Methods and compositions for bioproduction of pectolinarigenin

Abstract

The invention is directed to materials and methods for bioproduction of pectolinarigenin. The invention provides methods and materials for cell-based and cell-free production of pectolinarigenin.

Description

[0001] METHODS AND COMPOSITIONS FOR BIOPRODUCTION OF PECTOLINARIGENIN

[0002] I. Field of Invention

[0003] The invention is directed to materials and methods for bioproduction of pectolinarigenin. The invention provides methods and materials for cell-based and cell-free production of pectolinarigenin.

[0004] II. Reference to Sequence Listing

[0005] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named DEBU-023-01WO.xml, created on December 26, 2024, which is 107 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety

[0006] III. Background

[0007] Nature is recognized as a source of molecules with relevant potential pharmaceutical applications. Among the various phytochemicals, flavonoids have attracted and are still attracting most of the attention due to their notable biological benefits. Flavonoids have been associated with the role of preventing and managing current diseases such as cancers, diabetes, and cardiovascular disorders. More than 13,000 flavonoids were isolated and identified from plants, some of which like quercetin, kaempferol and scuteliarin showed potent pharmacological effects and, therefore, are promising for new drugs.

[0008] The glycosylated flavone pectolinarin was first isolated from Linaria vulgaris, a known medicinal Chinese herb used for the internal treatment of digestion problems and urinary disorders, in the external treatment of hemorrhoids, venous skin ulcer, as well as for the washing of festering wounds and skin 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, were identified as the major constituents in many medicinal herbs from different genera around the world.

[0009] Over the last 113 years from its first report, pectolinarin was isolated in most cases from the aerial parts of 87 plants belonging to 29 different genera distributing widely around the world. Most of these plants are used in folk medicine in different parts of the world. Pectolinarigenin is the aglycone part of pectolinarin, which is obtained by hydrolysis reaction. It is also a natural product, isolated and identified from 136 plants of 71 different genera. Pectolinarigenin proved to be more distributed in the vegetable kingdom than pectolinarin, and this evidence can be explained assuming that not all plants have the O-glycosylation enzymes. In most works, pectolinarigenin was isolated from the non-polar fractions of the plant extracts, mainly working on the aerial parts.

[0010] IV. Summary of the Invention

[0011] The conventional methods for production of pectolinarigenin involve isolation from plants. This increases the cost of production because of the purification required from these mixtures. Moreover, these factors can vary in the source plant from year to year and are influenced by weather and environmental factors. Thus, another challenge for the commercial production of polyphenols from plants is that harvest is often limited to once a year. This means a large volume of extract has to be prepared and stored for an extended time to supply the needs of the food industry throughout the year.

[0012] Thus, new methods for production of pectolinarigenin are required that are reliable and economically viable. The invention provides methods and compositions for bioproduction of pectolinarigenin. The methods of the invention may lead to production of pectolinarigenin in a host organism comprising certain genetic modifications. The methods of the invention also provide cell-free bioproduction of pectolinarigenin from one or more substrates through one or more substrates. The methods of the invention are beneficial because they provide an economically viable method for production of pectolinarigenin. Advantageously, the methods of the invention are also reliable and are not dependent on the available plants for preparations of pectolinarigenin.

[0013] Advantageously, the methods of the invention provide a cell-based or cell-free platform for production of pectolinarigenin. The platform provided in the invention is beneficial because it provides multiple pathways for preparation of pectolinarigenin from naringenin. The availability of different pathways for the production of pectolinarigenin is beneficial because it provides an avenue to prepare and isolate one or more intermediates in the biosynthesis pathway. Thus, the platform provided in the invention unlocks the option of preparation of one or more intermediates which might not otherwise be readily accessible for manufacturing. Beneficially, the pathway for production of pectolinarigenin could 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, which does not require that particular enzyme, may be selected for production of pectolinarigenin. Thus, the modularity of the biosynthetic pathway is especially beneficial.

[0014] In certain aspects, the invention provides a method for bioproduction of pectolinarigenin, the method comprising providing one or more enzymes, wherein the one or more enzymes result in transformation of naringenin to pectolinarigenin through one or more intermediates. The method for production of pectolinarigenin includes cell -based and cell-free methods.

[0015] In certain aspects of the invention, the method for production of pectolinarigenin comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of naringenin to pectolinarigenin through one or more intermediates. In certain embodiments, the genetic modifications in the host cell may also include one or more genetic modifications to enhance the production of naringenin. In certain embodiments, the engineered host cell comprises genetic modifications to enhance the production of naringenin or any precursor leading to production of naringenin in the engineered host cell. The additional genetic modifications leading to an increase in production of naringenin may be beneficial for production of pectolinarigenin because the naringenin may be used a substrate for production of pectolinarigenin. The enhanced amount of naringenin would lead to higher yield of pectolinarigenin.

[0016] In certain aspects of the invention, the method for production of pectolinarigenin is a cell- free production of pectolinarigenin, wherein the one or more enzymes in a cell-free medium result in transformation of naringenin to pectolinarigenin through one or more intermediates. The ingredients of the components in the cell-free reaction may be generated in a host cell. For example, the substrate, such as naringenin, may be generated in a cell and the subsequent conversion to pectolinarigenin may be conducted in a cell-free medium. In certain embodiments, the one or more intermediates generated in conversion of naringenin to pectolinarigenin may be generated in an engineered host cell, and those one or more intermediates may be subsequently converted to pectolinarigenin by one or more enzymes in a cell-free medium. In certain aspects, the method comprises a combination of: (i) an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of naringenin to pectolinarigenin or one or more intermediates, and (ii) cell-free production comprising one or more enzymes resulting in transformation of naringenin or a substrate to pectolinarigenin or one or more intermediates for synthesis of pectolinarigenin. In certain embodiments, the engineered host cell comprises genetic modifications to enhance the production of naringenin or any precursor leading to production of naringenin in the engineered host cell.

[0017] In certain embodiments of the invention, the one or more intermediates are selected from a group consisting of: apigenin, scutellarein, hispidulin, isosakuranetin, carthamidin, 6- m ethoxy carthamidin, scutellarein, 4-methoxy carthamidin, acacetin, 4 ’,6-meth oxy naringenin, 4’- methoxyscutellarein, and any combination thereof. FIG. 1 provides exemplary schematic pathways for conversion of naringenin to pectolinarigenin. FIG. 1 also provides the enzymes mediating the conversion of naringenin to one or more intermediates, or the conversion of one of more intermediates to other intermediates, or the conversion of one or more intermediates to pectolinarigenin. As evident from FIG. 1, the methods of the invention provide a modular pathway for conversion of naringenin to pectolinarigenin. The pathway selected for production of pectolinarigenin will determine the identity of the one or more intermediates selected in the process. Each individual segment of the pathway may be controlled by the enzymes selected for that conversion. In certain embodiments, the methods of the invention provide the use of one or more enzymes to mediate the conversion of the starting material to one or more intermediates, or pectolinarigenin. In certain embodiments, the one or more enzymes selected from the group consisting of: flavone synthase (FNSI), flavone hydroxylase cytochrome P450 (P450), O- methyltransferase (OMT), and any combination thereof.

[0018] For example, in certain embodiments, the intermediates are apigenin, scutellarein, and hispidulin. In certain embodiments, the method provides a pathway, wherein, naringenin is converted to apigenin; apigenin is converted to scutellarein; scutellarein is converted to hispidulin; and hispidulin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. For example, as demonstrated in FIG. 2, the pathway provided comprises flavone synthase (FNSI) mediated conversion of naringenin to apigenin; flavone hydroxylase cytochrome P450 (P450) mediated conversion of apigenin to scutellarein; O- methyltransferase (OMT) mediated conversion of scutellarein to hispidulin; and O- methyltransferase (OMT) mediated conversion of hispidulin to pectolinarigenin. Any of these aforementioned steps may be conducted either in an engineered host cell or a cell-free medium.

