Methods and compositions for bioproduction of phloroglucinol

Abstract

The current invention is directed to methods and compositions for bioproduction of phloroglucinol.

Description

[0001]Patent Application METHODS AND COMPOSITIONS FOR BIOPRODUCTION OF PHLOROGLUCINOL I. Field of Invention: The invention is related to materials and methods for production of phloroglucinol. The invention provides methods and materials for cell-free or cell-based production of phloroglucinol. II. Background Phloroglucinol is a polyphenolic compound that chemical structure includes an aromatic phenyl ring with three hydroxyl groups. Phloroglucinol (1,3,5-trihydroxybenzene) and its derivatives are widely used in commerce. Phloroglucinol and its derivatives, e.g., trimethylphloroglucinol, are used as pharmaceutical agents, e.g., as antispasmodics. Phloroglucinol is used as a starting material or intermediate in pharmaceutical, microbicide, and other organic syntheses. Phloroglucinol is used as a stain for microscopy samples that contain lignin (e.g., wood samples), and it is used in the manufacture of dyes, including leather, textile, and hair dyes. It is used in the manufacture of adhesives and as an epoxy resin curing agent, and in the preparation of explosives, e.g., the thermally- and shock-stable high explosive, 1,3,4- triamino-2,4,6-trinitrobenzene (TATB). Phloroglucinol also functions as an antioxidant, stabilizer, and corrosion resistance agent, and is utilized as a coupling agent for photosensitive duplicating paper, as a substitute for silver iodide in rain-making, as a bone sample decalcifying agent, and as a floral preservative. Phloroglucinol can also be converted to resorcinol by catalytic hydrogenation. III. Summary of the Invention Currently, phloroglucinol is commercially produced by chemical organic synthesis using caustics and high temperatures, beginning with petroleum-derived starting materials and creating much environmentally problematic waste. As a result, it would be beneficial to provide more efficient and cleaner processes for the production of phloroglucinol. One possible solution might be to provide a biosynthetic route for production of phloroglucinol. Biosynthetic production of compounds related to phloroglucinol has been reported in plants, algae, and microbes, e.g.: acetyl phloroglucinols from Pseudomonas spp.; hyperforins, hyperfoliatins, hyperjovinols, and hyperatomarins from Hypericum spp.; pallidusol, dehydropallidusol, pallidol, mallopallidol, and Patent Application homomallopallidol from Mallotus spp.; garcinielliptones from Garcinia spp.; flavaspidic acids from Dryopteris spp.; macrocarpals and sideroxylonals from Eucalyptus spp.; 1,3,5- trimethoxybenzene from Rosa spp.; as well as phloroglucinol-containing glycosides and phlorotannins. However, there are no known commercially viable methods for production of phloroglucinols from these sources. However, production of phloroglucinol is reported in such plants and microbes as merely a degradation product of more complex, and thus less abundant and / or more costly, starting materials. See, e.g.: L. Schoefer et al., Appl. Environ. Microbiol.70(10):6131-37 (2004); D. Baas & J. Rétey, Eur. J. Biochem.265:896-901 (1999). In addition, microbial biosynthetic production of di-acetyl phloroglucinols has been proposed as a means for improving the anti-fungal activity of recombinant bacteria to be released into the agricultural environment as biocontrol agents against phytopathogens. See U.S. Pat. No.6,051,383, Thomashow et al., issued Apr.18, 2000; and M. G. Bangera & L. S. Thomashow, J Bact.181(10):3155-63 (1999). Yet, a route of anabolic biosynthetic production of phloroglucinol, e.g., from inexpensive starting materials such as glucose, is not shown. Moreover, there is no known cell-free method for bioproduction of phloroglucinol. The methods provided in the current invention disclose cell-free production of phloroglucinol by converting a substrate to phloroglucinol. In certain embodiments, the substrate is phloretin. In certain other embodiments, the substrate is naringenin chalcone. The methods provided in the invention related to cell-free production of phloroglucinol are economic and reliable as compared to other conventional methods of production of phloroglucinol. In addition, the methods provided in the invention provide higher titer values of phloroglucinol from these processes as compared to phloroglucinol produced from other conventional methods. Moreover, phloroglucinol is toxic to cells. Thus, the cell-free production methods of the current invention are beneficial because they are cell-free and thus there is no concern for toxicity of phloroglucinol due to the cells. The methods of the invention are also environmentally friendly, and they have a smaller manufacturing footprint as compared to the other conventional methods of production of phloroglucinol. Patent Application In certain embodiments, the methods of the invention also result in production of phloretic acid and / or p-coumaric acid. Phloretic acid and p-coumaric acid may be purified from the reaction mixture. In certain aspects, the method of the present invention provides a method for bioproduction of phloroglucinol, the method comprising providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of a substrate to phloroglucinol. In certain embodiments, the substrate is phloretin. In certain embodiments, the substrate is naringenin chalcone. In certain embodiments, the method results in production