PRIORITY

This Application claims the benefit of, and priority to, US Application Nos. 62/645,443 filed March 20, 2018 and 62/656,678 filed April 12, 2018, each of which is hereby incorporated by reference in its entirety.

BACKGROUND

Ar onychia derived compounds, including 3-(4-famesyloxyphenyl)propionic acid (FOPPA) have been described for use as antioxidants, antibacterials, anthelmintics, anti inflammatories, cancer chemopreventatives, food additives, and/or fragrance components. See US 2011/0318439, which is hereby incorporated by reference in its entirety. Additionally, US Patent 4,939,171, which is hereby incorporated by reference in its entirety, discloses the use of compounds, such as FOPPA, to provide antiseborrhoeic properties. US Patent 9,814,659, which is hereby incorporated by reference in its entirety, discloses that FOPPA can be used to provide skin lightening and photo-protective effects when used on skin.

Given the many potential uses of FOPPA, the compound needs to be produced quickly and efficiently. Moreover, there is a growing need to provide sustainable, and environmental-friendly methods of manufacturing compounds, such as FOPPA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structure of 3-(4-famesyloxyphenyl)propionic acid (FOPPA). FIG. 1 shows the two primary components of FOPPA, a compound derived from famesyl-PP or famesol, and phloretate (3 -(4-hydroxy phenyl)propionic acid.

FIG. 2 shows summarizes biosynthetic routes to obtain the phloretate precursor of FOPPA. The first approach reconstructs and improves upon plant pathways, including a 7-step pathway from L-phenylalanine or a 6-step pathway from L-tyrosine. The second approach involves the creation of a shortcut pathway using bacterial enzymes, which includes a 3 -step pathway from L-tyrosine.

FIG. 3 depicts a biosynthetic pathway to phloretate and FOPPA via phloretin.

FIG. 4 depicts a bacteria-based biosynthetic pathway to phloretate and FOPPA DESCRIPTION OF THE INVENTION

The present invention provides methods of producing and making 3-(4- famesyloxyphenyl)propionic acid (FOPPA), which is also known as 3-(4- famesyloxyphenyl)propanoic acid, 3-(p-famesyloxyphenyl)propionic acid, and 3-(p- famesyloxyphenyl)propanoic acid. FOPPA was originally found in fruit from Acronychia spp. and, specifically, Acronychia acidula (lemon aspen). FOPPA has many beneficial characteristics and can be used in a variety of medical, cosmetic, and food related applications. For example, FOPPA has utility as an agent for skin lightening and photo-protective effects.

The present invention provides methods of producing FOPPA resulting from unique biosynthetic pathways, including biosynthetic pathways based on the phenylalanine/tyrosine biosynthetic branch and biosynthetic pathways based on bacteria metabolism. In particular, the present invention provides methods of producing FOPPA in microbial cells. These methods provide a low-cost, sustainable, and environmentally friendly source for FOPPA.

In some aspects, the present invention provides a microbial cell producing 3-(4- famesyloxyphenyl)propionic acid (FOPPA), or a derivative thereof. The microbial cell comprises an enzyme pathway for the synthesis of a first substrate that is selected from famesyl pyrophosphate, famesyl-phosphate, or famesol; and an enzyme pathway for the synthesis of a second substrate that is selected from phloretate or an analog thereof. The microbial cell further comprises a transferase enzyme forming FOPPA, or a derivative thereof, from the first substrate and the second substrate. As shown in Figure 1, FOPPA is composed of a compound derived from famesyl-PP or famesol and phloretate.

In various embodiments, an analog of phloretate is produced. The analog of phloretate is selected from cinnamic acid, hydrocinnamic acid, and /i-coumaric acid.

In various embodiments, the enzyme pathway for the synthesis of the first substrate comprises one or more famesyl diphosphate synthases (FPPS). In some embodiments, the FPPS enzyme is a Saccharomyces cerevisiae famesyl pyrophosphate synthase (ScFPPS), which comprises the amino acid sequence of SEQ ID NO: 1, or a derivative thereof. In various embodiments, the derivative comprises an amino acid sequence having at least about 50% identity to SEQ ID NO: l, or in other embodiments, at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: l. Alternatively, the FPPS is E. coli ispA, or a variant thereof. Numerous alternative FPPS enzymes are known in the art, and may be employed for conversion of IPP and/or DMAPP to famesyl diphosphate in accordance with this aspect.

In some embodiments, the FPPS comprises an amino acid sequence having from 1 to 20 amino acid modifications or having from 1 to 10 amino acid modifications with respect to SEQ ID NO: 1, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.

In various embodiments, the enzyme pathway for the synthesis of the first substrate comprises one or more overexpressed enzymes of the methylerythritol phosphate (MEP) pathway or mevalonic acid (MV A) pathway. In such embodiments, the MEP or MVA pathway is engineered to increase carbon flux to famesyl diphosphate or famesol.

The microbial cell will produce MEP or MVA products, which act as substrates for the enzyme pathway. The MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, also called the MEP/DOXP (2-C-methyl-D-erythritol 4-phosphate/l-deoxy-D-xylulose 5- phosphate) pathway or the non-mevalonate pathway or the mevalonic acid-independent pathway refers to the pathway that converts glyceraldehyde-3-phosphate and pyruvate to IPP and DMAPP. The pathway, which is present in bacteria, typically involves action of the following enzymes: l-deoxy-D-xylulose-5-phosphate synthase (Dxs), l-deoxy-D- xylulose-5-phosphate reductoisomerase (IspC), 4-diphosphocytidyl-2-C-methyl-D- erythritol synthase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), l-hydroxy-2-methyl-2- (E)-butenyl 4-diphosphate synthase (IspG), and isopentenyl diphosphate isomerase (IspH). The MEP pathway, and the genes and enzymes that make up the MEP pathway, are described in US 8,512,988, which is hereby incorporated by reference in its entirety. For example, genes that make up the MEP pathway include dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, and ispA. In some embodiments, the host cell expresses or overexpresses one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA, or modified variants thereof, which results in the increased production of IPP and DMAPP. In some embodiments, the FPP or famesol is produced at least in part by metabolic flux through an MEP pathway, and wherein the host cell has at least one additional gene copy of one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA, or modified variants thereof.

The MVA pathway refers to the biosynthetic pathway that converts acetyl-CoA to IPP. The mevalonate pathway, which will be present in yeast, typically comprises enzymes that catalyze the following steps: (a) condensing two molecules of acetyl-CoA to acetoacetyl-CoA (e.g., by action of acetoacetyl-CoA thiolase); (b) condensing acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoenzymeA (HMG- CoA) (e.g., by action of HMG-CoA synthase (HMGS)); (c) converting HMG-CoA to mevalonate (e.g., by action of HMG-CoA reductase (HMGR)); (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by action of mevalonate kinase (MK)); (e) converting mevalonate 5 -phosphate to mevalonate 5 -pyrophosphate (e.g., by action of phosphomevalonate kinase (PMK)); and (f) converting mevalonate 5 -pyrophosphate to isopentenyl pyrophosphate (e.g., by action of mevalonate pyrophosphate decarboxylase (MPD)). The MVA pathway, and the genes and enzymes that make up the MVA pathway, are described in US 7,667,017, which is hereby incorporated by reference in its entirety. In some embodiments, the host cell expresses or overexpresses one or more of acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, and MPD or modified variants thereof, which results in the increased production of IPP and DMAPP. In some embodiments, FPP or famesol is produced at least in part by metabolic flux through an MVA pathway, and wherein the host cell has at least one additional gene copy of one or more of acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, or modified variants thereof.

In some embodiments, the host cell is a bacterial host cell engineered to increase production of IPP and DMAPP from glucose as described in US 2018/0245103 and US 2018/0216137, the contents of which are hereby incorporated by reference in their entireties. For example, in some embodiments the host cell overexpresses MEP pathway enzymes, with balanced expression to push/pull carbon flux to IPP and DMAP. In some embodiments, the host cell is engineered to increase the availability or activity of Fe-S cluster proteins, so as to support higher activity of IspG and IspH, which are Fe-S enzymes. In some embodiments, the host cell is engineered to overexpress IspG and IspH, so as to provide increased carbon flux to l-hydroxy-2-methyl-2-(E)-butenyl 4- diphosphate (HMBPP) intermediate, but with balanced expression to prevent accumulation of HMBPP at an amount that reduces cell growth or viability, or at an amount that inhibits MEP pathway flux and/or terpenoid production. In some embodiments, the host cell exhibits higher activity of IspH relative to IspG. In some embodiments, the host cell is engineered to downregulate the ubiquinone biosynthesis pathway, e.g., by reducing the expression or activity of IspB, which uses IPP and FPP substrate. In various embodiments, the enzyme pathway for the synthesis of the second substrate comprises tyrosine ammonia lyase (TAL) and phenolic acid reductase (PAR), or variants thereof. The enzyme pathway may further comprise phenylalanine ammonia lyase (PAL) and cinnamate-4-hydroxylase (C4H), or variants thereof.

