Methods and compositions for the bioproduction of salicornin

Abstract

This invention relates to materials and methods for the bioproduction of scallop flavonoids. The invention provides methods and materials for cell-based and cell-free production of scallop flavonoids.

Description

Technical Field

[0001] This invention relates to materials and methods for the bioproduction of scallop flavonoids. The invention provides methods and materials for cell-based and cell-free production of scallop flavonoids.

[0002] References to sequence lists

[0003] This application is submitted together with an electronic sequence list. The sequence list is provided as a 107-kilobyte file named DEBU-023-01WO.xml, created on December 26, 2024. The information in the electronic sequence list is incorporated herein by reference in its entirety. Background Technology

[0004] Nature is considered a source of molecules with relevant potential pharmaceutical applications. Among various phytochemicals, flavonoids have attracted and continue to attract the majority of attention due to their significant biological benefits. Flavonoids are associated with the prevention and management of current diseases such as cancer, diabetes, and cardiovascular disease. More than 13,000 flavonoids have been isolated and identified from plants, some of which, such as quercetin, kaempferol, and scutellarin, have shown effective pharmacological effects and therefore hold promise as new drugs.

[0005] Glycosylated flavonoid pectolinarin was first isolated from *Linaria vulgaris*, a known medicinal herb used internally for digestive and urinary tract disorders, externally for hemorrhoids and venous skin ulcers, and for cleaning purulent wounds and rashes. (Cheriet et al., I. Isolation and Biological Properties of the Natural Flavonoids Pectolinarin and Pectolinarigenin—A Review. *Antibiotics*, 2020, 9, 417.) Pectolinarin and its aglycone, pectolinarigenin, have been identified as major components in many medicinal herbs from various genera worldwide.

[0006] In the 113 years since its first report, sennaroside has been isolated, in most cases, from the aerial parts of 87 plants belonging to 29 different genera, widely distributed throughout the world. Most of these plants are used in folk medicine in different parts of the world. Sennaroside is the aglycone of sennaroside, obtained through hydrolysis. Sennaroside has also been isolated and identified from 136 plants of 71 different genera. Sennaroside has been shown to be more widely distributed in the plant kingdom than sennaroside, and this evidence can be explained by the assumption that not all plants possess O-glycosylation enzymes. In most works, sennaroside has been isolated from the nonpolar fraction of plant extracts, acting primarily on the aerial parts. Summary of the Invention

[0007] Conventional methods for producing senna flavonoids involve isolation from plants. This increases production costs because purification is required from these mixtures. Furthermore, these factors vary from year to year in the source plants and are affected by weather and environmental factors. Therefore, another challenge to the commercial production of polyphenols from plants is that harvesting is typically limited to once a year. This means that large quantities of extracts must be prepared and stored for extended periods to meet the food industry's year-round needs.

[0008] Therefore, there is a need for a reliable and cost-effective new method for producing sennae. This invention provides methods and compositions for the biological production of sennae. The methods of this invention can induce the production of sennae in a host organism containing certain genetic modifications. The methods of this invention also provide the production of sennae from one or more substrates via cell-free organisms. The methods of this invention are advantageous because they provide cost-effective methods for producing sennae. Advantageously, the methods of this invention are also reliable and independent of the available plants for the preparation of sennae.

[0009] Advantageously, the method of the present invention provides a cell-based or cell-free platform for the production of senna flavin. The platform provided by the present invention is advantageous because it offers multiple pathways for the preparation of senna flavin from naringenin. The availability of different pathways for the production of senna flavin is beneficial because it provides pathways for the preparation and isolation of one or more intermediates in the biosynthetic pathway. Therefore, the platform provided in the present invention unlocks the option of preparing one or more intermediates that might otherwise be difficult to use in manufacturing. Advantageously, the pathway for the production of senna flavin can also be selected based on the availability of various enzymes for that particular reaction pathway. In other words, if an enzyme is unavailable (or not readily available), a different pathway that does not require that particular enzyme can be selected to produce senna flavin. Therefore, the modularity of the biosynthetic pathway is particularly advantageous.

[0010] In some aspects, the present invention provides a method for the biological production of sennaroflavin, the method comprising providing one or more enzymes, wherein the one or more enzymes cause naringenin to be converted into sennaroflavin via one or more intermediates. The method for producing sennaroflavin includes cell-based methods and cell-free methods.

[0011] In some aspects of the invention, a method for producing scallopeflavin includes an engineered host cell containing one or more gene modifications for expressing one or more enzymes that induce the conversion of naringenin to scallopeflavin via one or more intermediates. In some embodiments, these gene modifications in the host cell may further include one or more gene modifications for enhancing naringenin production. In some embodiments, the engineered host cell contains gene modifications for enhancing the production of naringenin or any precursors that induce naringenin production in the engineered host cell. Additional gene modifications that increase naringenin production can be beneficial for scallopeflavin production because naringenin can be used as a substrate for scallopeflavin production. An increase in the amount of naringenin will result in a higher scallopeflavin yield.

[0012] In certain aspects of the invention, a method for producing scalloped flavonoids is a cell-free production of scalloped flavonoids, wherein one or more enzymes in a cell-free medium cause naringenin to be converted to scalloped flavonoids via one or more intermediates. Components of the components in the cell-free reaction can be produced in host cells. For example, substrates, such as naringenin, can be produced in cells, and the subsequent conversion to scalloped flavonoids can be carried out in a cell-free medium. In some embodiments, one or more intermediates generated in the conversion of naringenin to scalloped flavonoids can be produced in engineered host cells, and these one or more intermediates can subsequently be converted to scalloped flavonoids in a cell-free medium via one or more enzymes.

[0013] In some aspects, the method comprises a combination of: (i) an engineered host cell containing one or more gene modifications for expressing one or more enzymes that induce the conversion of naringenin to sennaroxen, or one or more intermediates; and (ii) cell-free production comprising one or more enzymes that induce the conversion of naringenin or a substrate to sennaroxen, or one or more intermediates, to synthesize sennaroxen. In some embodiments, the engineered host cell contains gene modifications for enhancing the production of naringenin or any precursors that induce naringenin production in the engineered host cell.

[0014] In some embodiments of the invention, the one or more intermediates are selected from the group consisting of: apigenin, scutellarein, hispidulin, isosakuranetin, carthamidin, 6-methoxyscarmine, scutellarein, 4-methoxyscarmine, acacetin, 4',6-methoxynaringenin, 4'-methoxyscutellarein, and any combination thereof. Figure 1 An exemplary schematic pathway for the conversion of naringenin to saurolophytin is provided. Figure 1 It also provides enzymes that mediate the conversion of naringenin into one or more intermediates, or one or more intermediates into other intermediates, or one or more intermediates into saurolophytin. From Figure 1 As is evident, the method of the present invention provides a modular pathway for the conversion of naringenin to scalloped flavonoids. The choice of pathway for the production of scalloped flavonoids will determine the identity of one or more intermediates selected in the process. Each individual segment of the pathway can be controlled by an enzyme selected for the conversion. In some embodiments, the method of the present invention provides the use of one or more enzymes for mediating the conversion of starting material into one or more intermediates or scalloped flavonoids. In some embodiments, the one or more enzymes are selected from the group consisting of: flavonoid synthase (FNSI), flavonoid hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and any combination thereof.

[0015] For example, in some embodiments, these intermediates are apigenin, baicalin, and crocin. In some embodiments, the method provides a pathway in which naringenin is converted to apigenin; apigenin is converted to baicalin; baicalin is converted to crocin; and crocin is converted to sauroplasmin. In some embodiments, one or more of these steps can be performed by one or more enzymes. For example, as... Figure 2 As shown, the provided pathway includes: flavonoid synthase (FNSI)-mediated conversion of naringenin to apigenin; flavonoid hydroxylase cytochrome P450 (P450)-mediated conversion of apigenin to baicalin; O-methyltransferase (OMT)-mediated conversion of baicalin to crocin; and O-methyltransferase (OMT)-mediated conversion of crocin to saurolophytin. Any of these foregoing steps can be performed in engineered host cells or cell-free media.

[0016] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalinoflavin are apigenin, baicalin, and 4'-methoxybaicalin. This invention provides the conversion of naringenin to apigenin; the conversion of apigenin to baicalin; the conversion of baicalin to 4'-methoxybaicalin; and the conversion of 4'-methoxybaicalin to squalinoflavin. In some embodiments, one or more of these steps can be performed by one or more enzymes.

[0017] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are apigenin, baicalin, and psyllium. This invention provides the conversion of naringenin to apigenin; the conversion of apigenin to baicalin; the conversion of baicalin to psyllium; and the conversion of psyllium to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0018] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are apigenin, farnesin, and 4'-methoxybaicalin. This invention provides the conversion of naringenin to apigenin; the conversion of apigenin to farnesin; the conversion of farnesin to 4'-methoxybaicalin; and the conversion of 4'-methoxybaicalin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0019] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are isoporphyrin, 4'-methoxyscarmine, and 4',6-methoxynaringenin. This invention provides the conversion of naringenin to isoporphyrin; the conversion of isoporphyrin to 4'-methoxyscarmine; the conversion of 4'-methoxyscarmine to 4',6-methoxynaringenin; and the conversion of 4',6-methoxynaringenin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0020] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are isopyroxin, 4'-methoxyscarmine, and 4'-methoxybaicalin. This invention provides the conversion of naringenin to isopyroxin; the conversion of isopyroxin to 4'-methoxyscarmine; the conversion of 4'-methoxyscarmine to 4'-methoxybaicalin; and the conversion of 4'-methoxybaicalin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0021] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are isopyroxin, farnesin, and 4'-methoxybaicalein. This invention provides the conversion of naringenin to isopyroxin; the conversion of isopyroxin to farnesin; the conversion of farnesin to 4'-methoxybaicalein; and the conversion of 4'-methoxybaicalein to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0022] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, 6-methoxysafflowerin, and physalisin. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to 6-methoxysafflowerin; the conversion of 6-methoxysafflowerin to physalisin; and the conversion of physalisin to squalene. In some embodiments, one or more of these steps can be performed by one or more enzymes.

