur naturally.

[0004] Most naturally occurring cytokinins exist as characteristic structural derivatives or forms, such as free bases, ribosides, and nucleotides, or as conjugates with glucose, xylose, or amino acid residues. In plants, free cytokinin bases are considered the most biologically active form, while glucose conjugates are thought to be either permanently inactive or reversibly stored forms, depending on the glycosylation site.

[0005] Zeatin is the primary form of cytokinin in plants. Zeatin is a derivative of adenine (N). 6 It is an adenine derivative having a hydroxylated isoprene-derived side chain at the 1 / 2 position and can exist in cis or trans configuration (Figure 1). Trans-zeatin is one of the most effective naturally occurring cytokinins. Many previous studies have been unable to distinguish between cis- and trans-zeatin by analytical methods, and the understanding of the presence and role of both compounds in observed physiological processes has been ambiguous.

[0006] Despite their structural similarity, cis-zeatin is synthesized via the tRNA pathway, while trans-zeatin, as well as its biosynthetically related compounds iP and DZ, are synthesized in plant cells via the de novo (or AMP) biosynthesis pathway.

[0007] In the tRNA pathway, cis-zeatin is a recycled product of the degradation of isopentenylated tRNA. In almost all organisms except archaea (Schaefer et al. 2015), cis-zeatin is synthesized at a very low rate by tRNA-isopentenyltransferase (tRNA-IPT), which catalyzes the prenylation of adenine 37 on specific (UNN-)tRNAs, resulting in the formation of isopentenyladenine (IP)-containing tRNAs. In the de novo cytokinin biosynthesis pathway, the first step is the N-prenylation of adenosine 5'-phosphate (AMP, ADP, or ATP) by dimethylallyl diphosphate (DMAPP), catalyzed by adenylate isopentenyltransferase (IPT, EC 2.5.1.27), which produces isopentenyladenine nucleotides (iP). In plants, the iP produced by IPT subsequently undergoes hydroxylation of the prenyl side chain to produce tZ-nucleotides. In Arabidopsis thaliana, two cytochrome P450 monooxygenases, CYP735A1 and CYP735A2, catalyze the hydroxylation reaction. CYP735A selectively utilizes iP-nucleotides over iP-nucleosides and iP. Because this reaction is stereospecific, CYP735A produces tZ-nucleotides (Takei, Yamaya, and Sakakibara 2004). In the final stages of both the de novo and tRNA pathways, the cytokinin activating enzyme cytokinin riboside 5'-monophosphate phosphoribohydrolase "Lonely Guy" (LOG, EC 3.2.2.n1) removes the ribosyl moiety, converting cytokinin nucleotides into active nucleic acid bases (Kurakawa et al. 2007). All four cytokinin nucleoside monophosphates—iPRMP, tZRMP, DZRMP, and cZRMP—are utilized by LOG.

[0008] In plant infections caused by plant pathogenic bacteria such as Agrobacterium tumefaciens, tZ biosynthesis is initiated, promoting infection. During the infection process, tZ or iP biosynthesis may occur within the bacterial cell, or a bacterial IPT gene homolog may be incorporated into the host nuclear genome and expressed in infected plant cells.

[0009] Importantly, bacterial IPTs and higher plant IPTs differ in substrate specificity (Kakimoto 2001; Sakakibara 2005). In the first aspect, bacterial IPTs such as Tmr and Tzs from A. tumefaciens use only AMP as an acceptor (whereas plant IPT enzymes selectively use ADP and ATP), forming trans-zeatin riboside 5'-monophosphate (tZRMP) (Sakakibara 2006; Kamada-Nobusada and Sakakibara 2009). Furthermore, Agrobacterium IPTs, Tzs and Tmr, can use either DMAPP or 1-hydroxy-2-methyl-2-butenyl 4-bisphosphate (HMBDP), a hydroxylation precursor of DMAPP in the methylerythritol phosphate (MEP) pathway, as side-chain donors. When DMAPP is used as a substrate, the main product is iPRMP, but when IPT utilizes HMBDP, tZRMP is formed. Therefore, the expression of Agrobacterium IPT Tmr in the chloroplasts of plant cells during infection creates a metabolic bypass for the direct synthesis of tZRMP, and an efficient supply of the isoprenoid precursor HMBDP is sequestered into the plant cell plastids. Similar to the original pathway, LOG phosphoribohydrolase ultimately releases the ribose monophosphate moiety from iP- or tZ-nucleoside monophosphates (iPRMP, tZRMP) to produce the biologically active molecules iP and tZ.

[0010] The tumor-inducing plant pathogenic bacterium A. tumefaciens possesses two IPTs, Tmr and Tzs. Tmr and Tzs are homologous proteins, both DMAPP:AMP isopentenyltransferases, but they differ from plant adenylate IPTs in the amino acids essential for substrate recognition (Chu et al., 2010). The tmr(ipt) gene is located in the T region of the Ti plasmid and mediates infection of host plants. The tmr gene is introduced from the bacterium into the plant genome, causing the host plant to produce cytokinin and thereby inducing tumorigenesis. In vitro experiments have demonstrated that Tmr transfers both DMAPP and HMBDP to AMP at similar Km values ​​(Sakakibara et al. 2005). Nopalin-producing strains of A. tumefaciens possess tzs, another gene for DMAPP:AMP isopentenyltransferase, which resides in the vir region of the Ti plasmid and does not translocate to plant cells. Tzs enables high levels of cytokinin production and secretion by these A. tumefaciens strains (Morris et al. 1993), and HMBDP or DMAPP can also be used for the production of iPRMP and tZRMP. Tzs has 51.3% protein sequence identity with Tmr. Genes encoding DMAPP:AMP isopentenyltransferase homologous to tmr / tzs are present in other Agrobacterium species such as A. vitis and A. rhizogenes, as well as in other plant pathogenic bacteria such as Pseudomonas syringae pv. savastanoi, Pseudomonas solanacearum, Pantoea agglomerans, and Rhodococcus fascians (Kakimoto, 2003).

[0011] The isopentenyl donor dimethylallyl diphosphate (DMAPP) and its precursor 1-hydroxy-2-methyl-2-butenyl 4-diphosphate (HMBDP) are produced in plant chloroplasts and bacteria, as well as in Bacillus subtilis, via the methylerythritol phosphate (MEP) pathway. In the MEP pathway, pyruvate and glyceraldehyde-3-phosphate are condensed by the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (Dxs, EC 2.2.1.7) into a metabolic cascade that ultimately produces HMBDP, DMAPP, and isoprene or larger terpenoid compounds. The first step of the DXS-mediated MEP pathway is the rate-limiting step in isoprenoid production in plants and bacteria (Julsing et al. 2007).

[0012] Summary of the Invention The natural biosynthesis rate of cytokinins via the de novo pathway during infection in plant pathogenic bacteria is very low. This relies on the expression of bacterial-derived IPT enzymes, LOG enzymes, and building blocks supplied by the host cell's metabolism. Such infection-based systems cannot be easily transitioned to industrial-scale production. Due to the high activity and usability of cytokinin hormones in agricultural applications, there is a need for the efficient and sustainable production of cytokinins such as iP, tZ, and ribosides tZR and iPR in genetically and biotechnically suitable host strains.

[0013] The object of the present invention is to provide means that enable the more efficient production of cytokinins having isoprenoid side chains (isoprenoid cytokinins), such as tZ and iP, and their ribosides, tZR and iPR. More specifically, the object of the present invention is to provide means that enable the production of cytokinins having isoprenoid side chains (isoprenoid cytokinins), such as tZ and iP, and their ribosides, tZR and iPR, in higher nominal yields.

[0014] This is achieved by the inventors who manipulated a bacterial strain modified to express a heterologous polypeptide having an adenylate isopentenyltransferase activity and optionally to increase the protein expression of a polypeptide having a cytokinin riboside 5'-monophosphate phosphoribohydrolase activity. As shown in the examples, such manipulated bacterial strains surprisingly exhibit an unusually high titer of isoprenoid cytokinins exceeding 10 mg / L in the supernatant. As a result, this means that the situation of plant cell infection is no longer required, and biosynthetic substrates and cofactors for the efficient biosynthesis of isoprenoid cytokinins such as tZ and iP, and their ribosides tZR and iPR, are effectively supplied by the manipulated bacterial cells.

[0015] Thus, in a first aspect, the present invention provides a bacterium expressing a heterologous polypeptide having an adenylate isopentenyltransferase activity. More specifically, the present invention provides a bacterium that expresses a heterologous polypeptide having an adenylate isopentenyltransferase activity and optionally is modified such that the protein expression of a polypeptide having a cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is increased as compared to a bacterium that is otherwise identical but has no modification.

[0016] In a second aspect, the present invention further provides a method for producing a cytokinin or a riboside derivative thereof, particularly an isoprenoid cytokinin or a riboside derivative thereof, the method comprising culturing a bacterium according to the present invention in a suitable culture medium under suitable culture conditions.

[0017] The present invention can be summarized by the following items:

[0018] 1. A bacterium expressing a heterologous polypeptide having an adenylate isopentenyltransferase activity.

[0019] 2. The bacterium according to item 1, wherein the polypeptide having isopentenyl transferase activity of adenylic acid is selected from the group consisting of: i) a polypeptide containing any one of the amino acid sequences of SEQ ID NOs: 1 to 33; and ii) a polypeptide containing an amino acid sequence having at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 33.

[0020] 3. The bacterium according to item 1, wherein the polypeptide having isopentenyl transferase activity of adenylic acid is selected from the group consisting of: i) a polypeptide containing any one of the amino acid sequences of SEQ ID NOs: 1 to 10; and ii) a polypeptide containing an amino acid sequence having at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 10.

[0021] 4. The bacterium according to item 1, wherein the polypeptide having isopentenyl transferase activity of adenylic acid is selected from the group consisting of: i) a polypeptide containing the amino acid sequence of SEQ ID NO: 1; and ii) a polypeptide containing an amino acid sequence having at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0022] 5. A bacterium described in any one of items 1 to 4, wherein the bacterium contains an exogenous nucleic acid molecule comprising a nucleotide sequence encoding the heterologous polypeptide described above.

[0023] 6. The bacterium described in item 5, further comprising a promoter in which an exogenous nucleic acid molecule functions to induce the production of mRNA molecules in the bacterium and is operably linked to a nucleotide sequence encoding the heterologous polypeptide.

[0024] 7. Bacteria as described in item 5 or 6, in which an exogenous nucleic acid molecule is the vector.

[0025] 8. Bacteria as described in item 5 or 6, in which exogenous nucleic acid molecules are stably incorporated into the bacterial genome.

[0026] 9. A bacterium described in any one of items 1 through 8, wherein the protein expression of a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is modified to be increased compared to the same bacterium, but without modification.

[0027] 10. The bacterium described in item 9, wherein increased protein expression of a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is achieved by increasing the copy number of the gene encoding the polypeptide.

[0028] 11. The bacteria described in item 10, wherein an increase in the copy number of a gene is achieved by introducing one or more exogenous nucleic acid molecules (e.g., one or more vectors) into the bacteria, which contain a gene that is manipulably linked to a promoter that functions to induce the production of mRNA molecules in the bacteria.

[0029] 12. A bacterium described in any one of items 9 to 11, wherein the bacterium contains an exogenous nucleic acid molecule (such as a vector) comprising a nucleotide sequence encoding a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity.

[0030] 13. The bacterium described in item 12, further comprising a promoter in which an exogenous nucleic acid molecule functions to induce the production of mRNA molecules in the bacterium and is operably linked to a nucleotide sequence encoding a polypeptide.

[0031] 14. A bacterium described in any one of items 11 through 13, in which an exogenous nucleic acid molecule is the vector.

[0032] 15. Bacteria in which an exogenous nucleic acid molecule is stably incorporated into the bacterial genome, as described in any one of items 11 to 13.

[0033] 16. A bacterium described in any one of items 9 to 15, wherein increased protein expression of a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is achieved by modifying the ribosome binding site.

[0034] 17. A bacterium described in any one of items 9 to 16, wherein increased protein expression of a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is achieved by increasing the strength of a promoter manipulably linked to the gene encoding the polypeptide.

[0035] 18. A bacterium according to any one of items 9 to 17, wherein the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is selected from the group consisting of i) a polypeptide comprising any one amino acid sequence of SEQ ID NOs. 34 to 62, and ii) a polypeptide comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one amino acid sequence of SEQ ID NOs. 34 to 62.

[0036] 19. A bacterium according to any one of items 9 to 18, wherein the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is selected from the group consisting of i) a polypeptide comprising any one amino acid sequence of SEQ ID NOs. 34 to 44; and ii) a polypeptide comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one amino acid sequence of SEQ ID NOs. 34 to 44.

[0037] 20. A bacterium according to any one of items 9 to 18, wherein the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is selected from the group consisting of i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 34; and ii) a polypeptide comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 34.

[0038] 21. A bacterium according to any one of items 9 to 20, wherein the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is a bacterial polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity.

[0039] 22. A bacterium described in any one of items 1 to 21, wherein the protein expression of a polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is further modified to be increased compared to the same bacterium, but without modification.

[0040] 23. The bacterium described in item 22, wherein increased protein expression of a polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is achieved by increasing the copy number of the gene encoding the polypeptide.

[0041] 24. The bacteria described in item 23, in which an increase in the copy number of a gene is achieved by introducing one or more exogenous nucleic acid molecules (e.g., one or more vectors) into the bacteria, which contain a gene that is manipulably linked to a promoter that functions to induce the production of mRNA molecules in the bacteria.

[0042] 25. A bacterium described in any one of items 22 to 24, wherein the bacterium contains an exogenous nucleic acid molecule (such as a vector) comprising a nucleotide sequence encoding a polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity.

[0043] 26. The bacterium described in item 25, further comprising a promoter in which an exogenous nucleic acid molecule functions to induce the production of mRNA molecules in the bacterium and is operably linked to a nucleotide sequence encoding a polypeptide.

[0044] 27. A bacterium described in any one of items 24 through 26, in which an exogenous nucleic acid molecule is the vector.

[0045] 28. Bacteria described in any one of items 24 to 26, in which an exogenous nucleic acid molecule is stably incorporated into the bacterial genome.

[0046] 29. A bacterium described in any one of items 22 to 28, wherein increased protein expression of a polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is achieved by modifying the ribosome binding site.

[0047] 30. A bacterium described in any one of items 22 to 29, wherein increased protein expression of a polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is achieved by increasing the strength of a promoter manipulably linked to the gene encoding the polypeptide.

[0048] 31. A bacterium according to any one of items 22 to 30, wherein the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is selected from the group consisting of i) polypeptides comprising any one amino acid sequence of SEQ ID NOs. 63 to 70; and ii) polypeptides comprising amino acid sequences having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one amino acid sequence of SEQ ID NOs. 63 to 70.

[0049] 32. A bacterium according to any one of items 22 to 31, wherein the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is selected from the group consisting of i) a polypeptide comprising any one amino acid sequence of SEQ ID NOs. 63 to 65; and ii) a polypeptide comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one amino acid sequence of SEQ ID NOs. 63 to 65.

[0050] 33. A bacterium according to any one of items 22 to 32, wherein the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is selected from the group consisting of i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 63; and ii) a polypeptide comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 63.

[0051] 34. A bacterium according to any one of items 22 to 32, wherein the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is selected from the group consisting of i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 64; and ii) a polypeptide comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 64.

[0052] 35. A bacterium described in any one of items 22 to 34, wherein the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is a bacterial polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity.

[0053] 36. A bacterium described in any one of items 1 to 35, wherein the expression and / or activity of at least one enzyme involved in the purine nucleotide biosynthesis pathway (e.g., at least one enzyme involved in the adenosine monophosphate biosynthesis pathway) is further modified to be increased compared to the same bacterium, but without modification.

[0054] 37. The bacterium described in item 36, wherein at least one enzyme involved in the purine nucleotide biosynthesis pathway is selected from the group consisting of an enzyme having ribose-phosphate diphosphokinase activity, an enzyme having amide phosphoribosyltransferase activity, an enzyme having formyltetrahydrofolate deformylase activity, an enzyme having adenylosuccinate lyase activity, an enzyme having phosphoribosylaminoimidazole-carboxamideformyltransferase activity, an enzyme having adenylosuccinate synthase activity, and an enzyme having adenosine kinase activity.

[0055] 38. A bacterium described in any one of items 1 through 37, wherein the expression and / or activity of at least one endogenous enzyme involved in the purine nucleotide degradation pathway is further modified to be reduced compared to the same bacterium, but without modification.

[0056] 39. The bacterium described in item 38, wherein at least one endogenous enzyme involved in the purine nucleotide degradation pathway is selected from the group consisting of enzymes having purine nucleoside phosphorylase activity and enzymes having adenosine-phosphoribosyltransferase activity.

[0057] 40. A bacterium as described in item 38 or 39, wherein at least one endogenous enzyme involved in the purine nucleotide degradation pathway is an enzyme having purine nucleoside phosphorylase activity.

[0058] 41. A bacterium described in any one of items 38 to 40, wherein at least one endogenous enzyme involved in the purine nucleotide degradation pathway is an enzyme having adenosine-phosphoribosyltransferase activity.

[0059] 42. A bacterium described in any one of items 1 through 41, wherein the expression and / or activity of at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is further modified to be reduced compared to the same bacterium, but without modification.

[0060] 43. The bacterium described in item 42, wherein at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is selected from the group consisting of enzymes having IMP dehydrogenase activity and enzymes having GMP synthetase activity.

[0061] 44. A bacterium as described in item 42 or 43, wherein at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is an enzyme having IMP dehydrogenase activity.

[0062] 45. A bacterium described in any one of items 42 to 44, wherein at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is an enzyme having GMP synthetase activity.

[0063] 46. ​​A bacterium described in any one of items 1 through 45, wherein the protein expression of a polypeptide having cytochrome P450 monooxygenase (CYP450) activity is further modified to be increased compared to the same bacterium, but without modification.

[0064] 47. The bacterium described in item 46, wherein increased protein expression of a polypeptide having cytochrome P450 monooxygenase (CYP450) activity is achieved by increasing the copy number of the gene encoding the polypeptide.

[0065] 48. The bacteria described in item 47, in which an increase in the copy number of a gene is achieved by introducing one or more exogenous nucleic acid molecules (e.g., one or more vectors) into the bacteria, which contain a gene that is manipulably linked to a promoter that functions to induce the production of mRNA molecules in the bacteria.

[0066] 49. A bacterium described in any one of items 1 through 48, wherein the bacterium contains an exogenous nucleic acid molecule (such as a vector) comprising a nucleotide sequence encoding a polypeptide having cytochrome P450 monooxygenase (CYP450) activity.

[0067] 50. The bacterium described in item 49, further comprising a promoter in which an exogenous nucleic acid molecule functions to induce the production of mRNA molecules in the bacterium and is operably linked to a nucleotide sequence encoding a polypeptide.

[0068] 51. A bacterium described in any one of items 48 through 50, in which an exogenous nucleic acid molecule is the vector.

[0069] 52. Bacteria described in any one of items 48 to 50, in which an exogenous nucleic acid molecule is stably incorporated into the bacterial genome.

[0070] 53. A bacterium according to any one of items 46 to 52, wherein the polypeptide having cytochrome P450 monooxygenase (CYP450) activity is selected from the group consisting of i) a polypeptide comprising any one amino acid sequence of SEQ ID NOs. 93 to 95, and ii) a polypeptide comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one amino acid sequence of SEQ ID NOs. 93 to 95.

[0071] 54. A bacterium described in any one of items 1 through 53, wherein the bacterium belongs to a family selected from the group consisting of Enterobacteriaceae, Bacillus, Lactobacillus, and Corynebacterium.

[0072] 55. A bacterium described in any one of items 1 through 53, wherein the bacterium belongs to a family selected from the group consisting of Bacillus and Corynebacterium.

[0073] 56. A bacterium belonging to the Bacillus family, as described in any one of items 1 through 53.

[0074] 57. A bacterium belonging to the family Corynebacterium, described in any one of items 1 through 53.

[0075] 58. A bacterium listed in any one of items 1 through 53, whose bacterium belongs to the genera Bacillus, Lactococcus, Lactobacillus, Clostridium, Corynebacterium, Geobacillus, Thermoanaerobacterium, Streptococcus, Pseudomonas, Streptomyces, Escherichia, Sigella, Acinetobacter, Citrobacter, Salmonella, Klebsiella, Enterobacter, Erwinia, Klebella, Serratia, Sedesea, Morganella, Hafnia, Edwardsiella, Providencia, Proteus, or Yersinia.

[0076] 59. A bacterium described in any one of items 1 through 53, wherein the bacterium belongs to the genus Bacillus or Corynebacterium.

[0077] 60. A bacterium belonging to the genus Bacillus, as described in any one of items 1 through 53.

[0078] 61. A bacterium belonging to the genus Corynebacterium, as described in any one of items 1 through 53.

[0079] 62. A bacterium that is Bacillus subtilis, as described in any one of items 1 through 53.

[0080] 63. A bacterium described in any one of items 1 through 53, wherein the bacterium is Corynebacterium stationis.

[0081] 64. A method for producing isoprenoid cytokinin or its riboside derivative, comprising culturing a bacterium described in any one of items 1 to 63 in a suitable culture medium under suitable culture conditions.

[0082] 65. Isoprenoid cytokinin or its riboside derivatives are trans-zeatin (tZ), trans-zeatin riboside (tZR), N 6The method described in item 64, selected from the group consisting of -(D2-isopentenyl)adenine (iP), N(6)-(dimethylallyl)adenosine (iPR), dihydrozeatin (DZ), ribosyldihydrozeatin (DZR), and combinations thereof.

[0083] 66. The method according to item 65, wherein the isoprenoid cytokinin or its riboside derivative is trans-zeatin (tZ) and trans-zeatin riboside (tZR), respectively.