In various embodiments, the enzyme pathway for the synthesis of the second substrate comprises the enzymes tyrosine ammonia lyase (TAL), phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl-CoA double bond reductase (HCDBR) and/or phenolic acid reductase (PAR), chalcone synthase (CHS), and phloretin hydrolase (PH).

An exemplary enzyme pathway for the synthesis of a second substrate that uses one or more of TAL, PAL, C4H, PAR, 4CL, HCDBR, CHS, and PH is provided in the plant-derived biosynthetic pathway shown in Figure 3. In this pathway, the amino acids L-tyrosine or L-phenylalanine are used as precursors. When L-phenylalanine is the precursor substrate, it is converted by a phenylalanine ammonia lyase (PAL) to cinnamic acid. Alternatively, L-tyrosine can be converted to p-Coumaric acid by a tyrosine ammonia lyase. The cinnamic acid is converted to / coumaric acid by a cinnamate-4- hydroxylase (C4H). A 4-coumaric acid CoA ligase (4CL) converts />coumaric acid to p- coumaroyl-CoA, which is converted by hydroxycinnamoyl-CoA double bond reductase (HCDBR) and nicotinamide adenine dinucleotide phosphate (NADPH) to p- dihydrocoumaroyl-CoA. The / dihydrocoumaroyl-CoA is converted to phloretin by chalcone synthase (CHS) and malonyl-CoA. Phloretin is broken down to phloretate and phloroglucinol by phloretin hydrolase (PH). Using famesyl pyrophosphate, famesyl- phosphate, or famesol, the phloretate is famesylated via a transferase, such as prenyltransferase, to create FOPPA.

Yet another exemplary pathway for the synthesis of a second substrate is provided in the alternative biosynthetic pathway shown in Figure 4. In this pathway, the amino acid L-tyrosine is the precursor substrate. The L-tyrosine is converted to p- coumaric acid by a tyrosine ammonia lyase (TAL). The / coumaric acid is then converted to phloretate by a phenolic acid reductase (PAR) and NAD+ cofactor. With famesyl pyrophosphate, famesyl-phosphate, or famesol, the phloretate is famesylated via a transferase, such as prenyltransferase, to make FOPPA.

In various embodiments, the PAR enzyme comprises an amino acid sequence that has at least about 50% amino acid sequence identity with a wild type PAR enzyme from Clostridium spp., such as Clostridium orbiscindens, or Lactobacillus spp., such as Lactobacillus plantarum. In various embodiments, the PAR enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity to PAR from Clostridium orbiscindens or Lactobacillus plantarum.

In various embodiments, the TAL enzyme comprises the amino acid sequence of a wild type TAL enzyme (or derivative thereof) from Rhodobacter spp., e.g., Rhodobacter sphaeroides Rhodotorula spp., e.g., Rhodotorula glutinis Herpatosiphon spp., e.g., Herpatosiphon auranticus Flavobacterium spp., e.g., Flavobacterium johnsoniae Saccharothrix spp., e.g., Saccharothrix espanaensis Amaranthus spp., e.g. Amaranthus hypocondriacus Amborella spp., e.g. Amborella trichopoda; Aquilegia spp., e.g. Aquilegia coerulea Arabidopsis spp., e.g Arabidopsis thaliana Azadirachta spp., e.g. Azadiractha indica Bambusa spp., e.g., Bambusa vulgaris, Beta spp., e.g. Beta vulgaris, Cannabis spp., e.g. Cannabis sativa; Capsicum spp., e.g., Capsicum annuum, Carica spp., e.g. Carica papaya, Catharanthus spp., e.g., Catharanthus roseus; Cistanche spp., e.g., Cistanche deserticola; Citrus spp., e.g. Citrus sinensis; Cucumis spp., e.g. Cucumis melo; Elaeis spp., e.g., Elaeis guineensis; Eucalyptus spp., e.g. Eucalyptus grandis; Glycine spp., e.g. Glycine max; Gossypium spp., e.g. Gossypium Raimondi; Helianthus spp., e.g., Helianthus tuberosus; Kalanchoe spp., e.g. Kalanchoe fedtschenkoi; Linum spp., e.g. Linum usitatissimum; Malus spp., e.g. Malus x domestica; Manihot spp., e.g. Manihot esculenta; Mimulus spp., e.g. Mimulus guttatus; Musa spp., e.g. Musa acuminate; Nelumbo spp., e.g. Nelumbo nucifera; Nicotiana spp., e.g., Nicotiana tabacum; Oryza spp., e.g. Oryza sativa; Petroselinum spp., e.g., Petroselinum crispum; Phalaenopsis spp., e.g. Phalaenopsis equestris; Phyllostacys spp., e.g. Phyllostacys edulis; Physcomitrella spp., e.g., Physcomitrella patens; Pisum spp., e.g. Pisum sativum; Pinus spp., e.g. Pinus taeda; Populus spp., e.g., Populus trichocarpa; Selaginella spp., e.g. Selaginella moellendorfli; Sesamum spp., e.g. Sesamum indicum; Spirodela spp., e.g. Spirodela polyrhiza; Stevia spp., e.g. Stevia rebaudiana; Thapsia spp., e.g. Thapsia villosa; Triticum spp., e.g. Triticum aestivum; Utricularia spp., e.g. Utricularia gibba; Vigna spp., e.g., Vigna radiate; Vitis spp., e.g. Vitis vinifera; or Zea spp., e.g. Zea mays. In various embodiments, the TAL enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity to a TAL enzyme of a species disclosed in this paragraph.

In various embodiments, the PAL enzyme comprises an amino acid of a wild type PAL enzyme (or derivative thereof) from Brevibacillus spp., e.g., Brevibacillus laterosporus Streptomyces spp.; Dictyostelium spp., e.g., Dictyostelium discoideum; Photorhabdus spp., e.g Photorhabdus luminescens Amaranthus spp., e.g. Amaranthus hypocondriacus Amborella spp., e.g. Amborella trichopoda; Aquilegia spp., e.g. Aquilegia coerulea Arabidopsis spp., e.g Arabidopsis thaliana Azadirachta spp., e.g. Azadiractha indica Bambusa spp., e.g., Bambusa vulgaris, Beta spp., e.g. Beta vulgaris, Cannabis spp., e.g. Cannabis sativa; Capsicum spp., e.g., Capsicum annuum, Carica spp., e.g. Carica papaya, Catharanthus spp., e.g., Catharanthus roseus; Cistanche spp., e.g., Cistanche deserticola; Citrus spp., e.g. Citrus sinensis; Cucumis spp., e.g. Cucumis melo; Elaeis spp., e.g., Elaeis guineensis; Eucalyptus spp., e.g. Eucalyptus grandis; Glycine spp., e.g. Glycine max; Gossypium spp., e.g. Gossypium Raimondi; Helianthus spp., e.g., Helianthus tuberosus; Kalanchoe spp., e.g. Kalanchoe fedtschenkoi; Linum spp., e.g. Linum usitatissimum; Malus spp., e.g. Malus x domestica; Manihot spp., e.g. Manihot esculenta; Mimulus spp., e.g. Mimulus guttatus; Musa spp., e.g. Musa acuminate; Nelumbo spp., e.g. Nelumbo nucifera; Nicotiana spp., e.g., Nicotiana tabacum; Oryza spp., e.g. Oryza sativa; Petroselinum spp., e.g., Petroselinum crispum; Phalaenopsis spp., e.g. Phalaenopsis equestris; Phyllostacys spp., e.g. Phyllostacys edulis; Physcomitrella spp., e.g., Physcomitrella patens; Pisum spp., e.g. Pisum sativum; Pinus spp., e.g. Pinus taeda; Populus spp., e.g., Populus trichocarpa; Selaginella spp., e.g. Selaginella moellendorfli; Sesamum spp., e.g. Sesamum indicum; Spirodela spp., e.g. Spirodela polyrhiza; Stevia spp., e.g. Stevia rebaudiana; Thapsia spp., e.g. Thapsia villosa; Triticum spp., e.g. Triticum aestivum; Utricularia spp., e.g. Utricular ia gibba; Vigna spp., e.g., Vigna radiate; Vitis spp., e.g. Vitis vinifera; or Zea spp., e.g. Zea mays. In various embodiments, the PAL enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity to a PAL enzyme of a species disclosed in this paragraph.