[0023] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, 6-methoxysafflowerin, and 4',6-methoxynaringenin. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to 6-methoxysafflowerin; the conversion of 6-methoxysafflowerin to 4',6-methoxynaringenin; and the conversion of 4',6-methoxynaringenin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0024] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, 6-methoxysafflowerin, and 4'-methoxybaicalin. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to 6-methoxysafflowerin; the conversion of 6-methoxysafflowerin to 4'-methoxybaicalin; and the conversion of 4'-methoxybaicalin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0025] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, baicalin, and psyllium. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to baicalin; the conversion of baicalin to psyllium; and the conversion of psyllium to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0026] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, baicalein, and 4'-methoxybaicalein. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to baicalein; the conversion of baicalein to 4'-methoxybaicalein; and the conversion of 4'-methoxybaicalein to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0027] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, 4'-methoxysafflowerin, and 4',6-methoxynaringenin. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to 4'-methoxysafflowerin; the conversion of 4'-methoxysafflowerin to 4',6-methoxynaringenin; and the conversion of 4',6-methoxynaringenin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0028] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, 4'-methoxysafflowerin, and 4'-methoxybaicalin. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to 4'-methoxysafflowerin; the conversion of 4'-methoxysafflowerin to 4'-methoxybaicalin; and the conversion of 4'-methoxybaicalin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0029] In some embodiments, the present invention provides a method for producing sardine flavonoids, the method comprising an engineered host cell containing one or more gene modifications. In some embodiments, the engineered host cell may be prepared from yeast, bacteria, or mammalian cells. In some embodiments, the engineered host cell is *Escherichia coli* (E. coli). In some embodiments, one or more enzymes are introduced into the host organism by integration into the genome of the host organism or integration into a plasmid. In some embodiments, one or more gene modifications in the engineered host cell are any of the modifications described above. For example, one or more gene modifications may be overexpression or expression of a variant of an enzyme involved in the conversion of naringenin to sardine flavonoids. These gene modifications include, but are not limited to, overexpression or expression of variants of flavonoid synthase (FNSI), flavonoid hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and / or any combination thereof.

[0030] In some embodiments, one or more gene modifications in the host cell may include any gene modification for increasing the availability of naringenin. For example, one or more gene modifications in the engineered host cell may be selected from the group consisting of acetyl-CoA carboxylase (ACC), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and / or any combination thereof. In some embodiments, the gene modification may include variants expressing one or more of these enzymes. In some embodiments, one or more gene modifications for increasing the production of naringenin are provided in PCT / US2022 / 024591, which is incorporated herein by reference in its entirety.

[0031] In some embodiments, the engineered host cell contains one or more genetic modifications for increasing the production of naringenin and / or squalene. In some embodiments, the engineered host cell contains one or more genetic modifications for increasing the production of naringenin. In these embodiments, the starting material for producing naringenin is glycerol. Therefore, the method of the present invention advantageously provides engineered host cells for converting glycerol to squalene. The engineered host cell may optionally contain genetic modifications optimized for converting glycerol to naringenin via one or more intermediates and subsequently converting naringenin to squalene via one or more intermediates. Exemplary genetic modifications for producing naringenin are provided in WO2022 / 221392, which is incorporated herein by reference in its entirety.

[0032] in addition, Figure 6 An overview of the conversion of glycerol to naringenin via one or more intermediates is provided. In some preferred embodiments, engineered host cells for enhanced naringenin production contain one or more gene modifications selected from the group consisting of ACC, TAL, 4CL, CHS, and CHI overexpression. In some embodiments, the engineered host cells may contain additional gene modifications for downregulating and / or deleting one or more enzymes that convert naringenin to other substrates in the bioproduction pathway that does not involve squalene.

[0033] Therefore, in some aspects, the present invention provides engineered host cells for converting glycerol to saurolophus via a plurality of intermediates. In some embodiments, the engineered host cells may comprise a plurality of gene modifications for the enzymatic conversion of glycerol to saurolophus via a plurality of intermediates. In some embodiments, the plurality of gene modifications are used to convert glycerol to naringenin. In some embodiments, the gene modifications may be selected from the group consisting of overexpression or variant expression of the following: ACC, TAL, 4CL, CHS, and CHI. In some embodiments, the engineered host cells further comprise one or more gene modifications for converting naringenin to saurolophus. In some embodiments, the engineered host cells comprise one or more gene modifications selected from the group consisting of overexpression or variant expression of the following: flavonoid synthase (FNSI), flavonoid hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and / or any combination thereof. In some embodiments, glycerol is derived from a carbon feedstock. In some embodiments, glycerol is crude glycerol. In some embodiments, glycerol is obtained as a byproduct of biodiesel processing.

[0034] In some aspects, the present invention provides a method for producing scallope flavonoids by converting glycerol via one or more intermediates. In some embodiments, the glycerol is enzymatically converted to scallope flavonoids via one or more intermediates. In some embodiments, the one or more intermediates are naringenin. In some embodiments, the gene modification may be selected from the group consisting of overexpression or variant expression of the following: ACC, TAL, 4CL, CHS, and CHI. In some embodiments, the engineered host cell further comprises one or more gene modifications for converting naringenin to scallope flavonoids. In some embodiments, the engineered host cell comprises one or more gene modifications selected from the group consisting of overexpression or variant expression of the following: flavonoid synthase (FNSI), flavonoid hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and / or any combination thereof. In some embodiments, the glycerol is derived from a carbon feedstock. In some embodiments, the glycerol is crude glycerol. In some embodiments, the glycerol is obtained as a byproduct of biodiesel processing.

[0035] In some aspects, the present invention provides a method for the cell-free production of naringenin. The cell-free medium used for the conversion of naringenin to naringenin is a cell lysate. In some embodiments, the cell lysate is a cell lysate from cells of a host organism expressing one or more enzymes. The one or more enzymes may be enzymes involved in the conversion of naringenin to naringenin. In some embodiments, the host organism expressing one or more enzymes is cultured until a predetermined amount of biomass is produced to generate the required amount of one or more enzymes. In some embodiments, the method of the present invention further comprises lysing cells and subsequently removing cell debris to produce a cell lysate for use in the cell-free medium for the cell-free production of naringenin.

[0036] In some embodiments, the cell-free medium comprises: buffer solution, magnesium chloride, one or more substrates, one or more cofactors, one or more enzymes, and / or water.

[0037] In some embodiments, the buffer in the cell-free medium maintains the pH of the reaction mixture within an optimal range. In some embodiments, the pH of the reaction mixture is from about 5.5 to about 10. In some embodiments, the pH of the reaction mixture is from about 6 to about 9.5. In some embodiments, the pH of the cell-free medium is from about 6 to about 9. In some embodiments, the pH of the cell-free medium is from about 6 to about 8. In some embodiments, the concentration of the buffer in the cell-free medium is from about 1 mM to about 1000 mM. In some embodiments, the concentration of the buffer is from about 2.5 mM to about 750 mM. In some embodiments, the concentration of the buffer is from about 5 mM to about 100 mM. In some embodiments, the buffer concentration is from about 20 mM to about 100 mM. In some embodiments, the buffer is a phosphate buffer.

[0038] In some embodiments, the method of the present invention includes converting one or more substrates into squalinoflavin. In some embodiments, the one or more substrates are present at a concentration of about 1 mM to about 500 mM. In some embodiments, the one or more substrates are present at a concentration of about 1 mM to about 200 mM. In some embodiments, the one or more substrates are present at a concentration of about 10 mM to about 100 mM. In some embodiments, the substrate used for producing squalinoflavin is naringenin.

[0039] In some embodiments, the cell-free medium comprises magnesium chloride. The magnesium chloride may be present in the range of about 1 mM to about 50 mM. In some embodiments, the cell-free medium contains magnesium chloride in the range of about 1 mM to about 25 mM. In some embodiments, the cell-free medium contains magnesium chloride in the range of about 1 mM to about 20 mM. In some embodiments, the cell-free medium contains magnesium chloride in the range of about 5 mM to about 25 mM.

[0040] In some embodiments, the cell-free medium comprises a lysate from a host microorganism having one or more enzymes for use in the cell-free reaction medium. In some embodiments, the cell-free medium comprises a lysate having one or more enzymes at a concentration of about 1% (v / v) to about 50% (v / v). In some embodiments, the cell-free medium comprises a lysate having one or more enzymes at a concentration of about 5% (v / v) to about 40% (v / v). In some embodiments, the cell-free medium comprises a lysate having one or more enzymes at a concentration of about 10% (v / v) to about 30% (v / v). In some embodiments, the cell-free medium further comprises one or more enzymes present in the range of about 0.1 μM to about 5 mM. In some embodiments, the one or more enzymes are present in the range of about 0.1 μM to about 1 mM. In some embodiments, the one or more enzymes are present in the range of about 1 μM to about 100 μM.

[0041] In some embodiments, the method of the present invention provides the use of one or more cofactors for promoting a reaction that induces the conversion of naringenin to squalene. In some embodiments, the one or more cofactors are present at a concentration of about 1 mM to about 500 mM. In some embodiments, the one or more cofactors are present at a concentration of about 1 mM to about 200 mM. In some embodiments, the one or more cofactors are present at a concentration of about 1 mM to about 10 mM.