[0019] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are apigenin, scutellarein, and 4’-methoxyscutellarein. The invention provides that naringenin is converted to apigenin; apigenin is converted to scutellarein; scutellarein is converted to 4’-methoxyscutellarein; 4’-methoxyscutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0020] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are apigenin, scutellarein, and hispidulin. The invention provides that naringenin is converted to apigenin; apigenin is converted to scutellarein; scutellarein is converted to hispidulin; hispidulin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0021] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are apigenin, acacetin, and 4’-methoxyscutellarein. The invention provides that naringenin is converted to apigenin; apigenin is converted to acacetin; acacetin is converted to 4’ -methoxy scutellarein; 4’-methoxyscutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0022] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are isosakuranetin, 4’-methoxycarthamidin, and 4’, 6- methoxynaringenin. The invention provides that naringenin is converted to isosakuranetin; isosakuranetin is converted to 4’ -methoxy carthamidin; 4’ -methoxy carthamidin is converted to 4’,6-methoxynaringenin; 4’,6-methoxynaringenin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are isosakuranetin, 4’ -methoxy carthamidin, and 4’- methoxyscutellarein. The invention provides that naringenin is converted to isosakuranetin; isosakuranetin is converted to 4’ -methoxy carthamidin; 4’-methoxy carthamidin is converted to 4’- methoxyscutellarein; 4’ -methoxy scutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0023] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are isosakuranetin, acacetin, and 4’ -methoxy scutellarein. The invention provides that naringenin is converted to isosakuranetin; isosakuranetin is converted to acacetin; acacetin is converted to 4’-methoxyscutellarein; 4’-methoxyscutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0024] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, 6-methoxycarthamidin, and hispidulin. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to 6- methoxy carthamidin; 6-methoxycarthamidin is converted to hispidulin; hispidulin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0025] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, 6-methoxycarthamidin, and 4’, 6- methoxynaringenin. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to 6-methoxycarthamidin; 6-methoxycarthamidin is converted to 4’, 6- methoxynaringenin; 4’,6-methoxynaringenin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, 6-methoxycarthamidin, and 4’- methoxyscutellarein. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to 6-methoxycarthamidin; 6-methoxycarthamidin is converted to 4’- methoxyscutellarein; 4’ -methoxy scutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0026] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, scutellarein, and hispidulin. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to scutellarein; scutellarein is converted to hispidulin; hispidulin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0027] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, scutellarein, and 4’ -methoxy scutellarein. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to scutellarein; scutellarein is converted to 4’-methoxyscutellarein; 4’-methoxyscutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0028] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, 4’ -methoxy carthamidin, and 4’, 6- methoxynaringenin. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to 4’-methoxycarthamidin; 4’-methoxycarthamidin is converted to 4’, 6- methoxynaringenin; 4’,6-methoxynaringenin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0029] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, 4’-methoxycarthamidin, and 4’- methoxyscutellarein. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to 4’-methoxycarthamidin; 4’-methoxycarthamidin is converted to 4’- methoxyscutellarein; 4’ -methoxyscutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0030] In certain embodiments, the invention provides method of production of pectolinarigenin comprises engineered host cells, wherein the host cells comprise one or more genetic modifications. In certain embodiments, the engineered host cell could be prepared from yeast, bacteria, or mammalian cells. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the one or more enzymes are introduced in the host organism by integration into genome of the host organism or on a plasmid. In certain embodiments, the one or more genetic modifications in the engineered host cell are any of the modifications described above. For example, the one or more genetic modification may be overexpression or endogenous enzymes or expression of a variant of an enzyme involved in the conversion of naringenin to pectolinarigenin. The genetic modifications include, without limitation, overexpression or expression of a variant of flavone synthase (FNSI), flavone hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and / or any combination thereof.

[0031] In certain embodiments, the one or more genetic modifications in the host cell may include any genetic modification to increase the availability of naringenin. For example, the one or more genetic modifications in an engineered host cell may be selected from a group consisting of: acetyl - CoA carboxylase (ACC), tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chaicone synthase (CHS), chaicone isomerase (CHI), and / or any combination thereof. In certain embodiments, the genetic modifications may comprise expressing a variant of one or more of these enzymes. In certain embodiments, the one or more genetic modifications to increase the production of naringenin are provided in PCT / US2022 / 024591, which is incorporated in its entirety by reference.

[0032] In certain embodiments, the engineered host cell comprises one or more genetic modifications to increase the production naringenin and / or pectolinarigenin. In certain embodiments, the engineered host cell comprises one or more genetic modifications to increase the production of naringenin. In these embodiments, the starting material for production of naringenin would is glycerol. Thus, the methods of the invention beneficially provide an engineered host cell for conversion of glycerol to pectolinarigenin. The engineered host cell may optionally comprise genetic modifications optimized for conversion of glycerol to naringenin through one or more intermediates and subsequent conversion of naringenin to pectolinarigenin through one or more intermediates. The exemplary genetic modifications for production of naringenin are provided in WO2022 / 221392, which is incorporated by reference in its entirety.

[0033] In addition, FIG. 6 provides an overview of the conversion of glycerol to naringenin through one or more intermediates. In certain preferred embodiments, the engineered host cell for enhanced production of naringenin comprises one or more genetic modifications selected from the group consisting of: overexpression of ACC, TAL, 4CL, CHS, and CHI. In certain embodiments, the engineered host cell may comprise additional genetic modifications for downregulation and / or deletion of one or more enzymes that convert naringenin to other substrates that are not involved in the pathway for bioproduction of pectolinarigenin.

[0034] Accordingly, in certain aspects, the invention provides an engineered host cell for conversion of glycerol to pectolinarigenin through one more intermediates. In certain embodiments, the engineered host cell may comprise one more genetic modifications for enzymatic transformation of glycerol to pectolinarigenin through one more intermediates. In certain embodiments, the one more genetic modifications for transformation of glycerol to naringenin. In certain embodiments, the genetic modifications may be selected from the group consisting of overexpression or expression of a variant of: ACC, TAL, 4CL, CHS, and CHI. In certain embodiments, the engineered host cell further comprises one or more genetic modifications for transformation of naringenin to pectolinarigenin. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from the group consisting of overexpression or expression of a variant of: flavone synthase (FNSI), flavone hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and / or any combination thereof. In certain embodiments, the glycerol is from a carbon feedstock. In some embodiments, the glycerol is crude glycerol. In certain embodiments, glycerol obtained as a byproduct of biodiesel processing.

[0035] In certain aspects, the invention provides a method for production of pectolinarigenin through transformation of glycerol through one or more intermediates. In certain embodiments, the glycerol is enzymatically transformed to pectolinarigenin through one or more intermediates. In certain embodiments, the one or more intermediates is naringenin. In certain embodiments, the genetic modifications may be selected from the group consisting of overexpression or expression of a variant of: ACC, TAL, 4CL, CHS, and CHI. In certain embodiments, the engineered host cell further comprises one or more genetic modifications for transformation of naringenin to pectolinarigenin. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from the group consisting of overexpression or expression of a variant of: flavone synthase (FNSI), flavone hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and / or any combination thereof. In certain embodiments, the glycerol is from a carbon feedstock. In some embodiments, the glycerol is crude glycerol. In certain embodiments, glycerol obtained as a byproduct of biodiesel processing.

[0036] In certain aspects, the invention provides methods of cell-free production of pectolinarigenin. The cell-free medium for the conversion of naringenin to pectolinarigenin is a cell lysate. In certain embodiments, the cell lysate is a cell lysate from cells from a host organism expressing the one or more enzymes. The one or more enzymes may be the enzymes involved in conversion of naringenin to pectolinarigenin. In certain embodiments, the host organisms expressing the one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes. In certain embodiments, the method of the invention further comprising lysing cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of pectolinarigenin.

[0037] In certain embodiments, cell-free medium comprises: buffer, magnesium chloride, one or more substrates, one or more cofactors, the one or more enzymes, and / or water.

[0038] In certain embodiments, the buffer in the cell-free medium maintains the pH of the reaction mixture in the optimal range. In certain embodiments, the pH of the reaction medium is from about 5.5 to about 10. In certain embodiments, the pH of the reaction medium is from about 6 to about 9.5. In certain embodiments, the pH of cell-free medium is from about 6 to about 9. In certain embodiments, the pH of cell-free medium is from about 6 to about 8. In certain embodiments, the concentration of the buffer in the cell-free medium is from about 1 mM to about 1000 mM. In certain embodiments, the concentration of the buffer is from about 2.5 mM to about 750 mM. In certain embodiments, the concentration of the buffer is from about 5 mM to about 100 mM. In certain embodiments, the buffer concentration is from about 20 mM to about 100 mM. In certain embodiments, the buffer is a phosphate buffer.

[0039] In certain embodiments, the method of the invention comprises conversion of one or more substrates to pectolinarigenin. In certain embodiments, the one or more substrates are present in a concentration of from about 1 mM to about 500 mM. In certain embodiments, the one or more substrates are present in a concentration of from about 1 mM to about 200 mM. In certain embodiments, the one or more substrates are present in a concentration of from about 10 mM to about 100 mM. In certain embodiments, the substrate used for production of pectolinarigenin is naringenin.

[0040] In certain embodiments, the cell-free medium comprises magnesium chloride. The magnesium chloride may be present in the range of from about 1 mM to about 50 mM. In certain embodiments, the cell-free medium comprises magnesium chloride in the range of from about 1 mM to about 25 mM. In certain embodiments, the cell-free medium comprises magnesium chloride in the range of from about 1 mM to about 20 mM. In certain embodiments, the cell-free medium comprises magnesium chloride in the range of from about 5 mM to about 25 mM.

[0041] In certain embodiments, the cell-free medium comprises the lysate from host microbe with the one or more enzymes for the cell-free reaction medium. In certain embodiments, the cell-free medium comprises lysate with the one or more enzymes in a concentration of from about 1% (v / v) to about 50% (v / v). In certain embodiments, the cell -free medium comprises lysate with the one or more enzymes in a concentration of from about 5% (v / v) to about 40% (v / v). In certain embodiments, the cell-free medium comprises lysate with the one or more enzymes in a concentration of from about 10% (v / v) to about 30% (v / v). In certain embodiments, the cellmedium further comprises the one or more enzymes are present in the range of from about 0.1 pM to about 5 mM. In certain embodiments, the one or more enzymes are present in the range of from about 0.1 pM to about 1 mM. In certain embodiments, the one or more enzymes are present in the range of from about 1 pM to about 100 pM.