In various embodiments, the C4H comprises an amino acid sequence of a wild type C4H enzyme (or derivative thereof) from Amaranthus spp., e.g. Amaranthus hypocondriacus; Amborella spp., e.g. Amborella trichopoda; Aquilegia spp., e.g. Aquilegia coerulea Arabidopsis spp., e.g Arabidopsis thaliana Azadirachta spp., e.g. Azadiractha indica Bambusa spp., e.g., Bambusa vulgaris, Beta spp., e.g. Beta vulgaris, Cannabis spp., e.g. Cannabis sativa; Capsicum spp., e.g., Capsicum annuum, Carica spp., e.g. Carica papaya, Catharanthus spp., e.g., Catharanthus roseus; Cistanche spp., e.g., Cistanche deserticola; Citrus spp., e.g. Citrus sinensis; Cucumis spp., e.g. Cucumis melo; Elaeis spp., e.g., Elaeis guineensis; Eucalyptus spp., e.g. Eucalyptus grandis; Glycine spp., e.g. Glycine max; Gossypium spp., e.g. Gossypium Raimondi; Helianthus spp., e.g., Helianthus tuberosus; Kalanchoe spp., e.g. Kalanchoe fedtschenkoi; Linum spp., e.g. Linum usitatissimum; Malus spp., e.g. Malus x domestica; Manihot spp., e.g. Manihot esculenta; Mimulus spp., e.g. Mimulus guttatus; Musa spp., e.g. Musa acuminate; Nelumbo spp., e.g. Nelumbo nucifera; Nicotiana spp., e.g., Nicotiana tabacum; Oryza spp., e.g. Oryza sativa; Petroselinum spp., e.g., Petroselinum crispum; Phalaenopsis spp., e.g. Phalaenopsis equestris; Phyllostacys spp., e.g. Phyllostacys edulis; Physcomitrella spp., e.g., Physcomitrella patens; Pisum spp., e.g. Pisum sativum; Pinus spp., e.g. Pinus taeda; Populus spp., e.g., Populus trichocarpa; Selaginella spp., e.g. Selaginella moellendorfli; Sesamum spp., e.g. Sesamum indicum; Spirodela spp., e.g. Spirodela polyrhiza; Stevia spp., e.g. Stevia rebaudiana; Thapsia spp., e.g. Thapsia villosa; Triticum spp., e.g. Triticum aestivum; Utricularia spp., e.g. Utricularia gibba; Vigna spp., e.g., Vigna radiate; Vitis spp., e.g. Vitis vinifera; or Zea spp., e.g. Zea mays. In various embodiments, the C4H enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity to a C4H enzyme of a species disclosed in this paragraph.

In various embodiments, the 4CL enzyme comprises an amino acid sequence of a wild type 4CL enzyme (or derivative thereof) from Amaranthus spp., e.g. Amaranthus hypocondriacus; Amborella spp., e.g. Amborella trichopoda; Aquilegia spp., e.g. Aquilegia coerulea; Arabidopsis spp., e.g ., Arabidopsis thaliana; Azadirachta spp., e.g. Azadiractha indica; Bambusa spp., e.g., Bambusa vulgaris; Beta spp., e.g. Beta vulgaris; Cannabis spp., e.g. Cannabis sativa; Capsicum spp., e.g., Capsicum annuum; Carica spp., e.g. Carica papaya; Catharanthus spp., e.g., Catharanthus roseus; Cistanche spp., e.g., Cistanche deserticola; Citrus spp., e.g. Citrus sinensis; Cucumis spp., e.g. Cucumis melo; Elaeis spp., e.g., Elaeis guineensis; Eucalyptus spp., e.g. Eucalyptus grandis; Glycine spp., e.g. Glycine max; Gossypium spp., e.g. Gossypium Raimondi; Helianthus spp., e.g., Helianthus tuberosus Kalanchoe spp., e.g. Kalanchoe fedtschenkoi; Linum spp., e.g. Linum usitatissimum Malus spp., e.g. Malus x domestica Manihot spp., e.g. Manihot esculenta Mimulus spp., e.g. Mimulus guttatus Musa spp., e.g. Musa acuminate, Nelumbo spp., e.g. Nelumbo nucifera, Nicotiana spp., e.g., Nicotiana tabacum Oryza spp., e.g. Oryza sativa Petroselinum spp., e.g., Petroselinum crispum Phalaenopsis spp., e.g. Phalaenopsis equestris Phyllostacys spp., e.g. Phyllostacys edulis Physcomitrella spp., e.g., Physcomitrella patens, Pisum spp., e.g. Pisum sativum, Pinus spp., e.g. Pinus taeda; Populus spp., e.g., Populus trichocarpa; Selaginella spp., e.g. Selaginella moellendorfii; Sesamum spp., e.g. Sesamum indicum; Spirodela spp., e.g. Spirodela polyrhiza, Stevia spp., e.g. Stevia rebaudiana; Thapsia spp., e.g. Thapsia villosa; Triticum spp., e.g. Triticum aestivum; Utricularia spp., e.g. Utricularia gibba; Vigna spp., e.g., Vigna radiate, Vitis spp., e.g. Vitis vinifera; or Zea spp., e.g. Zea mays. In various embodiments, the 4CL enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity to a 4CL enzyme of a species disclosed in this paragraph.

In various embodiments, the HCDBR enzyme comprises an amino acid sequence of a wild type HCDBR enzyme (or derivative thereof) from Amaranthus spp., e.g. Amaranthus hypocondriacus; Amborella spp., e.g. Amborella trichopoda; Aquilegia spp., e.g. Aquilegia coerulea; Arabidopsis spp., e.g., Arabidopsis thaliana; Azadirachta spp., e.g. Azadiractha indica; Bambusa spp., e.g., Bambusa vulgaris, Beta spp., e.g. Beta vulgaris, Cannabis spp., e.g. Cannabis sativa, Capsicum spp., e.g., Capsicum annuum, Carica spp., e.g. Carica papaya, Catharanthus spp., e.g., Catharanthus roseus; Cistanche spp., e.g., Cistanche deserticola; Citrus spp., e.g. Citrus sinensis; Cucumis spp., e.g. Cucumis melo; Elaeis spp., e.g., Elaeis guineensis; Eucalyptus spp., e.g. Eucalyptus grandis; Glycine spp., e.g. Glycine max; Gossypium spp., e.g. Gossypium Raimondi; Helianthus spp., e.g., Helianthus tuberosus; Kalanchoe spp., e.g. Kalanchoe fedtschenkoi; Linum spp., e.g. Linum usitatissimum; Malus spp., e.g. Malus x domestica; Manihot spp., e.g. Manihot esculenta; Mimulus spp., e.g. Mimulus guttatus; Musa spp., e.g. Musa acuminate; Nelumbo spp., e.g. Nelumbo nucifera; Nicotiana spp., e.g., Nicotiana tabacum; Oryza spp., e.g. Oryza sativa; Petroselinum spp., e.g., Petroselinum crispum; Phalaenopsis spp., e.g. Phalaenopsis equestris; Phyllostacys spp., e.g. Phyllostacys edulis; Physcomitrella spp., e.g., Physcomitrella patens; Pisum spp., e.g. Pisum sativum., Pinus spp., e.g. Pinus taeda Populus spp., e.g., Populus trichocarpa, Selaginella spp., e.g. Selaginella moellendorfli; Sesamum spp., e.g. Sesamum indicum Spirodela spp., e.g. Spirodela polyrhiza, Stevia spp., e.g. Stevia rebaudiana Thapsia spp., e.g. Thapsia villosa Triticum spp., e.g. Triticum aestivum Utricularia spp., e.g. Utricularia gibba Vigna spp., e.g., Vigna radiate, Vitis spp., e.g. Vitis vinifera; or Zea spp., e.g. Zea mays. In various embodiments, the HCDBR enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity to a HCDBR enzyme of a species disclosed in this paragraph.