[0084] 67. The method described in item 64, wherein the method is for producing trans-zeatin (tZ). [Brief explanation of the drawing]

[0085] [Figure 1] This figure shows isoprenoid cytokinins: A) Ribosides: N6-(D2-isopentenyl)adenine riboside (iPR), trans-zeatin riboside (tZR), cis-zeatin riboside (cZR), and dihydrozeatin riboside (DZR). B) Free bases: N6-(D2-isopentenyl)adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin (DZ). [Figure 2] This is a schematic diagram of the MEP metabolic pathway. [Figure 3] This figure shows the isoprenoid cytokinin biosynthesis pathway via heterologous expression of IPT (EC 2.5.1.27) and LOG (EC 3.2.2.n1). [Figure 4] This figure shows the production of trans-zeatin and related isoprenoid cytokinin in Bacillus subtilis strains in a shaker-scale experiment. VKPM B2116 - parent strain, TZAB14, TZAB15 - IPT-LOG. [Figure 5]This figure shows the production of trans-zeatin and related isoprenoid cytokinin in Bacillus subtilis strains using adenine sulfate in a shaker-scale experiment. VKPM B2116 - parent strain, TZAB14 - IPT-LOG. [Figure 6] This figure shows the production of trans-zeatin and related isoprenoid cytokinins by Bacillus subtilis strains possessing the IPT and LOG genes and the DXS gene in a shaker-scale experiment. VKPM B2116 - parent strain, TZAB15 - IPT-LOG, TZAB43 - IPT-LOG-DXS. [Figure 7] This figure shows the production of trans-zeatin and related isoprenoid cytokinins by Bacillus subtilis strains expressing IPT (SEQ ID NO: 1) and LOG (SEQ ID NO: 34) or IPT (SEQ ID NO: 2) and LOG (SEQ ID NO: 34) after 28 hours in a shaker-scale experiment. VKPM B2116 - Parent strain, TZAB1, TZAB2, TZAB3, TZAB4 - Strains expressing IPT (SEQ ID NO: 2) and LOG (SEQ ID NO: 34), TZAB14, TZAB15 - Strains expressing IPT (SEQ ID NO: 1) and LOG (SEQ ID NO: 34). [Figure 8] This figure shows the cytokinin production in Bacillus subtilis strain 168, which overexpresses IPT (SEQ ID NO: 1), LOG (SEQ ID NO: 34), and DXS (SEQ ID NO: 63), after 24 hours in a shaker-scale experiment. [Figure 9] This figure shows the cytokinin production in Bacillus subtilis strain RB50, which overexpresses IPT (SEQ ID NO: 1), LOG (SEQ ID NO: 34), and DXS (SEQ ID NO: 63), after 18 hours in a shaker-scale experiment. [Figure 10]This figure shows the cytokinin production in Bacillus subtilis strain VKPM B2116 with overexpression of IPT (SEQ ID NO: 1), LOG (SEQ ID NO: 34), and DXS (SEQ ID NO: 63) after 24 hours in a shaker-scale experiment. [Figure 11] This figure shows the cytokinin production in Escherichia coli strain BL21(DE3) with overexpression of IPT (SEQ ID NO: 1), LOG (SEQ ID NO: 34), and DXS (SEQ ID NO: 63) after 10 hours in a shaker-scale experiment. [Figure 12] This figure shows the cytokinin production in Corynebacterium stationis with overexpression of IPT (SEQ ID NO: 1), LOG (SEQ ID NO: 34), and DXS (SEQ ID NO: 63) after 48 hours in a shaker-scale experiment. [Figure 13] This figure shows cytokinin production in Bacillus subtilis strains expressing LOG8 (SEQ ID NO: 41) in combination with various IPTs (SEQ ID NO: 1, 6, 7, and 9) after 18 hours in a shaker-scale experiment. [Figure 14] This figure shows cytokinin production in Bacillus subtilis strains expressing IPT (SEQ ID NO: 1) in combination with various LOGs (SEQ ID NOs: 34-44) after 18 hours in a shaker-scale experiment.

[0086] The present invention will now be described in more detail below.

[0087] Detailed description of the invention Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of biochemistry, genetics, and microbiology.

[0088] All methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the present invention, and preferred methods and materials are described herein. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to limit unless otherwise specified.

[0089] Unless otherwise specified, the implementation of this invention will involve conventional methods of cell biology, cell culture, molecular biology, transgenic biology, microbiology, and recombinant DNA, within the scope of the skill of those skilled in the art. Such methods are adequately described in the literature. For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA);Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press);Oligonucleotide Synthesis (MJ Gait ed., 1984);Mullis et al. al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (BD Harries & SJ Higgins eds. 1984); Transcription And Translation (BD Hames & SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning See the series *Methods In ENZYMOLOGY* (1984) (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.).

[0090] The bacteria of the present invention As described above, the inventors have manipulated bacterial strains that are modified to a) express a heterologous polypeptide having adenylate isopentenyltransferase activity, and optionally b) increase the protein expression of a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity. As shown in the examples, such manipulated bacterial strains surprisingly exhibit abnormally high titers of isoprenoid cytokinins exceeding 10 mg / L in the supernatant. Consequently, this means that a plant cell infection condition is no longer required, and biosynthetic substrates and cofactors for the efficient biosynthesis of isoprenoid cytokinins such as tZ and iP, as well as their ribosides tZR and iPR, are effectively supplied by the manipulated bacterial cells.

[0091] Accordingly, in a first embodiment, the present invention provides a bacterium that expresses a heterologous polypeptide having adenylate isopentenyltransferase activity. More specifically, the present invention provides a bacterium that a) expresses a heterologous polypeptide having adenylate isopentenyltransferase activity, and optionally b) has been modified to increase the protein expression of a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity, while remaining unmodified compared to the same bacterium.

[0092] Adenyl isopentenyltransferase (IPT) is a clearly defined class of enzymes that catalyze the N-prenylation of adenosine 5'-phosphate (AMP, ADP, or ATP) by either dimethylallyl diphosphate (DMAPP) or 1-hydroxy-2-methyl-2-butenyl 4-diphosphate (HMBDP), which is the first step in the de novo cytokinin biosynthesis pathway. There are two types of IPT (EC 2.5.1.27 and EC 2.5.1.112). IPT enzymes are found in bacteria and plants. IPTs derived from bacteria such as Agrobacterium, which include two IPT homologs, Tzs and Tmr, use AMP as the prenyl acceptor and HMBDP or DMAPP as the donor (EC 2.5.1.27). The product of the reaction with HMBDP is the tZ nucleotide. Furthermore, bacterial IPT belongs to the Pfam family IPT (PF01745) in the Pfam database of protein families and domains (https: / / pfam.xfam.org / family / ipt). The IPT Pfam domain gene is phylogenetically dispersed and is found only in some members of Actinobacteria, Cyanobacteria, α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria, as well as the eukaryote Dictyostelium discoideum (Nishii et al., 2018). Higher plant IPT mainly uses ATP or ADP as the prenyl acceptor and DMAPP as the donor (EC 2.5.1.112) and belongs to the Pfam family IPPT (PF01715) (https: / / pfam.xfam.org / family / ippt). The product of the reaction is iP, which is hydroxylated at the prenyl side chain to produce a tZ nucleotide.

[0093] Agrobacterium tumefaciens Tzs (IPT(PF01745); EC 2.5.1.27; Sequence ID 1) consists of two domains: an N-terminal domain with five-stranded parallel β-sheets surrounded by three α-helices (α1-α3), and a C-terminal domain with five α-helices (α4-α8). The N-terminal domain contains a nucleotide-binding p-loop motif Gly-8-Pro-Thr-Cys-Ser-Gly-Lys-Thr-15 and is structurally related to the p-loop-containing nucleoside triphosphate hydrolase (pNTPase) superfamily. The C-terminal side of α8 extends to the N-terminus and attaches to it. The interface between the domains forms a solvent-exposed channel that binds AMP. The prenylation site of AMP binds to Asp-33 and Ser-45. DMAPP binds to Asp-173, Tyr-211, and His-214. Thr-10, Asp-33, and Arg-138 are completely conserved within IPT. In Tzs, the hydrophilic region formed by the side chain contains two key amino acid residues in the substrate-binding pocket, His-214 and Asp-173, which distinguish between the presence and absence of a hydroxyl group in the prenyl-donor substrate, thereby enabling the use of HMBDP (Sugawara et al. 2008).

[0094] Generally, polypeptides having adenylate isopentenyltransferase activity used in accordance with the present invention are heterologous to bacteria; that is, these polypeptides are not normally found in bacteria or produced (i.e., expressed) by bacteria, and originate from different species. Furthermore, polypeptides having adenylate isopentenyltransferase activity originate from or correspond to members of the Pfam family IPT (PF01745), and preferably are bacterial polypeptides having adenylate isopentenyltransferase activity. "Bacterial polypeptide having adenylate isopentenyltransferase activity" means that the polypeptide having adenylate isopentenyltransferase activity originates from bacteria such as Agrobacterium tumefaciens.

[0095] Polypeptides possessing adenylate isopentenyltransferase activity best suited for the biosynthesis of isoprenoid cytokinins, including tZ, iP, tZR, and iPR, are enzymes that can utilize both HMBDP and DMAPP as prenyl donors, such as Tzs or Tmr from Agrobacterium tumefaciens. Furthermore, these belong to the Pfam family IPT (PF01745) and contain an Asp residue at position 173 of SEQ ID NO: 1, a Tyr residue at position 211 of SEQ ID NO: 1, and / or a His residue at position 214 of SEQ ID NO: 1.

[0096] Polypeptides having adenylate isopentenyltransferase activity used in accordance with the present invention may be, for example, polypeptides having adenylate isopentenyltransferase activity selected from the group consisting of i) polypeptides comprising any one amino acid sequence of SEQ ID NOs: 1 to 33; and ii) polypeptides comprising amino acid sequences having at least about 50%, for example, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to any one amino acid sequence of SEQ ID NOs: 1 to 33.

[0097] Polypeptides having adenylate isopentenyltransferase activity used in accordance with the present invention may be, for example, polypeptides having adenylate isopentenyltransferase activity selected from the group consisting of i) polypeptides comprising any one amino acid sequence of SEQ ID NOs: 1 to 33; and ii) polypeptides comprising amino acid sequences having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to any one amino acid sequence of SEQ ID NOs: 1 to 33.

[0098] Polypeptides having adenylate isopentenyltransferase activity used in accordance with the present invention may be, for example, polypeptides having adenylate isopentenyltransferase activity selected from the group consisting of i) polypeptides comprising any one amino acid sequence of SEQ ID NOs: 1 to 10; and ii) polypeptides comprising amino acid sequences having at least about 85%, for example, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to any one amino acid sequence of SEQ ID NOs: 1 to 10.

[0099] Polypeptides having adenylate isopentenyltransferase activity used in accordance with the present invention may be, for example, polypeptides having adenylate isopentenyltransferase activity selected from the group consisting of i) polypeptides comprising any one amino acid sequence of SEQ ID NOs: 1 to 10; and ii) polypeptides comprising amino acid sequences having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to any one amino acid sequence of SEQ ID NOs: 1 to 10.

[0100] Polypeptides having adenylate isopentenyltransferase activity used in accordance with the present invention may be, for example, polypeptides having adenylate isopentenyltransferase activity selected from the group consisting of i) polypeptides comprising any one amino acid sequence of SEQ ID NOs: 1 to 10; and ii) polypeptides comprising amino acid sequences having at least about 85%, for example, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to any one amino acid sequence of SEQ ID NOs: 1 to 10.

[0101] According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 50%, for example, at least 55%, sequence identity with amino acid sequence SEQ ID NO: 1. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 60%, for example, at least 65%, sequence identity with amino acid sequence SEQ ID NO: 1. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with amino acid sequence SEQ ID NO: 1. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with amino acid sequence SEQ ID NO: 1. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with amino acid sequence SEQ ID NO: 1. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 95%, for example, at least 97%, sequence identity with amino acid sequence SEQ ID NO: 1. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises the amino acid sequence of SEQ ID NO: 1.

[0102] According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 50%, for example, at least 55%, sequence identity with amino acid sequence SEQ ID NO: 2. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 60%, for example, at least 65%, sequence identity with amino acid sequence SEQ ID NO: 2. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with amino acid sequence SEQ ID NO: 2. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with amino acid sequence SEQ ID NO: 2. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with amino acid sequence SEQ ID NO: 2. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 95%, for example, at least 97%, sequence identity with amino acid sequence SEQ ID NO: 2. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises the amino acid sequence of SEQ ID NO: 2.

[0103] According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 50%, for example, at least 55%, sequence identity with amino acid sequence SEQ ID NO: 3. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 60%, for example, at least 65%, sequence identity with amino acid sequence SEQ ID NO: 3. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with amino acid sequence SEQ ID NO: 3. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with amino acid sequence SEQ ID NO: 3. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with amino acid sequence SEQ ID NO: 3. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 95%, for example, at least 97%, sequence identity with amino acid sequence SEQ ID NO: 3. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises the amino acid sequence of SEQ ID NO: 3.

[0104] According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 50%, for example, at least 55%, sequence identity with amino acid sequence SEQ ID NO: 4. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 60%, for example, at least 65%, sequence identity with amino acid sequence SEQ ID NO: 4. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with amino acid sequence SEQ ID NO: 4. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with amino acid sequence SEQ ID NO: 4. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with amino acid sequence SEQ ID NO: 4. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 95%, for example, at least 97%, sequence identity with amino acid sequence SEQ ID NO: 4. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises the amino acid sequence of SEQ ID NO: 4.

[0105] According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 50%, for example, at least 55%, sequence identity with amino acid sequence SEQ ID NO: 5. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 60%, for example, at least 65%, sequence identity with amino acid sequence SEQ ID NO: 5. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with amino acid sequence SEQ ID NO: 5. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with amino acid sequence SEQ ID NO: 5. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with amino acid sequence SEQ ID NO: 5. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 95%, for example, at least 97%, sequence identity with amino acid sequence SEQ ID NO: 5. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises the amino acid sequence of SEQ ID NO: 5.

[0106] According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 50%, for example, at least 55%, sequence identity with amino acid sequence SEQ ID NO: 6. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 60%, for example, at least 65%, sequence identity with amino acid sequence SEQ ID NO: 6. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with amino acid sequence SEQ ID NO: 6. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with amino acid sequence SEQ ID NO: 6. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with amino acid sequence SEQ ID NO: 6. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 95%, for example, at least 97%, sequence identity with amino acid sequence SEQ ID NO: 6. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises the amino acid sequence of SEQ ID NO: 6.

[0107] According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 50%, for example, at least 55%, sequence identity with amino acid sequence SEQ ID NO: 7. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 60%, for example, at least 65%, sequence identity with amino acid sequence SEQ ID NO: 7. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with amino acid sequence SEQ ID NO: 7. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with amino acid sequence SEQ ID NO: 7. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with amino acid sequence SEQ ID NO: 7. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 95%, for example, at least 97%, sequence identity with amino acid sequence SEQ ID NO: 7. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises the amino acid sequence of SEQ ID NO: 7.

[0108] According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 50%, for example, at least 55%, sequence identity with amino acid sequence SEQ ID NO: 8. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 60%, for example, at least 65%, sequence identity with amino acid sequence SEQ ID NO: 8. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with amino acid sequence SEQ ID NO: 8. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with amino acid sequence SEQ ID NO: 8. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with amino acid sequence SEQ ID NO: 8. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 95%, for example, at least 97%, sequence identity with amino acid sequence SEQ ID NO: 8. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises the amino acid sequence of SEQ ID NO: 8.

[0109] According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity includes an amino acid sequence having at least 50%, for example, at least 55%, sequence identity with amino acid sequence SEQ ID NO: 9. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity includes an amino acid sequence having at least 60%, for example, at least 65%, sequence identity with amino acid sequence SEQ ID NO: 9. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity includes an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with amino acid sequence SEQ ID NO: 9. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity includes an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with amino acid sequence SEQ ID NO: 9. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity includes an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with amino acid sequence SEQ ID NO: 9. According to some embodiments, the polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 95%, for example, at least 97%, sequence identity with amino acid sequence SEQ ID NO: 9. According to some embodiments, the polypeptide having adenylate isopentenyltransferase activity comprises the amino acid sequence of SEQ ID NO: 9.

[0110] According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity includes an amino acid sequence having at least 50%, for example, at least 55%, sequence identity with amino acid sequence SEQ ID NO: 10. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity includes an amino acid sequence having at least 60%, for example, at least 65%, sequence identity with amino acid sequence SEQ ID NO: 10. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity includes an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with amino acid sequence SEQ ID NO: 10. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity includes an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with amino acid sequence SEQ ID NO: 10. According to several embodiments, a polypeptide having adenylate isopentenyltransferase activity includes an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with amino acid sequence SEQ ID NO: 10. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises an amino acid sequence having at least 95%, for example, at least 97%, sequence identity with amino acid sequence SEQ ID NO: 10. According to some embodiments, a polypeptide having adenylate isopentenyltransferase activity comprises the amino acid sequence of SEQ ID NO: 10.

[0111] Methods for determining adenylate isopentenyltransferase activity are known to those skilled in the art. Exemplary methods are described, for example, in Takei, Sakakibara, and Sugiyama (2001) and Frebortova, Greplova et al, (2015). Adenylate isopentenyltransferase activity can be determined, for example, by one of the following assays:

[0112] (1) The enzyme is incubated at 25°C for 20 minutes in a reaction mixture containing 1 mM AMP and 340 μM DMAPP (1 M betaine, 20 mM triethanolamine, 50 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 1 mg / ml bovine serum albumin, pH 8.0). The reaction is stopped by adding 1 / 4 volume of 10% acetate and the mixture is centrifuged at 18,000 × g for 20 minutes. The supernatant is subjected to cytokinin analysis. One unit of IPT activity is defined as the amount of enzyme that produces 1 μmol of iPMP per minute under the reaction conditions (Takei, Sakakibara, and Sugiyama 2001).

[0113] (2) The activity assay is carried out overnight at 25°C in 200 μl of reaction mixture (100 mM Tris / HCl buffer, pH 7.5, containing 10 mM MgCl2) containing 100 μM AMP and 100 μM DMAPP (Echelon BioSciences, Salt Lake City, UT, USA) as substrates, and 100 μl of purified enzyme. To evaluate the substrate preference of IPT, ADP or ATP is used as the isoprene chain acceptor substrate, and isopentenyl diphosphate or HMBPP (Echelon Biosciences) is used as the isoprene chain donor substrate. The reaction is initiated by the addition of the isoprenoid substrate and stopped by heating at 95°C for 5 minutes to inactivate the enzyme. The IPT activity assay is based on the determination of the reaction product by HPLC or capillary electrophoresis and UV detection at 268 nm. Cytokinin ribosides and their corresponding monophosphates were determined using a Symmetry C18 column (2.1 × 150 mm, 5 μm; Waters, Milford, MA, USA) connected to an Alliance 2695 high-performance liquid chromatograph (Waters). The column was eluted by a linear gradient of 15 mM ammonium formate, pH 4.0 (A) and methanol (B) using the following solvent mixture: 0–25 min, 5–60% B; 25–26 min, 60–100% B; 26–27 min, 100% B. A linear gradient of 15 mM ammonium formate, pH 4.0 (A) and acetonitrile (B) was used for the analysis of oligoribonucleotide hydrolysates (0–30 min, 5–24% B; 30–31 min, 24–100% B). The flow rate was 0.25 ml / min and the column temperature was 30°C. The concentration of the product is determined by a calibration curve using true standard compounds (Olchemim, Olomouc, Czech Republic). Capillary electrophoresis is used to determine cytokinin diphosphate and triphosphate (Frebortova, Greplova et al, 2015).

[0114] In addition to expressing a heterologous polypeptide having adenyl isopentenyltransferase activity, the bacteria of the present invention may optionally be further modified to increase the protein expression of a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity, even if the expression is otherwise unmodified, compared to the same bacteria.

[0115] Accordingly, according to some embodiments, the bacteria of the present invention are modified to express a heterologous polypeptide having adenyl isopentenyltransferase activity, and the protein expression of a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is increased compared to the same bacteria, although the expression of the polypeptide is otherwise unmodified.

[0116] "Increased protein expression" means that the amount of polypeptides with cytokinin riboside 5'-monophosphate phosphoribohydrolase activity produced by these modified bacteria is increased compared to unmodified bacteria that are otherwise identical. More specifically, "increased expression" means that the amount of polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity produced by the thus modified bacteria is increased by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%, at least about 9000%, or at least about 10000% compared to unmodified but otherwise identical bacteria. The amount of protein in a given cell can be determined by any suitable quantification technique known in the art, such as ELISA, immunohistochemistry, or Western blotting.

[0117] Increased protein expression can be achieved by any suitable means known to those skilled in the art. For example, increased protein expression can be achieved by increasing the copy number of the gene encoding the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity in bacteria by introducing an exogenous nucleic acid into bacteria, such as a vector containing a gene encoding a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity, which is operably linked to a promoter that functions to induce the production of mRNA molecules in bacteria, for example.

[0118] Increased protein expression can also be achieved by incorporating at least a second copy of the gene encoding a polypeptide with cytokinin riboside 5'-monophosphate phosphoribohydrolase activity into the bacterial genome.

[0119] Increased protein expression can also be achieved by increasing the strength of a promoter manipulably linked to a gene encoding a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity, for example, by replacing the native promoter with a promoter that allows for higher expression and overproduction of the polypeptide compared to the native promoter. Potential promoters include natural promoters derived from Bacillus subtilis, Bacillus amyloliquefaciens, etc., such as P43, P15, Pveg, Pylb, PgroES, PsigX, PtrnQ, Ppst, PsodA, PrpsF, PlepA, PliaG, PrpsF, Ppst, PfusA, PsodA, Phag, as well as artificial promoters active in Bacillus subtilis, or inducible Bacillus subtilis promoters, such as PmtlA, Pspac, PxylA, PsacB. Further examples include natural promoters derived from Corynebacterium, such as P CP_2454, Ptuf, and Psod; natural promoters derived from E. coli, such as T7, ParaBAD, Plac, Ptac, and Ptrc; and promoter P F1 derived from Corynepage BFK20.

[0120] Increased protein expression can also be achieved by modifying the ribosome binding site on mRNA molecules encoding polypeptides with cytokinin riboside 5'-monophosphate phosphoribohydrolase activity. By modifying the sequence of the ribosome binding site, the translation initiation rate can be increased, thereby improving translation efficiency.

[0121] According to several embodiments, an increase in the copy number of a gene is achieved by introducing one or more (e.g., two or three) exogenous nucleic acid molecules (e.g., one or more vectors) into bacteria, which contain a gene that is operably linked to a promoter that functions to induce the production of mRNA molecules in a host cell.

[0122] According to several embodiments, the bacteria of the present invention comprises an exogenous nucleic acid molecule (vector, etc.) comprising one or more (e.g., two, three, or four) nucleotide sequences encoding a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity. Preferably, the exogenous nucleic acid molecule further comprises a promoter that functions to induce the production of an mRNA molecule in the bacterium and is operably ligated to the nucleotide sequence encoding the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity. According to several embodiments, the exogenous nucleic acid molecule is stably incorporated into the bacterial genome.

[0123] Polypeptides possessing cytokinin riboside 5'-monophosphate phosphoribohydrolase activity are cytokinin-activating enzymes that function as cytokinin riboside 5'-monophosphate phosphoribohydrolase. Such polypeptides, also known as lonely-guy (LOG) proteins, are encoded in the genomes of a wide range of organisms, with the majority of LOG proteins belonging to prokaryotes. Enzymes from several organisms, including Oryza sativa, Arabidopsis thaliana, Claviceps purpurea, Mycobacterium tuberculosis, and Corynebacterium glutamicum, have been characterized as LOGs through biochemical and functional studies. LOG proteins were initially characterized as plant hormone-activating plant enzymes, and bacterial LOG homologs were mistakenly designated as putative lysine decarboxylases (LDCs) without experimental evidence. The true enzymatic activity of these proteins has recently been confirmed by functional analysis of the Corynebacterium glutamicum homolog (Seo et al. 2016). In bacteria, two types of LOG proteins have been identified: dimeric type I LOG and hexameric type II LOG. Type II LOG proteins differ in their oligomeric state and the residues at the prenyl binding site. Type I LOGs can be further classified into two subgroups: type Ia and type Ib. Type Ia includes dimeric LOGs from most organisms, while type Ib includes dimeric LOGs from the Actinomycetes order. Type II LOGs are classified into type IIa, which includes hexameric LOGs from most organisms, and type IIb, which includes LOGs from higher plants (Seo and Kim 2017).