[0042] In certain embodiments, the methods of the invention provide a use of one or more cofactors to facilitate the reactions leading to conversion of naringenin to pectolinarigenin. In certain embodiments, wherein the one or more cofactors are present in a concentration of about 1 mM to about 500 mM. In certain embodiments, the one or more cofactors are present in a concentration of about 1 mM to about 200 mM. In certain embodiments, the one or more cofactors are present in a concentration of about 1 mM to about 10 mM.

[0043] In certain embodiments, the one or more cofactors for methods of the invention are selected from the group consisting of: 2-oxoglutarate, ascorbic acid, iron(II), FAD, FMN, NADPH, SAM, ATP or any combination thereof. In certain embodiments, the one or more cofactors are 2-oxoglutarate. In certain embodiments, the one or more cofactors are ascorbic acid. In certain embodiments, the one or more cofactors are iron(II). In certain embodiments, the one or more cofactors are heme. In certain embodiments, the one or more cofactors are FAD. In certain embodiments, the one or more cofactors are FMN. In certain embodiments, the one or more cofactors are NADPH. In certain embodiments, the one or more cofactors are s-adenosyl methionine (SAM). In certain embodiments, the one or more cofactors are a divalent metal such as magnesium or manganese. In certain embodiments, the one or more cofactors are ATP.

[0044] In certain embodiments, the cell-free reaction for production of polyphenols is conducted for the requisite duration till the desired quantity of the polyphenols are produced in the said reaction. In certain embodiments, the reaction for cell-free production of polyphenols is carried out for a duration of from about 0.1 hours to about 20 hours. In certain embodiments, the reaction for cell -free production of polyphenols is carried out for a duration of from about 0.5 hours to about 20 hours. In certain embodiments, reaction for cell-free production polyphenols is carried out for a duration of from about 1 hours to about 15 hours.

[0045] In certain embodiments, the reaction of the cell-free medium is adjusted for an optimal yield of polyphenols to be produced. In certain embodiments, the temperature of the cell-free medium is from about 20 °C to about 40 °C.

[0046] In certain embodiments, the reaction is conducted in a bubble column reactor, wherein the one or more enzymes are in a solution. In certain embodiments, the reaction is conducted in a packed bed reactor, wherein the one or more enzymes are immobilized.

[0047] In certain aspects, the invention provides A method for bioproduction of pectolinarigenin, the method comprising: providing one or more enzymes, wherein the one or more enzymes result in transformation of one or more substrates to pectolinarigenin optionally through one or more intermediates. In certain embodiments, one or more substrates are selected from the group consisting of naringenin, apigenin, scutellarein, hispidulin, or any combination thereof. In certain embodiments, the substrate is apigenin. In certain embodiments, the substrate is scutellarein. In certain embodiments, the substrate is hispidulin.

[0048] In certain aspects, the method comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of the one or more substrate to pectolinarigenin optionally through one or more intermediates. In certain embodiments, the invention provides a method for cell-free production of pectolinarigenin, wherein the one or more enzymes in a cell -free medium result in transformation of the one or more substrate to pectolinarigenin optionally through one or more intermediates.

[0049] In certain aspects, the invention provides a composition for bioproduction of pectolinarigenin, wherein the composition comprises one or more enzymes, wherein the one or more enzymes result in transformation of one or more substrates to pectolinarigenin optionally through one or more intermediates. In certain embodiments, one or more substrates are selected from the group consisting of naringenin, apigenin, scutellarein, hispidulin, or any combination thereof. In certain embodiments, one or more substrates are selected from the group consisting of naringenin, apigenin, scutellarein, hispidulin, or any combination thereof. In certain embodiments, the substrate is apigenin. In certain embodiments, the substrate is scutellarein. In certain embodiments, the substrate is hispidulin. In certain embodiments, the composition comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of the one or more substrate to pectolinarigenin optionally through one or more intermediates. In certain embodiments, the composition comprises one or more enzymes in a cell-free medium that results in transformation of the one or more substrates to pectolinarigenin optionally through one or more intermediates.

[0050] In certain aspects, the invention provides a method for production of pectolinarigenin, the method comprises: providing an engineered host cell that comprises one or more genetic modifications, wherein the one or more genetic modifications result in enzymatic transformation of glycerol to pectolinarigenin through one or more intermediates. In certain embodiments, the engineered host cell comprises one or more genetic modifications to enhance the production of naringenin from glycerol the engineered host cell. In certain embodiments, the engineered host cell comprises one or more genetic modifications to enhance the transformation of naringenin to pectolinarigenin in the engineered host cell through one or more intermediates. In certain embodiments, the one or more genetic modification is expression or overexpression of one or more enzymes. In certain embodiments, one or more enzymes are selected from the group consisting of: FNSI, P450, OMT, and any combination thereof. In certain embodiments, one or more enzymes are selected from the group consisting of expression or overexpression of: FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI, and any combination thereof. In certain embodiments, the engineered host cell is E. coli.

[0051] In certain aspects, the invention provides a composition for production of pectolinarigenin, wherein the composition comprises an engineered host cell that comprises one or more genetic modifications, wherein the one or more genetic modifications result in enzymatic transformation of glycerol to pectolinarigenin through one or more intermediates. In certain embodiments, the engineered host cell comprises one or more genetic modifications to enhance the production of naringenin from glycerol the engineered host cell. In certain embodiments, the engineered host cell comprises one or more genetic modifications to enhance the transformation of naringenin to pectolinarigenin in the engineered host cell through one or more intermediates. In certain embodiments, the one or more genetic modification is expression or overexpression of one or more enzymes. In certain embodiments, one or more enzymes are selected from the group consisting of: FNSI, P450, OMT, and any combination thereof. In certain embodiments, one or more enzymes are selected from the group consisting of expression or overexpression of: FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI, and any combination thereof. In certain embodiments, the engineered host cell is E. coli.

[0052] In certain embodiments, the flavone synthase (FNSI) is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to any one of enzymes selected from the group consisting of:

[0053]

[0054] In certain embodiments, flavone hydroxylase cytochrome P450 is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to any one of enzymes selected from the group consisting of:

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

[0056] The methods of production of pectolinarigenin provided in the invention are beneficial because they provide for an economic and efficient pathway for synthesis of pectolinarigenin. Advantageously, the methods of the invention do not extensive processes for extraction and / or purification of pectolinarigenin from the plants or roots. The methods requiring extraction of the product from plants has complications, such as the processes leading to low yields, a higher number of by-products, and expensive purification and separation protocols.

[0057] The methods of the current invention beneficially provide cell-based and cell-free approaches for bioproduction of pectolinarigenin. In particular, the product titers for pectolinarigenin produced by the methods of the invention are higher than the product titer produced by any other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least two-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least five-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least ten-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least hundred-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least five- hundred-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least thousand-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least five-thousand-fold higher than other known methods for production of pectolinarigenin.

[0058] In other beneficial aspects of the method of the invention, the methods of the invention provide economically efficient methods of production of pectolinarigenin. Because the methods of the invention also provide for cell-free production of pectolinarigenin, it leads to decreased costs of purification of the reaction products. Moreover, in certain aspects of the invention, the one or more enzymes are included in the same reaction mixture, i.e., the enzymes do not need to be isolated after being expressed in the host organisms. The one or more enzymes could be expressed in the host organisms at the same time and be utilized for the cell-free production without the need of any additional purification and / or separation steps.

[0059] In certain other beneficial aspects of the invention, the use of the one or more enzymes for the cell-free production of polyphenols leads to high yield of the product to be produced. The one more enzyme may be further modified to optimize the yield of the product being produced in the cell-free reaction.

[0060] In certain aspects, the invention provides compositions for the production of pectolinarigenin from one or more substrates. In certain embodiments, the compositions provided in the invention are compositions suitable for preparing pectolinarigenin from one or more substrates. In certain embodiments, the compositions of the invention comprise engineered host organisms, wherein the engineered host organisms comprise genetic modifications for production of pectolinarigenin from naringenin through one or more intermediates. In certain embodiments, the compositions of the invention comprise a cell-free medium, which may comprise one or more enzymes, for production of pectolinarigenin from naringenin through one or more intermediates. In certain embodiments, the compositions of the invention comprise a combination of engineered host cells and a cell-free medium for preparation of pectolinarigenin.

[0061] V. Brief Description of Drawings

[0062] FIG. 1 provides a description of the modular pathways for conversion of naringenin to pectolinarigenin through one or more intermediates.

[0063] FIG. 2 provides a schematic pathway for conversion of naringenin to pectolinarigenin through apigenin, scutellarein, and hispidulin as the intermediates.

[0064] FIGS. 3A and 3B provide HPLC chromatograms for the cell-free production of hispidulin and pectolinaringenin.