In various embodiments, the CHS enzyme comprises an amino acid sequence of a wild type CHS enzyme (or derivative thereof) from Amaranthus spp., e.g. Amaranthus hypocondriacus; Amborella spp., e.g. Amborella trichopoda; Aquilegia spp., e.g. Aquilegia coerulea; Arabidopsis spp., e.g ., Arabidopsis thaliana; Azadirachta spp., e.g. Azadiractha indica; Bambusa spp., e.g., Bambusa vulgaris, Beta spp., e.g. Beta vulgaris, Cannabis spp., e.g. Cannabis sativa; Capsicum spp., e.g., Capsicum annuum, Carica spp., e.g. Carica papaya, Catharanthus spp., e.g., Catharanthus roseus; Cistanche spp., e.g., Cistanche deserticola; Citrus spp., e.g. Citrus sinensis; Cucumis spp., e.g. Cucumis melo; Elaeis spp., e.g., Elaeis guineensis; Eucalyptus spp., e.g. Eucalyptus grandis; Glycine spp., e.g. Glycine max; Gossypium spp., e.g. Gossypium Raimondi; Helianthus spp., e.g., Helianthus tuberosus; Kalanchoe spp., e.g. Kalanchoe fedtschenkoi; Linum spp., e.g. Linum usitatissimum; Malus spp., e.g. Malus x domestica; Manihot spp., e.g. Manihot esculenta; Mimulus spp., e.g. Mimulus guttatus; Musa spp., e.g. Musa acuminate; Nelumbo spp., e.g. Nelumbo nucifera; Nicotiana spp., e.g., Nicotiana tabacum; Oryza spp., e.g. Oryza sativa; Petroselinum spp., e.g., Petroselinum crispum; Phalaenopsis spp., e.g. Phalaenopsis equestris; Phyllostacys spp., e.g. Phyllostacys edulis; Physcomitrella spp., e.g., Physcomitrella patens; Pisum spp., e.g. Pisum sativum; Pinus spp., e.g. Pinus taeda; Populus spp., e.g., Populus trichocarpa; Selaginella spp., e.g. Selaginella moellendorfli; Sesamum spp., e.g. Sesamum indicum; Spirodela spp., e.g. Spirodela polyrhiza; Stevia spp., e.g. Stevia rebaudiana; Thapsia spp., e.g. Thapsia villosa; Triticum spp., e.g. Triticum aestivum; Utricularia spp., e.g. Utricularia gibba; Vigna spp., e.g., Vigna radiate; Vitis spp., e.g. Vitis vinifera; or Zea spp., e.g. Zea mays. In various embodiments, the CHS enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity to a CHS enzyme of a species disclosed in this paragraph.

In various embodiments, the PH enzyme comprises an amino acid sequence of a wild type PH enzyme (or derivative thereof) from Acidaminococcus spp., e.g. Acidaminococcus fermentans strain ATCC 25085; Anaerovibrio spp., e.g. Anaerovibrio lipolyticus Aspergillus spp., e.g. Aspergillus nidulans Butyricicoccus spp., e.g. Butyricicoccus pullicaecorum Canis spp., e.g. Canis lupus, Clostridium spp., e.g. Clostridium aurantibutyricum Dialister spp., e.g. Dialister succinatiphilus Erwinia spp., e.g. Erwinia herbicola Eubacterium spp., e.g. Eubacterium ramulus Flavonifractor spp., e.g. Flavonifractor sp. Anil 2; Homo spp., e.g. Homo sapiens, Lachnospira spp., e.g. Lachnospira multipara, Megasphaera spp., e.g. Megasphaera elsdenii; Mus spp., e.g. Mus musculus; Oribacterium spp., e.g. Oribacterium sp. P6A1; Oryctolagus spp., e.g. Oryctolagus cuniculus; Pantoea spp., e.g. Pantoea agglomerans; Parasporobacterium spp., e.g. Parasporobacterium paucivorans; Propionispira spp., e.g. Propionispira arboris; Ratus spp., e.g. Ratus norvegicus; Roseburia spp., e.g. Roseburia sp. CAG:50; Selenomonas spp., e.g. Selenomonas ruminantium, or Sharpea spp., e.g. Sharpea azabuensis. In various embodiments, the PH enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity to a PH enzyme of a species disclosed in this paragraph.

In some embodiments, the enzyme pathway for the synthesis of the second substrate may comprise one or more cytochrome P450 reductases (CPR). In some embodiments, the CPR comprises an amino acid sequence identity with a wild type CPR from Saccharomyces spp., e.g. Saccharomyces cerevisiae; Amaranthus spp., e.g. Amaranthus hypocondriacus; Amborella spp., e.g. Amborella trichopoda; Aquilegia spp., e.g. Aquilegia coerulea; Arabidopsis spp., e.g ., Arabidopsis thaliana; Azadirachta spp., e.g. Azadiractha indica; Bambusa spp., e.g., Bambusa vulgaris, Beta spp., e.g. Beta vulgaris, Cannabis spp., e.g. Cannabis sativa; Capsicum spp., e.g., Capsicum annuum, Carica spp., e.g. Carica papaya, Catharanthus spp., e.g., Catharanthus roseus; Cistanche spp., e.g., Cistanche deserticola; Citrus spp., e.g. Citrus sinensis; Cucumis spp., e.g. Cucumis melo; Elaeis spp., e.g., Elaeis guineensis; Eucalyptus spp., e.g. Eucalyptus grandis; Glycine spp., e.g. Glycine max; Gossypium spp., e.g. Gossypium Raimondi, Helianthus spp., e.g., Helianthus tuberosus Kalanchoe spp., e.g. Kalanchoe fedtschenkoi Linum spp., e.g. Linum usitatissimum Mains spp., e.g. Malus x domestica Manihot spp., e.g. Manihot esculenta Mimulus spp., e.g. Mimulus guttatus Musa spp., e.g. Musa acuminate, Nelumbo spp., e.g. Nelumbo nucifera, Nicotiana spp., e.g., Nicotiana tabacum; Oryza spp., e.g. Oryza sativa; Petroselinum spp., e.g., Petroselinum crispum; Phalaenopsis spp., e.g. Phalaenopsis equestris; Phyllostacys spp., e.g. Phyllostacys edulis; Physcomitrella spp., e.g., Physcomitrella patens, Pisum spp., e.g. Pisum sativum, Pinus spp., e.g. Pinus taeda; Populus spp., e.g., Populus trichocarpa; Selaginella spp., e.g. Selaginella moellendorflr, Sesamum spp., e.g. Sesamum indicum; Spirodela spp., e.g. Spirodela polyrhiza, Stevia spp., e.g. Stevia rebaudiana; Thapsia spp., e.g. Thapsia villosa; Triticum spp., e.g. Triticum aestivum; Utricularia spp., e.g. Utricularia gibba; Vigna spp., e.g., Vigna radiate, Vitis spp., e.g. Vitis vinifera; or Zea spp., e.g. Zea mays. In various embodiments, the CPR enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity to a CPR enzyme of a species disclosed in this paragraph.

In various embodiments, the enzyme pathway for the synthesis of the second substrate comprises an enzyme, a pathway, and/or reaction that converts / coumaric acid to phloretate in Lactobacillus plantarum. Exemplary enzymes, pathways, and reactions are disclosed in Barthelmebs et al, Applied and Environmental Microbiology, 66(8): 3368-75 (August 2000), the contents of which are hereby incorporated by reference in their entirety.

In various embodiments, the enzyme pathway for the synthesis of the second substrate comprises an enzyme, a pathway, and/or reaction for the production of phloretate from tyrosine by Clostridium spp. Exemplary enzymes, pathways, and reactions are disclosed in Mead, G., Journal of General Microbiology , 67: 47-56 (1971); Elsden et al, Arch. Microbiol., 107: 283-88 (1976); and/or Jellet et al, Can. J. Microbiol., 26: 448-53 (1980), the contents of which are hereby incorporated by reference in their entireties.