[0042] In some embodiments, the one or more cofactors used in the method of the present invention are selected from the group consisting of: 2-oxoglutarate, ascorbic acid, iron(II), FAD, FMN, NADPH, SAM, ATP, or any combination thereof. In some embodiments, the one or more cofactors are 2-oxoglutarate. In some embodiments, the one or more cofactors are ascorbic acid. In some embodiments, the one or more cofactors are iron(II). In some embodiments, the one or more cofactors are heme. In some embodiments, the one or more cofactors are FAD. In some embodiments, the one or more cofactors are FMN. In some embodiments, the one or more cofactors are NADPH. In some embodiments, the one or more cofactors are S-adenosylmethionine (SAM). In some embodiments, the one or more cofactors are divalent metals, such as magnesium or manganese. In some embodiments, the one or more cofactors are ATP.

[0043] In some embodiments, the cell-free reaction for producing polyphenols is carried out for the necessary duration until the desired amount of polyphenols is produced in the reaction. In some embodiments, the reaction for producing polyphenols in a cell-free manner is carried out for a duration of about 0.1 hours to about 20 hours. In some embodiments, the reaction for producing polyphenols in a cell-free manner is carried out for a duration of about 0.5 hours to about 20 hours. In some embodiments, the reaction for producing polyphenols in a cell-free manner is carried out for a duration of about 1 hour to about 15 hours.

[0044] In some embodiments, the reaction in the cell-free medium is adjusted to achieve the optimal yield of the polyphenols to be produced. In some embodiments, the temperature of the cell-free medium is from about 20°C to about 40°C.

[0045] In some embodiments, the reaction is carried out in a bubble column reactor, wherein one or more enzymes are in solution. In some embodiments, the reaction is carried out in a packed bed reactor, wherein one or more enzymes are immobilized.

[0046] In some aspects, the present invention provides a method for the bioproduction of scallopeflavin, the method comprising: providing one or more enzymes, wherein the one or more enzymes cause one or more substrates optionally to be converted into scallopeflavin via one or more intermediates. In some embodiments, the one or more substrates are selected from the group consisting of: naringenin, apigenin, baicalin, psyllium husk, or any combination thereof. In some embodiments, the substrate is apigenin. In some embodiments, the substrate is baicalin. In some embodiments, the substrate is psyllium husk.

[0047] In some aspects, the method includes an engineered host cell containing one or more gene modifications for expressing one or more enzymes that induce the conversion of one or more substrates optionally via one or more intermediates into squalinoflavin. In some embodiments, the invention provides a method for cell-free production of squalinoflavin, wherein one or more enzymes in a cell-free medium induce the conversion of one or more substrates optionally via one or more intermediates into squalinoflavin.

[0048] In some aspects, the present invention provides a composition for the bioproduction of scallop flavonoids, wherein the composition comprises one or more enzymes, wherein the one or more enzymes cause one or more substrates optionally to be converted into scallop flavonoids via one or more intermediates. In some embodiments, the one or more substrates are selected from the group consisting of naringenin, apigenin, baicalin, psyllium, or any combination thereof. In some embodiments, the substrate is apigenin. In some embodiments, the substrate is baicalin. In some embodiments, the substrate is psyllium. In some embodiments, the substrate is psyllium. In some embodiments, the composition comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expressing one or more enzymes, which cause one or more substrates optionally to be converted into scallop flavonoids via one or more intermediates. In some embodiments, the composition comprises one or more enzymes in a cell-free medium that cause the one or more substrates to be optionally converted to saurolophus flavonoids via one or more intermediates.

[0049] In some aspects, the present invention provides a method for producing naringin, the method comprising: providing an engineered host cell comprising one or more gene modifications, wherein the one or more gene modifications cause glycerol to be enzymatically converted to naringin via one or more intermediates. In some embodiments, the engineered host cell comprises one or more gene modifications for enhancing the production of naringenin from glycerol in the engineered host cell. In some embodiments, the engineered host cell comprises one or more gene modifications for enhancing the conversion of naringenin to naringin via one or more intermediates in the engineered host cell. In some embodiments, the one or more gene modifications are the expression or overexpression of one or more enzymes. In some embodiments, the one or more enzymes are selected from the group consisting of FNSI, P450, OMT, and any combination thereof. In some embodiments, the one or more enzymes are selected from the group consisting of the expression or overexpression of FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI, and any combination thereof. In some embodiments, the engineered host cell is *Escherichia coli*.

[0050] In some aspects, the present invention provides a composition for producing naringin, wherein the composition comprises an engineered host cell containing one or more gene modifications, wherein the gene modifications cause glycerol to be enzymatically converted to naringin via one or more intermediates. In some embodiments, the engineered host cell contains one or more gene modifications for enhancing the production of naringenin from glycerol in the engineered host cell. In some embodiments, the engineered host cell contains one or more gene modifications for enhancing the conversion of naringenin to naringin via one or more intermediates in the engineered host cell. In some embodiments, the gene modifications are the expression or overexpression of one or more enzymes. In some embodiments, the enzymes are selected from the group consisting of FNSI, P450, OMT, and any combination thereof. In some embodiments, the enzymes are selected from the group consisting of the expression or overexpression of FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI, and any combination thereof. In some embodiments, the engineered host cell is *Escherichia coli*.

[0051] In some embodiments, flavonoid synthase (FNSI) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any enzyme selected from the group consisting of:

[0052]

[0053] In some embodiments, the flavonoid hydroxylase cytochrome P450 is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any one of the enzymes selected from the group consisting of:

[0054]

[0055] In some embodiments, the O-methyltransferase is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any enzyme selected from the group consisting of:

[0056]

[0057] The methods for producing sennaroflavin provided in this invention are advantageous because they offer an economical and efficient route for the synthesis of sennaroflavin. Advantageously, the methods of this invention do not involve extensive processes for extracting and / or purifying sennaroflavin from plants or roots. These methods, which require extraction of the product from plants, are complex, resulting in low yields, large amounts of byproducts, and expensive purification and separation procedures.

[0058] The method of the present invention advantageously provides cell-based and cell-free methods for the biological production of scallopeflavin. Specifically, the scallopeflavin produced by the method of the present invention has a higher product titer than that produced by any other known method for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least twice that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least five times that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least ten times that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least one hundred times that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least five hundred times that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least one thousand times that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least five thousand times that of other known methods for producing saural flavonoids.

[0059] In other advantageous aspects of the method of the present invention, the method provides an economical and efficient method for producing scallop flavonoids. Because the method of the present invention also provides cell-free production of scallop flavonoids, this results in reduced purification costs of the reaction products. Furthermore, in some aspects of the invention, one or more enzymes are included in the same reaction mixture, i.e., these enzymes do not need to be isolated after expression in the host organism. One or more enzymes can be simultaneously expressed in the host organism and used for cell-free production without any additional purification and / or isolation steps.

[0060] In certain other advantageous aspects of the invention, the use of one or more enzymes for cell-free production of polyphenols results in high yields of the products to be produced. Multiple enzymes can be further modified to optimize the yield of products produced in cell-free reactions.

[0061] In some aspects, the present invention provides compositions for the production of squalene from one or more substrates. In some embodiments, the compositions provided by the present invention are suitable for the preparation of squalene from one or more substrates. In some embodiments, the compositions of the present invention comprise engineered host organisms, wherein these engineered host organisms contain genetic modifications for the production of squalene from naringenin via one or more intermediates. In some embodiments, the compositions of the present invention comprise cell-free media, which may contain one or more enzymes for the production of squalene from naringenin via one or more intermediates. In some embodiments, the compositions of the present invention comprise a combination of engineered host cells for the preparation of squalene and cell-free media. Attached Figure Description

[0062] Figure 1 A modular pathway for converting naringenin to saurolophytin via one or more intermediates is described.

[0063] Figure 2 A schematic pathway for converting naringenin into saurolophytin using apigenin, baicalin, and crocin as intermediates is provided.

[0064] Figure 3A and 3B HPLC chromatograms of cell-free production of psyllium and saurolophytin are provided.

[0065] Figure 4 HPLC chromatograms of saurolophytin and apigenin produced by glycerol cells are provided.

[0066] Figure 5 An HPLC chromatogram of willow flavin produced by arborescent cells is provided.

[0067] Figure 6 An overview of the enzymatic conversion of glycerol to naringenin is provided. Detailed Implementation

[0068] This application provides compositions and methods for producing scallop flavin in cell-based or cell-free media, wherein one or more enzymes induce the conversion of one or more substrates into scallop flavin. The one or more enzymes may be engineered. The engineered enzymes may be non-naturally occurring.

[0069] When used to refer to enzymes, the term "non-natural" is intended to mean that a nucleic acid or polypeptide includes at least one genetic modification in a polypeptide or nucleic acid sequence that is not normally present in naturally occurring organisms. Naturally occurring nucleic acids and polypeptides may be referred to as "wild-type" or "original." Host cells, organisms, or microorganisms that include at least one genetically modified organism produced through human intervention may also be referred to as "non-natural," "engineered," "genetically engineered," or "recombinant."

[0070] As used herein, the singular forms “a / an” and “the” are intended to also include the plural forms, unless the context clearly indicates otherwise. Furthermore, where the terms “including / include,” “having / has / with,” or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0071] As used herein, “reaction solution” can refer to all components necessary for enzyme-based chemical transformations. This is generally not limited to buffers, salts, cofactors, and substrates (starting materials).