[0065] FIG. 4 provides HPLC chromatograms for cell production of pectolinarigenin and apigenin from glycerol.

[0066] FIG. 5 provides HPLC chromatograms for cell production of pectolinarigenin from hispidulin.

[0067] FIG. 6 provides an overview for the enzymatic conversion of glycerol to naringenin.

[0068] VI. Detailed Description

[0069] The present application provides compositions and methods for production of pectolinarigenin in a cell-based or cell-free medium, wherein the one or more enzymes result in transformation of one or more substrates to pectolinarigenin. The one or more enzymes may be engineered. The engineered enzyme may be non-naturally occurring.

[0070] The term “non-naturally occurring”, when used in reference to an enzyme is intended to mean that nucleic acids or polypeptides include at least one genetic alteration not normally found in a naturally occurring polypeptide or nucleic acid sequence. Naturally occurring nucleic acids, and polypeptides can be referred to as “wild-type” or “original”. A host cell, organism, or microorganism that includes at least one genetic modification generated by human intervention can also be referred to as “non-naturally occurring”, “engineered”, “genetically engineered,” or “recombinant”. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0071] As used herein, “reaction solution” may refer to all components necessary for enzymebased chemical transformation. This is typically, but not limited to, buffering agent, salts, cofactor, and substrate (starting material).

[0072] As used herein, “reaction mixture” may refer to all components from the “reaction solution” plus the enzyme(s) and / or products from the reaction. In some embodiments, the “reaction mixture” may refer to just the reaction solution without any enzymes or reaction products. In some embodiments, “reaction solution” and “reaction mixture” may be used interchangeably.

[0073] As used herein, “buffering agents” may refer to chemicals added to water-based solutions that resist changes in pH by the action of acid-base conjugate components.

[0074] As used herein, “cofactors” may refer to a non-protein chemical compound that may bind to a protein and assist with a biological chemical reaction. Non-limiting examples of cofactors may include but are not limited to NADPH and NADH.

[0075] In the cell-free systems described herein, the critical components of the cell, namely cofactors and enzymes, are used in a chemical reaction without cellular components that can directly or indirectly inhibit the desired biochemical reaction. The same enzymes found in plants and other organisms may be created in vivo (typically through protein overexpression in hosts such as bacteria), isolated via chromatography and / or any other methods, and then added into a bioreactor with a substrate (starting material). The enzymes may also be used directly from plants without any isolation. The enzymes transform the substrate in the same way that occurs in the original organism without the organism’s complexity. Additionally, the biochemical reaction may be enhanced by the addition of co-solvents, detergents, or both, which would not be tolerated by, or simply would not work in a whole cell-based manufacturing method. In this way, natural products can be created without the plant, cell, or chemical synthesis.

[0076] Pectolinarigenin: Pectolinarigenin is a flavonoid compound that belongs to the flavone 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 widely studied for their antioxidant, anti-inflammatory, anticancer, and neuroprotective properties.

[0077] The chemical structure of pectolinarigenin is provided below:

[0078] The research regarding the potential applications for pectolinarigenin is still in early stages, and the early data is from in vitro and animal studies. For example, pectolinarigenin is known to have antioxidant, anticancer, anti-inflammatory, and neuroprotective properties.

[0079] In spite of the promising biological activity, there have been no known economically efficient method for the production of pectolinarigenin. In conventional methods, pectolinarigenin is specifically derived from the plant Pectis papposa, which is commonly known as the Guayule plant. This plant is native to the southwestern United States and northern Mexico and has been traditionally used in folk medicine for its medicinal properties. These methods are not cost efficient, and require various steps, including harvesting the plants, preparation of the harvested materials (such as leaves, flowers, or roots), extraction (subjecting the extracted materials to various processes and / or chemicals to bring out pectolinarigenin), and purification (isolating pectolinarigenin from other chemicals). This is an expensive and time-consuming process. The processes involving chemical synthesis of pectolinarigenin are not commercially viable. Thus, a need exists for an economic and efficient process for production of pectolinarigenin.

[0080] Production of pectolinarigenin:

[0081] Thus, new methods for production of pectolinarigenin are required that are reliable and economically viable. The methods of the invention provides methods and compositions for bioproduction of pectolinarigenin. The methods of the invention may lead to production of pectolinarigenin in a host organism comprising certain genetic modifications. The methods of the invention also provide cell-free bioproduction of pectolinarigenin from one or more substrates through one or more substrates. The methods of the invention are beneficial because they provide an economically viable method for production of pectolinarigenin. Advantageously, the methods of the invention are also reliable and are not dependent on the available plants for preparations of pectolinarigenin.

[0082] Advantageously, the methods of the invention provide a cell-based or cell-free platform for production of pectolinarigenin. The platform provided in the invention is beneficial because it provides multiple pathways for preparation of pectolinarigenin from naringenin. The availability of different pathways for the production of pectolinarigenin is beneficial because it provides an avenue to prepare and isolate one or more intermediates in the biosynthesis pathway. Thus, the platform provided in the invention unlocks the option of preparation of one or more intermediates which might not otherwise be readily accessible for manufacturing. Beneficially, the pathway for production of pectolinarigenin could 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, which does not require that particular enzyme, may be selected for production of pectolinarigenin. Thus, the modularity of the biosynthetic pathway is especially beneficial.

[0083] In certain aspects, the invention provides method for bioproduction of pectolinarigenin, the method comprising providing one or more enzymes, wherein the one or more enzymes result in transformation of naringenin to pectolinarigenin through one or more intermediates. The method for production of pectolinarigenin includes cell-based and cell-free methods.

[0084] In certain aspects of the invention, the method for production of pectolinarigenin comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of naringenin to pectolinarigenin through one or more intermediates. In certain embodiments, the genetic modifications in the host cell may also include one or more genetic modifications to enhance the production of naringenin. In certain embodiments, engineered host cell comprises genetic modifications to enhance the production of naringenin or any precursor leading to production of naringenin in the engineered host cell. The additional genetic modifications leading to increase in production of naringenin may be beneficial for production of pectolinarigenin because the naringenin may be used a substrate for production of pectolinarigenin. The enhanced amount of naringenin would lead to higher yield of pectolinarigenin.

[0085] In certain aspects of the invention, the method for production of pectolinarigenin is a cell- free production of pectolinarigenin, wherein the one or more enzymes in a cell-free medium result in transformation of naringenin to pectolinarigenin through one or more intermediates. The ingredients of the components in the cell-free reaction may be generated in a host cell. For example, the substrate, such as naringenin, may be generated in a cell and the subsequent conversion to pectolinarigenin may be conducted in a cell-free medium. In certain embodiments, the one or more intermediates generated in conversion of naringenin to pectolinarigenin may be generated in an engineered host cell, and those one or more intermediates may be subsequently converted to pectolinarigenin by one or more enzymes in a cell-free medium.

[0086] In certain aspects, the method comprises a combination of: (i) an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of naringenin to pectolinarigenin or one or more intermediates, and (ii) cell-free production comprising one or more enzymes resulting in transformation of naringenin or a substrate to pectolinarigenin or one or more intermediates for synthesis of pectolinarigenin. In certain embodiments, engineered host cell comprises genetic modifications to enhance the production of naringenin or any precursor leading to production of naringenin in the engineered host cell.

[0087] In certain embodiments of the invention, the one or more intermediates are selected from a group consisting of: apigenin, scutellarein, hispidulin, isosakuranetin, carthamidin, 6- methoxycarthamidin, scutellarein, 4-methoxycarthamidin, acacetin, 4’,6-methoxynaringenin, 4’- methoxyscutellarein, and any combination thereof. FIG. 1 provides exemplary schematic pathways for conversion of naringenin to pectolinarigenin. As evident from FIG. 1, the methods of the invention provide a modular pathway for conversion of naringenin to pectolinarigenin. The pathway selected for production of pectolinarigenin will determine the identity of the one or more intermediates selected in the process. Each individual segment of the pathway may be controlled by the enzymes selected for that conversion. In certain embodiments, the methods of the invention provide the use of one or more enzymes to mediate the conversion of the starting material to one or more intermediates, or pectolinarigenin. In certain embodiments, the one or more enzymes selected from the group consisting of: flavone synthase (FNSI), flavone hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and any combination thereof.

[0088] For example, in certain embodiments, the intermediates are apigenin, scutellarein, and hispidulin. In certain embodiments, the method provides a pathway, wherein, naringenin is converted to apigenin; apigenin is converted to scutellarein; scutellarein is converted to hispidulin; and hispidulin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. For example, as demonstrated in FIG. 2, the pathway provided comprises flavone synthase (FNSI) mediated conversion of naringenin to apigenin; flavone hydroxylase cytochrome P450 (P450) mediated conversion of apigenin to scutellarein; O- methyltransferase (OMT) mediated conversion of scutellarein to hispidulin; and O- methyltransferase (OMT) mediated conversion of hispidulin to pectolinarigenin. Any of these aforementioned steps may be conducted either in an engineered host cell or a cell-free medium.