In various embodiments, the enzyme pathway for the synthesis of the second substrate comprises an enzyme, a pathway, and/or reaction for the production of phloretate by Clostridium orbiscindens . Exemplary enzymes, pathways, and reactions are disclosed in Steed et al, Science, 357: 498-502 (August 4, 2017), the contents of which are hereby incorporated by reference in their entirety.

In various embodiments, the enzyme pathway for the synthesis of the second substrate comprises an enzyme, a pathway, and/or reaction disclosed in PCT Pub. No. WO 2016/193504, the contents of which are hereby incorporated by reference in their entirety.

In various embodiments, the transferase enzyme comprises an amino acid sequence of a Aspergillus terreus aromatic Prenyl Transferase (AtaPT) enzyme having an accession number selected from KP893683, EAU39348, EAU39467, EAU36097, EAU36020, EAU31601, EAU29429, EAU29303 and a variant thereof. Examples of such a transferase enzyme are disclosed in Chen et al., Nature Chemical Biology, 13(2): 226-34 (December 19, 2016), the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the transferase enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity with any one of the AtaPT enzymes having the accession number selected from KP893683, EAU39348, EAU39467, EAU36097, EAU36020, EAU31601, EAU29429, and EAU29303.

In various embodiments, the transferase enzyme comprises an amino acid sequence selected from SEQ ID NOs: 2-22, or a variant thereof. In some embodiments, the transferase enzyme comprises an amino acid sequence that has at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity with any of SEQ ID NOs: 2-22.

In various embodiments, the transferase enzyme comprises the amino acid sequence of SEQ ID NO: 2 with one or more of the following modifications: deletion of amino acids corresponding to amino acids 1-10 of SEQ ID NO: 2 and a substitution at a position corresponding to H88, E91, S177, or W397 of SEQ ID NO: 2. In some embodiments, the transferase comprises a substitution selected from H88A, E91A, E91Q, E91D, S177A, and W397A.

In various embodiments, the transferase enzyme comprises the amino acid sequence of SEQ ID NO: 3 with one or more substitutions at positions corresponding to W97, E123, F170, A173, and F189 of SEQ ID NO: 3. In some embodiments, the transferase enzyme comprises a substitution selected from W97Y and A173M. In various embodiments, the transferase enzyme comprises the amino acid sequence of SEQ ID NO: 4 with one or more substitutions at positions corresponding to Y80, W157, and M159 of SEQ ID NO: 4. In some embodiments, the transferase enzyme comprises a substitution selected from Y80W and M159A.

In various embodiments, at least one enzyme is a circular permutant. Circular permutant strategies for engineering enzymes are described in WO 2016/073740, which is hereby incorporated by reference in its entirety.

In various embodiments, the derivative of FOPPA is selected from 3-(4- famesyloxyphenyl)-propionic acid methyl ester, 4-famesyloxycinnamic acid methyl ester, and 4-famesyloxycinnamic acid. Exemplary FOPPA derivatives are disclosed in in US Patent Nos. 4,939,171 and 9,814,659, US Publication No. 2011/0318439, and PCT Publication No. WO 2016/193501, the contents of which are hereby incorporated by reference in their entireties.

In various embodiments, the microbial cell is prokaryotic or eukaryotic. In some embodiments the microbial cell is a bacteria cell. In some embodiments, the microbial cell is a yeast cell. In some embodiments, the microbial host cell is a bacteria selected from Escherichia spp., Bacillus spp., Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Vibrio spp., and Pseudomonas spp. For example, in some embodiments, the bacterial host cell is a species selected from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides , Zymomonas mobilis, Vibrio natriegens, or Pseudomonas putida. In some embodiments, the bacterial host cell is E. coli. Alternatively, the microbial cell may be a yeast cell, such as but not limited to a species of Saccharomyces, Pichia, or Yarrowia, including Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica.

In some aspects, the invention provides a method for making FOPPA, or a derivative thereof, comprising: culturing the microbial cell as discussed herein, and recovering FOPPA, or a derivative thereof, from the cells or from the culture.

In some apsects, the invention provides a method for making FOPPA, or a derivative thereof, comprising: contacting a first substrate and a second substrate with a prenyltransferase to make FOPPA, or a derivative thereof, wherein the first substrate is selected from famesyl pyrophosphate, famesyl-phosphate, or famesol; wherein the second substrate is selected from phloretate or a precursor or analog thereof. In some embodiments, the prenyltransferase is selected from Aspergillus terreus aromatic Prenyl Transferase (AtaPT) enzyme having an accession number selected from KP893683, EAU39348, EAU39467, EAU36097, EAU36020, EAU31601, EAU29429, and EAU29303, a variant thereof, or is selected from a transferase enzyme comprising an amino acid sequence selected from SEQ ID NOs: 2-22, or a variant thereof. Exemplary prenyltransferases are disclosed in Chen et al, Nature Chemical Biology, 13(2): 226-34 (December 19, 2016), the contents of which are hereby incorporated by reference in their entirety. In various embodiments, the precursor or analog of phloretate is selected from cinnamic acid, hydrocinnamic acid, and /i-coumaric acid.

In various embodiments, the prenyltransferase enzyme comprises an amino acid sequence having at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity with any one of the enzymes having the accession number selected from KP893683, EAU39348, EAU39467, EAU36097, EAU36020, EAU31601, EAU29429, and EAU29303.

In various embodiments, the prenyltransferase enzyme comprises an amino acid sequence having has at least about 60% identity, or at least about 70% identity, or at least about 80% identity, or at least about 90% identity, or at least about 95% identity, or at least about 97%, 98%, or 99% amino acid sequence identity with any one of SEQ ID NOs: 2-22.

In various embodiments, the prenyltransferase enzyme comprises the amino acid sequence of SEQ ID NO: 2 with one or more of the following modifications: deletion of amino acids corresponding to amino acids 1-10 of SEQ ID NO: 2 and a substitution at a position corresponding to H88, E91, S177, or W397 of SEQ ID NO: 2. In some embodiments, the prenyltransferase comprises a substitution selected fromH88A, E91A, E91Q, E91D, S177A, and W397A.

In various embodiments, the prenyltransferase enzyme comprises the amino acid sequence of SEQ ID NO: 3 with one or more substitutions at positions corresponding to W97, E123, F170, A173, and F189 of SEQ ID NO: 3. In some embodiments, the prenyltransferase enzyme comprises a substitution selected from W97Y and A173M.

In various embodiments, the prenyltransferase enzyme comprises the amino acid sequence of SEQ ID NO: 4 with one or more substitutions at positions corresponding to Y80, W157, and M159 of SEQ ID NO: 4. In some embodiments, the prenyltransferase enzyme comprises a substitution selected from Y80W and M159A. In various embodiments, the prenyltransferase is expressed in a microbe and contacted with the first substrate and the second substrate in the form of whole cells expressing the prenyltransferase, cellular extract, or in purified form.

In various embodiments, the prenyltransferase is expressed in a microbe, wherein the microbe overexpresses an enzyme in the pathway for the synthesis of the first substrate.

In various embodiments, the phloretate or an analog thereof is fed to the culture or reaction.

In various embodiments, the phloretate, or a derivative thereof, is prepared from a phloretate precursor selected from L-phenylalanine, cinnamic acid, tyrosine, p- coumaric acid, / coumaroyl-CoA. /i-diliydrocoumaroyl-CoA. phloretin, p- hydroxyphenylpyruvic acid, and / hydro\y phenyl lactic acid by a reaction with one or more enzymes for producing the phloretate or a derivative thereof (as described herein). In some embodiments, the contacting of the phloretate, or a derivative thereof, and the famesyl pyrophosphate, famesyl-phosphate, and/or famesol with a prenyltransferase occurs in a cell free system. In some embodiments, the prenyltransferase and/or the famesyl pyrophosphate, famesyl-phosphate, or famesol are provided in the form of a cellular extract. In some embodiments, the cellular extract is an extract of a microbe overexpressing the prenyltransferase, and optionally overexpressing an enzyme to increase production of famesyl pyrophosphate, famesyl-phosphate, or famesol.

In various embodiments, the famesyl pyrophosphate, famesyl-phosphate, and/or famesol are provided in a cell free system comprising the prenyltransferase and at least one microbial cell engineered to produce the phloretate, or a derivative thereof.

In various embodiments, the phloretate is prepared from a precursor through an enzymatic pathway disclosed herein.

In various embodiments, the method further comprises harvesting the FOPPA from the cell culture or reaction.