[0072] As used herein, "reaction mixture" can refer to all components of a "reaction solution" plus enzymes and / or products from the reaction. In some embodiments, "reaction mixture" can refer only to a reaction solution that does not contain any enzymes or reaction products. In some embodiments, "reaction solution" and "reaction mixture" can be used interchangeably.

[0073] As used in this article, "buffer" can refer to a chemical added to a water-based solution that resists changes in pH through the action of an acid-base conjugate.

[0074] As used herein, "cofactor" can refer to a non-protein compound that can bind to proteins and contribute to biochemical reactions. Non-limiting examples of cofactors include, but are not limited to, NADPH and NADH.

[0075] In the cell-free system described herein, key cellular components, namely cofactors and enzymes, are used in the chemical reaction without any cellular components that can directly or indirectly inhibit the desired biochemical reaction. The same enzymes found in plants and other organisms can be produced in vivo (typically through protein overexpression in a host such as bacteria), isolated by chromatography and / or any other method, and then added to a bioreactor containing the substrate (starting material). Enzymes can also be used directly from plants without any isolation. The enzymes transform the substrate in the same manner as in the original organism without complicating the organism. Additionally, the biochemical reaction can be enhanced by adding a cosolvent, detergent, or both, which is not permissible or even ineffective in whole-cell-based manufacturing methods. In this way, natural products can be produced without the need for plants, cells, or chemical synthesis.

[0076] Squid flavonoids:

[0077] Scutellarin is a flavonoid compound belonging to the flavonoid subclass of flavonoids. Flavonoids are a diverse group of phytonutrients found in various fruits, vegetables, and herbs. They are known for their potential health benefits and have been extensively studied for their antioxidant, anti-inflammatory, anticancer, and neuroprotective properties.

[0078] The chemical structure of saurolophytin is provided below:

[0079]

[0080] Research on the potential applications of saurolophus flavonoids is still in its early stages, and early data comes from in vitro and animal studies. For example, saurolophus flavonoids are known to have antioxidant, anticancer, anti-inflammatory, and neuroprotective properties.

[0081] Despite its promising biological activity, there is currently no known cost-effective method for producing sennaroflavin. In conventional methods, sennaroflavin is specifically derived from the plant *Pectis papposa*, commonly known as the silver senna plant. This plant is native to the southwestern United States and northern Mexico and has traditionally been used in folk medicine for its medicinal properties. These methods are not cost-effective and require various steps, including harvesting the plant, preparing the harvested material (such as leaves, flowers, or roots), extraction (substituting the extracted material with various processes and / or chemicals to produce sennaroflavin), and purification (separating sennaroflavin from other chemicals). This is an expensive and time-consuming process. Methods involving the chemical synthesis of sennaroflavin are commercially unfeasible. Therefore, a cost-effective method for producing sennaroflavin is needed.

[0082] Production of sardine flavonoids:

[0083] Therefore, there is a need for a reliable and cost-effective new method for producing sennae. The method of the present invention provides a method and composition for the biological production of sennae. The method of the present invention can induce the production of sennae in a host organism containing certain genetic modifications. The method of the present invention also provides the cell-free production of sennae from one or more substrates. The methods of the present invention are advantageous because they provide an economically feasible method for producing sennae. Advantageously, the methods of the present invention are also reliable and independent of the available plants for the preparation of sennae.

[0084] Advantageously, the method of the present invention provides a cell-based or cell-free platform for the production of senna flavin. The platform provided by the present invention is advantageous because it offers multiple pathways for the preparation of senna flavin from naringenin. The availability of different pathways for the production of senna flavin is beneficial because it provides pathways for the preparation and isolation of one or more intermediates in the biosynthetic pathway. Therefore, the platform provided in the present invention unlocks the option of preparing one or more intermediates that might otherwise be difficult to use in manufacturing. Advantageously, the pathway for the production of senna flavin can also be selected based on the availability of various enzymes for that particular reaction pathway. In other words, if an enzyme is unavailable (or not readily available), a different pathway that does not require that particular enzyme can be selected to produce senna flavin. Therefore, the modularity of the biosynthetic pathway is particularly advantageous.

[0085] In some aspects, the present invention provides a method for the biological production of scallopeflavin, the method comprising providing one or more enzymes, wherein the one or more enzymes cause naringenin to be converted into scallopeflavin via one or more intermediates. The method for producing scallopeflavin includes cell-based methods and cell-free methods.

[0086] In some aspects of the invention, a method for producing scallopeflavin includes an engineered host cell containing one or more gene modifications for expressing one or more enzymes that induce the conversion of naringenin to scallopeflavin via one or more intermediates. In some embodiments, these gene modifications in the host cell may further include one or more gene modifications for enhancing naringenin production. In some embodiments, the engineered host cell contains gene modifications for enhancing the production of naringenin or any precursors that induce naringenin production in the engineered host cell. Additional gene modifications that increase naringenin production can be beneficial for scallopeflavin production because naringenin can be used as a substrate for scallopeflavin production. An increase in the amount of naringenin will result in a higher scallopeflavin yield.

[0087] In certain aspects of the invention, a method for producing scalloped flavonoids is a cell-free production of scalloped flavonoids, wherein one or more enzymes in a cell-free medium cause naringenin to be converted to scalloped flavonoids via one or more intermediates. Components of the components in the cell-free reaction can be produced in host cells. For example, substrates, such as naringenin, can be produced in cells, and the subsequent conversion to scalloped flavonoids can be carried out in a cell-free medium. In some embodiments, one or more intermediates generated in the conversion of naringenin to scalloped flavonoids can be produced in engineered host cells, and these one or more intermediates can subsequently be converted to scalloped flavonoids in a cell-free medium via one or more enzymes.

[0088] In some aspects, the method comprises a combination of: (i) an engineered host cell containing one or more gene modifications for expressing one or more enzymes that induce the conversion of naringenin to sennaroxen, or one or more intermediates; and (ii) cell-free production comprising one or more enzymes that induce the conversion of naringenin or a substrate to sennaroxen, or one or more intermediates, to synthesize sennaroxen. In some embodiments, the engineered host cell contains gene modifications for enhancing the production of naringenin or any precursors that induce naringenin production in the engineered host cell.

[0089] In some embodiments of the invention, the one or more intermediates are selected from the group consisting of: apigenin, baicalin, physalisin, isopyroside, safflowerin, 6-methoxysafflowerin, baicalin, 4-methoxysafflowerin, acaciain, 4',6-methoxynaringenin, 4'-methoxybaicalin, and any combination thereof. Figure 1 An exemplary schematic pathway for the conversion of naringenin to saurolophytin is provided. From Figure 1 As is evident, the method of the present invention provides a modular pathway for the conversion of naringenin to scalloped flavonoids. The choice of pathway for the production of scalloped flavonoids will determine the identity of one or more intermediates selected in the process. Each individual segment of the pathway can be controlled by an enzyme selected for the conversion. In some embodiments, the method of the present invention provides the use of one or more enzymes for mediating the conversion of starting material into one or more intermediates or scalloped flavonoids. In some embodiments, the one or more enzymes are selected from the group consisting of: flavonoid synthase (FNSI), flavonoid hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and any combination thereof.

[0090] For example, in some embodiments, these intermediates are apigenin, baicalin, and crocin. In some embodiments, the method provides a pathway in which naringenin is converted to apigenin; apigenin is converted to baicalin; baicalin is converted to crocin; and crocin is converted to sauroplasmin. In some embodiments, one or more of these steps can be performed by one or more enzymes. For example, as... Figure 2 As shown, the provided pathway includes: flavonoid synthase (FNSI)-mediated conversion of naringenin to apigenin; flavonoid hydroxylase cytochrome P450 (P450)-mediated conversion of apigenin to baicalin; O-methyltransferase (OMT)-mediated conversion of baicalin to crocin; and O-methyltransferase (OMT)-mediated conversion of crocin to saurolophytin. Any of these foregoing steps can be performed in engineered host cells or cell-free media.

[0091] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are apigenin, baicalin, and 4'-methoxybaicalin. This invention provides the conversion of naringenin to apigenin; the conversion of apigenin to baicalin; the conversion of baicalin to 4'-methoxybaicalin; and the conversion of 4'-methoxybaicalin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0092] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are apigenin, baicalin, and psyllium. This invention provides the conversion of naringenin to apigenin; the conversion of apigenin to baicalin; the conversion of baicalin to psyllium; and the conversion of psyllium to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0093] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are apigenin, farnesin, and 4'-methoxybaicalin. This invention provides the conversion of naringenin to apigenin; the conversion of apigenin to farnesin; the conversion of farnesin to 4'-methoxybaicalin; and the conversion of 4'-methoxybaicalin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0094] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are isoporphyrin, 4'-methoxyscarmine, and 4',6-methoxynaringenin. This invention provides the conversion of naringenin to isoporphyrin; the conversion of isoporphyrin to 4'-methoxyscarmine; the conversion of 4'-methoxyscarmine to 4',6-methoxynaringenin; and the conversion of 4',6-methoxynaringenin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0095] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are isopyroxin, 4'-methoxyscarmine, and 4'-methoxybaicalin. This invention provides the conversion of naringenin to isopyroxin; the conversion of isopyroxin to 4'-methoxyscarmine; the conversion of 4'-methoxyscarmine to 4'-methoxybaicalin; and the conversion of 4'-methoxybaicalin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0096] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are isopyroxin, farnesin, and 4'-methoxybaicalein. This invention provides the conversion of naringenin to isopyroxin; the conversion of isopyroxin to farnesin; the conversion of farnesin to 4'-methoxybaicalein; and the conversion of 4'-methoxybaicalein to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0097] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, 6-methoxysafflowerin, and physalisin. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to 6-methoxysafflowerin; the conversion of 6-methoxysafflowerin to physalisin; and the conversion of physalisin to squalene. In some embodiments, one or more of these steps can be performed by one or more enzymes.