[0124] LOG proteins are N proteins in cytokinin precursors such as iPRMP or trans-zeatin riboside 5'-monophosphate (tZRMP). 6Active cytokinin is generated by dephosphoribosylation, which is the hydrolysis of the bond between the substituted base and ribose 5'-monophosphate. C. glutamicum LOG is composed of two identical monomers, with a central β-sheet formed by seven parallel β-strands, surrounded by eight α-helices. The LOG protein contains a "PGGXGTXXE" motif that contributes to the formation of the active site. The active site is formed in a dimer pocket, and the conserved "PGGXGTXXE" motif is present on the surface of the pocket. The "PGGXGTXXE" motif is a nucleotide binding site, and conserved residues stabilize the bound AMP (Seo et al. 2016). This motif is highly conserved in all LOG enzymes (Seo and Kim 2017).

[0125] Polypeptides having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity may originate from the same species of bacteria that express it, or from a different species (i.e., heterogeneous). According to some embodiments, polypeptides having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity originate from the same species of bacteria that express it. According to some embodiments, polypeptides having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity originate from a different species (i.e., heterogeneous) of bacteria that express it.

[0126] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is a bacterial polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity. "Bacterial polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity" means that the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is naturally derived from bacteria such as Corynebacterium glutamicum.

[0127] Polypeptides having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity used in accordance with the present invention may be, for example, polypeptides having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity, selected from the group consisting of i) polypeptides comprising any one amino acid sequence of SEQ ID NOs. 34 to 62; and ii) polypeptides comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to any one amino acid sequence of SEQ ID NOs. 34 to 62.

[0128] Polypeptides having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity used in accordance with the present invention may be, for example, polypeptides having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity, selected from the group consisting of i) polypeptides comprising any one amino acid sequence of SEQ ID NOs. 34 to 44; and ii) polypeptides comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to any one amino acid sequence of SEQ ID NOs. 34 to 44.

[0129] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 34. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 34. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 34. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 34.

[0130] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 35. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 35. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 35. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 35.

[0131] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 36. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 36. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 36. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 36.

[0132] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 37. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 37. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 37. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 37.

[0133] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 38. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 38. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 38. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 38.

[0134] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 39. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 39. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 39. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 39.

[0135] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 40. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 40. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 40. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 40.

[0136] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 41. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 41. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 41. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 41.

[0137] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 42. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 42. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 42. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 42.

[0138] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 43. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 43. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 43. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 43.

[0139] According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 44. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 44. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 44. According to several embodiments, the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity includes the amino acid sequence of SEQ ID NO: 44.

[0140] Methods for determining cytokinin riboside 5'-monophosphate phosphoribohydrolase activity are known to those skilled in the art. Exemplary methods are described, for example, in Seo et al. (2016). Cytokinin riboside 5'-monophosphate phosphoribohydrolase activity can be determined, for example, by the following method:

[0141] Phosphoribohydrolase activity is determined by detecting adenine ring compounds separated by thin-layer chromatography (TLC). The enzymatic reaction is carried out at 30°C in a mixture of 20 mM AMP, 36 mM Tris-HCl, pH 8.0, and 23 μM purified enzyme, and the reaction is stopped by heating the mixture at 95°C for 1.5 minutes. The reaction mixture is then spot-deposited onto a PEI-cellulose-F plastic TLC sheet (Merck Millipore). The mobile phase is 1 M sodium chloride. After development in a TLC chamber, the sheet is completely dried. Compounds containing adenine rings are detected by a UV lamp (290 nm) (Seo et al. 2016).

[0142] To increase the supply of isopentenyl side chain precursors of isoprenoid cytokinins, the metabolic flux via the methylerythritol 4-phosphate (MEP) pathway may be increased. This is mainly achieved by overexpression of 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS).

[0143] Accordingly, according to some embodiments, the bacteria of the present invention are characterized in that the protein expression of polypeptides having 1-deoxy-D-xylulose-5-phosphate synthase activity is increased compared to the same bacteria, although the expression of polypeptides having 1-deoxy-D-xylulose-5-phosphate synthase activity is otherwise modified.

[0144] "Increased protein expression" means that the amount of polypeptide with 1-deoxy-D-xylulose-5-phosphate synthase activity produced by these modified bacteria is increased compared to unmodified bacteria that are otherwise identical. More specifically, "increased expression" means that the amount of polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity produced by the modified bacteria is increased by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%, at least about 9000%, or at least about 10000% compared to bacteria that are otherwise identical but not modified. The amount of protein in a given cell can be determined by any suitable quantification technique known in the art, such as ELISA, immunohistochemistry, or Western blotting.

[0145] Increased protein expression can be achieved by any suitable means known to those skilled in the art. For example, increased protein expression can be achieved by increasing the copy number of the gene encoding the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity in bacteria by introducing an exogenous nucleic acid into bacteria, such as a vector containing a gene encoding a polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity, which is operably linked to a promoter that functions to induce the production of mRNA molecules in bacteria.

[0146] Increased protein expression can also be achieved by incorporating at least a second copy of the gene encoding a polypeptide with 1-deoxy-D-xylulose-5-phosphate synthase activity into the bacterial genome.

[0147] Increased protein expression can also be achieved by increasing the strength of a promoter manipulatively linked to a gene encoding a polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity, for example, by replacing the native promoter with a promoter that allows for higher polypeptide expression and overproduction compared to the native promoter. Potential promoters include natural promoters derived from Bacillus subtilis, Bacillus amyloliquefaciens, etc., such as P43, P15, Pveg, Pylb, PgroES, PsigX, PtrnQ, Ppst, PsodA, PrpsF, PlepA, PliaG, PrpsF, Ppst, PfusA, PsodA, Phag, as well as artificial promoters active in Bacillus subtilis, or inducible Bacillus subtilis promoters, such as PmtlA, Pspac, PxylA, PsacB. Further examples include natural promoters derived from Corynebacterium, such as P CP_2454, Ptuf, and Psod; natural promoters derived from E. coli, such as T7, ParaBAD, Plac, Ptac, and Ptrc; and promoter P F1 derived from Corynepage BFK20.

[0148] Increased protein expression can also be achieved by modifying the ribosome binding site on mRNA molecules encoding polypeptides with 1-deoxy-D-xylulose-5-phosphate synthase activity. By modifying the sequence of the ribosome binding site, the translation initiation rate can be increased, thereby improving translation efficiency.

[0149] According to several embodiments, an increase in the copy number of a gene is achieved by introducing one or more (e.g., two or three) exogenous nucleic acid molecules (e.g., one or more vectors) into bacteria, which contain a gene that is operably linked to a promoter that functions to induce the production of mRNA molecules in the bacteria.

[0150] According to several embodiments, the bacteria of the present invention comprises an exogenous nucleic acid molecule (vector, etc.) comprising one or more (e.g., two, three, or four) nucleotide sequences encoding a polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity. Preferably, the exogenous nucleic acid molecule further comprises a promoter that functions to induce mRNA molecule production in the bacterium and is operably ligated to the nucleotide sequence encoding the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity. According to several embodiments, the exogenous nucleic acid molecule is stably incorporated into the bacterial genome.

[0151] Polypeptides possessing 1-deoxy-D-xylulose-5-phosphate synthase activity are enzymes that catalyze the condensation between D-glyceraldehyde 3-phosphate and pyruvate to produce 1-deoxy-D-xylulose 5-phosphate (DXP) (DXS; EC2.2.1.7). DXS catalyzes the initial enzymatic step in the isoprenoid biosynthesis of HMBDP and DMAPP in the methylerythritol phosphate (MEP) pathway, and its kcat / Km value is significantly lower than that of other enzymes in this pathway (Kuzuyama et al. 2000). DXS exists as a single copy in eubacteria, while green algae and higher plants possess two or more genes encoding DXS, forming three distinct groups. In higher plants, the expression of different DXS isoenzymes is determined by tissue type and developmental stage.

[0152] DXS is highly conserved in bacteria and plants. Its protein sequence has approximately 20% identity with the transketolase and pyruvate dehydrogenase E1 subunits. All three enzymes catalyze similar reactions and require the coenzyme thiamine pyrophosphate (TPP). DXS derived from E. coli contains three domains (I, II, and III), which are homologous to the equivalent domains in the transketolase and pyruvate dehydrogenase E1 subunits. Two DXS monomers are arranged side by side in a dimer. Domain I (residues 1-319) is located above domains II (residues 320-495) and III (residues 496-629) of the same monomer. All three domains have an α / β fold, with a central, nearly parallel β-sheet between the α-helices. Domain I contains a quintuple parallel β-sheet, Domain II contains a hexaple parallel β-sheet, and Domain III contains a quintuple β-sheet, with the first chain being antiparallel to the other four chains. Domain I has several extended surface segments at its N-terminus (residues 1-49), after the first chain (residues 81-122), and at the junction between the fourth and fifth chains (residues 184-250).

[0153] The active site of DXS is located at the interface of domains I and II within the same monomer. The C-terminuses of the central parallel β-sheets of the two domains face each other, and the TPP coenzyme is located at the bottom of the pocket at this interface. The aminopyrimidine ring of TPP interacts with domain II, and the pyrophosphate group interacts with domain I. The C2 atom of the thiazolium ring is exposed at the substrate binding site. The C2 atom is involved in the reaction. The pyrophosphate group of TPP has numerous polar interactions with the enzyme. The active site consists of a magnesium ion bound between the two phosphate groups and the side chains Asp154, Asn183, and Met185. The Gly153-Asp-Gly155-Asn183 sequence of DXS corresponds to the TPP binding motif ≫GDG-X(25-30)-N≪. C2 constitutes the pyruvate binding site. GAP is located within the pocket (Xiang et al., 2007).

[0154] Several studies have shown that DXP formation is the rate-limiting step in the MEP pathway, and therefore, increasing DXS activity is recognized as the most effective strategy for increasing terpenoid biosynthesis in B. subtilis, as in many other species (Yang et al. 2019). Single amino acid mutations in Dxs of E. coli and Deinococcus radiodurans increase their catalytic activity. The E. coli Dxs mutation Y392F increased relative catalytic activity 2.5-fold compared to the wild type (Xiang et al. 2012). DXS is regulated by a negative feedback mechanism involving IPP and DMAPP, the end products of the MEP pathway (Banerjee et al. 2013). DXS is a target for site-directed mutagenesis to mitigate the negative feedback inhibition of IPP and DMAPP. Mutations in Populus trichocarpa DXS at A147G / A352G slightly reduced its IPP binding affinity but increased Km for TPP and pyruvate, reducing the enzyme's catalytic efficiency by approximately 15 times (Banerjee et al., 2016). Overexpression of several other enzymes in pathways other than DXS has also been tested, yielding various results in different bacteria. Furthermore, overexpression of the entire MEP pathway as an artificial operon (operon dxs-ispD-ispF-ispH and ispC / dxr-ispE-ispG-ispA) has been tested in B. subtilis (Xue et al. 2015).

[0155] Polypeptides having 1-deoxy-D-xylulose-5-phosphate synthase activity may originate from the same species of bacteria that express it, or from a different species (i.e., heterogeneous). According to some embodiments, polypeptides having 1-deoxy-D-xylulose-5-phosphate synthase activity originate from the same species of bacteria that express it. According to some embodiments, polypeptides having 1-deoxy-D-xylulose-5-phosphate synthase activity originate from a different species (i.e., heterogeneous) of bacteria that express it.

[0156] Preferably, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is a bacterial polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity. "Bacterial polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity" means that the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is naturally derived from bacteria such as Bacillus subtilis.

[0157] Polypeptides having 1-deoxy-D-xylulose-5-phosphate synthase activity used in accordance with the present invention may be, for example, polypeptides having 1-deoxy-D-xylulose-5-phosphate synthase activity, selected from the group consisting of i) polypeptides comprising any one amino acid sequence of SEQ ID NOs. 63 to 70; and ii) polypeptides comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to any one amino acid sequence of SEQ ID NOs. 63 to 70.

[0158] According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 63. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 63. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 63. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 63.

[0159] According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 64 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 64. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 64 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 64. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 64 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 64. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 64.

[0160] According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 65. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 65. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 65. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 65.

[0161] According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 66 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 66. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 66 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 66. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 66 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 66. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 66.

[0162] According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 67. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 67. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 67. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 67.

[0163] According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 68. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 68. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 68. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes the amino acid sequence of SEQ ID NO: 68.

[0164] As described above, modification of the wild-type DXS sequence can increase catalytic activity. Therefore, the present invention particularly intends to use mutant DXS having increased activity or an inactivated negative feedback mechanism compared to the wild-type DXS enzyme from which it is derived. Non-limiting examples of such mutant DXS enzymes are described in SEQ ID NOs. 69 and 70.

[0165] According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with the amino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 69, provided that the amino acid at position 392 is not Y, but preferably F. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with the amino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 69, provided that the amino acid at position 392 is not Y, but preferably F. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with the amino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 69, provided that the amino acid at position 392 is not Y, but preferably F. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity comprises the amino acid sequence of SEQ ID NO: 69.

[0166] According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 70, provided that the amino acid at position 389 is not Y, but preferably F. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 70, provided that the amino acid at position 389 is not Y, but preferably F. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity includes an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 70, provided that the amino acid at position 389 is not Y, but preferably F. According to several embodiments, the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity comprises the amino acid sequence of SEQ ID NO: 70.

[0167] Methods for determining 1-deoxy-D-xylulose-5-phosphate synthase activity are known to those skilled in the art. Exemplary methods are described, for example, in Kuzuyama et al (2000) and Kudoh et al (2017). 1-deoxy-D-xylulose-5-phosphate synthase activity can be determined, for example, by the following method:

[0168] (1) Determine the activity of 1-deoxy-D-xylulose-5-phosphate synthase by DXS assay (Kuzuyama et al., 2000): The standard assay system consists of 100 mM Tris-HCl (pH 8.0) containing 0.5 ml of final volume 1 mM MgCl2, 2 mM dl-dithiothreitol, 1 mM sodium pyruvate, 2 mM dl-glyceraldehyde 3-phosphate, and 150 μM thiamine diphosphate. The reaction is initiated by adding the enzyme solution to the complete assay mixture at 37°C, followed by incubation for 10 minutes, and then cessation of the reaction by incubation at 100°C for 1 minute. Next, the reaction mixture is treated with alkaline phosphatase at 56°C for 60 minutes to completely dephosphorylate the reaction product, DXP. The formation of the resulting dephosphorylated compound, 1-deoxyxylose (DX), is monitored using a refractive index spectrometer (Model RI-71; Showa Denko, Tokyo, Japan) with a Shodex KS-801 (8mm × 300mm) column (Showa Denko) eluted with H2O at 80°C and a flow rate of 1 ml / min. Under these conditions, DX elutes in 8.6 minutes. The amount of DX produced is accurately estimated using chemically synthesized DX as a standard. One unit of DXS activity is defined as the amount of enzyme that produces 1 μmol of DXP per minute at 37°C. The formation of DXP by DXS is monitored at 195 nm by high-performance liquid chromatography using a Senshu Pak NH2-1251-N (4.6mm × 250mm) column (Senshu Science Co., Ltd., Tokyo, Japan) eluted with 100 mM KH2PO4 (pH 3.5) at a flow rate of 1 ml / min. Under these conditions, DXP ​​elutes in 8.1 minutes.

[0169] (2) Conjugated enzyme assay of DXS (Kudoh et al., 2017): DXS activity is measured using a conjugated enzyme assay with DXR derived from E. coli as the conjugated enzyme. In this assay, DXP generated by DXS activity is further converted to MEP. At this stage, NADPH is consumed, and the entire reaction can be spectrophotometrically measured at 340 nm. The assay mixture contains 100 mM Tris / HCl (pH 7.8), 10 mM MgCl2, 0.3 mM thiamine pyrophosphate (TPP), 1 mM dithiothreitol (DTT), 0.3 mM nicotinamide adenine dinucleotide phosphate (NADPH), various concentrations of sodium pyruvate (0.05 - 5 mM) and D,L-GAP (0.2 - 2.0 mM), and DXR (100 or 50 mg / ml). The mixture is incubated at 30 °C for 2 minutes in a temperature-controlled spectrophotometer (model UV-1800, Shimadzu Corporation, Kyoto, Japan), added to the DXS sample (final concentration 50 or 25 mg / ml), and the reaction is initiated. The reaction is monitored by monitoring the absorption at 340 nm at 30 °C.

[0170] Purine nucleotides are components of the structures of DNA and RNA, energy carriers (i.e., ATP and GTP), cofactors of enzymes (i.e., NAD + and NADP +Purine nucleotides are essential metabolites for cell physiology. The synthesis of purine nucleotides begins with the synthesis of 5'-phosphoribosyl-pyrophosphate (PRPP) from D-ribose 5-phosphate and ATP. The enzyme PRPP synthase (ribose-phosphate diphosphokinase; EC 2.7.6.1) catalyzes the transfer of the diphosphoryl group of ATP to D-ribose 5-phosphate, and AMP is formed simultaneously. PRPP synthase is ubiquitous in free-living organisms. Most bacteria have one gene encoding PRPP synthase, but eukaryotes have two or more genes. The next step is the synthesis of 5-phospho-β-D-ribosylamine from PRPP and glutamine, catalyzed by glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidetransferase (amidephosphoribosyltransferase; EC 2.4.2.14), which is encoded by purF in B. subtilis. This is the rate-limiting reaction in purine de novo synthesis. The further 10 steps leading to the synthesis of inosine-5-phosphate (IMP) from 5-phospho-β-D-ribosylamine are catalyzed by enzymes encoded by the pur operon. The pur operon is negatively regulated at the transcriptional level by the PurR repressor encoded by purR, and also by PurBox, a specific DNA sequence located in the upstream regulatory region of the affected gene. IMP is the branching point for the synthesis of AMP and GMP. AMP is synthesized from IMP in two enzymatic steps catalyzed by PurA and PurB, while GMP is synthesized from IMP by GuaB and GuaA. The expression of genes or operons encoding the enzymes of purine synthesis is regulated by purine bases and nucleosides in the growth medium. The enzymes PRPP synthetase, PRPP amidetransferase, adenylosuccinate synthetase, and IMP dehydrogenase are regulated by feedback inhibition of the final product of the pathway. The PurR repressor inhibits transcription initiation. The salvage pathway is also involved in generating the corresponding mononucleotides AMP and GMP using hypoxanthine, guanine, and adenine.

[0171] The purine nucleotide biosynthesis pathway is well-studied due to the role of purine nucleotides in primary metabolism. This includes both de novo and salvage pathways. Deregulation of the purine nucleotide biosynthesis pathway at the transcriptional and metabolic levels enhances the metabolic flow through this pathway, resulting in increased yields of products directly derived from it: inosine, guanosine, adenosine, and riboflavin. Various modifications, including gene overexpression, gene deletion, and enzyme deregulation through mutation, have been successfully employed to increase the yield of purine nucleotide biosynthesis products. Exemplary modifications in the B. subtilis enzyme that positively impact purine flux are listed in Table 1 below.

[0172] [Table 1-1] [Table 1-2] [Table 1-3]

[0173] Accordingly, according to some embodiments, the bacteria of the present invention are characterized in that the expression and / or activity of at least one enzyme involved in the purine nucleotide biosynthesis pathway is (further) modified to be increased compared to the same bacteria, but without modification.

[0174] According to several embodiments, the bacteria of the present invention are characterized in that the expression and / or activity of at least one enzyme involved in the adenosine monophosphate biosynthesis pathway is (further) modified to be increased compared to the same bacteria, but without modification.

[0175] At least one enzyme involved in the purine nucleotide biosynthesis pathway (e.g., at least one enzyme involved in the adenosine monophosphate biosynthesis pathway) may originate from the same species of bacterium in which it is expressed, or from a different species (i.e., heterogeneous). According to some embodiments, at least one enzyme involved in the purine nucleotide biosynthesis pathway (e.g., at least one enzyme involved in the adenosine monophosphate biosynthesis pathway) may originate from the same species of bacterium in which it is expressed. According to some embodiments, at least one enzyme involved in the purine nucleotide biosynthesis pathway (e.g., at least one enzyme involved in the adenosine monophosphate biosynthesis pathway) may originate from a different species (i.e., heterogeneous) of bacterium in which it is expressed.

[0176] "Increased protein expression" means that the amount of enzymes involved in the purine nucleotide biosynthesis pathway (for example, enzymes involved in the adenosine monophosphate biosynthesis pathway) produced by these modified bacteria is increased compared to bacteria that are not modified but otherwise identical. More specifically, "increased expression" means that the amount of enzymes involved in the purine nucleotide biosynthesis pathway (e.g., enzymes involved in the adenosine monophosphate biosynthesis pathway) produced by the modified bacteria is increased by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%, at least about 9000%, or at least about 10000% compared to bacteria without the modification but otherwise identical. The amount of protein in a given cell can be determined by any suitable quantification technique known in the art, such as ELISA, immunohistochemistry, or Western blotting.

[0177] Increased protein expression can be achieved by any suitable means known to those skilled in the art. For example, increased protein expression can be achieved by increasing the copy number of genes encoding enzymes involved in the purine nucleotide biosynthesis pathway (e.g., enzymes involved in the adenosine monophosphate biosynthesis pathway) in bacteria by introducing exogenous nucleic acids, such as vectors containing genes encoding enzymes involved in the purine nucleotide biosynthesis pathway (e.g., enzymes involved in the adenosine monophosphate biosynthesis pathway), into bacteria, for example, by operably linking them to a promoter that functions to induce the production of mRNA molecules in bacteria.

[0178] Increased protein expression can also be achieved by incorporating at least a second copy of the gene encoding an enzyme involved in the purine nucleotide biosynthesis pathway (for example, an enzyme involved in the adenosine monophosphate biosynthesis pathway) into the bacterial genome.

[0179] Increased protein expression can also be achieved by increasing the strength of a promoter manipulatively linked to a gene encoding an enzyme involved in the purine nucleotide biosynthesis pathway (e.g., an enzyme involved in the adenosine monophosphate biosynthesis pathway), for example, by replacing the native promoter with a promoter that allows for higher expression and overproduction of the enzyme compared to the native promoter. Potential promoters include natural promoters derived from Bacillus subtilis, Bacillus amyloliquefaciens, etc., such as P43, P15, Pveg, Pylb, PgroES, PsigX, PtrnQ, Ppst, PsodA, PrpsF, PlepA, PliaG, PrpsF, Ppst, PfusA, PsodA, Phag, as well as artificial promoters active in Bacillus subtilis, or inducible Bacillus subtilis promoters, such as PmtlA, Pspac, PxylA, PsacB. Further examples include natural promoters derived from Corynebacterium, such as P CP_2454, Ptuf, and Psod; natural promoters derived from E. coli, such as T7, ParaBAD, Plac, Ptac, and Ptrc; and promoter P F1 derived from Corynepage BFK20.