In some aspects, the invention provides methods for making a product comprising FOPPA, or a derivative thereof, comprising producing FOPPA, or a derivative thereof, according to a method discussed above, and incorporating the FOPPA, or a derivative thereof, into the product. In some embodiments, the product is a skin-lightening composition. In other embodiments, the product is an anti-seborrheic composition.

In various embodiments, the product is a composition for use in an application selected from antioxidant, antibacterial, anthelmintic, anti-inflammatory, cancer chemopreventative, food additive, and fragrance component. The product may be a cosmetic composition, a pharmaceutical composition, or a nutraceutical composition. Exemplary applications are disclosed in US Pub. No. 2011/0318439, the contents of which are hereby incorporated by reference in their entirety. All cited references are herein expressly incorporated by reference in their entirety.

SEQUENCE LISTING

SEQ ID NO: 1

> Saccharomyces cerevisiae farnesyl pyrophosphate synthase (ScFPPS) :

MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEACDWYAHSLNYNTPGGKLNRGLSWD TY AILSNKTVEQLGQEEYEKVAILGWCIELLQAYFLVADDMMDKSITRRGQPCWYKVPEVGE IAI NDAFMLEAAIYKLLKSHFRNEKYYIDITELFHEVTFQTELGQLMDLITAPEDKVDLSKFS LKK HSFIVTFKTAYYSFYLPVALAMYVAGITDEKDLKQARDVLIPLGEYFQIQDDYLDCFGTP EQI GKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGKKDSVAEAKCKKIFNDLKIEQLYH EYE ESIAKDLKAKISQVDESRGFKADVLTAFLNKVYKRSK

SEQ ID NO: 2

>AtaPT (Aspergillus terreus aromatic Prenyl Transferase,

AMB20850.1)

MLPPSDSKDPRPWQILSQALGFPNYDQELWWQNTAETLNRVLEQCDYSVHLQYKYLAFYH KYI LPSLGPFRRPGVEPEYISGLSHGGHPLEISVKIDKSKTICRLGLQAIGPLAGTARDPLNS FGD RELLKNLATLLPHVDLRLFDHFNAQVGLDRAQCAVATTKLIKESHNIVCTSLDLKDGEVI PKV YFSTIPKGLVTETPLFDLTFAAIEQMEVYHKDAPLRTALSSLKDFLRPRVPTDASITPPL TGL IGVDCIDPMLSRLKVYLATFRMDLSLIRDYWTLGGLLTDAGTMKGLEMVETLAKTLKLGD EAC ETLDAERLPFGINYAMKPGTAELAPPQIYFPLLGINDGFIADALVEFFQYMGWEDQANRY KDE LKAKFPNVDISQTKNVHRWLGVAYSETKGPSMNIYYDWAGNVARV

SEQ ID NO: 3

>TleC ( Streptomyces blastmyceticus tryptophan

dimethylallyltrans ferase , BAP27943.1)

MESAGPGTGPQPPRTSGDFTPDTGVIAEMTGRPMRFDSDRYRPTDTYAEVACDKVCRAYE GLG ADGGDRESLLAFLRDLTDPWGELPVGTPPEDACWVSIDGMPLETSVAWAGRKAGVRLSLE SPR GPAKRRMEDGMALTRRLAGRPGVSVDPCLRVEDLFTDDDPQGYFTIAHAVAWTPGGHPRY KIF LNPAVRGREQAAARTEEAMIRLGLEQPWRALTEHLGGAYGPEHEPAALAMDLVPGDDFRV QVY LAHSGVSAEAIDAKSAVAADHVPGSFARALRGINGADDTPEWKRKPPVTAFSFGPGRAVP GAT LYVPMIPVHGSDAAARDRVAAFLRSEGMDAVGYEAVLDAISDRSLPESHTQNFISYRGGD SPR FSVYLAPGVYREA SEQ ID NO: 4

>MpnD (Marinactinospora thermotolerans aromatic

prenyltrans ferase , AF085455.1) MAGDPFVDNGTVSSQRPLRAVPGRYPPGATHLDAAVDTLVRCHAALGRAPSEAEAAVCLL RRL

WGRWGNTPVERPGWRSYVAVDGSPFELSAAWNGDGPAEVRVTVEATADPPTPEGNQE AGWEYL

RGLSRHPGAATARVLALEDLFRPQTPHDRCWIMHGMASRPGADPLFKVYLDPDARGA AEAPSV

LDEAMDRLGVRAAWQGLRGWLDEHGGSGRIGSLALDLADTDDARVKVYVQHAGLDWA DIDRQA

AVARGHVPGAFSAALEEITGTEVPPHKPPVTCFAFHRGVGVPTAATLYIPMPAGVPE SDARRR

SAAFMRRSGLDSAAYLAFLAAATGDGEGVRALQNFVAYRPAAPGGRPRFACYVAPGL YR

SEQ ID NO: 5

>PfIACE ( Pestalotiopsis fici prenyltransferase, APC57597.1)

MAISTPSNGVSHVAKPLPNLKEVNKGIETDSEDRAFWWGALSEPLASLLEANHYTKE VQLHYL

RWFYQWILPALGPRPLDGKPYYGSWITHDLSPFEYSLNWKEKSSKQTIRFTIEAVTK QSGTAS

DPINQLGAKEFLEAVSKDVPGMDLTRFNQFLEATNVPNDCVDDAIAKHPAHFPRSRV WIAFDL

EHSGNLMAKSYFLPHWRAIQSGISANTI IGDTVKECNKADGSSYDGSLNAIESYLATFTRPEE

APQMGLLSNDCVAETPGSRLKVYFRSSADTLAKAKDMYNLGGRLKGPKMDASLKGIS DFWYHL

FGLDSSDPASDDKVCIGNHKCIFVYEMRSSQGSEPDIDVKFHIPMWQLGKTDGQISE LLASWF

ESHGHPDLASRYKSDLGTAFPKHNITGKSVGTHTYISITHTPKTGLYMTMYLSPKLP EFYY

SEQ ID NO: 6

>NphB ( Streptomyces sp. CNZ306 aromatic prenyltransferase, PJJ47653.1)

MIGIDFLECLVSEGIEAEGLYSAIEESARMVDAPFSRDKVWPILSAFGGGFSDAGGVIFS LQA

GKDVPEMEYSAQISAEVGDPYAHALATGVLNETDHPVSTVLAEIVSLAPTSEHYIDC GIVGGF

KKIYANFPHDQQKVSRLADLPAMPRAVGANAEFFDRYGLDNVALIGVDYRNKTINLY FQAPAE

TAGNLDPKTVSAMLRETGMSTPSEEMVAYADRAYRIYATLGWDSPEVMRLAFAPQPR RSIDLA

ELPARLEPRIEQFMRATPHKYPGALINATAAKWSKKHEVLDLAAYYQVSALHLKAIQ AEEGQS

S

SEQ ID NO: 7

>ScFLT ( Streptomyces cinnamonensis flaviolin linalyltranferase, A2AXG5.1)

MMSGTADLAGVYAAVEESAGLLDVSCAREKVWPILAAFEDVLPTAVIAFRVATNARH EGEFDC

RFTVPGSIDPYAVALDKGLTHRSGHPIETLVADVQKHCAVDSYGVDFGWGGFKKIWV YFPGG

RHESLAHLGEIPSMPPGLAATEGFFARYGLADKVDLIGVDYASKTMNVYFAASPEWS APTVL

AMHREIGLPDPSEQMLDFCSRAFGVYTTLNWDSSKVERIAYSVKTEDPLELSARLGS KVEQFL

KSVPYGIDTPKMVYAAVTAGGEEYYKLQSYYQWRTDSRLNLSYIGGRS SEQ ID NO: 8

>StrFBP ( Streptomyces sp . KO-3988 furaquinocin biosynthesis prenyltrans ferase , Q2L6E3.1)

MPGTDDVAVDVASVYSAIEKSAGLLDVTAAREWWPVLTAFEDVLEQAVIAFRVATNARHE GD

FDVRFTVPEEVDPYAVALSRSLIAKTDHPVGSLLSDIQQLCSVDTYGVDLGVKSGFK KVWVYF

PAGEHETLARLTGLTSMPGSLAGNVDFFTRYGLADKVDVIGIDYRSRTMNVYFAAPS ECFERE

TVLAMHRDIGLPSPSEQMFKFCENSFGLYTTLNWDTMEIERISYGVKTENPMTFFAR LGTKVE

HFVKNVPYGVDTQKMVYAAVTSSGEEYYKLQSYYRWRSVSRLNAAYIAARDKEST

SEQ ID NO: 9

>BrePT (Aspergillus versicolor brevianamide F reverse

prenyltrans ferase , AFM09725.1)