[0089] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are apigenin, scutellarein, and 4 ’-methoxy scutellarein. The invention provides that naringenin is converted to apigenin; apigenin is converted to scutellarein; scutellarein is converted to 4’-methoxyscutellarein; 4’-methoxyscutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0090] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are apigenin, scutellarein, and hispidulin. The invention provides that naringenin is converted to apigenin; apigenin is converted to scutellarein; scutellarein is converted to hispidulin; hispidulin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0091] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are apigenin, acacetin, and 4’-methoxyscutellarein. The invention provides that naringenin is converted to apigenin; apigenin is converted to acacetin; acacetin is converted to 4 ’-methoxy scutellarein; 4’-methoxyscutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0092] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are isosakuranetin, 4’-methoxycarthamidin, and 4’, 6- m ethoxy naringenin. The invention provides that naringenin is converted to isosakuranetin; isosakuranetin is converted to 4’ -methoxy carthamidin; 4’ -methoxy carthamidin is converted to 4’,6-methoxynaringenin; 4’,6-methoxynaringenin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0093] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are isosakuranetin, 4’ -methoxy carthamidin, and 4’- methoxyscutellarein. The invention provides that naringenin is converted to isosakuranetin; isosakuranetin is converted to 4’ -methoxy carthamidin; 4’-methoxy carthamidin is converted to 4’- methoxyscutellarein; 4’ -methoxy scutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0094] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are isosakuranetin, acacetin, and 4’ -methoxy scutellarein. The invention provides that naringenin is converted to isosakuranetin; isosakuranetin is converted to acacetin; acacetin is converted to 4’ -methoxy scutellarein; 4’-methoxyscutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0095] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, 6-methoxycarthamidin, and hispidulin. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to 6- methoxy carthamidin; 6-methoxycarthamidin is converted to hispidulin; hispidulin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0096] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, 6-methoxycarthamidin, and 4’,6- m ethoxy naringenin. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to 6-methoxycarthamidin; 6-methoxycarthamidin is converted to 4’,6- methoxynaringenin; 4’,6-methoxynaringenin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0097] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, 6-methoxycarthamidin, and 4’- methoxyscutellarein. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to 6-methoxycarthamidin; 6-methoxycarthamidin is converted to 4’- methoxyscutellarein; 4’ -methoxy scutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0098] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, scutellarein, and hispidulin. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to scutellarein; scutellarein is converted to hispidulin; hispidulin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0099] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, scutellarein, and 4’ -methoxy scutellarein. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to scutellarein; scutellarein is converted to 4’-methoxyscutellarein; 4’-methoxyscutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, 4’ -methoxy carthamidin, and 4’, 6- methoxynaringenin. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to 4’-methoxycarthamidin; 4’-methoxycarthamidin is converted to 4’, 6- methoxynaringenin; 4’,6-methoxynaringenin is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0100] In certain embodiments, the one or more intermediates involved in conversion of naringenin to pectolinarigenin are carthamidin, 4’-methoxycarthamidin, and 4’- methoxyscutellarein. The invention provides that naringenin is converted to carthamidin; carthamidin is converted to 4’-methoxycarthamidin; 4’-methoxycarthamidin is converted to 4’- methoxyscutellarein; 4’ -methoxy scutellarein is converted to pectolinarigenin. In certain embodiments, one or more these steps may be conducted by one or more enzymes. In certain embodiments, one or more these steps may be conducted by one or more enzymes.

[0101] In certain embodiments, the invention provides method of production of pectolinarigenin comprises engineered host cells, wherein the host cells comprise one or more genetic modifications. In certain embodiments, the engineered host cell could be prepared from yeast, bacteria, or mammalian cells. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the one or more enzymes are introduced in the host organism by integration into genome of the host organism or on a plasmid. In certain embodiments, the one or more genetic modifications in the engineered host cell are any of the modifications described above. For example, the one or more genetic modification may be overexpression or endogenous enzymes or expression of a variant of an enzyme involved in the conversion of naringenin to pectolinarigenin. The genetic modifications include, without limitation, overexpression or expression of a variant of : flavone synthase (FNSI), flavone hydroxylase cytochrome P450 (P450), O-methyltransferase (OMT), and / or any combination thereof.

[0102] In certain embodiments, the one or more genetic modifications in the host cell may include any genetic modification to increase the availability of naringenin. For example, the one or more genetic modifications in an engineered host cell may be selected from a group consisting of: acetyl - CoA carboxylase (ACC), tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chaicone synthase (CHS), chaicone isomerase (CHI), and / or any combination thereof. In certain embodiments, the genetic modifications may comprise expressing a variant of one or more of these enzymes. In certain embodiments, the one or more genetic modifications to increase the production of naringenin are provided in PCT7US2022 / 024591, which is incorporated in its entirety by reference.

[0103] In certain aspects, the invention provides methods of cell-free production of pectolinarigenin. The cell-free medium for the conversion of naringenin to pectolinarigenin is a cell lysate. In certain embodiments, the cell lysate is a cell lysate from cells from a host organism expressing the one or more enzymes. The one or more enzymes may be the enzymes involved in conversion of naringenin to pectolinarigenin. In certain embodiments, the host organisms expressing the one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes. In certain embodiments, the method of the invention further comprising lysing cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of pectolinarigenin.

[0104] In certain embodiments, cell-free medium comprises: buffer, magnesium chloride, one or more substrates, one or more cofactors, the one or more enzymes, and / or water.

[0105] In certain embodiments, the buffer in the cell-free medium maintains the pH of the reaction mixture in the optimal range. In certain embodiments, the pH of the reaction medium is from about 5.5 to about 10. In certain embodiments, the pH of the reaction medium is from about 6 to about 9.5. In certain embodiments, the pH of cell-free medium is from about 6 to about 9. In certain embodiments, the pH of cell-free medium is from about 6 to about 8. In certain embodiments, the concentration of the buffer in the cell-free medium is from about 1 mM to about 1000 mM. In certain embodiments, the concentration of the buffer is from about 2.5 mM to about 750 mM. In certain embodiments, the concentration of the buffer is from about 5 mM to about 100 mM. In certain embodiments, the buffer concentration is from about 20 mM to about 100 mM. In certain embodiments, the buffer is a phosphate buffer.

[0106] In certain embodiments, the method of the invention comprises conversion of one or more substrates to pectolinarigenin. In certain embodiments, the one or more substrates are present in a concentration of from about 1 mM to about 500 mM. In certain embodiments, the one or more substrates are present in a concentration of from about 1 mM to about 200 mM. In certain embodiments, the one or more substrates are present in a concentration of from about 10 mM to about 100 mM. In certain embodiments, the substrate used for production of pectolinarigenin is naringenin.

[0107] In certain embodiments, the cell-free medium comprises magnesium chloride. The magnesium chloride may be present in the range of from about 1 mM to about 50 mM. In certain embodiments, the cell-free medium comprises magnesium chloride in the range of from about 1 mM to about 25 mM. In certain embodiments, the cell-free medium comprises magnesium chloride in the range of from about 1 mM to about 20 mM. In certain embodiments, the cell-free medium comprises magnesium chloride in the range of from about 5 mM to about 25 mM.

[0108] In certain embodiments, the cell-free medium comprises the lysate from host microbe with the one or more enzymes for the cell-free reaction medium. In certain embodiments, the cell-free medium comprises lysate with the one or more enzymes in a concentration of from about 1% (v / v) to about 50% (v / v). In certain embodiments, the cell-free medium comprises lysate with the one or more enzymes in a concentration of from about 5% (v / v) to about 40% (v / v). In certain embodiments, the cell-free medium comprises lysate with the one or more enzymes in a concentration of from about 10% (v / v) to about 30% (v / v). In certain embodiments, the cellmedium further comprises the one or more enzymes are present in the range of from about 0.1 pM to about 5 mM. In certain embodiments, the one or more enzymes are present in the range of from about 0.1 pM to about 1 mM. In certain embodiments, the one or more enzymes are present in the range of from about 1 pM to about 100 pM.

[0109] In certain embodiments, the methods of the invention provide a use of one or more cofactors to facilitate the reactions leading to conversion of naringenin to pectolinarigenin. In certain embodiments, wherein the one or more cofactors are present in a concentration of about 1 mM to about 500 mM. In certain embodiments, the one or more cofactors are present in a concentration of about 1 mM to about 200 mM. In certain embodiments, the one or more cofactors are present in a concentration of about 1 mM to about 10 mM.

[0110] In certain embodiments, the cell-free reaction for production of polyphenols is conducted for the requisite duration till the desired quantity of the polyphenols are produced in the said reaction. In certain embodiments, the reaction for cell-free production of polyphenols is carried out for a duration of from about 0.1 hours to about 20 hours. In certain embodiments, the reaction for cell -free production of polyphenols is carried out for a duration of from about 0.5 hours to about 20 hours. In certain embodiments, reaction for cell-free production polyphenols is carried out for a duration of from about 1 hours to about 15 hours.

[0111] In certain embodiments, the reaction of the cell-free medium is adjusted for an optimal yield of polyphenols to be produced. In certain embodiments, the temperature of the cell-free medium is from about 20 °C to about 40 °C.