[0180] Increased protein expression can also be achieved by modifying the ribosome binding site on mRNA molecules encoding enzymes involved in the purine nucleotide biosynthesis pathway. By altering the sequence of the ribosome binding site, the translation initiation rate can be increased, thereby improving translation efficiency.

[0181] According to several embodiments, an increase in the copy number of a gene is achieved by introducing one or more (e.g., two or three) exogenous nucleic acid molecules (e.g., one or more vectors) into bacteria, which contain a gene that is operably linked to a promoter that functions to induce the production of mRNA molecules in a host cell.

[0182] According to several embodiments, the bacteria of the present invention comprises an exogenous nucleic acid molecule (vector, etc.) containing one or more (e.g., two, three, or four) nucleotide sequences encoding an enzyme involved in the purine nucleotide biosynthesis pathway (e.g., an enzyme involved in the adenosine monophosphate biosynthesis pathway). Preferably, the exogenous nucleic acid molecule further comprises a promoter that functions to induce the production of an mRNA molecule in the bacterium and is operably ligated to the nucleotide sequence encoding the enzyme involved in the purine nucleotide biosynthesis pathway (e.g., an enzyme involved in the adenosine monophosphate biosynthesis pathway). According to several embodiments, the exogenous nucleic acid molecule is stably incorporated into the bacterial genome.

[0183] At least one enzyme involved in the purine nucleotide biosynthesis pathway is an enzyme with ribose-phosphate diphosphokinase activity, an enzyme with amide phosphoribosyltransferase activity, an enzyme with formyltetrahydrofolate deformylase activity, an enzyme with phosphoribosylamine-glycine ligase activity, an enzyme with phosphoribosylglycinamideformyltransferase activity, an enzyme with phosphoribosylformylglycineamidine synthase activity, an enzyme with phosphoribosylformylglycineamidine cycloligase activity, an enzyme with N5-carboxyaminoimidazole ribonucleotide synthetase activity, or an N5-carboxy The enzyme may be selected from the group consisting of an enzyme having aminoimidazole ribonucleotide mutase activity, an enzyme having phosphoribosylaminoimidazole succinocarboxamide synthase activity, an enzyme having adenylosuccinate lyase activity, an enzyme having phosphoribosylaminoimidazole-carboxamide formyltransferase activity, an enzyme having IMP cyclohydrolase activity, an enzyme having adenylosuccinate synthase activity, an enzyme having adenylate kinase activity, an enzyme having ATP synthase activity, an enzyme having adenosine kinase activity, an enzyme having IMP dehydrogenase activity, and an enzyme having GMP synthase activity.

[0184] At least one enzyme involved in the adenosine monophosphate biosynthesis pathway is an enzyme with ribose-phosphate diphosphokinase activity, an enzyme with amide phosphoribosyltransferase activity, an enzyme with formyltetrahydrofolate deformylase activity, an enzyme with phosphoribosylamine-glycine ligase activity, an enzyme with phosphoribosylglycinamideformyltransferase activity, an enzyme with phosphoribosylformylglycineamidine synthase activity, an enzyme with phosphoribosylformylglycineamidine cycloligase activity, or an N5-carboxyaminoimidazole ribonucleotide synthetase The enzyme may be selected from the group consisting of an enzyme having -ase activity, an enzyme having N5-carboxyaminoimidazole ribonucleotide mutase activity, an enzyme having phosphoribosylaminoimidazole succinocarboxamide synthase activity, an enzyme having adenylosuccinate lyase activity, an enzyme having phosphoribosylaminoimidazole-carboxamide formyltransferase activity, an enzyme having IMP cyclohydrolase activity, an enzyme having adenylosuccinate synthase activity, an enzyme having adenylate kinase activity, an enzyme having ATP synthase activity, and an enzyme having adenosine kinase activity.

[0185] According to several embodiments, at least one enzyme involved in the purine nucleotide biosynthesis pathway (such as the adenosine monophosphate biosynthesis pathway) is selected from the group consisting of enzymes having ribose-phosphate diphosphokinase activity, enzymes having amide phosphoribosyltransferase activity, enzymes having formyltetrahydrofolate deformylase activity, enzymes having adenylosuccinate lyase activity, enzymes having phosphoribosylaminoimidazole-carboxamideformyltransferase activity, enzymes having adenylosuccinate synthase activity, and enzymes having adenosine kinase activity.

[0186] According to several embodiments, the bacteria of the present invention are (further) modified to have one or more of the modifications disclosed in Table 1 with respect to one or more of its endogenous enzymes involved in the purine nucleotide biosynthesis pathway. In particular, the bacteria may be the result of random mutagenesis to exhibit resistance to inhibitors of the enzyme in question.

[0187] According to several embodiments, the bacteria of the present invention are characterized in that the expression and / or activity of at least one endogenous enzyme involved in the purine nucleotide degradation pathway is (further) modified to be reduced compared to the same bacteria, but without modification.

[0188] According to several embodiments, the bacteria of the present invention may be modified such that the expression of at least one endogenous enzyme involved in the purine nucleotide degradation pathway is reduced compared to the same bacteria, but without modification.

[0189] According to several embodiments, the bacteria of the present invention may be modified such that the expression level of an endogenous gene encoding at least one endogenous enzyme involved in the purine nucleotide degradation pathway is reduced compared to otherwise identical bacteria, but without modification. The expression level of the endogenous gene may be reduced by, for example, at least 50%, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to otherwise identical bacteria.

[0190] According to some embodiments, the endogenous gene encoding the enzyme is inactivated, for example, by the deletion of part or all of the gene sequence.

[0191] According to several embodiments, the endogenous gene encoding the enzyme is inactivated by introducing or expressing a rare-cut endonuclease in a microorganism that can selectively inactivate the endogenous gene encoding the enzyme by DNA cleavage, preferably by double-strand breaks. The rare-cut endonuclease used according to the present invention to inactivate the endogenous gene may be, for example, a transcription activator-like effector (TALE) nuclease, a meganuclease, a zinc finger nuclease (ZFN), or an RNA-inducible endonuclease.

[0192] One method for inactivating the endogenous gene encoding the enzyme mentioned above is to use the CRISPRi system. The CRISPRi system was developed as a tool for targeted repression of gene expression or blocking of targeted locations on the genome. The CRISPRi system consists of a catalytically inactive "dead" Cas9 protein (dCas9) and a guide RNA that defines the binding site of dCas9 to DNA.

[0193] Therefore, according to some embodiments, the endogenous gene encoding the enzyme is inactivated by introducing or expressing in bacteria an RNA-induced endonuclease, such as a catalytically inactive Cas9 protein, and a single guide RNA (sgRNA) that specifically hybridizes (e.g., binds) to the genomic DNA encoding the enzyme under cellular conditions.

[0194] According to several embodiments, the expression of at least one endogenous enzyme involved in the purine nucleotide degradation pathway is reduced by inhibition.

[0195] Inhibition of the expression of the above-mentioned endogenous enzyme can be achieved by any suitable means known in the art. For example, expression can be inhibited by gene silencing techniques involving the use of inhibitory nucleic acid molecules such as antisense oligonucleotides, ribozymes, or interfering RNA (RNAi) molecules, such as microRNA (miRNA), small interfering RNA (siRNA), or short hairpin RNA (shRNA).

[0196] According to several embodiments, the expression of at least one endogenous enzyme involved in the purine nucleotide degradation pathway is reduced (e.g., inhibited) by transcriptional and / or translational repression of the endogenous gene encoding the polypeptide.

[0197] According to several embodiments, the expression of at least one endogenous enzyme involved in the purine nucleotide degradation pathway is inhibited by introducing or expressing an inhibitory nucleic acid molecule in bacteria. For example, the inhibitory nucleic acid molecule can be introduced by an exogenous nucleic acid molecule containing a nucleotide sequence encoding the inhibitory nucleic acid molecule, which is operably ligated to a promoter, such as an inducible promoter, that functions to induce the production of the inhibitory nucleic acid molecule in bacteria. Preferably, the inhibitory nucleic acid molecule specifically hybridizes (e.g., binds) to the cellular mRNA and / or genomic DNA encoding the endogenous enzyme under cellular conditions. Depending on the target, it inhibits the transcription of the coding genomic DNA and / or the translation of the coding mRNA.

[0198] According to some embodiments, the inhibitory nucleic acid molecule is an antisense oligonucleotide, ribozyme, or interfering RNA (RNAi) molecule. Preferably, such nucleic acid molecule comprises at least 10 consecutive nucleotides that complement the cellular mRNA and / or genomic DNA encoding the polypeptide or enzyme of interest (e.g., cellular mRNA and / or genomic DNA encoding a polypeptide).

[0199] According to some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide. Such an antisense oligonucleotide is a nucleic acid molecule (either DNA or RNA) that specifically hybridizes (e.g., binds) to cellular mRNA and / or genomic DNA encoding a polypeptide under cellular conditions.

[0200] According to several embodiments, the inhibitory nucleic acid molecule is a ribozyme, such as a hammerhead ribozyme. The ribozyme molecule is designed to catalytically cleave mRNA transcripts to prevent polypeptide translation.

[0201] According to several embodiments, the inhibitory nucleic acid molecule is an interfering RNA (RNAi) molecule. RNA interference is a biological process in which an RNA molecule inhibits expression, typically resulting in the disruption of a specific mRNA. Exemplary types of RNAi molecules include microRNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA). According to several embodiments, the RNAi molecule is miRNA. According to several embodiments, the RNAi molecule is siRNA. According to several embodiments, the RNAi molecule is shRNA.

[0202] According to several embodiments, the bacteria of the present invention are modified such that the activity of at least one endogenous enzyme involved in the purine nucleotide degradation pathway is reduced compared to the same bacteria, but without modification.

[0203] A reduction in the activity of endogenous enzymes involved in the purine nucleotide degradation pathway can be achieved by any suitable means known in the art. For example, activity can be reduced by introducing one or more mutations into the active site of the enzyme that result in reduced or lost activity. Thus, according to some embodiments, the activity of endogenous enzymes involved in the purine nucleotide degradation pathway is reduced by at least one active site mutation that results in reduced or lost activity. The at least one active site mutation may be, for example, at least one non-conservative amino acid substitution.

[0204] According to several embodiments, at least one enzyme involved in the purine nucleotide degradation pathway is selected from the group consisting of purine nucleoside phosphorylases and adenosine-phosphoribosyltransferases. According to several embodiments, at least one endogenous enzyme involved in the purine nucleotide degradation pathway is a purine nucleoside phosphorylase. According to several embodiments, at least one endogenous enzyme involved in the purine nucleotide degradation pathway is adenosine-phosphoribosyltransferase.

[0205] According to several embodiments, the bacteria of the present invention are (further) modified such that the expression and / or activity of at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is reduced, but otherwise unmodified, compared to the same bacteria.

[0206] According to several embodiments, the bacteria of the present invention may be modified such that the expression level of an endogenous gene encoding at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is reduced compared to otherwise identical bacteria, but without modification. The expression level of the endogenous gene may be reduced by, for example, at least 50%, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to otherwise identical bacteria.

[0207] According to some embodiments, the endogenous gene encoding the enzyme is inactivated, for example, by the deletion of part or all of the gene sequence.

[0208] According to several embodiments, the endogenous gene encoding the enzyme is inactivated by introducing or expressing a rare-cut endonuclease in a microorganism that can selectively inactivate the endogenous gene encoding the enzyme by DNA cleavage, preferably by double-strand breaks. The rare-cut endonuclease used according to the present invention to inactivate the endogenous gene may be, for example, a transcription activator-like effector (TALE) nuclease, a meganuclease, a zinc finger nuclease (ZFN), or an RNA-inducible endonuclease.

[0209] One method for inactivating the endogenous gene encoding the enzyme mentioned above is to use the CRISPRi system. The CRISPRi system was developed as a tool for targeted repression of gene expression or blocking of targeted locations on the genome. The CRISPRi system consists of a catalytically inactive "dead" Cas9 protein (dCas9) and a guide RNA that defines the binding site of dCas9 to DNA.

[0210] Therefore, according to some embodiments, the endogenous gene encoding the enzyme is inactivated by introducing or expressing in bacteria an RNA-induced endonuclease, such as a catalytically inactive Cas9 protein, and a single guide RNA (sgRNA) that specifically hybridizes (e.g., binds) to the genomic DNA encoding the enzyme under cellular conditions.

[0211] According to several embodiments, the expression of at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is reduced by inhibition.

[0212] Inhibition of the expression of the above-mentioned endogenous enzyme can be achieved by any suitable means known in the art. For example, expression can be inhibited by gene silencing techniques involving the use of inhibitory nucleic acid molecules such as antisense oligonucleotides, ribozymes, or interfering RNA (RNAi) molecules, such as microRNA (miRNA), small interfering RNA (siRNA), or short hairpin RNA (shRNA).

[0213] According to several embodiments, the expression of at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is reduced (e.g., inhibited) by transcriptional and / or translational repression of the endogenous gene encoding the polypeptide.

[0214] According to several embodiments, the expression of at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is inhibited by introducing or expressing an inhibitory nucleic acid molecule in bacteria. For example, the inhibitory nucleic acid molecule can be introduced by an exogenous nucleic acid molecule containing a nucleotide sequence encoding the inhibitory nucleic acid molecule, which is operably ligated to a promoter, such as an inducible promoter, that functions to induce the production of the inhibitory nucleic acid molecule in bacteria. Preferably, the inhibitory nucleic acid molecule specifically hybridizes (e.g., binds) to the cellular mRNA and / or genomic DNA encoding the endogenous enzyme under cellular conditions. Depending on the target, it inhibits the transcription of the coding genomic DNA and / or the translation of the coding mRNA.

[0215] According to some embodiments, the inhibitory nucleic acid molecule is an antisense oligonucleotide, ribozyme, or interfering RNA (RNAi) molecule. Preferably, such nucleic acid molecule comprises at least 10 consecutive nucleotides that complement the cellular mRNA and / or genomic DNA encoding the polypeptide or enzyme of interest (e.g., cellular mRNA and / or genomic DNA encoding a polypeptide).

[0216] According to some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide. Such an antisense oligonucleotide is a nucleic acid molecule (either DNA or RNA) that specifically hybridizes (e.g., binds) to cellular mRNA and / or genomic DNA encoding a polypeptide under cellular conditions.

[0217] According to several embodiments, the inhibitory nucleic acid molecule is a ribozyme, such as a hammerhead ribozyme. The ribozyme molecule is designed to catalytically cleave mRNA transcripts to prevent polypeptide translation.

[0218] According to several embodiments, the inhibitory nucleic acid molecule is an interfering RNA (RNAi) molecule. RNA interference is a biological process in which an RNA molecule inhibits expression, typically resulting in the disruption of a specific mRNA. Exemplary types of RNAi molecules include microRNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA). According to several embodiments, the RNAi molecule is miRNA. According to several embodiments, the RNAi molecule is siRNA. According to several embodiments, the RNAi molecule is shRNA.

[0219] According to several embodiments, the bacteria of the present invention are modified such that the activity of at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is reduced, but otherwise unmodified, compared to the same bacteria.

[0220] A reduction in the activity of endogenous enzymes involved in the guanosine monophosphate biosynthesis pathway can be achieved by any suitable means known in the art. For example, activity can be reduced by introducing one or more mutations into the active site of the enzyme that result in a decrease or loss of activity. Thus, according to some embodiments, the activity of endogenous enzymes involved in the guanosine monophosphate biosynthesis pathway is reduced by at least one active site mutation that results in a decrease or loss of activity. The at least one active site mutation may be, for example, at least one non-conservative amino acid substitution.

[0221] According to several embodiments, at least one enzyme involved in the guanosine monophosphate biosynthesis pathway is selected from the group consisting of IMP dehydrogenase and GMP synthetase.

[0222] According to some embodiments, at least one enzyme involved in the guanosine monophosphate biosynthesis pathway is IMP dehydrogenase.

[0223] According to several embodiments, at least one enzyme involved in the guanosine monophosphate biosynthesis pathway is GMP synthetase.

[0224] CYP450 is a diverse group of heme-containing enzymes that catalyze a wide range of oxidation reactions. Cytochrome P450 monooxygenases (CYP450) also catalyze the hydroxylation of isopentenyladenine cytokinins. In Arabidopsis thaliana, there are two cytochrome P450 monooxygenases, CYP735A1 and CYP735A2, that hydroxylate isopentenyladenine cytokinins. CYP735A catalyzes the stereospecific reaction of iP-nucleotide hydroxylation, catalyzing the hydroxylation of iP-nucleosides or iP with lower affinity to synthesize tZ (Takei et al., 2004b). Most plant-derived CYP450s are fixed to the endoplasmic reticulum (ER) membrane. Cytochrome P450 monooxygenases are also present in bacteria such as Rhodococcus fascians. Cytochrome P450s are generally classified into two main classes based on the properties of their accessory proteins. Class I P450s are found on mitochondrial membranes and in bacteria, while Class II cytochrome P450s are represented by liver microsomal enzymes in mammalian cells. Class I P450s are three-component systems containing flavin adenine dinucleotide (FAD)-containing reductase, iron-sulfur protein (ferredoxin), and P450. Class II cytochrome P450s consist of FAD-containing, flavin mononucleotide (FMN)-containing NADPH-dependent cytochrome P450 reductase and P450. Class III and Class IV CYP450s have also been reported in bacteria, but Class I is the most common CYP450 in bacteria. The most well-characterized bacterial cytochrome P450 monooxygenase system of Pseudomonas ptyda P450cam consists of three soluble proteins: ptydaredoxin reductase; ptydaredoxin, an intermediate iron-sulfur protein; and cytochrome P450cam. CYP450 Fas1 is encoded in the fas region of a linear plasmid and is thought to hydroxylate cytokinin produced by R. fascians (Frebort et al., 2011).

[0225] Cytochrome P450 monooxygenase (CYP450) can hydroxylate iP nucleotides and produce trans-zeatin from AMP and DMAPP in three enzymatic steps. This enzyme can create a bypass to tZRMP by stereospecifically hydroxylating the prenyl side chain of iPRMP, which can then be further activated to produce trans-zeatin.

[0226] Accordingly, according to some embodiments, the bacteria of the present invention are characterized in that the protein expression of polypeptides having cytochrome P450 monooxygenase (CYP450) activity is increased compared to the same bacteria, although the expression of such polypeptides is otherwise unmodified.

[0227] "Increased protein expression" means that the amount of polypeptides with cytochrome P450 monooxygenase (CYP450) activity produced by these modified bacteria is increased compared to unmodified bacteria that are otherwise identical. More specifically, “increased expression” means that the amount of polypeptides having cytochrome P450 monooxygenase (CYP450) activity produced by the thus modified bacteria is increased by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%, at least about 9000%, or at least about 10000% compared to unmodified but otherwise identical bacteria. The amount of protein in a given cell can be determined by any suitable quantification technique known in the art, such as ELISA, immunohistochemistry, or Western blotting.

[0228] Increased protein expression can be achieved by any suitable means known to those skilled in the art. For example, increased protein expression can be achieved by increasing the copy number of the gene encoding a polypeptide having cytochrome P450 monooxygenase (CYP450) activity in bacteria by introducing an exogenous nucleic acid into bacteria, such as a vector containing a gene encoding a polypeptide having cytochrome P450 monooxygenase (CYP450) activity, which is operably linked to a promoter that functions to induce the production of mRNA molecules in bacteria.

[0229] Increased protein expression can also be achieved by incorporating at least a second copy of the gene encoding a polypeptide with cytochrome P450 monooxygenase (CYP450) activity into the bacterial genome.

[0230] Increased protein expression can also be achieved by increasing the strength of a promoter manipulably linked to a gene encoding a polypeptide having cytochrome P450 monooxygenase (CYP450) activity. Potential promoters include natural promoters derived from Bacillus subtilis, Bacillus amyloliquefaciens, etc., such as P43, P15, Pveg, Pylb, PgroES, PsigX, PtrnQ, Ppst, PsodA, PrpsF, PlepA, PliaG, PrpsF, Ppst, PfusA, PsodA, Phag, as well as artificial promoters active in Bacillus subtilis, or inducible Bacillus subtilis promoters, such as PmtlA, Pspac, PxylA, PsacB, etc. Further examples include natural promoters derived from Corynebacterium, such as P CP_2454, Ptuf, and Psod; natural promoters derived from E. coli, such as T7, ParaBAD, Plac, Ptac, and Ptrc; and promoter P F1 derived from Corynepage BFK20.

[0231] Increased protein expression can also be achieved by modifying the ribosome binding site on mRNA molecules encoding polypeptides with cytochrome P450 monooxygenase (CYP450) activity. By modifying the sequence of the ribosome binding site, the translation initiation rate can be increased, thereby improving translation efficiency.

[0232] According to several embodiments, an increase in the copy number of a gene is achieved by introducing one or more (e.g., two or three) exogenous nucleic acid molecules (e.g., one or more vectors) into bacteria, which contain a gene that is operably linked to a promoter that functions to induce the production of mRNA molecules in a host cell.

[0233] According to several embodiments, the bacteria of the present invention comprises an exogenous nucleic acid molecule (vector, etc.) comprising one or more (e.g., two, three, or four) nucleotide sequences encoding a polypeptide having cytochrome P450 monooxygenase (CYP450) activity. Preferably, the exogenous nucleic acid molecule further comprises a promoter that functions to induce the production of an mRNA molecule in the bacterium and is operably ligated to the nucleotide sequence encoding the polypeptide having cytochrome P450 monooxygenase (CYP45) activity. According to several embodiments, the exogenous nucleic acid molecule is stably incorporated into the bacterial genome.

[0234] A polypeptide having cytochrome P450 monooxygenase (CYP450) activity may originate from the same species of bacterium that expresses it, or from a different species (i.e., heterogeneous). According to some embodiments, the polypeptide having cytochrome P450 monooxygenase (CYP450) activity originates from the same species of bacterium that expresses it. According to some embodiments, the polypeptide having cytochrome P450 monooxygenase (CYP450) activity originates from a different species (i.e., heterogeneous) of bacterium that expresses it. For example, if a bacterium does not have an endogenous gene encoding a polypeptide having cytochrome P450 monooxygenase (CYP450) activity, the polypeptide having cytochrome P450 monooxygenase (CYP450) activity expressed in the bacterium is heterogeneous to the bacterium.

[0235] According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity is a bacterial polypeptide having cytochrome P450 monooxygenase (CYP450) activity. "Bacterial polypeptide having cytochrome P450 monooxygenase (CYP450) activity" means that the polypeptide having cytochrome P450 monooxygenase (CYP450) activity is naturally derived from bacteria such as Rhodococcus fascians. A non-limiting example of a bacterial polypeptide having cytochrome P450 monooxygenase (CYP450) activity is described in Sequence ID No. 93.