MTAPELRAPAGHPQEPPARSSPAQALSSYHHFPTSDQERWYQETGSLCSRFLEAGQYGLH QQY QFMFFFMHHLIPALGPYPQKWRSTISRSGLPIEFSLNFQKGSHRLLRIGFEPVNFLSGSS QDP FNRIPIADLLAQLARLQLRGFDTQCFQQLLTRFQLSLDEVRQLPPDDQPLKSQGAFGFDF NPD GAILVKGYVFPYLKAKAAGVPVATLIAESVRAIDADRNQFMHAFSLINDYMQESTGYNEY TFL SCDLVEMSRQRVKIYGAHTEVTWAKIAEMWTLGGRLIEEPEIMEGLARLKQIWSLLQIGE GSR AFKGGFDYGKASATDQIPSPI IWNYEISPGSSFPVPKFYLPVHGENDLRVARSLAQFWDSLGW SEHACAYPDMLQQLYPDLDVSRTSRLQSWISYSYTAKKGVYMSVYFHSQSTYLWEED

SEQ ID NO: 10

>7-DMATS (Aspergillus fumigatus At293 7-dimethylallyltryptophan synthase, Q4WYG3.2)

MSIGAEIDSLVPAPPGLNGTAAGYPAKTQKELSNGDFDAHDGLSLAQLTPYDVLTAALPL PAP

ASSTGFWWRETGPVMSKLLAKANYPLYTHYKYLMLYHTHILPLLGPRPPLENSTHPS PSNAPW

RSFLTDDFTPLEPSWNVNGNSEAQSTIRLGIEPIGFEAGAAADPFNQAAVTQFMHSY EATEVG

ATLTLFEHFRNDMFVGPETYAALRAKIPEGEHTTQSFLAFDLDAGRVTTKAYFFPIL MSLKTG

QSTTKWSDSILHLALKSEVWGVQTIAAMSVMEAWIGSYGGAAKTEMISVDCVNEADS RIKIY

VRMPHTSLRKVKEAYCLGGRLTDENTKEGLKLLDELWRTVFGIDDEDAELPQNSHRT AGTIFN

FELRPGKWFPEPKVYLPVRHYCESDMQIASRLQTFFGRLGWHNMEKDYCKHLEDLFP HHPLSS

STGTHTFLSFSYKKQKGVYMTMYYNLRVYST

SEQ ID NO: 11

>CdpNPT (Aspergillus fumigatus cyclic dipeptide N- prenyltrans ferase , ABR14712.1)

MDGEMTASPPDISACDTSAVDEQTGQSGQSQAPIPKDIAYHTLTKALLFPDIDQYQHWHH VAP

MLAKMLVDGKYSIHQQYEYLCLFAQLVAPVLGPYPSPGRDVYRCTLGGNMTVELSQN FQRSGS TTRIAFEPVRYQASVGHDRFNRTSVNAFFSQLQLLVKSVNIELHHLLSEHLTLTAKDERN LNE EQLTKYLTNFQVKTQYWALDLRKTGIVAKEYFFPGIKCAATGQTGSNACFGAIRAVDKDG HL DSLCQLIEAHFQQSKIDDAFLCCDLVDPAHTRFKVYIADPLVTLARAEEHWTLGGRLTDE DAA VGLEI IRGLWSELGIIQGPLEPSAMMEKGLLPIMLNYEMKAGQRLPKPKLYMPLTGIPETKIA RIMTAFFQRHDMPEQAEVFMENLQAYYEGKNLEEATRYQAWLSFAYTKEKGPYLSIYYFW PE

SEQ ID NO: 12

>BAE61387 (Aspergillus oryzae RIB40 putative prenyltransferase,

BAE61387.1)

MSLRNDLDNGRPTKRLESWDIASMWLSDRKDEIQDWWDFSGPQLATLAHEAGYSTMTQIE LLL FFRSWLPRMGRFPDACRPRACAQSRSILTYDGSPIEYSWKWNNSANDHPEIRFCVEPVGD GL CADGIVGGKLRATDEILVQLAKRVPSTDLEWYHHFRDSFGLGHWTDGPLHEDAGTWQVRR PRM PVAFEFTPKGIVTKVYFTPPATLDDMPSFNMFADWRPIGDKDTTALDESMEYLSRDPVGA TL RPDVLAIDCISPLKSRIKLYAGTAMTTFTSAISVLTLGGRIPVTRHSIDEMWALFRMVLG LHD KFLQDEELPVQNPFQPSRAHPEDYYSGLLYYFNLAPGALLPDVKLYLPVIRYGRSDADIA LGL QRFMASRHRGQYVDGFQRAMEI ISQRHKSGNGHRIQTYIACSFDKDGSLSLTSYLNPGVYFSS ETVDV

SEQ ID NO: 13

>EAU34068 (Aspergillus terreus NIH2624 putative

prenyltransferase, EAU34068.1)

MLPPSDSKDPRPWQILSQALGFPNYDQELWWQNTAETLNRVLEQCDYSVHLQYKYLA FYHKYI LPSLGPFRRPGVEPEYISGLSHGGHPLEISVKIDKSKTICRLGLQAIGPLAGTARDPLNS FGD RELLKNLATLLPHVDLRLFDHFNAQVGLDRAQCAVATTKLIKESHNIVCTSLDLKDGEVI PKV YFSTIPKGLVTETPLFDLTFAAIEQMEVYHKDAPLRTALSSLKDFLRPRVPTDASITPPL TGL IGVDCIDPMLSRLKVYLATFRMDLSLIRDYWTLGGLLKDEGTMKGLEMVETLAKTLKLGD EAC ETLDAERLPFGINYAMKPGTAELAPPQIYFPLLGINDGFIADALVEFFQYMGWEDQASRY KDE LKAKFPNVDISQTKNVHRWLGVAYSETKGPSMNIYYDWAGNVARV

SEQ ID NO: 14

>FgaPT2 (Aspergillus fumigatus tryptophan prenyltransferase, AAX08549.1)

MKAANASSAEAYRVLSRAFRFDNEDQKLWWHSTAPMFAKMLETANYTTPCQYQYLITYKE CVI

PSLGCYPTNSAPRWLSILTRYGTPFELSLNCSNSIVRYTFEPINQHTGTDKDPFNTH AIWESL

QHLLPLEKSIDLEWFRHFKHDLTLNSEESAFLAHNDRLVGGTIRTQNKLALDLKDGR FALKTY

IYPALKAWTGKTIHELVFGSVRRLAVREPRILPPLNMLEEYIRSRGSKSTASPRLVS CDLTS PAKSRIKIYLLEQMVSLEAMEDLWTLGGRRRDASTLEGLSLVRELWDLIQLSPGLKSYPA PYL

PLGVIPDERLPLMANFTLHQNDPVPEPQVYFTTFGMNDMAVADALTTFFERRGWSEM ARTYET

TLKSYYPHADHDKLNYLHAYISFSYRDRTPYLSVYLQSFETGDWAVANLSESKVKCQ DAACQP

TALPPDLSKTGVYYSGLH

SEQ ID NO: 15

>FtmPTl (Aspergillus fumigatus Af293 brevianamide F

prenyltrans ferase 1, AAX56314.1)

MPPAPPDQKPCHQLQPAPYRALSESILFGSVDEERWWHSTAPILSRLLISSNYDVDVQYK YLS

LYRHLVLPALGPYPQRDPETGI IATQWRSGMVLTGLPIEFSNNVARALIRIGVDPVTADSGTA

QDPFNTTRPKVYLETAARLLPGVDLTRFYEFETELVITKAEEAVLQANPDLFRSPWK SQILTA

MDLQKSGTVLVKAYFYPQPKSAVTGRSTEDLLVNAIRKVDREGRFETQLANLQRYIE RRRRGL

HVPGVTADKPPATAADKAFDACSFFPHFLSTDLVEPGKSRVKFYASERHVNLQMVED IWTFGG

LRRDPDALRGLELLRHFWADIQMREGYYTMPRGFCELGKSSAGFEAPMMFHFHLDGS QSPFPD

PQMYVCVFGMNSRKLVEGLTTYRRVGWEEMASHYQGNFLANYPDEDFEKAAHLCAYV SFAYKN

GGAYVTLYNHSFNPVGDVSFPN

SEQ ID NO: 16

>AnaPT (Aspergillus fischeri NRRL 181 indole diterpene prenyltrans ferase , A1DN10.1)