[0112] As described above, the invention provides that all or a certain number of steps for the conversion of a substrate to naringenin may be carried out in an engineered host organism. The host organism may be a bacteria, yeast, or a mammalian cell. In certain embodiments, the engineered host organism are incubated in a culture medium. The methods can further include recovering pectolinarigenin, or one of the intermediates leading to formation of pectolinarigenin, from the culture medium, whole culture, or cells.

[0113] The culture comprises cells engineered for the production of pectolinarigenin in a culture medium. In various embodiments the engineered cells can be prokaryotic or eukaryotic cells. The culture medium includes at least one carbon source that is also an energy source. Exemplary carbon sources include glucose, glycerol, sucrose, fructose, and xylose. Such carbon sources may be purified or crude, including a biomass comprising glycerol, for example, crude glycerol produced as a byproduct of biodiesel production from com waste. In addition, the culture medium can include one or more other carbon sources or compounds to increase precursor generation or cofactor supply such as, without limitation, tyrosine, phenylalanine, coumaric acid, acetate, malonate, succinate, glycine, bicarbonate, biotin, naringenin, 5-aminolevulinic acid, thiamine, pantothenate, alpha-ketoglutarate, and ascorbate. In some embodiments, tyrosine and coumaric acid are provided in the culture medium. In some embodiments, tyrosine, alpha- ketoglutarate, 5-aminolevulinic acid, and ascorbate are provided in the culture medium.

[0114] Culture conditions can include aerobic, microaerobic or any combination alternating aerobic / microaerobic growth conditions. Further, culture conditions can include shake flasks, fermentation, and other large scale culture procedures. An exemplary growth condition for achieving a flavonoid product include aerobic or microaerobic fermentation conditions. The culture conditions can be scaled up and grown continuously for manufacturing flavonoid product. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation. In an exemplary batch fermentation protocol, the cells are grown in a bioreactor that is well controlled for growth temperature, oxygen, pH, carbon sources, and other compounds. The desired temperature can be from, for example, 20-37 °C, depending on the growth characteristics of the production cells and desired conditions for the fermented products. The pH of the bioreactor can be controlled to range from 5-8 or left uncontrolled in some cases. The batch fermentation period can last in the range of several hours to several days, for examples, 8 to 96 hours. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit to remove cells and cell debris. The cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. In certain embodiments, the engineered host organisms, and their genetic modifications are provided in PCT / US2022 / 024591, which is incorporated in its entirety by reference.

[0115] In certain embodiments, the reaction is conducted in a bubble column reactor, wherein the one or more enzymes are in a solution. In certain embodiments, the reaction is conducted in a packed bed reactor, wherein the one or more enzymes are immobilized.

[0116] In certain aspects, the methods provided in the invention may be carried out in any reactor suitable for carrying out the cell-free production of pectolinarigenin. In certain embodiments, the reaction for cell-free production of pectolinarigenin is conducted in a bubble column reactor / bioreactor. In certain embodiments, in the bubble column reactor / bioreactor, the one or more enzymes involved in cell-free production of pectolinarigenin are in a solution. In certain embodiments, the reaction for cell-free production of pectolinarigenin is conducted in a bubble column reactor / bioreactor comprises the lysate from the host organism. In certain embodiments, it is advantageous to use the bubble column reactor / bioreactor for cell-free production of pectolinarigenin when the reaction mixture involves the lysate (or lysate with cellular debris removed) from the host cell organisms in which the one or more enzymes responsible for cell-free production of pectolinarigenin were utilized. In certain embodiments, the reaction for cell-free production of pectolinarigenin is conducted in a packed bed reactor / bioreactor. In certain embodiments, the one or more enzymes are immobilized in the packed bed reactor / bioreactor. The packed bed reactors / bioreactors are preferred for the purified enzymes playing a role in cell-free production of pectolinarigenin. In certain embodiments, the one or more enzymes may be immobilized in a single reactor / bioreactor. In certain other embodiments, the one or more enzymes may be immobilized in different reactors / bioreactors, wherein these reactors / bioreactors are linked sequentially. In certain embodiments, the bioreactor system provided in PCT / US2021 / 064049, incorporated by reference in its entirety.

[0117] In certain aspects, the invention provides A method for bioproduction of pectolinarigenin, the method comprising: providing one or more enzymes, wherein the one or more enzymes result in transformation of one or more substrates to pectolinarigenin optionally through one or more intermediates. In certain embodiments, one or more substrates are selected from the group consisting of naringenin, apigenin, scutellarein, hispidulin, or any combination thereof. In certain embodiments, the substrate is apigenin. In certain embodiments, the substrate is scutellarein. In certain embodiments, the substrate is hispidulin.

[0118] In certain aspects, the method comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of the one or more substrate to pectolinarigenin optionally through one or more intermediates. In certain embodiments, the invention provides a method for cell-free production of pectolinarigenin, wherein the one or more enzymes in a cell-free medium result in transformation of the one or more substrate to pectolinarigenin optionally through one or more intermediates.

[0119] In certain aspects, the invention provides a composition for bioproduction of pectolinarigenin, wherein the composition comprises one or more enzymes, wherein the one or more enzymes result in transformation of one or more substrates to pectolinarigenin optionally through one or more intermediates. In certain embodiments, one or more substrates are selected from the group consisting of naringenin, apigenin, scutellarein, hispidulin, or any combination thereof. In certain embodiments, one or more substrates are selected from the group consisting of naringenin, apigenin, scutellarein, hispidulin, or any combination thereof. In certain embodiments, the substrate is apigenin. In certain embodiments, the substrate is scutellarein. In certain embodiments, the substrate is hispidulin. In certain embodiments, the composition comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of the one or more substrate to pectolinarigenin optionally through one or more intermediates. In certain embodiments, the composition comprises one or more enzymes in a cell-free medium that results in transformation of the one or more substrates to pectolinarigenin optionally through one or more intermediates.

[0120] In certain aspects, the invention provides a method for production of pectolinarigenin, the method comprises: providing an engineered host cell that comprises one or more genetic modifications, wherein the one or more genetic modifications result in enzymatic transformation of glycerol to pectolinarigenin through one or more intermediates. In certain embodiments, the engineered host cell comprises one or more genetic modifications to enhance the production of naringenin from glycerol the engineered host cell. In certain embodiments, the engineered host cell comprises one or more genetic modifications to enhance the transformation of naringenin to pectolinarigenin in the engineered host cell through one or more intermediates. In certain embodiments, the one or more genetic modification is expression or overexpression of one or more enzymes. In certain embodiments, one or more enzymes are selected from the group consisting of: FNSI, P450, OMT, and any combination thereof. In certain embodiments, one or more enzymes are selected from the group consisting of expression or overexpression of: FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI, and any combination thereof. In certain embodiments, the engineered host cell is E. colt.

[0121] In certain aspects, the invention provides a composition for production of pectolinarigenin, wherein the composition comprises an engineered host cell that comprises one or more genetic modifications, wherein the one or more genetic modifications result in enzymatic transformation of glycerol to pectolinarigenin through one or more intermediates. In certain embodiments, the engineered host cell comprises one or more genetic modifications to enhance the production of naringenin from glycerol the engineered host cell. In certain embodiments, the engineered host cell comprises one or more genetic modifications to enhance the transformation of naringenin to pectolinarigenin in the engineered host cell through one or more intermediates. In certain embodiments, the one or more genetic modification is expression or overexpression of one or more enzymes. In certain embodiments, one or more enzymes are selected from the group consisting of: FNSI, P450, OMT, and any combination thereof. In certain embodiments, one or more enzymes are selected from the group consisting of expression or overexpression of: FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI, and any combination thereof. In certain embodiments, the engineered host cell is E. coli.

[0122] The methods provided in the invention are advantageous over other conventional methods of production of polyphenols. In certain embodiments, the methods of the invention provide cell- free production of polyphenols. Because the methods of the invention are conducted in cell-free medium, they provide significant economic efficiency by reducing the cost of production of polyphenols in other conventional methods. In certain embodiments, because the reaction for production of polyphenols is conducted from the lysates from the host organisms expressing the one or more enzymes involved in the reaction, the methods of the invention are cost-efficient. In certain embodiments, the methods of the invention do not involve the purification of the one or more enzymes. Because purification of individual enzymes is not required in the methods of the invention, provides further economic efficiency by reducing the cost that would have been otherwise required in purifying individual enzymes.

[0123] In some embodiments, the isolated pectolinarigenin has a purity of 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%.

[0124] In other embodiments, the isolated pectolinarigenin has a purity of from about 10% to 95%, or from about 10% to 90%, or from about 10% to 80% or from about 10% to 70%, or from about 10% to 60%, or from about 10% to 50%, or from about 10% to 40%, or from about 20% to 95%, or from about 20% to 90%, or from about 20% to 80% or from about 20% to 70%, or from about 20% to 60%, or from about 20% to 50%, or from about 20% to 40%, or from about 50% to 95%, or from about 50% to 90%, or from about 50% to 80% or from about 50% to 70%, or from about 50% to 60%.