of phloroglucinol and phloretic acid. In certain embodiments, the method results in production of phloroglucinol and p-coumaric acid. In certain embodiments, the one or more enzyme is a hydrolase enzyme. In certain embodiments, the hydrolase enzyme is phloretin hydrolase. In certain embodiments of the invention, phloretin is transformed to phloroglucinol and phloretic acid by phloretin hydrolase enzyme. The reaction leading to transformation of phloretin to phloroglucinol and phloretic acid is provided in FIG.1. This method is a cell-free method of bioproduction of phloroglucinol. FIG. 2 provides the analytical standards for phloretin, phloroglucinol, and phloretic acid, along with their respective times of retention. FIG.3 provides the HPLC plots demonstrating that phloretin is transformed to phloroglucinol and phloretic acid. The plot on top demonstrates the composition of reaction mixture at 1 minute, and the bottom panel describes the reaction mixture after 60 minutes. As evident from the plot in the lower panel of FIG. 3, phloretin is transformed to phloroglucinol and phloretic acid after 60 minutes of the start of the reaction in presence of a phloretin hydrolase enzyme. In certain embodiments of the invention, naringenin chalcone is transformed to phloroglucinol and p-coumaric acid by phloretin hydrolase enzyme. The reaction leading to transformation of naringenin chalcone to phloroglucinol and p-coumaric acid is provided in FIG. 4. This method is also a cell-free method of bioproduction of phloroglucinol. In certain embodiments, the methods of the invention involve additional steps to increase the availability of naringenin chalcone, which is the substrate for bioproduction of phloroglucinol. The additional steps may include any of the steps provided in PCT / US2022 / 024591, which is incorporated by reference in its entirety. For example, the additional steps may include overexpression of chalcone Patent Application synthase (EC 2.3.1.74), which transforms malonyl-CoA and p-coumaroyl-CoA to naringenin chalcone. In certain embodiments, both chalcone synthase and phloretin hydrolase are expressed in the same host organism prior to lysis. In certain embodiments, chalcone synthase and phloretin hydrolase are expressed in different same host organism prior to lysis. In certain embodiments, the method further provides for removal of chalcone synthase from the reaction mixture. In certain embodiments, chalcone synthase is not removed from the reaction mixture after the lysing the cells. In certain embodiments, the cell-free medium 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. In certain embodiments, the host organism is selected from a group consisting of: bacteria, yeast, and / or mammalian cells. In certain embodiments, wherein 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 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 methods of the invention further comprise lysing of cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of phloroglucinol. In certain embodiments, the one or more enzymes are purified and / or separated from the cell lysate. The purified enzyme may be used for the cell-free reaction. In certain embodiments, the one or more enzymes are not purified from the cell lysate. In these embodiments, the cell lysate comprising the one or more enzymes would be used for conducting the cell-free reaction for the production of phloroglucinol. In certain embodiments, phloretin hydrolase is expressed in a host organism, wherein the host organism is selected from a group consisting of: bacteria, yeast, and / or mammalian cells. In certain embodiments, wherein the phloretin hydrolase enzyme introduced in the host organism by integration into genome of the host organism or on a plasmid. In certain embodiments, the host organisms expressing phloretin hydrolase enzyme are cultured until a pre-determined biomass is achieved to produce the requisite quantity of phloretin hydrolase enzyme. In certain embodiments, the methods of the invention further comprise lysing of cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of phloroglucinol. In Patent Application certain embodiments, phloretin hydrolase enzyme are purified and / or separated from the cell lysate. The purified phloretin hydrolase enzyme may be used for the cell-free reaction. In certain embodiments, phloretin hydrolase enzyme is not purified from the cell lysate. In these embodiments, the cell lysate comprising phloretin hydrolase enzyme would be used for conducting the cell-free reaction for the production of phloroglucinol. In certain embodiments, the one or more enzymes to produce phloroglucinol is phloretin hydrolase. In certain embodiments, phloretin hydrolase used for cell-free production of phloroglucinol is selected from the enzymes having at least 80%, 85%, or 90% amino acid sequence identity from the enzymes provided in Table 1 below. In certain embodiments, phloretin hydrolase used for cell-free production of phloroglucinol is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in Table 1 below. In certain embodiments, phloretin hydrolase used for cell-free production of phloroglucinol is selected from the enzymes having at least 98% amino acid sequence identity from the enzymes provided in Table 1 below. In certain embodiments, phloretin hydrolase used for cell-free production of phloroglucinol is selected from the enzymes provided in Table 1 below. Table 1: UniProt ID Organism Patent Application In certain embodiments, the