MSPLSMQTDSVQGTAENKSLETNGTSNDQQLPWKVLGKSLGLPTIEQEQYWLNTAPYFNN LLI QCGYDVHQQYQYLAFYHRHVLPVLGPFIRSSAEANYISGFSAEGYPMELSVNYQASKATV RLG CEPVGEFAGTSQDPMNQFMTREVLGRLSRLDPTFDLRLFDYFDSQFSLTTSEANLAASKL IKQ RRQSKVIAFDLKDGAI IPKAYFFLKGKSLASGIPVQDVAFNAIESIAPKQIESPLRVLRTFVT KLFSKPTVTSDVFILAVDCIVPEKSRIKLYVADSQLSLATLREFWTLGGSVTDSATMKGL EIA EELWRILQYDDAVCSHSNMDQLPLWNYELSSGSATPKPQLYLPLHGRNDEAMANALTKFW DY LGWKGLAAQYKKDLYANNPCRNLAETTTVQRWVAFSYTESGGAYLTVYFHAVGGMKGNL

SEQ ID NO: 17

>XhAPT (Xylona heveae TC161 aromatic prenyltransferase,

XP_018190780.1)

MAPSMTANYPYSQISEFSKTIATSSDLDPNFGGGVSFKPSSCGGITTARKPWQILQDALG FRN EDEHFWWETTASVLGCLLEKAGYDVHLQYQYLSLYYRYVLPSYGPRPLQPGVPHWKSFMC DDF SPFEPSWNWDGSKSIIRFSFEPINRASGTSADPFNQIKPREVLAEISDISAGLDTQWYDH FAR EFFLPSETASI IRSRLPEGEHMSQSFLAWDLNGGEASTKAYFFPILRSLETGRSTRDIWDAI TKLDSEKTSLRPSLTVLEDYMSSLPTEWQAKYEMIAIDCTDPSKSRIKIYVRMPSMAFNK VRD

MYCLGGRLHGPNVDAAMKILDDLWPRVLYIPEGTGPDDELPSNTHRTAGAIFNFELK PGNPLP DPKLYLPVRHYAKSDLDIARGLQSFFRLQGWDEMADSYVEDLKNIFPTHDLANTAGSHTY LSY

SYKKKTGAAVTMYYNPRIYECPPWDEVF

SEQ ID NO: 18

>PpPPT ( Penicillium polonicum putative prenyltrans ferase , OQD60174.1)

MTYSTATPKDSTPVSLLSLYLTFRSKDDKLWWDNTAPVIGGFLAAAHYKVASQFEFLLFY HKY

ILPSLGHYPSPENEGDRWKSFLYRRGEPLELSFNYQKDSNCTVRLALEPVGPNAGTK DDPLNE

FEAKILVEKIAQLDSNIDLQWVDFLDKEILLHNDELSQIKNTELEGSAHMSQRLVGV DFMSGG

MKIKPYFVPWLKSLVTGVPTLQLMFQAIRKLDSVGSFSNGLSEVEAYLASTDQLLWS EENYLS

FDCVDPGKSRIKLYVAEKVTCFNRIQSHWTLGGQLRSQANQEGLLLLKKLWNLLGYP GDPAQQ

TDRYLPFNFNWELRPSNPIPLPKVYFALGNEPDSLVSKALIGLFTELGWSDQIHAHK RSVEFA

FPDCNLEETTHVLTWITVTYEEEKGAYITTYCNAIGGGHKLQFR

SEQ ID NO: 19

>AtAPT (Aspergillus taichungensis aromatic prenyltransferase, PLN85696.1)

MLLSRTTSSQNPFHLLLSGTPRLPKMRPEQEPSIQAPSKKVPLPIADGDARPWQVLSLLL PFH NPDQKLWWDKVGPLIEIYLNCSGYNVGAQYRYLLMLHSIILPVLGPFPNSTRTHTSWPYF MNN GDPCDLSINYQGGSAPCVRLGIEPIGPMAGTNQDPMNEYAGRRLLEDLSRIQPGIDFQLF DHF RDTLTLSNYKARLCWHAVQEHGIKAQGHVALDLHEHSFKVKAYSIPLLRSLTSGVHYVRM MID SIKMISRDQAITIGLSKVDEYLAATKHLLVDSRSCFSFDCADLQHSRYKIYVGANVKSLG EAY DFWTLGGRLKGEAIDRGFQLMETIWKTMYARSLPDRKPREYIPFIWNWEVSPTDSDPIPK AYF LVLNDYDILVSEVINCLFGELGWTEHAMTHQI IQKMAYPNHDFGSSTEIYSWISLAYSQSKGP YITIYSNPAASL

SEQ ID NO: 20

>TgFT ( Trypanosoma grayi farnesyltransferase, XP_009314693.1)

MQLREELRDAVCVFYLVLRALDTVEDDMSLAVDLKLRELPVFHEHLRDPSWRMCGVG AGRERE

LLERFPHVTRVYARLGKAYQDVITDICARMASGMCEFLTRRVESRADYDLYCHYVAG LVGHGL

TRLYVSGGFEDPNLADDLTNANHMGLFLQKTNI IRDFYEDICESPPRIFWPREIWAQYTDDLH

AFKEEAHEAKALECLNAMVADALVHVPHVIEYMAALRDPSVFAFCAIPQLMAMATLA LVFNNR

NVFHSKVKLTRGSTCSI ILYSTQLQSAMQTMRTQAQNLLARTGPDDVCYDKIAELVGEAVRAV

DAHLQPETDGVARSMLTRYPALGGRLLYTLIDNWGYLGK

SEQ ID NO: 21

>CoFT ( Cutaneotrichosporon oleaginosum farnesyltransferase, KLT41078.1) MATLYPSIQSLQKFPYPGDGWSSTLTDQHDTEGLIADVLDEQPPAHVPRLGLQNATTTLD SV NHLKFIQGAMMSLPSGFVGLDASRPWLVFWTVHSLDLLGVLLPQNIRDRAVSTILHFLHP TGG FCGGAANTHMPHLLPTYASWSLAIVGNAGKGGGWERLVDARQDIYNFFMRCKRPDGGFWG D NCEVDVRGTYCLLWATLLDI ITPELLHNVDKAIAAGQTFEGGFACSSFTFKDGNRVAMSEAH GGYTSCSVFSHFLLSSVQPPRRLESLPESFPVPIDVDSWRWSAMMQGEAADGGGFRGRSN KL VDGCYSWWVGGTFPVLEELRRREAEVKTSPNGPTATKIVAVDDDGEDEWADEASMHALFN RGM CDSEVRLMAVALQEYTLLVAQSVTRGGLRDKPGKGPDLYHTCNNLSGLSVAQHRLTHTPE EVQ KQREAFKADRGLPAVKPTTPGGGWKSEEERQAARREVWANVRAWVEDESDTLWGGQMSQV NT TVPPFNMLEVRLQPFIDYFYCQ SEQ ID NO: 22

>SrFT ( Sapingoeca rosetta farnesyltranferase , EGD76967.1) MGYDGLVKLDPEQHLPYVTGGLGTLPSGFETLDASRPWLVYWSLNALVILGGTISPELKR RVI NTLRMCQAETGGFGGGVGQVAHAAPTYAAVNALAI IGTEEAWSI INREKLASWLSSLIEDDGS MHMHDDGEIDVRAVYCGASAARLCGLDVDTIFAKCPQWVARCQTYEGGFAAIPGLEAHGG YTF CGFAAMSILCSTHLIDIPRLTEWLANRQMPMSGGFQGRPNKLVDGCYSFWVGGCFPILAD LLE AQGLPGDWNAEALIDYWCVCQCPSGFRDKPGKRQDYYHTSYCLSGLASMKRFAPNHPILS Q LNATHPIHNVPPANAERMIQAMSSQTTTRH