[0098] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, 6-methoxysafflowerin, and 4',6-methoxynaringenin. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to 6-methoxysafflowerin; the conversion of 6-methoxysafflowerin to 4',6-methoxynaringenin; and the conversion of 4',6-methoxynaringenin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0099] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, 6-methoxysafflowerin, and 4'-methoxybaicalin. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to 6-methoxysafflowerin; the conversion of 6-methoxysafflowerin to 4'-methoxybaicalin; and the conversion of 4'-methoxybaicalin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0100] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, baicalin, and psyllium. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to baicalin; the conversion of baicalin to psyllium; and the conversion of psyllium to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0101] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, baicalein, and 4'-methoxybaicalein. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to baicalein; the conversion of baicalein to 4'-methoxybaicalein; and the conversion of 4'-methoxybaicalein to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0102] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, 4'-methoxysafflowerin, and 4',6-methoxynaringenin. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to 4'-methoxysafflowerin; the conversion of 4'-methoxysafflowerin to 4',6-methoxynaringenin; and the conversion of 4',6-methoxynaringenin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0103] In some embodiments, one or more intermediates involved in the conversion of naringenin to squalene are safflowerin, 4'-methoxysafflowerin, and 4'-methoxybaicalin. This invention provides the conversion of naringenin to safflowerin; the conversion of safflowerin to 4'-methoxysafflowerin; the conversion of 4'-methoxysafflowerin to 4'-methoxybaicalin; and the conversion of 4'-methoxybaicalin to squalene. In some embodiments, one or more of these steps can be performed using one or more enzymes.

[0104] In some embodiments, the present invention provides a method for producing sardine flavonoids, the method comprising an engineered host cell containing one or more gene modifications. In some embodiments, the engineered host cell may be prepared from yeast, bacteria, or mammalian cells. In some embodiments, the engineered host cell is *Escherichia coli*. In some embodiments, one or more enzymes are introduced into the host organism by integration into the genome of the host organism or integration into a plasmid. In some embodiments, one or more gene modifications in the engineered host cell are any of the modifications described above. For example, one or more gene modifications may be overexpression or expression of a variant of an enzyme involved in the conversion of naringenin to sardine flavonoids. These gene modifications include, but are not limited to, overexpression or expression of variants of flavonoid synthase (FNSI), flavonoid hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and / or any combination thereof.

[0105] In some embodiments, one or more gene modifications in the host cell may include any gene modification for increasing the availability of naringenin. For example, one or more gene modifications in the engineered host cell may be selected from the group consisting of acetyl-CoA carboxylase (ACC), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and / or any combination thereof. In some embodiments, the gene modification may include variants expressing one or more of these enzymes. In some embodiments, one or more gene modifications for increasing the production of naringenin are provided in PCT / US2022 / 024591, which is incorporated herein by reference in its entirety.

[0106] In some aspects, the present invention provides a method for the cell-free production of naringenin. The cell-free medium used for the conversion of naringenin to naringenin is a cell lysate. In some embodiments, the cell lysate is a cell lysate from cells of a host organism expressing one or more enzymes. The one or more enzymes may be enzymes involved in the conversion of naringenin to naringenin. In some embodiments, the host organism expressing one or more enzymes is cultured until a predetermined amount of biomass is produced to generate the required amount of one or more enzymes. In some embodiments, the method of the present invention further comprises lysing cells and subsequently removing cell debris to produce a cell lysate for use in the cell-free medium for the cell-free production of naringenin.

[0107] In some embodiments, the cell-free medium comprises: buffer solution, magnesium chloride, one or more substrates, one or more cofactors, one or more enzymes, and / or water.

[0108] In some embodiments, the buffer in the cell-free medium maintains the pH of the reaction mixture within an optimal range. In some embodiments, the pH of the reaction mixture is from about 5.5 to about 10. In some embodiments, the pH of the reaction mixture is from about 6 to about 9.5. In some embodiments, the pH of the cell-free medium is from about 6 to about 9. In some embodiments, the pH of the cell-free medium is from about 6 to about 8. In some embodiments, the concentration of the buffer in the cell-free medium is from about 1 mM to about 1000 mM. In some embodiments, the concentration of the buffer is from about 2.5 mM to about 750 mM. In some embodiments, the concentration of the buffer is from about 5 mM to about 100 mM. In some embodiments, the buffer concentration is from about 20 mM to about 100 mM. In some embodiments, the buffer is a phosphate buffer.

[0109] In some embodiments, the method of the present invention includes converting one or more substrates into squalinoflavin. In some embodiments, the one or more substrates are present at a concentration of about 1 mM to about 500 mM. In some embodiments, the one or more substrates are present at a concentration of about 1 mM to about 200 mM. In some embodiments, the one or more substrates are present at a concentration of about 10 mM to about 100 mM. In some embodiments, the substrate used for producing squalinoflavin is naringenin.

[0110] In some embodiments, the cell-free medium comprises magnesium chloride. The magnesium chloride may be present in the range of about 1 mM to about 50 mM. In some embodiments, the cell-free medium contains magnesium chloride in the range of about 1 mM to about 25 mM. In some embodiments, the cell-free medium contains magnesium chloride in the range of about 1 mM to about 20 mM. In some embodiments, the cell-free medium contains magnesium chloride in the range of about 5 mM to about 25 mM.

[0111] In some embodiments, the cell-free medium comprises a lysate from a host microorganism having one or more enzymes for use in the cell-free reaction medium. In some embodiments, the cell-free medium comprises a lysate having one or more enzymes at a concentration of about 1% (v / v) to about 50% (v / v). In some embodiments, the cell-free medium comprises a lysate having one or more enzymes at a concentration of about 5% (v / v) to about 40% (v / v). In some embodiments, the cell-free medium comprises a lysate having one or more enzymes at a concentration of about 10% (v / v) to about 30% (v / v). In some embodiments, the cell-free medium further comprises one or more enzymes present in the range of about 0.1 μM to about 5 mM. In some embodiments, the one or more enzymes are present in the range of about 0.1 μM to about 1 mM. In some embodiments, the one or more enzymes are present in the range of about 1 μM to about 100 μM.

[0112] In some embodiments, the method of the present invention provides the use of one or more cofactors for promoting a reaction that induces the conversion of naringenin to squalene. In some embodiments, the one or more cofactors are present at a concentration of about 1 mM to about 500 mM. In some embodiments, the one or more cofactors are present at a concentration of about 1 mM to about 200 mM. In some embodiments, the one or more cofactors are present at a concentration of about 1 mM to about 10 mM.

[0113] In some embodiments, the cell-free reaction for producing polyphenols is carried out for the necessary duration until the desired amount of polyphenols is produced in the reaction. In some embodiments, the reaction for producing polyphenols in a cell-free manner is carried out for a duration of about 0.1 hours to about 20 hours. In some embodiments, the reaction for producing polyphenols in a cell-free manner is carried out for a duration of about 0.5 hours to about 20 hours. In some embodiments, the reaction for producing polyphenols in a cell-free manner is carried out for a duration of about 1 hour to about 15 hours.

[0114] In some embodiments, the reaction in the cell-free medium is adjusted to achieve the optimal yield of the polyphenols to be produced. In some embodiments, the temperature of the cell-free medium is from about 20°C to about 40°C.

[0115] As described above, the present invention provides that all or a number of steps for converting a substrate to naringenin can be performed in an engineered host organism. The host organism can be bacteria, yeast, or mammalian cells. In some embodiments, the engineered host organism is incubated in a culture medium. These methods may further include the recovery of saurolophytin or one of the intermediates that induce saurolophytin formation from the culture medium, whole culture, or cells.

[0116] The culture comprises cells engineered for the production of saurolophus flavonoids in a culture medium. In various embodiments, these engineered cells may be prokaryotic or eukaryotic cells. The culture medium includes at least one carbon source, which is also an energy source. Exemplary carbon sources include glucose, glycerol, sucrose, fructose, and xylose. This carbon source may be purified or crude, including biomass containing glycerol, such as crude glycerol produced as a byproduct of biodiesel production from corn waste. Additionally, the culture medium may include one or more other carbon sources or compounds for increasing precursor production or cofactor supply, such as, but not limited to, tyrosine, phenylalanine, coumaric acid, acetate, malonate, succinate, glycine, bicarbonate, biotin, naringenin, 5-aminolevulinic acid, thiamine, pantothenate, α-ketoglutarate, and ascorbate. In some embodiments, tyrosine and coumaric acid are provided in the culture medium. In some embodiments, tyrosine, α-ketoglutarate, 5-aminolevulinic acid, and ascorbate are provided in the culture medium.

[0117] Culture conditions can include any combination of aerobic, microaerophilic, or alternating aerobic / microaerophilic growth conditions. Furthermore, culture conditions can include shake flask, fermentation, and other large-scale culture procedures. Exemplary growth conditions for achieving flavonoid products include aerobic or microaerophilic fermentation conditions. Culture conditions can be scaled up and used for continuous growth to prepare flavonoid products. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation. In an exemplary batch fermentation scheme, cells are grown in a bioreactor with well-controlled growth temperature, oxygen, pH, carbon source, and other compounds. The required temperature can be, for example, 20-37°C, depending on the growth characteristics of the producing cells and the desired conditions for the fermentation products. The pH of the bioreactor can be controlled in the range of 5-8, or in some cases uncontrolled. Batch fermentation periods can last from several hours to several days, for example, 8 hours to 96 hours. Once the culture period ends, the fermenter contents can be passed through a cell separation unit to remove cells and cell debris. Cells can be enzymatically or chemically dissolved or destroyed as needed before or after separation from the fermentation broth to release additional products. In some embodiments, engineered host organisms and their genetic modifications are provided in PCT / US2022 / 024591, which is incorporated herein by reference in its entirety.