invention provides engineered phloretin hydrolase enzyme for the cell-free production of phloroglucinol. In certain embodiments, the engineered phloretin hydrolase enzyme is optimized for cell-free production of phloroglucinol. In certain embodiments, engineered phloretin hydrolase enzyme includes genetic modifications. In certain embodiments, the genetic modifications may be selected from a group consisting of: point mutations, insertions, deletions, and / or any other modifications such that those enzymes result in efficient and optimal cell-free production of phloroglucinol. In certain embodiments, the phloretin hydrolase enzyme used in the cell-free production of phloroglucinol is any enzymes disclosed in this application. In certain embodiments, the cell-free medium may further comprise any other additional ingredients required for the cell-free production of phloroglucinol. For example, in certain embodiments, the cell-free medium comprises buffer, phloretin, organic solvent, ascorbic acid / ascorbate, cell lysate, and / or water. In certain embodiments, the buffer used in the cell-free reaction medium is any buffer suitable for the enzymatic reaction. In certain embodiments, the buffer maintains the pH of about 6 to about 12 in the reaction mixture. In certain embodiments, the buffer maintains the pH of about 7 to about 11 in the reaction mixture. In certain embodiments, the buffer maintains the pH of about 8 to about 10 in the reaction mixture. In certain embodiments, the buffer maintains the pH of about 9. In certain embodiments, the buffer maintains the pH of about 10. In certain preferred embodiments, the buffer is a phosphate buffer. In certain embodiments, the buffer is present at a concentration of about 1 mM to about 500 mM. In certain embodiments, the buffer is present at a concentration of about 10 mM to about 400 mM. In certain embodiments, the buffer is present in the reaction mixture at a concentration of about 50 mM to about 300 mM. In certain embodiments, the buffer is present in the reaction mixture at a concentration of about 100 mM to about 200 mM. In certain embodiments, ethanol is present in the reaction mixture in a concentration of about 1% to about 60% (v / v). In certain embodiments, ethanol is present in the reaction mixture in a concentration of about 1% to about 50% (v / v). In certain embodiments, ethanol is present in the reaction mixture in a concentration of about 5% to about 30% (v / v). Patent Application In certain embodiments, the concentration of ascorbic acid / ascorbate in the reaction mixture is from about 0.5 mM to about 30 mM. In certain embodiments, the concentration of ascorbic acid / ascorbate in the reaction mixture is from about 1 mM to about 20 mM. In certain embodiments, phloretin is present is the in the reaction mixture at a concentration of from about 1 mM to about 1000 mM. In certain embodiments, phloretin is present in the reaction mixture at a concentration of from about 1 mM to about 500 mM. In certain embodiments, phloretin is present in the reaction mixture at a concentration of from about 100 mM to about 400 mM. In certain embodiments, wherein the cell lysate is present in the reaction mixture at a concentration of about including the one or more enzymes is present at a concentration of from about 0.1% (v / v) to about 50% (v / v). In certain embodiments, wherein the cell lysate is present in the reaction mixture at a concentration of about including the one or more enzymes is present at a concentration of from about 1% (v / v) to about 40% (v / v). In certain embodiments, purified isolated phloretin hydrolase enzyme is present in the reaction mixture at a concentration of from about 0.1% (v / v) to about 50% (v / v). In certain embodiments, purified isolated phloretin hydrolase enzyme is present in the reaction mixture at a concentration of from about 1% (v / v) to about 40% (v / v). In certain embodiments, the reaction for production of phloroglucinol is conducted for a duration till the desired quantity of phloroglucinol is obtained. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 10 minutes to about 48 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 10 minutes to about 36 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 20 minutes to about 24 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 0.5 hours to about 20 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 30 minutes to about 20 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 1 hour to about 15 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 2 hour to about 10 hours. In certain preferred embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 2 hours to about 5 hours. In certain preferred Patent Application embodiments, the reaction for cell-free production of phloroglucinol is carried out for about 3 hours. In certain preferred embodiments, the reaction for cell-free production of phloroglucinol is carried out for about 4 hours. In certain preferred embodiments, the reaction for cell-free production of phloroglucinol is carried out for about 5 hours. In certain embodiments, the temperature of the reaction mixture for cell-free production of phloroglucinol is varied to obtain optimal results for the production of phloroglucinol. In certain embodiments, the temperature of the reaction mixture for cell-free production of phloroglucinol is from about 15 ℃ to about 45 ℃. In certain embodiments, the temperature of the reaction mixture for cell-free production of phloroglucinol is from about 20 ℃ to about 40 ℃. In certain embodiments, the temperature of the reaction mixture for cell-free production of phloroglucinol is from about 22.5 ℃ to about 37.5 ℃. In certain embodiments, the temperature of the reaction mixture for cell-free production of phloroglucinol is from about 25 ℃. In certain embodiments, the temperature of the reaction mixture for cell-free production of phloroglucinol is from about 30 ℃. The temperature of the reaction mixture for cell-free production of phloroglucinol may influence the rate of production of phloroglucinol. Consequently, the duration of reaction may be adjusted according to the temperature of the reaction mixture to obtain optimal yield of phloroglucinol in cell-free production of phloroglucinol. 