[0236] According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity is a plant polypeptide having cytochrome P450 monooxygenase (CYP450) activity. "Plant polypeptide having cytochrome P450 monooxygenase (CYP450) activity" means that the polypeptide having cytochrome P450 monooxygenase (CYP450) activity is naturally derived from plants such as Arabidopsis thaliana. Non-limiting examples of plant polypeptides having cytochrome P450 monooxygenase (CYP450) activity are described in Sequence IDs 94 and 95.

[0237] Polypeptides having cytochrome P450 monooxygenase (CYP450) activity used in accordance with the present invention may be, for example, polypeptides having cytochrome P450 monooxygenase (CYP450) activity selected from the group consisting of i) polypeptides comprising any one amino acid sequence of SEQ ID NOs. 93 to 95; and ii) polypeptides comprising an amino acid sequence having at least about 70%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one amino acid sequence of SEQ ID NOs. 93 to 95.

[0238] According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 93 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 93. According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 93 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 93. According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 93 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 93. According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 93.

[0239] According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 94 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 94. According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 94 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 94. According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 94 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 94. According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 94.

[0240] According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 95 or an amino acid sequence having at least 70%, for example, at least 75%, sequence identity with SEQ ID NO: 95. According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 95 or an amino acid sequence having at least 80%, for example, at least 85%, sequence identity with SEQ ID NO: 95. According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 95 or an amino acid sequence having at least 90%, for example, at least 95%, sequence identity with SEQ ID NO: 95. According to several embodiments, a polypeptide having cytochrome P450 monooxygenase (CYP450) activity includes the amino acid sequence of SEQ ID NO: 95.

[0241] The bacteria according to the present invention can be produced from any suitable bacteria. The bacteria may be Gram-positive or Gram-negative. Non-limiting examples of Gram-negative bacterial host cells include species from the genera Escherichia, Erwinia, Klebsiella, and Citrobacter. Non-limiting examples of Gram-positive bacterial host cells include species from the genera Bacillus, Lactococcus, Clostridium, Corynebacterium, Streptomyces, Streptococcus, and Cerulomonas. According to some embodiments, the bacteria of the present invention are Gram-positive. According to some embodiments, the bacteria of the present invention are Gram-negative.

[0242] According to several embodiments, the bacteria of the present invention are bacteria of a family selected from the group consisting of Enterobacteriaceae, Bacillus, Lactobacillus, and Corynebacterium. According to several embodiments, the recombinant host cell is a bacterium of the Enterobacteriaceae family. According to several embodiments, the recombinant host cell is a bacterium of the Bacillaceae family. According to several embodiments, the recombinant host cell is a bacterium of the Corynebacteraceae family.

[0243] According to several embodiments, the bacteria of the present invention may be bacteria belonging to the genera Bacillus, Lactococcus, Clostridium, Corynebacterium, Geobacillus, Thermoanaerobacterium, Streptococcus, Pseudomonas, Streptomyces, Escherichia, Sigella, Acinetobacter, Citrobacter, Salmonella, Klebsiella, Enterobacter, Erwinia, Klebera, Serratia, Sedesea, Morganella, Hafnia, Edwardsiella, Providencia, Proteus, or Yersinia.

[0244] According to several embodiments, the bacteria of the present invention are bacteria of the genus Bacillus. Non-limiting examples of bacteria of the genus Bacillus include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus mojavensis. According to several embodiments, the bacteria of the present invention is Bacillus subtilis.

[0245] According to several embodiments, the bacteria of the present invention are bacteria of the genus Corynebacterium. Non-limiting examples of bacteria of the genus Corynebacterium are Corynebacterium glutamicum and Corynebacterium stationis. According to some, the bacteria of the present invention is Corynebacterium glutamicum. According to some, the bacteria of the present invention is Corynebacterium stationis. In the context of the present invention, Corynebacterium stationis and Corynebacterium ammoniagenes refer to the same species and can be used interchangeably.

[0246] According to some embodiments, the bacteria of the present invention are bacteria of the genus Escherichia. A non-limiting example of bacteria of the genus Escherichia is Escherichia coli. According to some embodiments, the bacteria of the present invention are Escherichia coli.

[0247] As described above, the bacteria of the present invention are modified to express one or more polypeptides as detailed herein, which may mean that an exogenous nucleic acid molecule, such as a DNA molecule, containing a nucleotide sequence encoding the polypeptide has been introduced into the bacteria. For this reason, the bacteria of the present invention may contain an exogenous nucleic acid molecule, such as a DNA molecule, containing a nucleotide sequence encoding the polypeptide in question. Methods for introducing exogenous nucleic acid molecules, such as DNA molecules, into bacterial cells are known to those skilled in the art, and include transformation (e.g., heat shock or spontaneous transformation).

[0248] To promote the (over)expression of polypeptides in bacteria, the exogenous nucleic acid molecule may function to induce the production of mRNA molecules in bacterial cells and may include suitable regulatory elements such as promoters operably linked to the nucleotide sequence encoding the polypeptide.

[0249] A useful promoter according to the present invention is any known promoter that functions to induce the production of an mRNA molecule in a given host cell. Many such promoters are known to those skilled in the art. Such promoters include promoters that are typically associated with other genes, and / or promoters isolated from any bacterium. The use of promoters for protein expression is generally known to those skilled in the art of molecular biology; see, for example, Sambrook et al., Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. The promoter used may be inducible, for example, a temperature-inducible promoter (e.g., a pL or pR phage λ promoter that can be controlled by a temperature-sensitive λ repressor c1857, respectively). As used in the context of a promoter, the term “inducible” means that the promoter induces the transcription of a manipulably linked nucleotide sequence only in the presence of a stimulus such as a change in temperature or the presence of a chemical substance (“chemical inducer”). As used herein, “chemical inducement” according to the present invention refers to the physical application of an exogenous or endogenous substance (including macromolecules, e.g., proteins or nucleic acids) to a host cell. This has the effect of increasing the transcription rate of the target promoter present in the host cell. Alternatively, the promoter used may be constitutive. In the context of promoters, the term "constitutive" means that the promoter can induce the transcription of a manipulably linked nucleotide sequence in the absence of a stimulus (heat shock, chemical, etc.).

[0250] Temperature-inducible systems operate by using promoters that are suppressed, for example, by thermally unstable repressors. These repressors are active at lower temperatures, such as 30°C, but are inactive at 37°C because they cannot fold correctly. Therefore, by using such a circuit, target genes can also be directly regulated by integrating them into the genome along with the repressors. An example of such a temperature-inducible expression system is based on pL and / or pR λ phage promoters regulated by the thermally unstable cI857 repressor. Similar to genome-integrated DE3 systems, the expression of the T7 RNA polymerase gene can also be controlled using a temperature-controlled promoter system, and the expression of target genes can be controlled using the T7 promoter.

[0251] Non-limiting examples of promoters that function in bacteria include both constitutive and inductive promoters, such as the T7 promoter, β-lactamase and lactose promoter systems; alkaline phosphatase (phoA) promoter, tryptophan (trp) promoter systems, tetracycline promoter, λ-phage promoter, ribosomal protein promoter; and hybrid promoters such as the tac promoter. Other bacterial and synthetic promoters are also suitable.

[0252] Exogenous nucleic acid molecules may further include, in addition to the promoter, at least one regulatory element selected from the 5' untranslated region (5'UTR) and the 3' untranslated region (3'UTR). Many such 5'UTRs and 3'UTRs from prokaryotes and eukaryotes are known to those skilled in the art. Such regulatory elements include 5'UTRs and 3'UTRs that are usually associated with other genes, as well as 5'UTRs and 3'UTRs isolated from any bacterium.

[0253] Typically, the 5'UTR contains a ribosome-binding site (RBS), also known as the Shine-Dalgano sequence, which is usually located 3 to 10 base pairs upstream of the start codon.

[0254] Exogenous nucleic acid molecules can be vectors or parts of vectors, such as expression vectors. Typically, such vectors remain extrachromosomally in bacterial cells, i.e., outside the bacterial nucleus or nucleoid region.

[0255] The present invention also aims to stably incorporate exogenous nucleic acid molecules into the genome of bacteria. For example, means for stably incorporating them into the genome of a host cell by homologous recombination are known to those skilled in the art.

[0256] Method of the present invention The present invention also provides a method for producing isoprenoid cytokinin or its riboside derivative, comprising culturing the bacteria according to the present invention in a suitable culture medium under suitable culture conditions.

[0257] The method may further include recovering isoprenoid cytokinin or its riboside derivative from the culture medium.

[0258] According to several embodiments, isoprenoid cytokinin or its riboside derivative is trans-zeatin (tZ), trans-zeatin riboside (tZR), N 6 -(D2-isopentenyl)adenine (iP), N 6 - Selected from the group consisting of (dimethylallyl)adenosine (iPR), dihydrozeatin (DZ), ribosyldihydrozeatin (DZR), and combinations thereof.

[0259] According to some embodiments, the isoprenoid cytokinin or its riboside derivative is trans-zeatin (tZ) and trans-zeatin riboside (tZR), respectively.

[0260] The culture medium used can be any conventional medium suitable for culturing the bacterial cells in question and may be constructed according to the principles of the prior art. The medium typically contains all the nutrients necessary for the growth and survival of each bacterium, such as carbon and nitrogen sources, as well as other inorganic salts. Suitable media, such as minimal media or compound media, are available from commercial suppliers or can be prepared according to published prescriptions, such as the strain catalog of the American Type Culture Collection (ATCC). Non-limiting standard media known to those skilled in the art include Luria bertani (LB) broth, Sabouraud dextrose (SD) broth, MS broth, yeast peptone dextrose, BMMY, GMMY, or yeast malt extract (YM) broth, all of which are commercially available. Non-limiting examples of culture media suitable for bacterial cells such as B. subtilis, L. lactis, or E. coli cells include minimal and nutrient-rich media, such as Luria broth (LB), M9 medium, M17 medium, SA medium, MOPS medium, Terrific broth, and YT.

[0261] The carbon source can be any suitable carbon substrate known in the art, in particular any carbon substrate commonly used for microbial culture and / or fermentation. Non-limiting examples of suitable fermentable carbon substrates include carbohydrates (e.g., C5 sugars such as arabinose or xylose, or C6 sugars such as glucose), glycerol, glycerin, acetate, dihydroxyacetone, one-carbon sources, methanol, methane, oils, animal fats, animal oils, vegetable oils, fatty acids, lipids, phospholipids, glycerolipids, monoglycerides, diglycerides, triglycerides, renewable carbon sources, polypeptides (e.g., microbial or plant proteins or peptides), yeast extracts, components from yeast extracts, peptones, casamino acids, or any combination of two or more of the above.

[0262] As nitrogen sources, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, natural nitrogen sources such as peptone, soy hydrolysate, and digests of fermenting microorganisms can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, etc. can be used.

[0263] To further improve the production of isoprenoid cytokinins or their riboside derivatives, such as trans-zeatin (tZ) and trans-zeatin riboside (tZR), a source of adenine or adenosine, such as adenine sulfate, may be added to the culture medium. For this reason, according to some embodiments, the culture medium contains adenine sulfate. The concentration of adenine sulfate in the culture medium can generally range from about 0.1 g / L to about 4 g / L, for example, from about 1 g / L to about 3.5 g / L. According to some embodiments, the concentration of adenine sulfate in the culture medium is in the range of about 2.5 g / L to about 3.5 g / L. However, other sources of adenine or adenosine, such as yeast extract, are also intended for use in accordance with the present invention.

[0264] Culturing can be carried out at a temperature of about 20 to about 45°C, for example, about 30 to 38°C, for example, about 37°C, preferably under aerobic conditions, such as by shaking culture and agitated culture with aeration. According to some embodiments, culturing is carried out at a temperature of about 30 to 38°C, for example, about 37°C. The pH of the culture is usually greater than 5, for example, in the range of about 6 to about 8, preferably about 6.5 to about 7.5, more preferably about 6.8 to about 7.2. According to some embodiments, culturing is carried out at a pH of about 6 to about 8. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases and buffers. Culturing can be carried out over a period of 10 to 70 hours, for example, in the range of 10 to 24 hours or 10 to 48 hours.

[0265] After culturing, solid matter such as cells can be removed from the culture medium by centrifugation or membrane filtration. Isoprenoid cytokinins or their riboside derivatives can be recovered by conventional methods for isolating and purifying chemical compounds from the culture medium. Well-known purification procedures include, but are not limited to, centrifugation or filtration, precipitation, ion exchange, chromatography (e.g., ion exchange chromatography or gel filtration chromatography), and crystallization.

[0266] Therefore, the present invention provides isoprenoid cytokinins or their riboside derivatives that can be obtained by the methods detailed herein.

[0267] Abbreviation iP - N 6 -(D2-isopentenyl)adenine iPR - N 6 -(D2-isopentenyl)adenine riboside, also known as N 6 -(D2-isopentenyl)adenosine tZ-trans-zeatin tZR - trans-ribosylzeatin, also known as trans-zeatin riboside DZ - Dihydrozeatin DZR - Ribosyldihydrozeatin, also known as dihydrozeatin riboside cZ-cis-zeatin MVA pathway - Mevalonate biosynthesis pathway MEP pathway - Methylerythritol phosphate biosynthesis pathway DMAPP - Dimethylallyl diphosphate HMBDP-1-hydroxy-2-methyl-2-butenyl-4-diphosphate DXP-1-deoxy-D-xylulose-5-phosphate DXS - 1-deoxy-D-xylulose-5-phosphate synthase; DXP-synthase (EC 2.2.1.7) tZRMP - trans-zeatin riboside 5'-monophosphate iPRMP - N6 -(D2-Isopentenyl)adenosine riboside 5'-monophosphate DZRMP - Dihydrozeatin riboside 5'-monophosphate cZRMP - cis-Zeatin riboside 5'-monophosphate IPT - Adenylate isopentenyltransferase (EC 2.5.1.27) LOG - Cytokinin riboside 5'-monophosphate phosphoryribohydrolase "Lonely Guy" (EC 3.2.2.n1) CYP450 - Cytochrome P450 monooxygenase

[0268] Certain other definitions As used herein, a "polypeptide having adenylate isopentenyltransferase activity" ("polypeptide having adenylate isopentenyltransferase activity" or a "polypeptide, which has adenylate isopentenyltransferase activity") means a polypeptide that catalyzes the following reaction: dimethylallyl diphosphate + AMP <=> diphosphate + N(6)-(dimethylallyl)adenosine 5'-phosphate (EC 2.5.1.27) and optionally 1-hydroxy-2-methyl-2-butenyl 4-diphosphate + AMP <=> diphosphate + trans-zeatin riboside 5'-phosphate. Non-limiting examples of such polypeptides are shown in SEQ ID NOs: 1 to 33.

[0269] As used herein, “polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity” or “polypeptide which has cytokinin riboside 5'-monophosphate phosphoribohydrolase activity” means a polypeptide that catalyzes the following reaction: N(6)-(Δ(2)-isopentenyl)-adenosine 5'-phosphate + H(2)O<=> N(6)-(dimethylallyl)adenine + D-ribose 5'-phosphate (EC 3.2.2.n1). Non-limiting examples of such polypeptides are shown in SEQ ID NOs. 34-62.

[0270] As used herein, “polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity” or “polypeptide, which has 1-deoxy-D-xylulose-5-phosphate synthase activity” means a polypeptide that catalyzes the following reaction: pyruvate + D-glyceraldehyde 3-phosphate <=> 1-deoxy-D-xylulose 5-phosphate + CO(2) (EC 2.2.1.7). Non-limiting examples of such polypeptides are shown in SEQ ID NOs. 63-70.

[0271] As used herein, “enzyme having ribose-phosphate diphosphokinase activity” or an “enzyme, which has ribose-phosphate diphosphokinase activity” means an enzyme that catalyzes the following reaction: ATP + D-ribose 5-phosphate <=> AMP + 5-phospho-α-D-ribose 1-diphosphate (EC 2.7.6.1). Enzymes having ribose-phosphate diphosphokinase activity are encoded by bacterial genes prs or their orthologs.

[0272] As used herein, “enzyme having amidophosphoribosyltransferase activity” or an “enzyme, which has amidophosphoribosyltransferase activity” means an enzyme that catalyzes the following reaction: 5-phospho-β-D-ribosylamine + diphosphate + L-glutamic acid <=> L-glutamine + 5-phospho-α-D-ribose-1-diphosphate + H2O (EC 2.4.2.14). Enzymes having amidophosphoribosyltransferase activity are encoded in the bacterial gene purF or its ortholog.

[0273] As used herein, “enzyme having formyltetrahydrofolate deformylase activity” or an “enzyme, which has formyltetrahydrofolate deformylase activity” means an enzyme that catalyzes the following reaction: 10-formyltetrahydrofolate + H2O <=> formic acid + tetrahydrofolate (EC 3.5.1.10). Enzymes having amide phosphoribosyltransferase activity are encoded by the bacterial gene purU or its ortholog.

[0274] As used herein, “enzyme having phosphoribosylamine-glycine ligase activity” or “enzyme, which has phosphoribosylamine-glycine ligase activity” means an enzyme that catalyzes the following reaction: ATP + 5-phospho-β-D-ribosylamine + glycine <=> ADP + phosphate + N 1 -(5-phospho-β-D-ribosyl)glycinamide (EC 6.3.4.13). Enzymes with phosphoribosylamine-glycine ligase activity are encoded by the bacterial gene purD or its ortholog.

[0275] As used herein, “enzyme having phosphoribosylglycineamide formyltransferase activity” or an “enzyme, which has phosphoribosylglycineamide formyltransferase activity” means an enzyme that catalyzes the following reaction: 10-formyltetrahydrofolate + N 1 -(5-phospho-β-D-ribosyl)glycinamide<=>tetrahydrofolate+N 2 -Formyl-N 1 -(5-phospho-β-D-ribosyl)glycinamide (EC 2.1.2.2). The enzyme possessing phosphoribosylglycinamide formyltransferase activity is encoded by the bacterial gene purT or its ortholog.

[0276] As used herein, “enzyme having phosphoribosylformylglycinamidine synthase activity” or an “enzyme, which has phosphoribosylformylglycinamidine synthase activity” means an enzyme that catalyzes the following reaction: ATP+N 2 -Formyl-N 1 -(5-phospho-β-D-ribosyl)glycinamide + L-glutamine + H2O <=> ADP + phosphate + 2-(formamide)-N 1 -(5-phospho-β-D-ribosyl)acetamidine + L-glutamic acid (EC 6.3.5.3). The enzyme possessing phosphoribosylformylglycineamidine synthase activity is encoded by the bacterial gene purL or its ortholog.

[0277] As used herein, “enzyme having phosphoribosylformylglycineamidine cyclo-ligase activity” or “enzyme, which has phosphoribosylformylglycineamidine cyclo-ligase activity” means an enzyme that catalyzes the following reaction: ATP + 2-(formamide)-N 1 -(5-phospho-β-D-ribosyl)acetamidine <=> ADP + phosphate + 5-amino-1-(5-phospho-β-D-ribosyl)imidazole (EC 6.3.3.1). Enzymes possessing phosphoribosylformylglycineamidin cycloligase activity are encoded by the bacterial gene purM or its ortholog.

[0278] As used herein, “enzyme having N5-carboxyaminoimidazole ribonucleotide synthetase activity” or “enzyme, which has N5-carboxyaminoimidazole ribonucleotide synthetase activity” means an enzyme that catalyzes the following reaction: ATP + 5-amino-1-(5-phospho-D-ribosyl)imidazole + HCO3 - <=> ADP + phosphate + 5-carboxyamino-1-(5-phospho-D-ribosyl)imidazole (EC 6.3.4.18). The enzyme possessing N5-carboxyaminoimidazole ribonucleotide synthetase activity is encoded by the bacterial gene purK or its ortholog.

[0279] As used herein, “enzyme having N5-carboxyaminoimidazole ribonucleotide mutase activity” or an “enzyme, which has N5-carboxyaminoimidazole ribonucleotide mutase activity” means an enzyme that catalyzes the following reaction: 5-carboxyamino-1-(5-phospho-D-ribosyl)imidazole <=> 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylic acid (EC 5.4.99.18). Enzymes having N5-carboxyaminoimidazole ribonucleotide mutase activity are encoded in the bacterial gene purE or its ortholog.

[0280] As used herein, “enzyme having phosphoribosylaminoimidazolesuccinocarboxamide synthase activity” or an “enzyme, which has phosphoribosylaminoimidazolesuccinocarboxamide synthase activity” means an enzyme that catalyzes the following reaction: ATP + 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylic acid + L-aspartic acid <=> ADP + phosphate + (S)-2-(5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide) succinate (EC 6.3.2.6). The enzyme having phosphoribosylaminoimidazolesuccinocarboxamide synthase activity is encoded in the bacterial gene purC or its ortholog.

[0281] As used herein, “enzyme having adenylosuccinate lyase activity” or an “enzyme, which has adenylosuccinate lyase activity” means an enzyme that catalyzes the following reaction: (S)-2-(5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide)succinate <=> fumarate + 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide (EC 4.3.2.2). Enzymes having adenylosuccinate lyase activity are encoded in the bacterial gene purB or its ortholog.

[0282] As used herein, “enzyme having phosphoribosylaminoimidazole-carboxamide formyltransferase activity” or an “enzyme, which has phosphoribosylaminoimidazole-carboxamide formyltransferase activity” means an enzyme that catalyzes the following reaction: 10-formyltetrahydrofolate + 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide <=> tetrahydrofolate + 5-formamide-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide (EC 2.1.2.3). The enzyme having phosphoribosylaminoimidazole-carboxamide formyltransferase activity is encoded in the bacterial gene purH or its ortholog.

[0283] As used herein, “enzyme having IMP cyclohydrolase activity” or an “enzyme, which has IMP cyclohydrolase activity” means an enzyme that catalyzes the following reaction: IMP + H2O <=> 5-formamide-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide (EC 3.5.4.10). Enzymes having IMP cyclohydrolase activity are encoded, for example, in the bacterial gene purH or its ortholog.

[0284] As used herein, “enzyme having adenylosuccinate synthase activity” or “enzyme, which has adenylosuccinate synthase activity” means an enzyme that catalyzes the following reaction: GTP + IMP + L-aspartate <=> GDP + phosphate + N6 -(1,2-dicarboxyethyl)-AMP (EC 6.3.4.4). The enzyme possessing adenylosuccinate synthase activity is encoded by the bacterial gene purA or its ortholog.

[0285] As used herein, “enzyme having adenylate kinase activity” or an “enzyme, which has adenylate kinase activity” means an enzyme that catalyzes the following reaction: ATP + AMP <=> 2ADP (EC 2.7.4.3). Enzymes having adenylate kinase activity are encoded by the bacterial gene adk or its ortholog.

[0286] As used herein, “enzyme having ATP synthase activity” or “enzyme, which has ATP synthase activity” means an enzyme that catalyzes the following reaction: ATP + H2O + H2O + (Cytosol) <=> ADP + Phosphate + H + (Periplasm) (EC 3.6.3.14). Enzymes possessing ATP synthase activity are, for example, the bacterial ATP operon (containing the genes atpB, atpF, atpE, atpD, atpG, atpA, atpH, and atpC) or the ATP synthase F0 or F1 complex encoded by its ortholog.