[0118] In some embodiments, the reaction is carried out in a bubble column reactor, wherein one or more enzymes are in solution. In some embodiments, the reaction is carried out in a packed bed reactor, wherein one or more enzymes are immobilized.

[0119] In some aspects, the methods provided in this invention can be carried out in any reactor suitable for cell-free production of scallopeflavin. In some embodiments, the reaction for cell-free production of scallopeflavin is carried out in a bubble column reactor / bioreactor. In some embodiments, in the bubble column reactor / bioreactor, one or more enzymes involved in cell-free production of scallopeflavin are in solution. In some embodiments, the reaction for cell-free production of scallopeflavin is carried out in a bubble column reactor / bioreactor containing lysates from a host organism. In some embodiments, it is advantageous to use a bubble column reactor / bioreactor for cell-free production of scallopeflavin when the reaction mixture involves lysates from a host cell organism (or lysates from which cell debris has been removed), wherein one or more enzymes responsible for cell-free production of scallopeflavin are used. In some embodiments, the reaction for cell-free production of scallopeflavin is carried out in a packed bed reactor / bioreactor. In some embodiments, one or more enzymes are immobilized in a packed bed reactor / bioreactor. The packed bed reactor / bioreactor is preferably used to enable purified enzymes to function in cell-free production of scallopeflavin. In some embodiments, one or more enzymes may be immobilized in a single reactor / bioreactor. In some other embodiments, one or more enzymes may be immobilized in different reactors / bioreactors connected sequentially. In some embodiments, a bioreactor system is provided in PCT / US2021 / 064049, which is incorporated herein by reference in its entirety.

[0120] In some aspects, the present invention provides a method for the bioproduction of scallopeflavin, the method comprising: providing one or more enzymes, wherein the one or more enzymes cause one or more substrates optionally to be converted into scallopeflavin via one or more intermediates. In some embodiments, the one or more substrates are selected from the group consisting of: naringenin, apigenin, baicalin, psyllium husk, or any combination thereof. In some embodiments, the substrate is apigenin. In some embodiments, the substrate is baicalin. In some embodiments, the substrate is psyllium husk.

[0121] In some aspects, the method includes an engineered host cell containing one or more gene modifications for expressing one or more enzymes that induce the conversion of one or more substrates optionally via one or more intermediates into squalinoflavin. In some embodiments, the invention provides a method for cell-free production of squalinoflavin, wherein one or more enzymes in a cell-free medium induce the conversion of one or more substrates optionally via one or more intermediates into squalinoflavin.

[0122] In some aspects, the present invention provides a composition for the bioproduction of scallop flavonoids, wherein the composition comprises one or more enzymes, wherein the one or more enzymes cause one or more substrates optionally to be converted into scallop flavonoids via one or more intermediates. In some embodiments, the one or more substrates are selected from the group consisting of naringenin, apigenin, baicalin, psyllium, or any combination thereof. In some embodiments, the substrate is apigenin. In some embodiments, the substrate is baicalin. In some embodiments, the substrate is psyllium. In some embodiments, the substrate is psyllium. In some embodiments, the composition comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expressing one or more enzymes, which cause one or more substrates optionally to be converted into scallop flavonoids via one or more intermediates. In some embodiments, the composition comprises one or more enzymes in a cell-free medium that cause the one or more substrates to be optionally converted to saurolophus flavonoids via one or more intermediates.

[0123] In some aspects, the present invention provides a method for producing naringin, the method comprising: providing an engineered host cell comprising one or more gene modifications, wherein the one or more gene modifications cause glycerol to be enzymatically converted to naringin via one or more intermediates. In some embodiments, the engineered host cell comprises one or more gene modifications for enhancing the production of naringenin from glycerol in the engineered host cell. In some embodiments, the engineered host cell comprises one or more gene modifications for enhancing the conversion of naringenin to naringin via one or more intermediates in the engineered host cell. In some embodiments, the one or more gene modifications are the expression or overexpression of one or more enzymes. In some embodiments, the one or more enzymes are selected from the group consisting of FNSI, P450, OMT, and any combination thereof. In some embodiments, the one or more enzymes are selected from the group consisting of the expression or overexpression of FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI, and any combination thereof. In some embodiments, the engineered host cell is *Escherichia coli*.

[0124] In some aspects, the present invention provides a composition for producing naringin, wherein the composition comprises an engineered host cell containing one or more gene modifications, wherein the gene modifications cause glycerol to be enzymatically converted to naringin via one or more intermediates. In some embodiments, the engineered host cell contains one or more gene modifications for enhancing the production of naringenin from glycerol in the engineered host cell. In some embodiments, the engineered host cell contains one or more gene modifications for enhancing the conversion of naringenin to naringin via one or more intermediates in the engineered host cell. In some embodiments, the gene modifications are the expression or overexpression of one or more enzymes. In some embodiments, the enzymes are selected from the group consisting of FNSI, P450, OMT, and any combination thereof. In some embodiments, the enzymes are selected from the group consisting of the expression or overexpression of FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI, and any combination thereof. In some embodiments, the engineered host cell is *Escherichia coli*.

[0125] The methods provided in this invention are superior to other conventional methods for producing polyphenols. In some embodiments, the methods of this invention provide cell-free production of polyphenols. Because the methods of this invention are carried out in a cell-free medium, these methods provide significant economic benefits by reducing the cost of producing polyphenols in other conventional methods. In some embodiments, the methods of this invention are cost-effective because the reactions used to produce polyphenols are carried out from lysates of the host organism from one or more enzymes involved in the expression reaction. In some embodiments, the methods of this invention do not involve the purification of one or more enzymes. Because the purification of individual enzymes is not required in the methods of this invention, further economic efficiency is provided by reducing the cost required to purify individual enzymes.

[0126] In some embodiments, the purity of the isolated sauryflavin is about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%.

[0127] In other embodiments, the purity of the isolated saurolophus flavonoids is about 10% to 95%, or about 10% to 90%, or about 10% to 80%, or about 10% to 70%, or about 10% to 60%, or about 10% to 50%, or about 10% to 40%, or about 20% to 95%, or about 20% to 90%, or about 20% to 80%, or about 20% to 70%, or about 20% to 60%, or about 20% to 50%, or about 20% to 40%, or about 50% to 95%, or about 50% to 90%, or about 50% to 80%, or about 50% to 70%, or about 50% to 60%.

[0128] In some embodiments, the flavonoid synthase is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any enzyme selected from the group consisting of:

[0129]

[0130] In some embodiments, the flavonoid hydroxylase cytochrome P450 is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any one of the enzymes selected from the group consisting of:

[0131]

[0132] In some embodiments, the O-methyltransferase is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any enzyme selected from the group consisting of:

[0133]

[0134] In some embodiments, acetyl-CoA carboxylase (ACC) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any enzyme selected from the group consisting of:

[0135]

[0136] In some preferred embodiments, the acetyl-CoA carboxylase (ACC) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to the enzyme provided in SEQ ID NO: 70.

[0137] In some embodiments, the tyrosine amino lyase (TAL) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any enzyme selected from the group consisting of:

[0138]

[0139] In some preferred embodiments, the tyrosine amino lyase (TAL) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to the enzyme provided in SEQ ID NO: 71.

[0140] In some embodiments, 4-coumarate-CoA ligase (4CL) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any enzyme selected from the group consisting of:

[0141]

[0142] In some preferred embodiments, the 4-coumaric acid-CoA ligase (4CL) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to the enzymes provided in SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and / or SEQ ID NO: 76.

[0143] In some embodiments, chalcon synthase (CHS) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any enzyme selected from the group consisting of:

[0144]

[0145]

[0146] In some preferred embodiments, the chalcon synthase (CHS) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to the enzymes provided in SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 and / or SEQ ID NO: 81.

[0147] In some embodiments, the chalcone isomerase (CHI) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to any enzyme selected from the group consisting of:

[0148]

[0149] In some preferred embodiments, the chalcone isomerase (CHI) is an enzyme having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or completely identical to the enzymes provided in SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 and / or SEQ ID NO: 86.

[0150] The methods for producing sennaroflavin provided in this invention are advantageous because they offer an economical and efficient route for the synthesis of sennaroflavin. Advantageously, the methods of this invention do not involve extensive processes for extracting and / or purifying sennaroflavin from plants or roots. These methods, which require extraction of the product from plants, are complex, resulting in low yields, large amounts of byproducts, and expensive purification and separation procedures.

[0151] The method of the present invention advantageously provides cell-based and cell-free methods for the biological production of scallopeflavin. Specifically, the scallopeflavin produced by the method of the present invention has a higher product titer than that produced by any other known method for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least twice that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least five times that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least ten times that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least one hundred times that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least five hundred times that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least one thousand times that of other known methods for producing scallopeflavin. In some embodiments, the product titer produced by the method of the present invention is at least five thousand times that of other known methods for producing saural flavonoids.