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 phloroglucinol. In certain embodiments, the reaction for cell-free production of phloroglucinol 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 phloroglucinol are in a solution. In certain embodiments, the reaction for cell-free production of phloroglucinol 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 phloroglucinol 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 phloroglucinol were utilized. In certain embodiments, the reaction for cell-free production of phloroglucinol 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 Patent Application packed bed reactors / bioreactors are preferred for the purified enzymes playing a role in cell-free production of phloroglucinol. 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. The methods provided in the invention are advantageous over other conventional methods of production of phloroglucinol. In certain embodiments, the methods of the invention provide for cell-free production of phloroglucinol. Because the methods of the invention are conducted in cell- free medium, they provide significant economic efficiency by reducing the cost of production of phloroglucinol in other conventional methods. In certain embodiments, because the reaction for production of phloroglucinol 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 particular, 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, it results in economic efficiency by reducing the cost that would have been otherwise required in purifying individual enzymes. Advantageously, the cell-free production of phloroglucinol using the methods of the invention provides for a much higher concentration of substrates for production as compared to the conventional methods. Moreover, the cell-free production of phloroglucinol provided herein provides a significantly higher titer value for phloroglucinol as compared to the conventional methods. The higher titer values of phloroglucinol provide additional cost advantages for production of phloroglucinol because the higher titer phloroglucinol would provide efficiency in purifying and / or concentrating phloroglucinol from the reaction mixture. In certain embodiments, the methods of the invention provide phloroglucinol titer value of at least 5-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer values at least 10-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer value of at least 50-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer values at least 100-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer values at least 500- Patent Application fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer values at least 1000-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer values at least 5000-fold higher than the conventional methods. In certain aspects, the invention provides compositions for cell-free production of phloroglucinol. The compositions of the invention are utilized for cell-free production of phloroglucinol in accordance with the methods described above. In certain aspects, the invention provides that the phloretin used as a substrate for production of phloroglucinol is produced in a cell. In certain embodiments, phloretin used as a substrate for production of phloroglucinol is produced in an engineered host cell. In certain embodiments, the engineered host cell comprises one or more genetic modifications to increase the production of phloretin through transformation by one or more enzymes of a carbon source by the engineered host cell. In certain embodiments, the carbon source is selected from a group consisting of naringenin or p-coumaric acid. In certain embodiments, the carbon source is provided to the engineered cells of the invention by external supplementation. In certain embodiments, the carbon source may be added to the medium in which the cells are grown. In certain embodiments, the carbon source may be produced by the engineered cells, wherein the engineered cells include one or more genetic modifications designed to increase the production of the carbon source. The methods of the invention provide biosynthetic routes for conversion of naringenin or p-coumaric acid to phloretin through one or more intermediates. The methods of the invention also provide for engineered host cells wherein the host cells include one or more genetic modifications to optimize the production of phloretin and / or a precursor or ingredient used in the production of phloretin. The methods of the invention recognize that it would be beneficial to optimize the production of precursor and / or ingredient in the pathway for production of phloretin because it would increase the overall yield of phloretin. For example, in certain embodiments, the one or more genetic modifications in the engineered host cell may be genetic modifications targeted to increasing the production of naringenin or p-coumaric acid in the engineered host cell. In other embodiments, one or more genetic modifications in the engineered host cell may be genetic modifications targeted to decrease the degradation of naringenin or p-coumaric acid in the engineered host cell. Patent Application The invention further recognizes that one or more the steps involved in the conversion of naringenin or p-coumaric acid to phloretin may be conducted in a cell-free medium. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified flavonone / flavonol-cleaving reductase (FCR) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified enoate reductase (ER) or a homolog thereof, and (iii) any combinations thereof. In certain embodiments, the engineered host cell further comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified 4-coumarate-CoA ligase (4CL) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified chalcone synthase (CHS) or a homolog thereof, (iii) nucleic acid sequences encoding native or modified chalcone isomerase (CHI) or a homolog thereof, (iv) nucleic acid sequences encoding native or tyrosine ammonia lyase (TAL) or a homolog thereof and (v) any combinations thereof. The engineered host cell in the methods of the current invention is any host organism that is suitable for expression of the enzymes involved in the conversion of one or more substrates to phloretin. In certain preferred embodiments, the engineered host cell is E. coli or yeast. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the engineered host cell is yeast. In certain embodiments naringenin and / or naringenin chalcone are converted to phloretin in an engineered host cell through one or more chemical intermediates. In certain embodiments, the one or more enzymes is flavonone / flavonol-cleaving reductase (FCR), which plays a role in transformation of naringenin and / or naringenin chalcone to phloretin through one or more chemical intermediates. The engineered host cells used in the methods of the invention may include one or more genetic modifications listed in WO 2022 / 221392, which is incorporated herein by reference in its entirety. In certain embodiments, p-coumaric acid is converted to phloretin in an engineered host cell through one or more chemical intermediates. In certain embodiments, the one or more enzymes playing a role in transformation of p-coumaric acid to phloretin are: 4-coumarate-CoA ligase Patent Application (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and / or flavonone / flavonol-cleaving reductase (FCR). In certain embodiments, the one or more chemical intermediates formed in the transformation of p-coumaric acid to phloretin are p-coumaroyl-CoA, naringenin chalcone, and naringenin. In certain embodiments, 4-coumarate-CoA ligase (4CL) transforms p-coumaric acid to p-coumaroyl-CoA, which is subsequently transformed by chalcone synthase (CHS) and / or chalcone isomerase (CHI) to naringenin. Naringenin is subsequently transformed to phloretin by flavonone / flavonol-cleaving reductase (FCR). In certain embodiments, p-coumaric acid is converted to phloretin in an engineered host cell through one or more chemical intermediates. In certain embodiments, the one or more enzymes playing a role in transformation of p-coumaric acid to phloretin is eonate reductase (ER). The enzyme eonate reductase transforms p-coumaric acid to phloretic acid. The phloretic acid is subsequently converted to phloretin through one or more intermediates. In certain embodiments, 4-coumarate-CoA ligase (4CL), and / or chalcone synthase (CHS) also play a role in transformation of phloretic acid to phloretin through one or more chemical intermediates. In certain embodiments, the processes for production of phloretin are provided in PCT / US2024 / 039825, which is incorporated by herein by reference in its entirety. An overview of the method of production of phloretin is provided in FIG.5. For example, the method may further comprise wherein phloretin is produced in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of phloretin through transformation by one or more enzymes of a carbon source by the engineered host cell. In certain embodiments, the carbon source is selected from a group consisting of naringenin or p-coumaric acid. In certain embodiments, the carbon source is provided to the engineered cells of the invention by external supplementation. In certain embodiments, the carbon source may added to the medium in which the cells are grown. In certain embodiments, the carbon source may be produced by the engineered cells, wherein the engineered cells include one or more genetic modifications designed to increase the production of the carbon source. The methods of the invention also provide for engineered host cells wherein the host cells include one or more genetic modifications to optimize the production of phloretin and / or a precursor or ingredient used in the production of phloretin. The methods of the invention recognize that it would be beneficial to optimize the production of precursor and / or ingredient in the pathway for production of phloretin because it would increase Patent Application the overall yield of phloretin. For example, in certain embodiments, the one or more genetic modifications in the engineered host cell may be genetic modifications targeted to increasing the production of naringenin or p-coumaric