[0287] As used herein, “enzyme having adenosine kinase activity” or an “enzyme, which has adenosine kinase activity” means an enzyme that catalyzes the following reaction: ATP + adenosine <=> ADP + AMP (EC 2.7.1.20). Enzymes having adenosine kinase activity are encoded by the bacterial gene adk or its ortholog.

[0288] As used herein, an “enzyme having IMP dehydrogenase activity” or an “enzyme, which has IMP dehydrogenase activity” means an enzyme that catalyzes the following reaction: inosine 5′-phosphate + NAD + + H2O <=> xanthosine 5′-phosphate + NADH (EC 1.1.1.205). An enzyme having IMP dehydrogenase activity is encoded by the bacterial gene guaB or its ortholog.

[0289] As used herein, an “enzyme having GMP synthase activity” or an “enzyme, which has GMP synthase activity” means an enzyme that catalyzes the following reaction: ATP + XMP + L-glutamine + H2O <=> AMP + diphosphate + GMP + L-glutamic acid (EC 6.3.5.2). An enzyme having GMP synthase activity is encoded by the bacterial gene guaA or its ortholog.

[0290] As used herein, an “enzyme having purine nucleoside phosphorylase activity” or an “enzyme, which has purine nucleoside phosphorylase activity” means an enzyme that catalyzes the following reaction: purine nucleoside + phosphate <=> purine + α-D-ribose 1-phosphate (EC 2.4.2.1). An enzyme having purine nucleoside phosphorylase activity is encoded, for example, by the bacterial gene deoD or its ortholog.

[0291] As used herein, “enzyme having adenosine phosphoribosyltransferase activity” or an “enzyme, which has adenosine phosphoribosyltransferase activity” means an enzyme that catalyzes the following reaction: AMP + diphosphate <=> adenine + 5-phospho-α-D-ribose 1-diphosphate (EC 2.4.2.7). Enzymes having adenosine phosphoribosyltransferase activity are encoded, for example, in the bacterial gene apt or its ortholog.

[0292] As used herein, the term "purine nucleotide biosynthesis pathway" is understood to include both the de novo biosynthesis pathway and the salvage pathway through which nucleotides are synthesized.

[0293] As used herein, the “adenosine monophosphate biosynthesis pathway” is understood to include both the de novo biosynthesis pathway and the salvage pathway through which adenosine monophosphate is synthesized.

[0294] As used herein, the “guanosine monophosphate biosynthesis pathway” is understood to include both the de novo biosynthesis pathway and the salvage pathway through which guanosine monophosphate is synthesized.

[0295] As used herein, “polypeptide having cytochrome P450 monooxygenase (CYP450) activity” or “polypeptide, which has cytochrome P450 monooxygenase (CYP450) "Activity" refers to polypeptides that catalyze the following transhydroxylation reactions: 1) N6-(Δ2-isopentenyl)-adenosine 5'-monophosphate + reduced [NADPH-hemeprotein reductase] + oxygen → trans-zeatin riboside monophosphate + oxidized [NADPH-hemeprotein reductase] + H2O; 2) N6-(Δ2-isopentenyl)-adenosine 5'-triphosphate + reduced [NADPH-hemeprotein reductase] + oxygen → trans-zeatin riboside triphosphate + oxidized [NADPH-hemeprotein reductase] + H2O; 3) N6-(Δ2-isopentenyl)-adenosine 5'-diphosphate + reduced [NADPH-hemeprotein reductase] + oxygen → trans-zeatin riboside diphosphate + oxidized [NADPH-hemeprotein reductase] + H2O (EC Polypeptides possessing cytochrome P450 monooxygenase (CYP450) activity are encoded, for example, in the bacterial gene FAS1, or in the Arabidopsis thaliana (plant) genes CYP735A1 and CYP735A2, or their orthologs. Non-limiting examples include sequence numbers 93-95.

[0296] "Polypeptide" and "protein" are used herein without distinction to represent polymers of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). D- and L-amino acids, as well as mixtures of D- and L-amino acids, are included in this definition.

[0297] "Nucleic acid" or "polynucleotide" is used herein without distinction to represent a polymer of at least two nucleic acid monomer units or bases (e.g., adenine, cytosine, guanine, thymine) covalently linked by a phosphodiester bond, regardless of length or base modification.

[0298] For example, as used with respect to bacteria, nucleic acids, or polypeptides, “recombinant” or “non-spontaneous” refers to a substance or a substance corresponding to its natural or native form that has been modified in a manner not otherwise found in nature, or is identical to such a substance but produced or induced from a synthetic substance and / or through manipulation using recombinant technology. A non-limiting example is, in particular, recombinant bacterial cells that express genes not found in the cell’s native (non-recombinant) form, or that express native genes that would otherwise be expressed at different levels.

[0299] As used herein in the context of genes or nucleic acid molecules, “exogenous” or “exogenous” refers to a gene or nucleic acid molecule (i.e., a DNA or RNA molecule) that is not naturally present as part of the genome of the bacterium in which it exists, or that is located in a different position within the genome than where it would naturally exist. Thus, a “exogenous” or “exogenous” gene or nucleic acid molecule is introduced into the microorganism exogenously, rather than endogenously, to the bacterium. The DNA molecule of a “exogenous” gene or nucleic acid molecule may originate from a different organism, a different species, a different genus, or a different kingdom as host DNA.

[0300] As used herein in the context of polypeptides, “heterogeneous” means that the polypeptide is not normally found in or produced (i.e., expressed) by a host microorganism and originates from a different organism, a different species, a different genus, or a different kingdom.

[0301] As used herein, the term “ortholog” or “orthologs” refers to a gene that originates from a common ancestral gene but is present in different species, the nucleic acid molecule it encodes, i.e., mRNA, or the protein it encodes.

[0302] A "decrease in gene expression level" means that the amount of transcripts produced by the modified bacteria, and the amount of polypeptides encoded by the respective genes, is reduced compared to an unmodified but otherwise identical bacterium. More specifically, a "decrease in gene expression level" means that the amount of transcripts produced by the modified bacteria, and the amount of polypeptides encoded by the respective genes, is reduced by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, compared to an unmodified but otherwise identical bacterium. The level of gene expression can be determined by known methods, including PCR and Southern blotting. In addition, the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various known methods, including Northern blotting and quantitative RT-PCR. The amount of polypeptides encoded by the gene can be measured by known methods, including ELISA, immunohistochemistry, or Western blotting.

[0303] Gene expression can be reduced by introducing mutations into genes within the genome of a modified bacterium such that the intracellular activity of the polypeptide encoded by the gene is reduced compared to the same bacterium, but without the mutation. Mutations that result in reduced gene expression include substitutions of one or more nucleotides that cause amino acid substitutions in the polypeptide encoded by the gene (missense mutations), introduction of stop codons (nonsense mutations), deletion or insertion of nucleotides that cause frameshifts, insertion of drug resistance genes, or deletion of part or all of a gene (Qiu and Goodman, 1997; Kwon et al., 2000). Expression can also be reduced by modifying expression regulatory sequences such as promoters and Shine-Dalgano (SD) sequences. Gene expression can also be reduced by gene substitutions such as "λ-RED mediated gene substitution" (Datsenko and Wanner, 2000). λ-RED mediated gene substitution is a particularly preferred method for inactivating one or more genes as described herein.

[0304] When used in the context of genes, "inactivate," "deactivate," and "inactivated" mean that the gene in question no longer expresses a functional protein. Modified DNA regions may not be able to spontaneously express a gene due to partial or complete deletion of the gene sequence, shifting of the gene's reading frame, introduction of missense / nonsense mutations, or alteration of regulatory regions of the gene, including sequences that control gene expression, such as promoters, enhancers, attenuators, and ribosome binding sites. Preferably, the gene in question is inactivated by partial or complete deletion of the gene sequence, such as by gene substitution. Inactivation can also be achieved by introducing or expressing a rare-cut endonuclease that can selectively inactivate the gene in question by DNA breaks, preferably double-strand breaks. In the context of this invention, "rare-cut endonucleases" include transcription activator-like effector (TALE) nucleases, meganucleases, zinc finger nucleases (ZFNs), and RNA-inducible endonucleases.

[0305] The presence or absence of genes within a bacterial genome can be detected by known methods, including PCR and Southern blotting. In addition, gene expression levels can be estimated by measuring the amount of mRNA transcribed from genes using various known methods, including Northern blotting and quantitative RT-PCR. The amount of protein encoded by genes can be measured by known methods, including immunoblotting assays (Western blotting analysis) following SDS-PAGE.

[0306] An "increase in gene expression level" means that the amount of transcripts produced by the modified bacteria, and the amount of polypeptides encoded by the respective genes, increases compared to bacteria that are not modified but otherwise identical. More specifically, "increased gene expression level" means that the amount of transcript produced by the modified bacteria, the amount of polypeptide encoded by each of the genes, increases by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%, at least about 9000%, or at least about 10000% compared to bacteria that are otherwise identical but not modified. The level of gene expression can be determined by known methods, including PCR and Southern blotting. In addition, the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various known methods, including Northern blotting and quantitative RT-PCR. The amount of polypeptide encoded by the gene can be measured by known methods, including ELISA, immunohistochemistry, or Western blotting.

[0307] An "increase in polypeptide expression level" means that the amount of the polypeptide in question produced by a modified microorganism is increased compared to an unmodified but otherwise identical bacterium. More specifically, “increased expression level” of a polypeptide means that the amount of the polypeptide in question produced by the modified bacteria is increased by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%, at least about 9000%, or at least about 10000% compared to bacteria that are otherwise identical but not modified. The amount of polypeptide produced in a given cell can be determined by any suitable quantification technique known in the art, such as ELISA, immunohistochemistry, or Western blotting.

[0308] Increased polypeptide expression can be achieved by any suitable means known to those skilled in the art. For example, increased polypeptide expression can be achieved by increasing the copy number of the polypeptide-encoding gene in a microorganism, for example, by introducing an exogenous nucleic acid, such as a vector, containing the polypeptide-encoding gene, which is operably linked to a promoter that functions to induce the production of mRNA molecules in the microorganism. Increased polypeptide expression can also be achieved by incorporating at least a second copy of the polypeptide-encoding gene into the microbial genome. Increased polypeptide expression can also be achieved by increasing the strength of a promoter operably linked to the polypeptide-encoding gene. Increased polypeptide expression can also be achieved by modifying the ribosome binding site on the polypeptide-encoding mRNA molecule. By modifying the sequence of the ribosome binding site, the translation initiation rate can be increased, thereby improving translation efficiency.

[0309] As used herein, “reduced,” “decreased,” or “decreased” expression of a polypeptide (e.g., an enzyme involved in the purine nucleotide degradation pathway) means that the expression of the polypeptide in the modified bacteria is lower compared to the expression of the polypeptide in an unmodified but otherwise identical bacterium (control). The expression of the polypeptide in the modified bacteria may be reduced by at least about 10%, preferably at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, or by any percentage between 10% and 100% (e.g., 6%, 7%, 8%, etc.) compared to the expression of the polypeptide in an unmodified but otherwise identical bacterium (control). More specifically, "decreased," "reduced," or "reduced" polypeptide expression means that the amount of polypeptide in the modified bacteria is reduced by at least about 10%, preferably at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, or by any percentage in integers between 10% and 100% (e.g., 6%, 7%, 8%, etc.). Polypeptide expression or amount in microorganisms can be determined by any suitable means known in the art, including methods such as ELISA, immunohistochemistry, Western blotting, or flow cytometry.

[0310] As used herein, “reduced,” “decreased,” or “decreased” activity of a polypeptide (e.g., an enzyme involved in the purine nucleotide degradation pathway) means that the catalytic activity of the polypeptide in the modified bacteria is lower compared to the catalytic activity of the polypeptide in the unmodified but otherwise identical bacteria (control). The activity of the polypeptide in the modified bacteria may be reduced by at least about 10%, preferably at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, or by any percentage between 10% and 100% (e.g., 6%, 7%, 8%, etc.) compared to the expression of the polypeptide in the unmodified but otherwise identical bacteria (control). The activity of polypeptides in microorganisms can be determined by any suitable protein and enzyme activity assay.

[0311] As used herein, “enzyme inhibitor” refers to any natural or synthetic chemical compound that inhibits the catalytic activity of an enzyme. Enzyme inhibitors do not necessarily need to achieve 100% or complete inhibition. In this regard, enzyme inhibitors can induce any level of inhibition.

[0312] "Substitution" or "substituted" refers to the modification of a polypeptide by replacing one amino acid residue with another; for example, replacing a serine residue in a polypeptide sequence with a glycine or alanine residue is an amino acid substitution. When used in relation to polynucleotides, "substitution" or "substituted" refers to the modification of a polynucleotide by replacing one nucleotide with another; for example, replacing cytosine with thymine in a polynucleotide sequence is a nucleotide substitution.

[0313] When used in reference to polypeptides, “conservative substitution” refers to the substitution of an amino acid residue with a different residue having a similar side chain, and therefore typically includes substituting amino acids in a polypeptide with amino acids of the same or similar class. Examples, but not limited to, include: amino acids having an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; amino acids having a hydroxyl side chain may be substituted with another amino acid having a hydroxyl side chain, e.g., serine and threonine; amino acids having an aromatic side chain may be substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; amino acids having a basic side chain may be substituted with another amino acid having a basic side chain, e.g., lysine and arginine; amino acids having an acidic side chain may be substituted with another amino acid having an acidic side chain, e.g., aspartic acid or glutamic acid; and hydrophobic or hydrophilic amino acids may be replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0314] When used in relation to polypeptides, "non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid whose side-chain properties are significantly different. Non-conservative substitutions can use amino acids between defined groups rather than within them, and affect (a) the structure of the peptide backbone in the region of substitution (e.g., serine instead of glycine), (b) charge or hydrophobicity, or (c) the bulkiness of the side chain. Examples of non-conservative substitutions, though not limited to these, may include acidic amino acids substituted with basic or aliphatic amino acids; aromatic amino acids substituted with small amino acids; and hydrophilic amino acids substituted with hydrophobic amino acids.

[0315] "Expression" includes, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion, any steps involved in the production of a polypeptide (e.g., the encoded enzyme).

[0316] As used herein, “regulatory region” of a gene refers to a nucleic acid sequence that affects the expression of a coding sequence. Regulatory regions are known in the art and include, but are not limited to, promoters, enhancers, transcriptional terminators, polyadenylation sites, matrix attachment regions, and / or other elements that modulate the expression of a coding sequence.

[0317] As used herein, “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded nucleic acid loop to which an additional nucleic acid segment can be ligated. Certain vectors can induce the expression of a gene to which they are manipulably ligated. Such vectors are referred herein to as “expression vectors.” Certain other vectors can facilitate the insertion of exogenous nucleic acid molecules into the genome of bacteria. Such vectors are referred herein to as “transformation vectors.” Generally, vectors useful in recombinant nucleic acid technology are often in the form of plasmids. Since plasmids are the most commonly used form of vectors, “plasmid” and “vector” may be used interchangeably herein. Numerous suitable vectors are known to those skilled in the art and are commercially available.

[0318] As used herein, “promoter” refers to a DNA sequence, usually upstream (5') of the coding region of a structural gene, that controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors that may be required for transcription initiation. The choice of promoter depends on the nucleic acid sequence of interest. A suitable “promoter” is generally one that can support transcription initiation in the bacteria of the present invention and induce the production of mRNA molecules. Examples of "strong" promoters include natural promoters derived from Bacillus subtilis, Bacillus amyloliquefaciens, etc., which enable efficient expression and overproduction of proteins, such as P43, P15, Pveg, Pylb, PgroES, PsigX, PtrnQ, Ppst, PsodA, PrpsF, PlepA, PliaG, PrpsF, Ppst, PfusA, PsodA, Phag, as well as artificial promoters active in Bacillus subtilis, or inducible Bacillus subtilis promoters, such as PmtlA, Pspac, PxylA, PsacB. Further examples of "strong" promoters include natural promoters from Corynebacterium, e.g., P CP_2454, Ptuf, and Psod; natural promoters from E. coli, e.g., T7; and promoter P F1 from Corynepage BFK20.

[0319] As used herein, “operably ligated” refers to a juxtaposition that is in a relationship that enables the described components to function as intended. A regulatory sequence “operably ligated” to a coding sequence ligates the coding sequence so that its expression is achieved under conditions compatible with the regulatory sequence. A promoter sequence is “operably ligated” to a gene if it is close enough to the gene’s transcription start site to regulate the gene’s transcription.

[0320] "Percentage of sequence identity," "% sequence identity," and "percent identity" are used herein to refer to a comparison between an amino acid sequence and a reference amino acid sequence. As used herein, "% sequence identity" is calculated from two amino acid sequences as follows: Align the sequences using Genetic Computing Group's GAP (Global Alignment Program) version 9, with a default BLOSUM62 matrix, a gap-open penalty of -12 (for the first null in the gap) and a gap-extension penalty of -4 (for each additional null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference amino acid sequence.

[0321] A "reference sequence" or "reference amino acid sequence" refers to a specific sequence that is compared to another sequence. In the context of the present invention, the reference amino acid sequence may be, for example, the amino acid sequence described in SEQ ID NO: 1.

[0322] As used herein, the term "approximately" means plus or minus 10% of the numerical value in which it is used.

[0323] Where numerical limits or ranges are defined herein, the endpoints are included. Furthermore, all values ​​and subranges within the numerical limits or ranges are included as specifically as if they were explicitly written out.

[0324] As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more" unless the context clearly indicates otherwise.

[0325] As used herein, “comprising,” “including,” “having,” and their grammatical variations are deemed to specify a feature, process, or component described, but do not preclude the addition of one or more additional features, processes, components, or groups thereof.

[0326] While the present invention has been described in general terms, further understanding can be gained by referring to certain specific examples presented herein for illustrative purposes only and not intended to be limiting unless otherwise specified.

[0327] Examples Example 1: Selection of Starting Stock Various Bacillus strains can be used as starting stocks for isoprenoid cytokinin production engineering (Table 2). Bacillus species can be isolated from nature or obtained from culture collections. In particular, starting stocks for isoprenoid cytokinin production can be selected from Bacillus subtilis strains that have already been subjected to classical mutagenesis and selection methods to overproduce metabolites related to the purine nucleotide biosynthesis pathway. For example, strains that overproduce riboflavin, inosine, guanosine, or adenosine can be selected. Strains that have been subjected to random mutagenesis and toxic metabolic inhibitors from the purine nucleotide and riboflavin pathways are preferred, as listed in Table 3.

[0328] [Table 2]

[0329] [Table 3]

[0330] Bacillus subtilis VKPM B2116 is a hybrid strain of B. subtilis 168 (the most common B. subtilis host strain with a genome of approximately 4 Mbp) and a 6.4 kbp island of DNA derived from the B. subtilis W23 strain. This structure is common to most B. subtilis industrial strains and was obtained by transforming B. subtilis 168 (the tryptophan-requiring strain trpC) with W23 (prototrophic TrpC+) DNA. The 6.4 kbp W23 island in the genome of VKPM B2116 is identical to that of B. subtilis SMY, one of the publicly available B. subtilis legacy strains (Zeigler et al. 2008). B. subtilis VKPM B2116 is a direct descendant strain of B. subtilis SMY obtained by classical mutagenesis and selection. This strain is also known as B. subtilis VNII Genetika 304. The construction of the strain is described in Soviet Patent Invention No. 908092, filed in 1980. This mutation was obtained by subsequent mutagenesis and selection of metabolic inhibitors. Strain VKPM B2116 exhibits resistance to roseoflavin, a toxic analogue of vitamin B2, due to a mutation in the ribC gene encoding flavin kinase. This strain also exhibits resistance to 8-azaguanine, a toxic purine analogue.

[0331] Example 2: Synthesis of synthetic genes IPT SEQ ID NO: 1 and LOG SEQ ID NO: 34 for isoprenoid cytokinin biosynthesis optimized for Bacillus subtilis. The amino acid sequence of adenylate dimethylallyltransferase (IPT) Tzs derived from Agrobacterium tumefaciens (synonym Agrobacterium fabrum) (strain C58 / ATCC 33970) (gene tzs, EC2.5.1.27, UniProt:P58758) (SEQ ID NO: 1) and the amino acid sequence of cytokinin riboside 5'-monophosphate phosphoribohydrolase (LOG) derived from Corynebacterium glutamicum (strain ATCC 13032 / DSM 20300 / NCIMB) Using gene Cgl2379 (EC3.2.2.n1, UniProt:Q8NN34) (SEQ ID NO: 34), a codon-optimized nucleotide sequence for gene expression in B. subtilis was generated using GENEius (Eurofins). The synthetic DNA fragment IPT-LOG (SEQ ID NO: 71) was designed with an added RBS sequence and short adapter sequences at both ends of the synthetic fragment for further assembly of a synthetic isoprenoid cytokinin operon expression cassette.

[0332] Example 3: Synthesis of synthetic genes IPT SEQ ID NO: 2 and LOG SEQ ID NO: 34 for isoprenoid cytokinin biosynthesis optimized for Bacillus subtilis. The amino acid sequence of adenylate dimethylallyltransferase (IPT) Tmr derived from Agrobacterium tumefaciens (synonym Agrobacterium fabrum) (strain C58 / ATCC 33970) (gene izt, EC2.5.1.27, Uniprot:P0A3L5) (SEQ ID NO: 2) and Corynebacterium glutamicum Using the amino acid sequence (gene Cgl2379, EC3.2.2.n1, UniProt:Q8NN34) (SEQ ID NO: 34) of cytokinin riboside 5'-monophosphate phosphoribohydrolase (LOG) derived from glutamicum, a codon-optimized nucleotide sequence for gene expression in B. subtilis was generated using GENEius (Eurofins). The synthetic DNA fragment IPT-LOG (SEQ ID NO: 72) was designed with the addition of an RBS sequence and short adapter sequences at both ends of the synthetic fragment for further assembly of a synthetic isoprenoid cytokinin operon expression cassette.

[0333] Example 4: Assembly of a synthetic isoprenoid cytokinin operon containing IPT and LOG, and transformation into B. subtilis. Synthetic fragments containing the synthetic gene IPT-LOG for isoprenoid cytokinin biosynthesis (SEQ ID NOs. 71 and 72) were assembled into an artificial isoprenoid cytokinin operon. The leading and trailing fragments, containing gene integration homology, promoter sequence, and an erythromycin-selectable marker (SEQ ID NOs. 73), were designed and synthesized for stable genomic integration into the amyE locus in the B. subtilis genome.

[0334] The first step in the artificial operon assembly was PCR amplification of separate DNA fragments, performed using primer pairs of SEQ ID NO: 74 and 75, against the leading fragment of SEQ ID NO: 75, as well as the synthetic fragments IPT-LOG of SEQ ID NO: 71 and 72, which contain genes for isoprenoid-cytokinin biosynthesis. The primer sets of SEQ ID NO: 77 and 78 were used to amplify the final fragment of SEQ ID NO: 79.