[0152] In other advantageous aspects of the method of the present invention, the method provides an economical and efficient method for producing scallop flavonoids. Because the method of the present invention also provides cell-free production of scallop flavonoids, this results in reduced purification costs of the reaction products. Furthermore, in some aspects of the invention, one or more enzymes are included in the same reaction mixture, i.e., these enzymes do not need to be isolated after expression in the host organism. One or more enzymes can be simultaneously expressed in the host organism and used for cell-free production without any additional purification and / or isolation steps.

[0153] In certain other advantageous aspects of the invention, the use of one or more enzymes for cell-free production of polyphenols results in high yields of the products to be produced. Multiple enzymes can be further modified to optimize the yield of products produced in cell-free reactions.

[0154] In some aspects, the present invention provides compositions for the production of squalene from one or more substrates. In some embodiments, the compositions provided by the present invention are suitable for the preparation of squalene from one or more substrates. In some embodiments, the compositions of the present invention comprise engineered host organisms, wherein these engineered host organisms contain genetic modifications for the production of squalene from naringenin via one or more intermediates. In some embodiments, the compositions of the present invention comprise cell-free media, which may contain one or more enzymes for the production of squalene from naringenin via one or more intermediates. In some embodiments, the compositions of the present invention comprise a combination of engineered host cells for the preparation of squalene and cell-free media.

[0155] VII. Example

[0156] Example 1: Cell-free bioproduction of saurolophytin:

[0157] The following provides general reaction conditions for the cell-free production of saffron flavonoids. The substrate can be naringenin, or... Figure 1 Any of the other compounds shown. The enzyme can be any combination of one or more flavonoid synthases (FNSI), flavonoid hydroxylase cytochrome P450 (P450), and O-methyltransferase (OMT). Typically, the enzyme is expressed in BL21-DE3 cells using the pET28 vector. The starting culture is incubated overnight at 37°C and then diluted 1:100 in TB. Incubate at 37°C until OD. 600After adjusting the pH from 0.8 to 1.2, the culture was induced with IPTG (0.1 mM to 1 mM) and incubated overnight at 20°C to 30°C. The culture was then centrifuged and the supernatant was discarded. The cells were resuspended in lysis buffer (50 mM sodium phosphate, pH 7.5, 300 mM sodium chloride, and 20 mM imidazole). The resuspended cells were sonicated and centrifuged to produce clear lysates.

[0158]

[0159] Cell-free conversion of baicalin to high-purine: The reaction was carried out by combining 200 mM Tris-HCl pH 7.5, 2 mM MTT (dithiothreitol), 4 mM magnesium chloride, 0.5 mM SAM (S-adenosylmethionine), 50% v / v lysate of cells expressing OMT enzymes (SEQ ID 31 to 69), and 0.2 mM baicalin. The reaction solution was incubated at 30 °C for 1 h, and then quenched with an equal volume of methanol and filtered. HPLC analysis of the samples confirmed the production of up to 79 μM of high-purine.

[0160] Cell-free conversion of arbutin to safflower flavonoids: by combining 25 mM HEPES pH 7.5, 5 mM magnesium chloride, 0.2 mM SAM (S-adenosylmethionine), and 0.5 mM NAD. + The reaction was performed using 25% v / v lysates of cells expressing OMT enzymes (SEQ ID 31 to 69), 20% v / v lysates of cells expressing SAHH enzymes (S-adenosylhomocysteine ​​hydrolase, EC 3.13.2.1), and 0.3 mM of crocin. The reaction solution was incubated overnight at 25°C, then quenched with an equal volume of methanol and filtered. HPLC analysis of the samples confirmed the production of up to 130 µM of crocin.

[0161] Figure 3 shows the HPLC chromatograms of the formation of psyllium and sauropsidin. Figure 3A An HPLC chromatogram confirming the production of high-purine from baicalein is provided. Figure 3B An HPLC chromatogram confirming the production of crocin from crocin is provided.

[0162] Example 2: Cellular Bioproduction of Naringin: Cellular Production of Naringin from Glycerol: *E. coli* cells derived from MG1655 were engineered to produce naringin. Cells could be further genetically modified to include overexpression of ACC, TAL, 4CL, CHS, and CHI. For this strain, flavonoid synthase (FNSI), flavonoid hydroxylase cytochrome P450 (P450), and two O-methyltransferases (OMT) were added to the chromosome to produce naringin when glycerol was supplied in the medium. Cells with OD 2.0 were cultured for 22 hours in 48-well plates at 30°C with shaking at 600 RPM in minimal medium supplied with 2% glycerol, trace elements, 65 mg / L 5-aminolevulinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were extracted with 50% (v / v) methanol and centrifuged for 15 minutes. The flavonoids and pathway intermediates in the supernatant were analyzed by HPLC. Figure 4 The following are HPLC chromatograms at 288 nm: (a) standards; (b) starting strains producing NAR titers of up to 440 μM; (c) NAR-producing strains with FNSI to produce apigenin titers of up to 210 μM and NAR residues of 152 μM; and (d) NAR-producing strains with FNSI, P450 and two OMTs to produce the final product saurolophytin titers of up to 30 μM and farnesin produced as a byproduct of 38 μM.

[0163] Production of apigenin from glycerol-containing cells: *E. coli* cells derived from MG1655 were engineered to produce naringenin. The cells optionally underwent further genetic modifications including overexpression of ACC, TAL, 4CL, CHS, and CHI. For these cells, FNSI was added to the chromosome to produce apigenin when glycerol was supplied in the culture medium. Cells with an OD of 2.0 were cultured for 22 h in 48-well plates at 30°C with shaking at 600 RPM in minimal medium supplied with 2% glycerol, trace elements, 65 mg / L 5-aminolevulinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. The cell culture was extracted with 50% (v / v) methanol and centrifuged for 15 min. Apigenin and pathway intermediates in the supernatant were analyzed by HPLC. Figure 4The following are HPLC chromatograms at 288 nm: (a) standards; (b) starting strains producing NAR titers of up to 440 μM; (c) NAR-producing strains with FNSI to produce apigenin titers of up to 210 μM and 152 μM NAR residues; and (d) NAR-producing strains with FNSI, P450 and two OMTs to produce the final product saurolophytin titers of up to 30 μM and 38 μM farnesin produced as a byproduct.

[0164] Production of crocin from crocin-producing cells: *E. coli* BL21 cells were engineered to express OMT. For this purpose, crocin was fed into the culture. Cells with an OD of 2.0 were cultured for 22 h in minimal medium supplied with 2% glycerol, trace elements, and 500 µM crocin at 600 RPM in 48-well plates at 30°C. The cell culture was extracted with 50% (v / v) methanol and centrifuged for 15 min. Crocin and pathway intermediates in the supernatant were analyzed by HPLC. Figure 5 The following are HPLC chromatograms at 330 nm: (a) PNAR standard; (b) cyproterin standard; (c) BL21 cells; and (d) BL21 cells with OMT to produce PNAR titers of up to 250 μM.

[0165] The table below provides exemplary sequences of one or more enzymes that can be used according to the method of the present invention.

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190] By incorporating via reference

[0191] Throughout this disclosure, references and citations have been made to other documents, such as patents, patent applications, patent publications, magazines, books, papers, web content, and publicly available databases. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0192] equivalent

[0193] Based on the entire contents of this document, including references to scientific and patent literature cited herein, various modifications to the invention and many other embodiments thereof will become apparent to those skilled in the art, in addition to those shown and described herein. The subject matter of this document contains important information, illustrations, and guidance that can be adapted to practice the invention in its various embodiments and equivalents.

Claims

1. A method for the biological production of scallopeflavin, the method comprising providing one or more enzymes, wherein the one or more enzymes cause naringenin to be converted into scallopeflavin via one or more intermediates.

2. The method of claim 1, wherein the method comprises an engineered host cell, wherein the engineered host cell comprises one or more gene modifications for expressing one or more enzymes, the one or more enzymes causing naringenin to be converted to saurolophytin via one or more intermediates.

3. The method according to claim 1, wherein the method is for cell-free production of scallope flavonoids, wherein the one or more enzymes in the cell-free medium cause naringenin to be converted into scallope flavonoids via one or more intermediates.

4. The method of claim 1, wherein the method comprises a combination of: (i) engineered host cells, wherein the engineered host cells comprise one or more gene modifications for expressing one or more enzymes that cause naringenin to be converted into saurolophytin, or one or more intermediates; and (ii) cell-free production, wherein the cell-free production comprises one or more enzymes that cause naringenin or a substrate to be converted into saurolophytin, or one or more intermediates, to synthesize saurolophytin.

5. The method of claim 2, wherein the engineered host cell comprises a gene modification for enhancing the production of naringenin or any precursor that induces naringenin production in the engineered host cell.

6. The method according to any one of claims 1 to 4, wherein the one or more intermediates are selected from the group consisting of: apigenin, scutellarein, hispidulin, isosakuranetin, carthamidin, 6-methoxyscarmine, scutellarein, 4-methoxyscarmine, acacetin, 4',6-methoxynaringenin, 4'-methoxyscutellarein, and any combination thereof.

7. The method according to any one of claims 1 to 4, wherein the intermediate is apigenin, baicalin, and physalisin.

8. The method according to any one of claims 1 to 4, wherein the one or more enzymes are selected from the group consisting of: flavonoid synthase (FNSI), flavonoid hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT) and any combination thereof.

9. The method according to any one of claims 1 to 4, wherein one or more intermediates are apigenin, baicalin and 4'-methoxybaicalin.

10. The method according to any one of claims 1 to 4, wherein one or more intermediates are apigenin, baicalin, and physalisin.

11. The method according to any one of claims 1 to 4, wherein one or more intermediates are apigenin, farnesin and 4'-methoxybaicalin.