[0125] In certain embodiments, the flavone synthase is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to any one of enzymes selected from the group consisting of:

[0126]

[0127] In certain embodiments, flavone hydroxylase cytochrome P450 is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to any one of enzymes selected from the group consisting of:

[0128] In certain embodiments, O-methyltransferase is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to any one of enzymes selected from the group consisting of:

[0129] In certain embodiments, acetyl-CoA carboxylase (ACC) is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to any one of enzymes selected from the group consisting of:

[0130] In certain preferred embodiments, acetyl-CoA carboxylase (ACC) is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to the enzyme provided in SEQ ID NO: 70. In certain embodiments, tyrosine ammonia lyase (TAL) is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to any one of enzymes selected from the group consisting of:

[0131] In certain preferred embodiments, tyrosine ammonia lyase (TAL) is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to the enzyme provided in SEQ ID NO: 71.

[0132] In certain embodiments, 4-coumarate-CoA ligase (4CL) is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to any one of enzymes selected from the group consisting of:

[0133] In certain preferred embodiments, 4-coumarate-CoA ligase (4CL) is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to the enzyme provided in SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and / or SEQ ID NO: 76.

[0134] In certain embodiments, chaicone synthase (CHS) is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to any one of enzymes selected from the group consisting of:

[0135] In certain preferred embodiments, chaicone synthase (CHS) is an enzyme with an an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to the enzyme provided in SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and / or SEQ ID NO: 81.

[0136] In certain embodiments, chaicone isomerase (CHI) is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to any one of enzymes selected from the group consisting of:

[0137] In certain preferred embodiments, chaicone isomerase (CHI) is an enzyme with an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or completely identical to the enzyme provided in SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, and / or SEQ ID NO: 86.

[0138] The methods of production of pectolinarigenin provided in the invention are beneficial because they provide for an economic and efficient pathway for synthesis of pectolinarigenin. Advantageously, the methods of the invention do not extensive processes for extraction and / or purification of pectolinarigenin from the plants or roots. The methods requiring extraction of the product from plants has complications, such as the processes leading to low yields, a higher number of by-products, and expensive purification and separation protocols.

[0139] The methods of the current invention beneficially provide cell-based and cell-free approaches for bioproduction of pectolinarigenin. In particular, the product titers for pectolinarigenin produced by the methods of the invention are higher than the product titer produced by any other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least two-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least five-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least ten-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least hundred-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least five- hundred-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least thousand-fold higher than other known methods for production of pectolinarigenin. In certain embodiments, the product titers produced by methods of the invention are at least five-thousand-fold higher than other known methods for production of pectolinarigenin.

[0140] In other beneficial aspects of the method of the invention, the methods of the invention provide economically efficient methods of production of pectolinarigenin. Because the methods of the invention also provide for cell-free production of pectolinarigenin, it leads to decreased costs of purification of the reaction products. Moreover, in certain aspects of the invention, the one or more enzymes are included in the same reaction mixture, i.e., the enzymes do not need to be isolated after being expressed in the host organisms. The one or more enzymes could be expressed in the host organisms at the same time and be utilized for the cell-free production without the need of any additional purification and / or separation steps.

[0141] In certain other beneficial aspects of the invention, the use of the one or more enzymes for the cell-free production of polyphenols leads to high yield of the product to be produced. The one more enzyme may be further modified to optimize the yield of the product being produced in the cell-free reaction.

[0142] In certain aspects, the invention provides compositions for the production of pectolinarigenin from one or more substrates. In certain embodiments, the compositions provided in the invention are compositions suitable for preparing pectolinarigenin from one or more substrates. In certain embodiments, the compositions of the invention comprise engineered host organisms, wherein the engineered host organisms comprise genetic modifications for production of pectolinarigenin from naringenin through one or more intermediates. In certain embodiments, the compositions of the invention comprise a cell-free medium, which may comprise one or more enzymes, for production of pectolinarigenin from naringenin through one or more intermediates.

[0143] In certain embodiments, the compositions of the invention comprise a combination of engineered host cells and a cell-free medium for preparation of pectolinarigenin.

[0144] VII. Examples Example 1: Cell-Free bioproduction of pectolinarigenin:

[0145] The general reaction conditions for cell-free production of pectolinarigenin are provided below. The substrate can be naringenin, or any of the other compounds shown in FIG. 1. The enzymes can be any combination of one or more flavone synthase (FNSI), flavone hydroxylase cytochrome P450 (P450), and O-methyltransferase (OMT). Generally, enzymes were expressed from a pET28 vector in BL21-DE3 cells. Starter cultures were incubated overnight at 37 °C, and then diluted 1 : 100 in TB. After incubation at 37 °C until ODeoo = 0.8 to 1.2, cultures were induced with IPTG (0.1 to 1 mM) and incubated at 20 to 30 °C overnight. Cultures were then centrifuged and the supernatant discarded. Cells were resuspended in lysis buffer (50 mM sodium phosphate pH 7.5, 300 mM sodium chloride, and 20 mM imidazole). Resuspended cells were sonicated and centrifuged to generate clarified lysate.

[0146] Cell-free conversion of scutellarein to hispidulin: Reactions were performed by combining 200 mM Tris-HCl pH 7.5, 2 mM DTT (dithiothreitol), 4 mM magnesium chloride, 0.5 mM SAM (S- adenosyl methionine), 50% v / v lysate of cells expressing an OMT enzyme (SEQ ID 31 to 69), and 0.2 mM scutellarein. Reactions were incubated for 1 hour at 30 °C and then quenched using an equal volume of methanol and filtered. Samples were analyzed by HPLC, demonstrating production of up to 79 pM hispidulin.

[0147] Cell-free conversion of hispidulin to pectolinarigenin: Reactions were performed by combining 25 mM HEPES pH 7.5, 5 mM magnesium chloride, 0.2 mM SAM (S-adenosyl methionine), 0.5 mM NAD+, 25% v / v lysate of cells expressing an OMT enzyme (SEQ ID 31 to 69), 20% v / v lysate of cells expressing an SAHH enzyme (S-adenosyl homocysteine hydrolase, EC 3.13.2.1), and 0.3 mM hispidulin. Reactions were incubated overnight at 25 °C and then quenched using an equal volume of methanol and filtered. Samples were analyzed by HPLC, demonstrating production of up to 130 pM pectolinarigenin.

[0148] FIG. 3 provides HPLC chromatograms for the formation of hispidulin and pectolinarigenin. FIG. 3A provides the HPLC plot demonstrating the production of hispidulin from scutellarein. FIG. 3B provides the HPLC plot demonstrating the production of pectolinarigenin from hispidulin.

[0149] Example 2: Cellular bioproduction of pectolinarigenin: Cell-based production of pectolinarigenin from glycerol: An A. coli cell derived from MG1655 was engineered to produce naringenin. The cells may further comprise the genetic modifications comprising overexpression of ACC, TAL, 4CL, CHS, and CHI. To this strain, flavone synthase (FNSI), flavone hydroxylase cytochrome P450 (P450), and two O-methyltransferase (OMT) were added on the chromosome, to produce pectolinarigenin when glycerol was supplied in the culture medium. Cells of an OD 2.0 were cultured in a 48-well plate at 30 degrees for 22 hours with a shaking speed of 600 RPM in minimal medium supplied with 2% glycerol, trace elements, 65 mg / L 5-aminoleuvinic 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 supernatant was analyzed for pectolinarigenin and pathway intermediates by HPLC. FIG. 4 shows the HPLC chromatograms at 288nm of (a) standards, (b) starting strain producing NAR with titers up to 440 pM, (c) NAR- producing strain with an FNSI to make apigenin at a titer of up to 210 pM with 152 pM NAR leftover, and (d) NAR-producing strain with an FNSI, a P450 and two OMTs to make the final product pectolinarigenin at a titer of up to 30 pM, with acacetin produced as a byproduct at 38 pM.

[0150] Cell production of apigenin from glycerol: An E. coli cell derived from MG1655 was engineered to produce naringenin. The cells optionally further comprise the genetic modifications comprising overexpression of ACC, TAL, 4CL, CHS, and CHI. To this cell, FNSI was added on the chromosome to produce apigenin when glycerol was supplied in the culture medium. Cells of an OD 2.0 were cultured in a 48-well plate at 30 degrees for 22 hours with a shaking speed of 600 RPM in minimal medium supplied with 2% glycerol, trace elements, 65 mg / L 5-aminoleuvinic 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 supernatant was analyzed for apigenin and pathway intermediates by HPLC. FIG. 4 shows the HPLC chromatograms at 288nm of (a) standards, (b) starting strain producing NAR with titers up to 440 pM, (c) NAR- producing strain with an FNSI to make apigenin at a titer of up to 210 pM with 152 pM NAR leftover, and (d) NAR-producing strain with an FNSI, a P450 and two OMTs to make the final product pectolinarigenin at a titer of up to 30 pM, with acacetin produced as a byproduct at 38 pM.