[0335] The fragments were amplified for 30 cycles in a final volume of 50 μl using Eppendorf cycler and Phusion polymerase (Thermo Fisher) with 200 μM dNTPs, 0.5 μM primers, and approximately 10 ng of template added in the manufacturer's buffer. The PCR cycle conditions were as follows: 30 cycles at 98°C for 30 seconds (30 seconds at 98°C, 25 seconds at 68.5°C, and 23 / 25 seconds at 72°C), followed by 5 minutes at 72°C and a hold at 10°C.

[0336] The PCR reaction products of each fragment were electrophoresed on a 0.8% agarose gel, excised from the gel, and extracted from the gel using the GeneJET Gel Extraction Kit (Thermo Fisher) according to the manufacturer's protocol. The fragments were assembled into an artificial operon through repeated restriction and ligation steps. By using a combination of SpeI (BcuI) and XbaI restriction sites, restriction ends suitable for successful ligation were obtained. After each ligation step, the combined fragments were used as a new template for the next PCR amplification. Restriction was performed in 50 μl volumes by adding 5 μl of FD green buffer (Thermo Fisher Scientific), 2-3 μl of selective enzyme (SpeI (BcuI) and XbaI, Thermo Fisher), and up to approximately 1500 ng of PCR fragment. After restriction digestion, the digested DNA fragments were cleaned using the Wizard SV Gel and PCR Clean-up system according to the manufacturer's protocol. The first two fragments were mixed with 5% PEG 4000 in the manufacturer's buffer, and both fragments were mixed in a 1:1 molar ratio to a final volume of 15 μl, which was then used for ligation with 2.5 U of T4 DNA ligase (Thermo Fisher). In the next step, 1 μl of inactivated ligation was used as a template for a new 50 μL PCR using the primer sets of SEQ ID NOs. 80 and 81, with the same PCR mix and PCR cycle conditions as before, but with a longer extension time. Restriction digestion, cleaning, and ligation steps were repeated for ligation of the final fragment. PCR was performed on a 0.8% agarose gel, the fragment was excised from the gel, digested and cleaned as before, and ligated as before. The final operon, containing amy E homology, promoter with RBS sequence, IPT and LOG genes, and erythromycin resistance cassette, was amplified using the primer pairs of SEQ ID NOs. 76 and 77, and cleaned and ligated as described above.Synthetic trans-zeatin operons containing IPT-LOGs from SEQ ID NOs. 82 and 83 were used to transform Bacillus subtilis VKPM B2116. Transformation using the IPT-LOG operon from SEQ ID NOs. 83 yielded transformant strains TZAB1, TZAB2, TZAB3, and TZAB4. Transformation using the IPT-LOG operon from SEQ ID NOs. 82 yielded transformant strains TZAB14 and TZAB15. Accurate integration of the artificial operon at the amyE integration site was confirmed by cPCR.

[0337] [Table 4]

[0338] Example 5: Increased isoprenoid cytokinin biosynthesis by increasing isoprenoid precursor supply using dxs overexpression. The isoprenoid side chain for isoprenoid cytokinin biosynthesis is synthesized via the MEP pathway in B. subtilis. Using the protein sequence of 1-deoxyxylulose-5-phosphate synthase (DXS, EC2.2.1.7) derived from B. subtilis, the synthetic nucleotide sequence of dxs was generated using the codon optimization feature GENEius (Eurofins) for gene expression in B. subtilis. Synthetic DNA fragments of dxs (SEQ ID NOs. 84 and 85) were designed with two overlapping portions and joined by overlap PCR. The entire joined synthetic gene dxs (SEQ ID NOs. 86) was assembled into an artificial operon. The leading and trailing fragments, containing lacA homology, the promoter sequence, and the spectinomycin-selectable marker (SEQ ID NO. 87), were designed and synthesized for stable genomic integration into the B. subtilis genome. The fragments were assembled as described in Example 4.

[0339] The first step in the artificial operon assembly was PCR amplification of separate DNA fragments using the primer set of SEQ ID NO: 74 and SEQ ID NO: 75 against the combined synthetic gene dxs of the leading fragment of SEQ ID NO: 88, the final fragment of SEQ ID NO: 89, and SEQ ID NO: 86.

[0340] Fragments were amplified for 30 cycles in a final volume of 50 μl using Eppendorf cycler and Phusion polymerase (Thermo Fisher) with 200 μM dNTPs, 0.5 μM primers, and approximately 10 ng of template added in the manufacturer's buffer. The PCR cycle conditions used were 30 cycles at 98°C for 30 seconds (30 seconds at 98°C, 25 seconds at 71°C, and 23 / 25 seconds at 72°C), followed by a hold at 72°C for 5 minutes and a hold at 10°C.

[0341] The PCR reaction products of each fragment were electrophoresed on a 0.8% agarose gel, excised from the gel, and extracted from the gel using the GeneJET Gel Extraction Kit (Thermo Fisher) according to the manufacturer's protocol. The fragments were assembled into an artificial operon through repeated restriction and ligation steps. By using a combination of SpeI (BcuI) and XbaI restriction sites, restriction ends suitable for successful ligation were obtained. After each ligation step, the combined fragments were used as new templates for subsequent PCR amplification. Restriction was performed in 50 μl volumes by adding 5 μl of FD green buffer (Thermo Fisher Scientific), 2-3 μl of selective enzyme (SpeI (BcuI) and XbaI, Thermo Fisher), and up to approximately 1500 ng of PCR fragment. After restriction digestion, the digested DNA fragments were cleaned using the Wizard SV Gel and PCR Clean-up system according to the manufacturer's protocol. The first two fragments were mixed in a 1:1 molar ratio in the manufacturer's buffer to a final volume of 15 μl and used for ligation with 2.5 U of T4 DNA ligase (Thermo Fisher). In the next step, 1 μl of inactivated ligation was used as a template with the primer set of SEQ ID NO: 74 and SEQ ID NO: 75, using the same PCR mix and PCR cycle conditions as before, but with a longer extension time, for a new 50 μL PCR. Restriction digestion, cleaning, and ligation steps were repeated for ligation of the final fragment. In the final step, 1 μl of inactivated ligation was used as a template for the complete synthetic dxs operon of SEQ ID NO: 90, using the primers of SEQ ID NO: 91 and SEQ ID NO: 92, using the same PCR mix and PCR cycle conditions as before, but with a longer extension time, for a new 50 μL PCR. PCR was performed on a 0.8% agarose gel, the fragment was excised from the gel, cleaned as before, and ligated as before.The constructed synthetic dxs operon was used to transform Bacillus subtilis TZAB15, which has an IPT-LOG operon in amyE (SEQ ID NO: 80). Transformation using the artificial DXS operon (SEQ ID NO: 90) yielded the transformed strain TZAB43. Accurate integration of the artificial operon at the lacA integration site was confirmed by cPCR.

[0342] [Table 5]

[0343] Example 6: Culture of Bacillus subtilis strain for isoprenoid cytokinin production All constructed and control strains were cultured according to this procedure. Frozen stocks of strains VKPM B2116, TZAB1, TZAB2, TZAB3, TZAB4, TZAB14, TZAB15, and TZAB43, stored in 20% glycerol at -80°C, were streaked onto solid seed medium containing appropriate concentrations of erythromycin and lincomycin and incubated at 37°C for approximately 1 day. For further testing, 1 to 5 plugs of cultures from the solid seed plate were inoculated into vegetative stage medium in each 250 mL baffled Erlenmeyer flask containing 25 mL of nutrient medium and an appropriate amount of antibiotic. The cultures were incubated at 37°C for 18 to 20 hours at 220 RPM. The cultures in the nutrient medium were used as seed cultures and inoculated into the production medium. 10% inoculum was used (2.5 mL per 25 mL of production medium in the 250 mL Erlenmeyer flask). The culture medium was used as described above, or modified with adenine sulfate (final concentration 3 g / L). The cultures were incubated at 30°C or 37°C for up to 48 hours at 220 RPM. The fermented cultures were sampled and analyzed as described in Example 7. The titers of trans-zeatin, trans-zeatin riboside, and isopentenyl adenine were measured using LC / MS as described in Example 7.

[0344] [Table 6]

[0345] [Table 7]

[0346] The components of the nutrient medium (Table 7) were mixed, and the pH was adjusted to 7.2-7.4. Then, KH2PO4-K2HPO4 solution was added at final concentrations of 1.5 g / L of KH2PO4 and 3.5 g / L of K2HPO4. The medium was divided into Erlenmeyer flasks (25 ml / 250 ml baffled Erlenmeyer flasks) and autoclaved at 121°C for 30 minutes. Sterile glucose was added after autoclaving at a final concentration of 7.5 g / L. Antibiotics were added before inoculation.

[0347] [Table 8]

[0348] The components of the production medium (Table 8) were mixed and the pH was adjusted to 7.2-7.4. Then, KH2PO4-K2HPO4 solution was added at concentrations of 1.5 g / L of KH2PO4 and 3.5 g / L of K2HPO4. The medium was autoclaved at 121°C for 30 minutes. Sterile urea solution (20 ml of stock solution, final concentration 6 g / L) and sterile glucose solution (500 ml of stock solution, final concentration 100 g / L glucose) were added after autoclaving to obtain 1 L of production medium. Appropriate antibiotics were added before inoculation. Then, the medium was distributed into sterile Erlenmeyer flasks (25 ml / 250 ml baffled Erlenmeyer flasks).

[0349] Example 7: Analysis of isoprenoid cytokinins All strains cultured according to the procedure described in Example 6 were analyzed according to this procedure. Fermented production medium was sampled and immediately frozen at -20°C. For the extraction of metabolites, the fermented production medium was diluted 1:1 with an extraction buffer consisting of a 1:1 mixture of methanol and 100 mM ammonium acetate pH 4. The sample was extracted at room temperature for 1 hour with constant stirring, centrifuged at 4000-4500 RPM for 15 minutes, and filtered (0.22 μm). The sample was analyzed immediately by LC / MS or stored at -20°C until analysis.

[0350] Samples were analyzed using a Thermo Accela 1250 HPLC instrument combined with a Thermo TSQ Quantum Access MAX MS / MS-capable mass spectrometer. The method was based on a Thermo Accucore C30, 150 × 4.6 mm, 2.6 μm particle size column, maintained at 60°C, with mobile phase A - 0.1% formic acid in water and mobile phase B - methanol, using a gradient program at a flow rate of 1 ml / min, starting with 95% A, increasing the percentage of B to 50% in a linear gradient over 10 minutes, and stabilizing to the initial conditions for 5 minutes. The mass spectrometer was equipped with an hESI ion source and operated in positive (+) mode, with the spray voltage set to 4600 V, vaporizer temperature to 350°C, impact pressure to 1.0 Torr, and impact energy to 10 V. Trans-zeatin was observed in MRM mode during the transition from the parent ion at 219.9 m / z to daughter ions at 185.2, 148.0, and 136.0.

[0351] trans-zeatin riboside (tZR) was observed in MRM mode during the transition from the parent ion at 352.5 m / z to daughter ions at 220.1, 202.1, 148.0, and 136.1. Isopentenyl adenine (iP) was observed in MRM mode during the transition from the parent ion at 204.4 m / z to daughter ions at 148.4, 136.3, and 119.2. Isopentenyl adenine riboside (iPR) was observed in MRM mode during the transition from the parent ion at 336.5 m / z to daughter ions at 204.1, 148.0, and 113.1.

[0352] Example 8: Production of trans-zeatin and related isoprenoid cytokinins by Bacillus subtilis strains exhibiting heterologous expression of IPT and LOG. Culturing was performed as described in Example 6. Extraction and analysis were performed as described in Example 7. The yield of detected isoprenoid cytokinin is shown in Figure 4. Strains expressing IPT-LOG of SEQ ID NO: 82 produce isoprenoid cytokinin at a maximum amount of 10 mg / L (see Figure 4).

[0353] Example 9: Production of trans-zeatin and related isoprenoid cytokinins by Bacillus subtilis strains that heterologously express IPT and LOG and use adenine sulfate as the growth medium. Culturing was carried out as described in Example 6. The production medium was prepared as described in Example 6, or modified with adenine sulfate at a final concentration of 3 g / L. The fermentation broth was extracted and analyzed as described in Example 7. The results are shown in Figure 5. Isoprenoid cytokinin production in strain TZAB14, which has IPT-LOG of SEQ ID NO: 82, increased in a medium containing adenine sulfate compared to a medium without adenine sulfate.

[0354] Example 10: Production of trans-zeatin and related isoprenoid cytokinins by a Bacillus subtilis strain with heterologous expression of IPT-LOG of SEQ ID NO: 82 and overexpression of DXS. Culturing was carried out as described in Example 6. Extraction and analysis were carried out as described in Example 7. The yields are shown in Figure 6. Isoprenoid cytokinin production is increased in strains having the IPT-LOG operon of SEQ ID NO: 82 and the DXS operon of SEQ ID NO: 90.

[0355] Example 11: Analysis of trans-zeatin and related isoprenoid cytokinin production by Bacillus subtilis strain containing IPT-LOG (SEQ ID NO: 83) Culturing was performed as described in Example 6. Extraction and analysis were performed as described in Example 7. Strains TZAB1, TZAB2, TZAB3, and TZAB4, which have the IPT-LOG of SEQ ID NO: 83, and strains TZAB14 and TZAB15, which have the IPT-LOG of SEQ ID NO: 82, produced isoprenoid cytokinin. The results are shown in Figure 7.

[0356] Example 12: Production of trans-zeatin and related isoprenoid cytokinins by heterologous expression of IPT-LOG operon, IPT, and DXS in Bacillus subtilis strain. A synthetic DNA fragment containing the gene IPT1-LOG1 (SEQ ID NO: 71) for isoprenoid cytokinin biosynthesis was assembled into an artificial isoprenoid cytokinin operon as described in Example 4.

[0357] An IPT1 expression cassette containing only IPT1 (SEQ ID NO: 1) was assembled by adding 200 μM dNTPs, 0.5 μM primers, and approximately 10 ng of template to the manufacturer-provided buffer using Eppendorf cyclers and Phusion polymerase (Thermo Fisher) to the first part of the IPT1-LOG1 fragment (SEQ ID NO: 82) using primers SEQ ID NO: 123 and 183, and separately to the last part of IPT1-LOG1 fragment (SEQ ID NO: 82) using primers SEQ ID NO: 123 and 128, and then adding 200 μM dNTPs, 0.5 μM primers, and approximately 10 ng of template to a final volume of 50 μl and performing 30 cycles of PCR amplification under the following PCR cycle conditions: 30 seconds at 98°C, (30 seconds at 98°C, 25 seconds at 68.5°C, 25 seconds at 72°C) for 30 cycles, 5 minutes at 72°C, and hold at 10°C. PCR reaction products were electrophoresed on a 0.8% agarose gel, excised from the gel, and extracted from the gel using the GeneJET Gel Extraction Kit (Thermo Fisher) according to the manufacturer's protocol. The fragments were assembled onto an artificial IPT1 expression cassette by restriction and ligation. Using an XbaI restriction site, a suitable restriction end was obtained for successful ligation. After ligation, the assembled fragments were used as a new template for subsequent PCR amplification. Restriction was performed in 50 μl volumes by adding 5 μl of FD green buffer (Thermo Fisher Scientific), 2-3 μl of selective XbaI (Thermo Fisher) restriction enzyme, and up to approximately 1500 ng of PCR fragment. After restriction digestion, the digested DNA fragments were cleaned using the Wizard SV Gel and PCR Clean-up system according to the manufacturer's protocol. The first two fragments were mixed with 5% PEG 4000 in the manufacturer's buffer, and both fragments were mixed in a 1:1 molar ratio to a final volume of 15 μl, which was then used for ligation with 2.5 U of T4 DNA ligase (Thermo Fisher).In the next step, 1 μl of inactivated ligation was used as a template, and the primer sets of SEQ ID NO: 123 and SEQ ID NO: 128 were used. The same PCR mix and PCR cycle conditions as before were used, but the extension time was increased, and a new 50 μl was used for PCR. A final expression cassette containing the IPT fragment (SEQ ID NO: 184) was generated.

[0358] Next, novel transformed strains, shown in Table 9, were developed by using the final expression cassette constructed for a trans-zeatin operon containing IPT1-LOG1 (SEQ ID NO: 82) or IPT fragment (SEQ ID NO: 184) to transform Bacillus subtilis VKPM B2116, Bacillus subtilis 168, and Bacillus subtilis RB50 strains. The precise integration of the artificial operon at the amyE integration site was confirmed by cPCR.

[0359] A synthetic fragment containing the dxs gene (SEQ ID NO: 86) was assembled into an expression cassette as described in Example 5. The constructed DXS expression cassette (SEQ ID NO: 90) was used to transform strains derived from Bacillus subtilis VKPM B2116, Bacillus subtilis 168, and Bacillus subtilis RB50, resulting in the development of novel transformant strains shown in Table 9. Accurate integration of the artificial operon at the lacA integration site was confirmed by cPCR.

[0360] [Table 9]

[0361] All constructed and control strains were cultured according to the procedure described in Example 6. Frozen stocks of the strains, stored in 20% glycerol at -80°C, were streaked onto solid seed medium containing appropriate concentrations of erythromycin and lincomycin and incubated at 37°C for approximately 1 day. For further testing, 1 to 5 plugs of culture from the solid seed plate were inoculated into trophic stage medium in each baffled 250 mL Erlenmeyer flask containing 25 mL of nutrient medium and an appropriate amount of antibiotic. The cultures were incubated at 37°C for 8 to 20 hours at 220 RPM. The cultures in the nutrient medium were used as seed cultures and inoculated into the production medium. 10% inoculum was used (2.5 mL per 25 mL of production medium in a 250 mL Erlenmeyer flask). The production medium was modified with tryptophan (final concentration 50 mg / L) to evaluate the strain derived from Bacillus subtilis 168. The cultures were incubated at 30°C, 34°C, or 37°C for up to 48 hours at 220 RPM. Fermented cultures were sampled and analyzed as described in Example 7. The titers of trans-zeatin (tZ), trans-zeatin riboside (tZR), isopentenyl adenine riboside (iPR), and isopentenyl adenine (iP) were measured using LC / MS as described in Example 7.

[0362] Extraction and analysis were performed as described in Example 7. The yields of detected isoprenoid cytokinins are shown in Table 17, Figures 8, 9, and 10. Strains expressing IPT-LOG of SEQ ID NO: 82 produced isoprenoid cytokinins in a total amount of up to 60 mg / L (see Figure 10), and isoprenoid cytokinin production was further increased overall in strains having the IPT-LOG operon (SEQ ID NO: 82) and the DXS operon (SEQ ID NO: 90).

[0363] Example 13: Production of trans-zeatin and related isoprenoid cytokinins by heterologous expression of IPT, IPT1-LOG1 operon and DXS in Escherichia coli BL21(DE3) strain. Two sets of expression plasmids were constructed to evaluate Escherichia coli as a potential isoprenoid cytokinin-producing strain.

[0364] The first set consisted of plasmids for heterologous cytokinin gene / operon expression of the synthetic gene IPT (SEQ ID NO: 179) and the IPT-LOG operon (SEQ ID NO: 82). The IPT gene or IPT-LOG operon was assembled into a pBBR1 plasmid vector. The pBBR1 vector was a heterologous expression system based on a promoter inducible with isopropyl-β-D-1-thiogalactopyranoside (IPTG): LacI Q / P lacUV5 It has -T7 and includes a chloramphenicol-resistant cassette as a selective marker.

[0365] A second plasmid was cloned for heterologous expression of DXS (SEQ ID NO: 90), which increases the supply of isoprenoid precursors. The DXS gene was assembled into a p15A plasmid vector. The p15A vector has an XylS / Pm-based expression system, where XylS is a positive regulator of the m-toluylate-inducible Pm promoter and includes a kanamycin-resistant cassette as a selection marker.

[0366] The first step in expression plasmid assembly was PCR amplification of the insertion fragments: IPT-LOG and DXS, and the vector backbone: pBBR1 and p15A. IPT DNA fragments were amplified using primer sets SEQ ID NO: 163 and 182, while IPT-LOG DNA fragments were amplified using primer sets SEQ ID NO: 163 and 164 for IPT amplification, and SEQ ID NO: 165 and 166 for LOG amplification. Primer sets SEQ ID NO: 167 and 168 were used for amplification of the pBBR1 vector backbone. DXS fragments were amplified using primer sets SEQ ID NO: 169 and 170, and primer sets SEQ ID NO: 171 and 172 were used for amplification of the p15A vector backbone. The fragments were amplified for 30 cycles at a final volume of 50 μl using Eppendorf cyclers and Phusion polymerase (Thermo Fisher), with 200 μM dNTPs, 0.5 μM primers, and approximately 10 ng of template added in manufacturer-provided buffer. PCR reaction products of each fragment were run on a 0.8% agarose gel, excised from the gel, and extracted from the gel using the GeneJET Gel Extraction Kit (Thermo Fisher) according to the manufacturer's protocol. The mass of each fragment was measured using a NanoDrop instrument and absorbance at 260 nm. The fragments were assembled into a final expression construct using 10 μl of HIFI assembly reaction (NEBuilder HiFi DNA Assembly Master Mix). The reaction was performed on ice: fragments were added to 5 μl of HiFi DNA Assembly Master Mix in a DNA molar ratio of vector:insert = 1:2 (total fragment amount was a maximum of 0.2 pmol). The reaction mixture was filled to a final volume of 10 μl with nuclease-free water. The sample was incubated in a thermocycler at 50°C for 60 minutes. In the next step, 1 μl of the cooled assembly product was used for transformation of competent E. coli (E. coli) BL21 (DE3) cells.Plasmid DNA was isolated from the obtained strains, and accurate assembly was confirmed by sequencing.

[0367] Transformation with the p15A plasmid for DXS gene expression yielded transformant TZ3077. Transformation with the pBBR1 plasmid for IPT gene expression yielded transformant TZAB3079. Transformation with the pBBR1 plasmid for IPT-LOG operon expression yielded transformant TZ3082. Furthermore, IPT gene and IPT-LOG operon expression plasmids were used to transform TZ3077 (E. coli BL21(DE3) strain possessing the DXS expression plasmid). The resulting strain, possessing two plasmids—one for DXS gene expression and the other for IPT gene expression—was saved as TZAB3087. The resulting strain, possessing two plasmids—one for DXS gene expression and the other for IPT-LOG operon expression—was saved as TZ3091. All transformants were confirmed by cPCR and are listed in Table 10 below.