12. The method according to any one of claims 1 to 4, wherein one or more intermediates are isopyroside, 4'-methoxysafflowerin and 4',6-methoxynaringin.

13. The method according to any one of claims 1 to 4, wherein one or more intermediates are isopyroside, 4'-methoxysafflowerin and 4'-methoxybaicalin.

14. The method according to any one of claims 1 to 4, wherein one or more intermediates are isopyroside, acaciaside, and 4'-methoxybaicalin.

15. The method according to any one of claims 1 to 4, wherein one or more intermediates are safflowerin, 6-methoxysafflowerin, and psyllium husk.

16. The method according to any one of claims 1 to 4, wherein one or more intermediates are safflowerin, 6-methoxysafflowerin and 4',6-methoxynaringin.

17. The method according to any one of claims 1 to 4, wherein one or more intermediates are safflowerin, 6-methoxysafflowerin and 4'-methoxybaicalin.

18. The method according to any one of claims 1 to 4, wherein one or more intermediates are safflower extract, baicalin, and physalis extract.

19. The method according to any one of claims 1 to 4, wherein one or more intermediates are safflowerin, baicalin and 4'-methoxybaicalin.

20. The method according to any one of claims 1 to 4, wherein one or more intermediates are safflowerin, 4'-methoxysafflowerin and 4',6-methoxynaringin.

21. The method according to any one of claims 1 to 4, wherein one or more intermediates are safflowerin, 4'-methoxysafflowerin and 4'-methoxybaicalin.

22. The method of claim 2, wherein the engineered host cell is a bacterial, yeast, or mammalian cell.

23. The method of claim 3, wherein the cell-free medium is a cell lysate.

24. The method of claim 23, wherein the cell lysate is a cell lysate from a host organism expressing one or more of the enzymes.

25. The method of claim 24, wherein the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells.

26. The method of claim 24, wherein the one or more enzymes are introduced into the host organism by integration into the genome of the host organism or integration into a plasmid.

27. The method of claim 26, wherein the host organism expressing the one or more enzymes is cultured until a predetermined amount of biomass is produced to generate the required amount of the one or more enzymes.

28. The method of claim 27, further comprising lysing cells and subsequently removing cell debris to produce cell lysates for use in the cell-free medium for cell-free production of saural flavin.

29. The method of claim 3, wherein the cell-free medium comprises: buffer solution, magnesium chloride, one or more substrates, one or more cofactors, one or more enzymes, and / or water.

30. The method of claim 29, wherein the buffer solution is a phosphate buffer solution.

31. The method of claim 29, wherein the pH of the cell-free medium is about 5.5 to about 10.

32. The method of claim 29, wherein the pH of the cell-free medium is about 6.5 to about 9.

33. The method according to 29, wherein the buffer concentration is from about 10 mM to about 500 mM.

34. The method according to 29, wherein the buffer concentration is from about 50 mM to about 300 mM.

35. The method of claim 29, wherein the buffer concentration is from about 20 mM to about 100 mM.

36. The method of claim 29, wherein magnesium chloride is present in the range of about 1 mM to about 50 mM.

37. The method of claim 29, wherein magnesium chloride is present in the range of about 5 mM to about 25 mM.

38. The method of claim 29, wherein the substrate is naringenin.

39. The method of claim 29, wherein the one or more substrates are present at a concentration of about 1 mM to about 500 mM.

40. The method of claim 29, wherein the one or more substrates are present at a concentration of about 1 mM to about 200 mM.

41. The method of claim 29, wherein the one or more substrates are present at a concentration of about 10 mM to about 100 mM.

42. The method of claim 29, wherein the one or more cofactors are present at a concentration of about 1 mM to about 500 mM.

43. The method of claim 29, wherein the one or more cofactors are present at a concentration of about 1 mM to about 200 mM.

44. The method of claim 29, wherein the one or more cofactors are present at a concentration of about 1 mM to about 10 mM.

45. The method of claim 29, wherein the one or more enzymes are present in the range of about 0.1 µM to about 5 mM.

46. ​​The method of claim 29, wherein the one or more enzymes are present in the range of about 0.1 µM to about 1 mM.

47. The method of claim 29, wherein the one or more enzymes are present in the range of about 1 µM to about 100 µM.

48. The method of claim 29, wherein the reaction for cell-free production of saurolophus lasts for a duration of about 0.1 hours to about 20 hours.

49. The method of claim 29, wherein the reaction for cell-free production of saurolophus lasts for a duration of about 1 hour to about 10 hours.

50. The method of claim 29, wherein the temperature of the cell-free medium is from about 20°C to about 40°C.

51. The method of claim 3, wherein the reaction is carried out in a bubble column reactor, wherein the one or more enzymes are in solution.

52. The method of claim 3, wherein the reaction is carried out in a packed bed reactor, wherein the one or more enzymes are immobilized.

53. The method according to any one of claims 1 to 4, wherein the enzyme is a flavonoid synthase having at least 95% identical amino acid sequence to any enzyme selected from the group consisting of SEQ ID 1 to 15.

54. The method according to any one of claims 1 to 4, wherein the enzyme is a flavonoid hydroxylase cytochrome P450 enzyme having at least 95% identical amino acid sequence to any enzyme selected from the group consisting of SEQ ID 16 to 30.

55. The method according to any one of claims 1 to 4, wherein the enzyme is an O-methyltransferase having at least 95% identical amino acid sequence to any enzyme selected from the group consisting of SEQ ID 31 to 69.

56. A method for the biological production of scallop flavonoids, the method comprising: providing one or more enzymes, wherein the one or more enzymes cause one or more substrates to be converted into scallop flavonoids optionally via one or more intermediates.

57. The method of claim 56, wherein the one or more substrates are selected from the group consisting of: naringenin, apigenin, baicalin, psyllium husk, or any combination thereof.

58. The method of claim 56, wherein one or more substrates are apigenin.

59. The method of claim 56, wherein one or more substrates are baicalin.

60. The method of claim 56, wherein one or more substrates are phytoestrogen.

61. The method according to any one of claims 56 to 60, wherein the method comprises an engineered host cell, wherein the engineered host cell comprises one or more gene modifications for expressing one or more enzymes, the one or more enzymes causing the one or more substrates to be optionally converted to saurolophus flavonoids via one or more intermediates.

62. The method according to any one of claims 56 to 60, wherein the method is a cell-free production of scallop flavonoids, wherein the one or more enzymes in the cell-free medium cause the one or more substrates to be optionally converted to scallop flavonoids via one or more intermediates.

63. A composition for the bioproduction of scallop flavonoids, wherein the composition comprises one or more enzymes, wherein the one or more enzymes cause one or more substrates to be converted into scallop flavonoids optionally via one or more intermediates.

64. The composition of claim 63, wherein the one or more substrates are selected from the group consisting of: naringenin, apigenin, baicalin, psyllium husk, or any combination thereof.

65. The composition according to claim 63, wherein one or more substrates are apigenin.

66. The composition of claim 63, wherein one or more substrates are baicalin.

67. The composition of claim 63, wherein one or more substrates are psyllium husk.

68. The composition according to any one of claims 63 to 67, wherein the composition comprises an engineered host cell, wherein the engineered host cell comprises one or more gene modifications for expressing one or more enzymes, the one or more enzymes causing the one or more substrates to be optionally converted to saurolophus flavonoids via one or more intermediates.

69. The composition according to any one of claims 63 to 67, wherein the composition is used for cell-free production of scallop flavonoids, wherein the one or more enzymes in the cell-free medium cause the one or more substrates to be optionally converted to scallop flavonoids via one or more intermediates.

70. A method for producing scallop flavonoids, the method comprising: providing an engineered host cell containing one or more gene modifications, wherein the one or more gene modifications cause glycerol to be enzymatically converted into scallop flavonoids via one or more intermediates.

71. The method of claim 70, wherein the engineered host cell comprises one or more gene modifications for enhancing the engineered host cell to produce naringenin from glycerol.

72. The method of claim 71, wherein the engineered host cell comprises one or more gene modifications for enhancing the conversion of naringenin in the engineered host cell to saurolophytin via one or more intermediates.

73. The method according to claim 71 or 72, wherein the one or more gene modifications are the expression or overexpression of one or more enzymes.

74. The method of claim 73, wherein the one or more enzymes are selected from the group consisting of FNSI, P450, OMT and any combination thereof.

75. The method of claim 74, wherein the one or more enzymes are selected from the group consisting of expression or overexpression of the following: FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI and any combination thereof.

76. The method according to any one of claims 70 to 75, wherein the engineered host cell is Escherichia coli.

77. A composition for producing scallop flavonoids, wherein the composition comprises an engineered host cell including one or more genetic modifications, wherein the one or more genetic modifications cause glycerol to be enzymatically converted to scallop flavonoids via one or more intermediates.

78. The composition of claim 77, wherein the engineered host cell comprises one or more gene modifications for enhancing the engineered host cell to produce naringenin from glycerol.

79. The composition of claim 72, wherein the engineered host cell comprises one or more gene modifications for enhancing the conversion of naringenin in the engineered host cell to saurolophytin via one or more intermediates.

80. The composition according to claim 78 or 79, wherein the one or more gene modifications are the expression or overexpression of one or more enzymes.

81. The composition of claim 80, wherein the one or more enzymes are selected from the group consisting of: FNSI, P450, OMT and any combination thereof.

82. The composition of claim 80, wherein the one or more enzymes are selected from the group consisting of expression or overexpression of the following: FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI and any combination thereof.

83. The composition according to any one of claims 70 to 75, wherein the engineered host cell is Escherichia coli.

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