[0151] Cell production of pectolinarigenin from hispidulin: An E. coli BL21 cell was engineered to express an OMT. To this, hispidulin was fed to the culture. Cells of an OD 2.0 were cultured in a 48-well plate at 30 degrees for 22 hours with a shaking speed of 600 RPM in minimal medium supplied with 2% glycerol, trace elements and 500 pM of hispidulin. Cell cultures were extracted with 50% (v / v) methanol and centrifuged for 15 minutes. The supernatant was analyzed for pectolinarigenin and pathway intermediates by HPLC. FIG. 5 shows the HPLC chromatograms at 330 nm of (a) PNAR standard, (b) hispidulin standard, (c) BL21 cells, and (d) BL21 cells with OMT to produce PNAR at a titer up to 250 pM.

[0152] The table below provides exemplary sequences for one or more enzymes that may be used in accordance with the methods of the invention.

[0153] Incorporation by Reference

[0154] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, publicly accessible databases, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0155] Equivalents

[0156] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

CLAIMS:

1. A method for bioproduction of pectolinarigenin, the method comprising providing one or more enzymes, wherein the one or more enzymes result in transformation of naringenin to pectolinarigenin through 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 genetic modifications for expression of one or more enzymes resulting in transformation of naringenin to pectolinarigenin through one or more intermediates.

3. The method of claim 1, wherein the method is a cell-free production of pectolinarigenin, wherein the one or more enzymes in a cell-free medium result in transformation of naringenin to pectolinarigenin through one or more intermediates.

4. The method of claim 1, wherein the method comprises a combination of: (i) an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of naringenin to pectolinarigenin or one or more intermediates, and (ii) cell-free production comprising one or more enzymes resulting in transformation of naringenin or a substrate to pectolinarigenin or one or more intermediates for synthesis of pectolinarigenin.

5. The method of claim 2, wherein the engineered host cell comprises genetic modifications to enhance the production of naringenin or any precursor leading to production of naringenin in the engineered host cell.

6. The method of any of claims 1-4, wherein the one or more intermediates are selected from a group consisting of: apigenin, scutellarein, hispidulin, isosakuranetin, carthamidin, 6-methoxycarthamidin, scutellarein, 4-methoxy carthamidin, acacetin, 4’, 6- methoxynaringenin, 4’ -metehoxy scutellarein, and any combination thereof.

7. The method of any of claims 1-4, wherein the intermediates are apigenin, scutellarein, and hispidulin.

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

9. The method of any of claims 1-4, wherein the one or more intermediates are apigenin, scutellarein, and 4’-methoxyscutellarein.

10. The method of any of claims 1-4, wherein the one or more intermediates are apigenin, scutellarein, and hispidulin.

11. The method of any of claims 1-4, wherein the one or more intermediates are apigenin, acacetin, and 4’ -methoxy scutellarein.

12. The method of any of claims 1-4, wherein the one or more intermediates are isosakuranetin, 4’-methoxycarthamidin, and 4’,6-methoxynaringenin.

13. The method of any of claims 1-4, wherein the one or more intermediates are isosakuranetin, 4’-methoxycarthamidin, and 4 ’-meth oxy scutellarein.

14. The method of any of claims 1-4, wherein the one or more intermediates are isosakuranetin, acacetin, and 4’-methoxyscutellarein.

15. The method of any of claims 1-4, wherein the one or more intermediates are carthamidin, 6-methoxycarthamidin, and hispidulin.

16. The method of any of claims 1-4, wherein the one or more intermediates are carthamidin, 6-methoxycarthamidin, and 4’,6-methoxynaringenin.

17. The method of any of claims 1-4, wherein the one or more intermediates are carthamidin, 6-methoxycarthamidin, and 4’ -methoxy scutellarein.

18. The method of any of claims 1-4, wherein the one or more intermediates are carthamidin, scutellarein, and hispidulin.

19. The method of any of claims 1-4, wherein the one or more intermediates are carthamidin, scutellarein, and 4’-methoxyscutellarein.

20. The method of any of claims 1-4, wherein the one or more intermediates are carthamidin, 4’-methoxycarthamidin, and 4’,6-methoxynaringenin.

21. The method of any of claims 1-4, wherein the one or more intermediates are carthamidin, 4’-methoxycarthamidin, and 4’-methoxyscutellarein.

22. The method of claim 2, wherein the engineered host cell is bacteria, yeast, and / or mammalian cells.

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 cells from a host organism expressing the one or more enzymes.

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

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

27. The method of claim 26, wherein the host organisms expressing the one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes.

28. The method of claim 27, further comprising lysing cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of pectolinarigenin.

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

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

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

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

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

34. The method of 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 from about 1 mM to about 50 mM.

37. The method of claim 29, wherein magnesium chloride is present in the range of from 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 in a concentration of about 1 mM to about 500 mM.

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

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

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

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

44. The method of claim 29, wherein the one or more cofactors are present in 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 from about 0.1 pM to about 5 mM.

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

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

48. The method of claim 29, wherein reaction for cell-free production of pectolinarigenin is conducted for a duration of from about 0.1 hours to about 20 hours.

49. The method of claim 29, wherein reaction for cell-free production of pectolinarigenin is conducted for a duration of from about 1 hour to about 10 hours.

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

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

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

53. The method of any of claims 1-4, wherein the enzyme is flavone synthase enzyme with an amino acid sequence at least 95% identical to any one of enzymes selected from the group consisting of SEQ ID 1 to 15.

54. The method of any of claims 1-4, wherein the enzyme is flavone hydroxylase cytochrome P450 enzyme with an amino acid sequence at least 95% identical to any one of enzymes selected from the group consisting of SEQ ID 16 to 30.

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

56. A method for bioproduction of pectolinarigenin, the method comprising: providing one or more enzymes, wherein the one or more enzymes result in transformation of one or more substrates to pectolinarigenin optionally through one or more intermediates.

57. The method of claim 56, wherein the one or more substrates is selected from the group consisting of naringenin, apigenin, scutellarein, hispidulin, or any combination thereof.

58. The method of claim 56, wherein the one or more substrate is apigenin.

59. The method of claim 56, wherein the one or more substrate is scutellarein.

60. The method of claim 56, wherein the one or more substrate is hispidulin.

61. The method of any of claims 56 - 60, wherein the method comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of the one or more substrate to pectolinarigenin optionally through one or more intermediates.

62. The method of any of claims 56 - 60, wherein the method is a cell-free production of pectolinarigenin, wherein the one or more enzymes in a cell-free medium result in transformation of the one or more substrate to pectolinarigenin optionally through one or more intermediates.

63. A composition for bioproduction of pectolinarigenin, wherein the composition comprises one or more enzymes, wherein the one or more enzymes result in transformation of one or more substrates to pectolinarigenin optionally through one or more intermediates.

64. The composition of claim 63, wherein the one or more substrates is selected from the group consisting of naringenin, apigenin, scutellarein, hispidulin, or any combination thereof.

65. The composition of claim 63, wherein the one or more substrate is apigenin.

66. The composition of claim 63, wherein the one or more substrate is scutellarein.

67. The composition of claim 63, wherein the one or more substrate is hispidulin.

68. The composition of any of claims 63 - 67, wherein the composition comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for expression of one or more enzymes resulting in transformation of the one or more substrate to pectolinarigenin optionally through one or more intermediates.

69. The composition of any of claims 63 - 67, wherein the composition is for a cell-free production of pectolinarigenin, wherein the one or more enzymes in a cell-free medium result in transformation of the one or more substrate to pectolinarigenin optionally through one or more intermediates.

70. A method for production of pectolinarigenin, the method comprises: providing an engineered host cell that comprises one or more genetic modifications, wherein the one ormore genetic modifications result in enzymatic transformation of glycerol to pectolinarigenin through one or more intermediates.

71. The method of claim 70, wherein the engineered host cell comprises one or more genetic modifications to enhance the production of naringenin from glycerol the engineered host cell.

72. The method of claim 71, wherein the engineered host cell comprises one or more genetic modifications to enhance the transformation of naringenin to pectolinarigenin in the engineered host cell through one or more intermediates.

73. The method of claims 71 or 72, wherein the one or more genetic modification is expression or overexpression of one or more enzymes.

74. The method of claim 73, wherein the one or more enzymes 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 selected from the group consisting of expression or overexpression of: FNSI, P450, OMT, ACC, TAL, 4CL, CHS, CHI, and any combination thereof.

76. The method of any of claims 70 - 75, wherein the engineered host cell is E. coli.

77. A composition for production of pectolinarigenin, wherein the composition comprises an engineered host cell that comprises one or more genetic modifications, wherein the one or more genetic modifications result in enzymatic transformation of glycerol to pectolinarigenin through one or more intermediates.

78. The composition of claim 77, wherein the engineered host cell comprises one or more genetic modifications to enhance the production of naringenin from glycerol the engineered host cell.

79. The composition of claim 72, wherein the engineered host cell comprises one or more genetic modifications to enhance the transformation of naringenin to pectolinarigenin in the engineered host cell through one or more intermediates.

80. The composition of claims 78 or 79, wherein the one or more genetic modification is expression or overexpression of one or more enzymes.

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

83. The composition of any of claims 70 - 75, wherein the engineered host cell is E. coli.

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