[0368] [Table 10]

[0369] The constructed strains (TZ3077~TZ3091) and the control strain Escherichia coli BL21(DE3) were stored in 20% glycerol at -80°C. The strains were always cultured in the presence of an appropriate concentration of selective antibiotic (kanamycin / chloramphenicol). Initially, these were streaked onto solid seed medium-2YT agar plates (Table 11) and incubated at 37°C for approximately 17 hours. For further testing, a single colony from the culture on the solid seed plate was inoculated into vegetative stage medium-2YT in a 250 mL Erlenmeyer flask containing 50 mL of nutrient medium and an appropriate amount of antibiotic. The cultures were incubated at 37°C for 17 hours at 220 RPM. Cultures in nutrient medium (Table 12) were used as seed cultures: 2.5% inoculum was used to inoculate 50 mL of production medium in a 250 mL Erlenmeyer flask. 2YT with added glucose was used as the production medium (Table 13) (sterile glucose was added at a final concentration of 25 g / L after autoclaving). The cultures were incubated at 37°C for 10 hours at 220 RPM. After 2 hours of fermentation, strains with expression plasmids were induced. Strains with the IPT / IPT-LOG expression plasmid were induced with IPTG at a final concentration of 150 μM. Strains with the DXS expression plasmid were induced with m-toluyl acid at a final concentration of 1 mM. Strains with both expression systems (DXS and IPT / IPT-LOG) were induced with both inducers (150 μM IPTG and 1 mM m-toluyl acid). After 10 hours of fermentation, the cultures were sampled for analysis of trans-zeatin and related isoprenoid cytokinins. The extraction protocol used was as described in Example 7. The titers of trans-zeatin, trans-zeatin riboside, isopentenyl adenine, and isopentenyl adenine riboside were measured using LC / MS as described in Example 7.

[0370] [Table 11]

[0371] [Table 12]

[0372] [Table 13]

[0373] The yield of detected isoprenoid cytokinins is shown in Figure 11. Since strain growth differs in the presence of one or two selective antibiotics and different inducers used, cytokinin measurements were normalized by dividing each culture by its optical density at 600 nm (OD600).

[0374] Strains expressing IPT produce isoprenoid cytokinin at a maximum level of 2.8 mg / L (see Figure 11 and Table 17). Strains expressing IPT-LOG produce isoprenoid cytokinin at a maximum level of 3.4 mg / L. Isoprenoid cytokinin production increases to 3.4 mg / L in strains expressing IPT-LOG and additionally expressing DXS for isoprenoid precursor supply.

[0375] Example 14: Assembly of the IPT1-LOG1 operon, IPT1 and DXS genes into a cloning vector, and transformation of the plasmid into the bacterium Corynebacterium stationis. Synthetic fragments containing the IPT1-LOG1 gene (SEQ ID NO: 178) for isoprenoid cytokinin biosynthesis were amplified from the previously constructed synthetic trans-zeatin operon of SEQ ID NO: 82. For the SEQ ID NO: 178 fragment, the primer set of SEQ ID NO: 173 and SEQ ID NO: 174 was used. For the expression of the IPT1 gene (SEQ ID NO: 180), the primer set of SEQ ID NO: 173 and SEQ ID NO: 177 was used to construct the SEQ ID NO: 180 fragment. The fragments were amplified for 30 cycles in a final volume of 50 μl using an Eppendorf cycler and Phusion polymerase (Thermo Fisher) with 200 μM dNTPs, 0.5 μM primers, and approximately 10 ng of template added in the manufacturer's buffer. The following PCR cycle conditions were used: 30 seconds at 98°C, (30 seconds at 98°C, 25 seconds at 63.3°C, 45 seconds at 72°C) for 30 cycles, 5 minutes at 72°C, and hold at 10°C.

[0376] The synthetic dxs gene (SEQ ID NO: 181) was amplified for 30 cycles using the following PCR cycle conditions in a final volume of 50 μl: 30 seconds at 98°C, 30 cycles at 72°C for 5 minutes, and 30 minutes at 10°C. The PCR reaction products of each fragment were run on a 0.8% agarose gel, excised from the gel, and extracted from the gel using the GeneJET Gel Extraction Kit (Thermo Fisher) according to the protocol provided by the manufacturer. This was done using the primer set of SEQ ID NO: 175 and SEQ ID NO: 176. The product was then excised and extracted from the gel using the GeneJET Gel Extraction Kit (Thermo Fisher).

[0377] The IPT1 gene and IPT1-LOG1 isoprenoid cytokinin operon were further cloned into the pVWEx6 plasmid (Henke et al. 2021), and the dxs gene expression cassette into the pECXT99APsyn plasmid, using HiFi assembly reactions with NEBuilder HiFi DNA Assembly Master Mix, according to the manufacturer's protocol. Both plasmids were pre-linearized by digestion with BamHI FD restriction enzyme. The HiFi reaction mixture was further used for electroporation of electrocompetent DH10β E. coli (E. coli) cells. Kanamycin selection markers allowed for selection of pVWEx6+IPT1-LOG1 and pVWEx6+IPT1 transformants, while tetracycline was used for selection of pECXT99APsyn+dxs transformants. Transformants were confirmed by colony PCR. For plasmid isolation, colonies were inoculated into 2TY medium containing appropriate antibiotics and incubated overnight at 37°C. Plasmids were isolated from cultures overnight using the GeneJET Plasmid Miniprep Kit (Thermo Fisher) plasmid extraction kit, following the manufacturer's protocol. The isolated plasmids were further analyzed by digestion with KpnI and XbaI FD restriction enzymes.

[0378] To transform Corynebacterium stationis DSM 20305 with the generated plasmid, electrocompetent cells were prepared according to Yili et al. 2015. Transformation with the isolated plasmid was performed using a BioRad electroporator in a 2 mm cuvette. Approximately 250 ng of plasmid was introduced into 50 μL aliquots of previously prepared electrocompetent cells, transferred to a 2 mm cuvette, and exposed to an electrical pulse. The cells were immediately transferred to a 2 mL Eppendorf tube containing 1 mL of regeneration medium. After 3 hours of incubation at 30°C and 200 rpm in a shaker, the transformants were plated onto recovery agar plates (LBHIS) containing appropriate concentrations of selection markers. The resulting transformants (shown in Table 14) were validated by colony PCR.

[0379] For transformants possessing both the pVWEx6+ipt1-log1 and pECXT99APsyn+dxs constructs, as well as the pVWEx6+ipt1 and pECXT99APsyn+dxs constructs, the confirmed transformants were used to generate novel electrocompetent cells for second-generation strains. Electrocompetent cells were transformed with additional plasmids and selected on plates containing both kanamycin and tetracycline. Colony PCR validation of the transformants was performed for both plasmids.

[0380] [Table 14]

[0381] Example 15: Production of trans-zeatin and related isoprenoid cytokinins by heterologous expression of Corynebacterium stationis All constructed and control strains were cultured according to this procedure. Frozen stocks of Corynebacterium stationis strains DSM 20305, TZ3136, TZ3138, TZ3139, TZ3142, and TZ3146, stored in 20% glycerol and kept at -80°C, were streaked onto solid seed medium containing appropriate concentrations of tetracycline and / or kanamycin and incubated at 30°C for approximately 1 day. For further testing, 5 plugs of cultures from solid seed plates were inoculated into trophic stage medium in each 250 mL baffled Erlenmeyer flask containing 25 mL of trophic stage medium and an appropriate amount of antibiotic. The cultures were incubated at 30°C for 18 hours at 200 RPM. The cultures in the trophic stage medium were used as seed cultures and inoculated into production medium. A 10% inoculum was used (2.5 mL per 25 mL of production medium in a 250 mL baffled Erlenmeyer flask). The culture was incubated at 30°C for a maximum of 48 hours at 200 RPM. The fermented culture was sampled and analyzed as described in Example 7. The titers of trans-zeatin, trans-zeatin riboside, isopentenyl adenine, and isopentenyl adenine riboside were measured using LC / MS as described in Example 7.

[0382] [Table 15]

[0383] [Table 16]

[0384] Example 16: Analysis of isoprenoid cytokinins by heterologous expression of Corynebacterium stationis Isoprenoid cytokinin production in Corynebacterium stationis transformants was tested in a fermentation process. Culturing was carried out as described in Example 15. Extraction and analysis were performed according to the procedure described in Example 7. The results are shown in Figure 12 and Table 17.

[0385] [Table 17]

[0386] Example 17: Enhancement of the purine nucleotide biosynthesis pathway in Bacillus subtilis by overexpression of the purA gene. A synthetic fragment containing the purA gene (SEQ ID NO: 98), used to enhance the purine nucleotide biosynthesis pathway, was assembled into an artificial gene expression cassette. The leading (SEQ ID NO: 96) and terminal (SEQ ID NO: 97) fragments, containing genetic integration homology, promoter sequence, and zeosin-selectable markers, were designed and synthesized for stable genomic integration into the yybN locus in the B. subtilis genome.

[0387] The first step in the artificial operon assembly was PCR amplification of separate DNA fragments, performed using primer pairs from SEQ ID NO: 99 and 100 for the leading fragment of SEQ ID NO: 96, and primer sets from SEQ ID NO: 101 and 102 for the terminal fragment of SEQ ID NO: 97. Primer sets from SEQ ID NO: 103 and 104 were used to amplify the fragment containing the purA overexpression gene of SEQ ID NO: 98. The fragments were amplified for 30 cycles using Eppendorf cycler and Phusion polymerase (Thermo Fisher) with 200 μM dNTPs, 0.5 μM primers, and approximately 10 ng of template added in manufacturer-provided buffer, in a final volume of 50 μl, using the following PCR cycle conditions: 30 seconds at 98°C, 30 cycles of (30 seconds at 98°C, 25 seconds at 65°C, 23 / 25 seconds at 72°C), 5 minutes at 72°C, and hold at 10°C. The PCR reaction products of each fragment were electrophoresed on a 0.8% agarose gel, excised from the gel, and extracted from the gel using the GeneJET Gel Extraction Kit (Thermo Fisher) according to the manufacturer's protocol. The fragments were assembled into an artificial operon through repeated restriction and ligation steps. By using a combination of SpeI (BcuI) and XbaI restriction sites, restriction ends suitable for successful ligation were obtained. After each ligation step, the combined fragments were used as a new template for the next PCR amplification. Restriction was performed in 50 μl volumes by adding 5 μl of FD green buffer (Thermo Fisher Scientific), 2-3 μl of selective enzyme (SpeI (BcuI) and XbaI, Thermo Fisher), and up to approximately 1500 ng of PCR fragment. After restriction digestion, the digested DNA fragments were cleaned using the Wizard SV Gel and PCR Clean-up system according to the manufacturer's protocol.The first two fragments were mixed with 5% PEG 4000 in the manufacturer's buffer, and both fragments were mixed in a 1:1 molar ratio to a final volume of 15 μl, which was then used for ligation with 2.5 U of T4 DNA ligase (Thermo Fisher). In the next step, 1 μl of inactivated ligation was used as a template for a new 50 μL PCR using the primer sets of SEQ ID NO: 102 and SEQ ID NO: 105, with the same PCR mix and PCR cycle conditions as before, but with a longer extension time. Restriction digestion, cleaning, and ligation steps were repeated for ligation of the terminal fragments. PCR was performed on a 0.8% agarose gel, the fragments were excised from the gel, digested and cleaned as before, and ligated as before. The final operon containing yybN homology, promoter with RBS sequence, purA gene, and zeosin resistance cassette was amplified using the primer pairs of SEQ ID NO: 106 and SEQ ID NO: 107, and cleaned and ligated as described above. The constructed synthetic operon was used to transform Bacillus subtilis VKPM B2116. Transformation using the purA operon of SEQ ID NO: 108 yielded the transformed strain BS19. Accurate integration of the artificial operon at the yybN integration site was confirmed by cPCR.

[0388] Example 18: Assembly of a synthetic isoprenoid cytokinin operon containing various homologs of the IPT gene combined with the LOG8 gene, and transformation into B. subtilis. Synthetic fragments containing a synthetic LOG gene (LOG8-SEQ ID NO: 116) for isoprenoid cytokinin biosynthesis and various IPT genes (SEQ ID NOs: 129, 154-156) were assembled into an artificial isoprenoid cytokinin operon. Leading and terminal fragments containing gene integration homology, promoter sequences, and an erythromycin-selectable marker (SEQ ID NO: 73) were designed and synthesized for stable genomic integration into the amyE locus in the B. subtilis genome.

[0389] The first step in the artificial operon assembly was PCR amplification of separate DNA fragments, performed using primer pairs of SEQ ID NOs. 74 and 75, for the leading fragment of SEQ ID NO: 76, as well as the synthetic fragment LOG8 (SEQ ID NO: 116), which contains the gene for isoprenoid-cytokinin biosynthesis, and fragments IPT1, IPT6, IPT7, and IPT9 (SEQ ID NOs. 179, 151, 152, and 153, respectively). The primer sets of SEQ ID NOs. 77 and 78 were used to amplify the terminal fragment of SEQ ID NO: 79. The fragments were amplified for 30 cycles using an Eppendorf cycler and Phusion polymerase (Thermo Fisher) in manufacturer-provided buffer with 200 μM dNTPs, 0.5 μM primers, and approximately 10 ng of template in a final volume of 50 μl under the following PCR cycle conditions: 30 seconds at 98°C, 30 cycles of (30 seconds at 98°C, 25 seconds at 65°C, 30 seconds at 72°C), 5 minutes at 72°C, and hold at 10°C. The PCR reaction products of each fragment were electrophoresed on a 0.8% agarose gel, excised from the gel, and extracted from the gel using the GeneJET Gel Extraction Kit (Thermo Fisher) according to the manufacturer-provided protocol. The fragments were assembled into an artificial operon by repeated restriction and ligation steps. Restriction ends suitable for successful ligation were obtained by using combinations of SpeI (BcuI), VspI, NdeI, and SnaBI restriction sites. After each step of the ligation process, the combined fragments were used as new templates for subsequent PCR amplification. Restriction was performed in 50 μl volumes using 5 μl of FD green buffer (Thermo Fisher Scientific), 2-3 μl of selective enzymes (SpeI (BcuI), VspI, NdeI, and SnaBI, Thermo Fisher), and up to approximately 1500 ng of PCR fragments. After restriction digestion, the digested DNA fragments were cleaned using the Wizard SV Gel and PCR Clean-up system according to the protocol provided by the manufacturer.The leading fragment and various IPT fragments (SEQ ID NOs. 179, 151, 152, and 153) were mixed with 5% PEG 4000 in a buffer provided by the manufacturer. Both fragments were mixed in a 1:1 molar ratio to a final volume of 15 μl, which was then used for ligation with 2.5 U of T4 DNA ligase (Thermo Fisher). In the next step, 1 μl of each inactivated ligate was used as a template, and the primer sets of SEQ ID NOs. 120 and 121 were used for a new 50 μL PCR using the same PCR mix and PCR cycle conditions, but with a longer extension time. The DNA fragments were extracted from the agarose gel as described above. Restriction digestion (VspI and NdeI), cleaning, and ligation steps were repeated for ligation of fragments containing the AmyE+IPT1, AmyE+IPT6, AmyE+IPT7, and AmyE+IPT9 operons (SEQ ID NOs. 129, 154, 155, and 156, respectively), as well as the LOG8 gene (SEQ ID NO: 116). Using 1 μl of inactivated ligation as a template, a new 50 μl was used for PCR with the same PCR mix and cycle conditions as before, but with a longer extension time, using the primer sets of SEQ ID NOs. 122 and 123. DNA fragments were extracted from the agarose gel as described above. The operons AmyE 0+IPT1 / 6 / 7 / 9+LOG8 (sequences 136, 157, 158, and 159, respectively) and EryR+AmyE END (sequence 140) were ligated using XbaI enzyme digestion, ligated first using the SnaBI restriction site, and PCR amplified with the primer sets of SEQ ID NO: 126 and 127.

[0390] The final operon, containing amy E homology, a promoter with an RBS sequence, various IPT genes and gene LOG8, and an erythromycin resistance cassette, was amplified using primer pairs of SEQ ID NO: 128 and 123, and cleaned and ligated as described above. The constructed synthetic trans-zeatin operon containing IPT1-LOG8, IPT6-LOG8, IPT7-LOG8, and IPT9-LOG8 (SEQ ID NOs: 147, 160, 161, and 162, respectively) was used to transform Bacillus subtilis BS19 (described in Example 17). All transformants were confirmed by cPCR and are listed in Table 18 below. All constructed strains were cultured as described in Example 6. Extraction and analysis were performed as described in Example 7, and the cytokinin yield is shown in Figure 13.

[0391] [Table 18]

[0392] Example 19: Assembly of a synthetic isoprenoid cytokinin operon containing IPT1 and various LOGs, and transformation into B. subtilis. Synthetic fragments containing the synthetic gene IPT1 (SEQ ID NO: 71) for isoprenoid cytokinin biosynthesis and various LOG genes (SEQ ID NOs: 110-119) were assembled into an artificial isoprenoid cytokinin operon. Leading and terminal fragments containing gene integration homology, promoter sequences, and erythromycin-selectable markers were designed and synthesized for stable genomic integration into the amyE locus in the B. subtilis genome.

[0393] The first step in the artificial operon assembly was PCR amplification of separate DNA fragments, performed using primer pairs of SEQ ID NOs: 74 and 75 for the leading fragment of SEQ ID NO: 76, as well as the synthetic fragment IPT1 of SEQ ID NO: 71 containing the gene for isoprenoid-cytokinin biosynthesis, and fragments LOG2-LOG11 (SEQ ID NOs: 110-119). The primer sets of SEQ ID NOs: 126 and 127 were used to amplify the terminal fragment containing the erythromycin-selectable marker (SEQ ID NO: 140). The fragments were amplified for 30 cycles using Eppendorf cycler and Phusion polymerase (Thermo Fisher) with 200 μM dNTPs, 0.5 μM primers, and approximately 10 ng of template added in manufacturer-provided buffer, in a final volume of 50 μl, using the following PCR cycle conditions: 30 seconds at 98°C, 30 cycles of (30 seconds at 98°C, 25 seconds at 65°C, 30 seconds at 72°C), 5 minutes at 72°C, and hold at 10°C. The PCR reaction products of each fragment were electrophoresed on a 0.8% agarose gel, excised from the gel, and extracted from the gel using the GeneJET Gel Extraction Kit (Thermo Fisher) according to the manufacturer's protocol. The fragments were assembled into an artificial operon through repeated restriction and ligation steps. By using combinations of SpeI(BcuI), VspI, NdeI, XbaI, and SnaBI restriction sites, restriction ends suitable for successful ligation were obtained. After each ligation step, the combined fragments were used as new templates for subsequent PCR amplification. Restriction was performed in 50 μl volumes by adding 5 μl of FD green buffer (Thermo Fisher Scientific), 2-3 μl of selective enzyme (SpeI(BcuI), VspI, NdeI, XbaI, and SnaBI, Thermo Fisher), and up to approximately 1500 ng of PCR fragment. After restriction digestion, the digested DNA fragments were cleaned using the Wizard SV Gel and PCR Clean-up system according to the manufacturer's protocol.

[0394] The first two fragments were mixed with 5% PEG 4000 in the manufacturer's buffer, and both fragments were mixed in a 1:1 molar ratio to a final volume of 15 μl, which was then used for ligation with 2.5 U of T4 DNA ligase (Thermo Fisher). In the next step, 1 μl of inactivated ligation was used as a template, and the primer sets of SEQ ID NOs. 120 and 121 were used for a new 50 μL PCR using the same PCR mix and PCR cycle conditions as before, but with a longer extension time. The DNA fragments were extracted from the agarose gel as described above. Restriction digestion (VspI and NdeI), cleaning, and ligation steps were repeated for ligation of fragments containing various LOG genes (SEQ ID NOs. 110-119). 1 μl of inactivated ligation was used as a template, and the primer sets of SEQ ID NOs. 122 and 123 were used for a new 50 μL PCR using the same PCR mix and PCR cycle conditions as before, but with a longer extension time. DNA fragments were extracted from the agarose gel as described above. XbaI enzyme digestion was performed, followed by ligating with the SnaBI restriction site. Operons AmyE 0+IPT1+LOG2~11 (sequence numbers 130~139) and EryR+AmyE END (sequence number 140), which were PCR amplified using the primer sets of SEQ ID NOs. 126 and 127, were then ligated.

[0395] The final operon, containing amyE homology, a promoter with an RBS sequence, IPT1, and various LOG genes and an erythromycin resistance cassette, was amplified using primer pairs of SEQ ID NOs. 128 and 123, and then cleaned and ligated as described above. The constructed synthetic trans-zeatin operon containing IPT1-LOG 2~11 (SEQ ID NOs. 141~150) was used to transform Bacillus subtilis BS19 (described in Example 17). All transformants were confirmed by cPCR and are listed in Table 19 below. All constructed strains were cultured as described in Example 6. Extraction and analysis were performed as described in Example 7, and the cytokinin yields are shown in Figure 14.

[0396] [Table 19]

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Claims

1. A Gram-positive bacterium expressing a heterologous polypeptide having adenylate isopentenyltransferase activity, wherein the bacterium exhibits increased protein expression of a polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity compared to an identical bacterium without any other modifications, and the bacterium belongs to the genus Bacillus.

2. The bacterium according to claim 1, wherein the bacterium is Bacillus subtilis.

3. The bacterium according to claim 1 or 2, wherein the polypeptide having adenylate isopentenyltransferase activity is selected from the group consisting of i) a polypeptide comprising any one amino acid sequence of SEQ ID NOs: 1 to 33; and ii) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with any one amino acid sequence of SEQ ID NOs: 1 to 33.

4. The bacterium according to any one of claims 1 to 3, wherein the protein expression of a polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is increased compared to the same bacterium that has not undergone any other modifications.

5. The bacterium according to claim 4, wherein the polypeptide having cytokinin riboside 5'-monophosphate phosphoribohydrolase activity is selected from the group consisting of i) a polypeptide comprising any one amino acid sequence of SEQ ID NOs. 34 to 62, and ii) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with any one amino acid sequence of SEQ ID NOs. 34 to 62.

6. The bacterium according to any one of claims 1 to 5, wherein the polypeptide having 1-deoxy-D-xylulose-5-phosphate synthase activity is selected from the group consisting of i) a polypeptide comprising any one amino acid sequence of SEQ ID NOs. 63 to 70; and ii) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with any one amino acid sequence of SEQ ID NOs. 63 to 70.

7. The bacterium according to any one of claims 1 to 6, wherein the expression and / or activity of at least one enzyme involved in the purine nucleotide biosynthesis pathway is increased compared to the same bacterium without any other modifications.

8. The bacterium according to any one of claims 1 to 7, wherein the expression and / or activity of at least one endogenous enzyme involved in the purine nucleotide degradation pathway is reduced compared to the same bacterium otherwise without any modifications.

9. The bacterium according to any one of claims 1 to 8, wherein the expression and / or activity of at least one endogenous enzyme involved in the guanosine monophosphate biosynthesis pathway is reduced compared to the same bacterium without any other modifications.

10. The bacterium according to any one of claims 1 to 9, wherein the protein expression of polypeptides having cytochrome P450 monooxygenase (CYP450) activity is increased compared to the same bacterium without any other modifications.

11. A method for producing isoprenoid cytokinin or its riboside derivative, comprising culturing a bacterium according to any one of claims 1 to 10 in a suitable culture medium under suitable culture conditions.

12. The isoprenoid cytokinin or its riboside derivative is trans-zeatin (tZ), trans-zeatin riboside (tZR), N ６ The method according to claim 11, selected from the group consisting of -(D2-isopentenyl)adenine (iP), N(6)-(dimethylallyl)adenosine (iPR), dihydrozeatin (DZ), ribosyldihydrozeatin (DZR), and combinations thereof.

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