acid in the engineered host cell. In other embodiments, one or more genetic modifications in the engineered host cell may be genetic modifications targeted to decrease the degradation of naringenin or p-coumaric acid in the engineered host cell. The invention further recognizes that one or more the steps involved in the conversion of naringenin or p-coumaric acid to phloretin may be conducted in a cell-free medium. In certain aspects, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of phloroglucinol through transformation by one or more enzymes of a carbon source by the engineered host cell. In certain embodiments, the carbon source is phloretin. In certain embodiments, the carbon source is transformed to phloroglucinol through one or more intermediates. In certain embodiments, one more intermediate is phloretin. In certain embodiments, the carbon source is selected from a group consisting of naringenin, naringenin chalcone, tyrosine, or p-coumaric acid. In certain embodiments, the carbon source is generated by the engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to enhance availability of the carbon source. In certain embodiments, one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under- expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified flavonone / flavonol-cleaving reductase (FCR) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified enoate reductase (ER) or a homolog thereof, (iii) nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof, and (iv) any combinations thereof. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified 4-coumarate-CoA ligase (4CL) enzyme or a homolog Patent Application thereof, (ii) nucleic acid sequences encoding native or modified chalcone synthase (CHS) or a homolog thereof, (iii) nucleic acid sequences encoding native or modified chalcone isomerase (CHI) or a homolog thereof, (iv) nucleic acid sequences encoding native or tyrosine ammonia lyase (TAL) or a homolog thereof, (v) nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof, and (vi) any combinations thereof. In certain embodiments, the engineered host cell comprises nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof. In certain embodiments, the engineered host cell is E. coli or yeast. In certain aspects, the invention provides a method for production of phloroglucinol in an engineered host cell. In certain embodiments, the engineered host cell comprises one or more genetic modifications to increase the production of phloroglucinol through transformation by one or more enzymes of a substrate by the engineered host cell. In certain embodiments, the substrate is phloretin. In certain embodiments, the substrate is naringenin chalcone. In certain embodiments, the engineered host cell expresses one or more enzymes for conversion of the substrate to phloroglucinol. In certain embodiments, the one or more enzymes expressed in the engineered host cell for conversion of a substrate to phloroglucinol is phloretin hydrolase. In certain embodiments, phloretin hydrolase expressed in the engineered host cell for production of phloroglucinol is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in Table 1 below. In certain embodiments, phloretin hydrolase expressed in the engineered host cells for production of phloroglucinol is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in Table 1 below. Table 1: UniProt ID Organism Patent Application Q715L4 Eubacterium ramulus B8NDM8 Aspergillus flavus In certain aspects, the invention provides a method for production of phloroglucinol in an engineered host cell wherein the engineered host cell comprises one or more genetic modifications to increase the production of phloretin through transformation by one or more enzymes of a carbon source by the engineered host cell. Thus, in certain embodiments, the engineered host cell comprises one or more genetic modifications to produce phloretin and phloroglucinol. This approach is advantageous because it would provide efficiency for conversion of a carbon source to phloroglucinol, wherein phloretin is an intermediate in the production of phloroglucinol. This approach provides cost benefits in the production of phloroglucinol because there would be no requirement for separation and / or purification of phloretin. In certain embodiments, the processes for production of phloroglucinol in an engineered host cell comprises one more genetic modification listed in U.S. Prov. Appl. No. 63 / 529,002, which is incorporated by herein by reference in its entirety. In addition to these genetic modifications, the engineered host cell may additionally include one or more genetic modifications for conversion of phloretin to phloroglucinol in the engineered host cell. In certain embodiments, the engineered host cell expresses one or more enzymes for conversion of the substrate to phloroglucinol. In certain embodiments, the one or more enzymes expressed in the engineered host cell for conversion of a substrate to phloroglucinol is phloretin hydrolase. In certain embodiments, phloretin hydrolase expressed in the engineered host cell for production of phloroglucinol is selected from the enzymes having at least 80%, 90%, or 95% amino acid sequence identity from the enzymes provided in Table 1 above. IV. Brief Description of the Drawings: FIG. 1 provides a schematic of enzymatically mediated conversion of phloretin to phloroglucinol and phloretic acid. Patent Application FIG.2 provides analytical standards for phloretin, phloroglucinol, and phloretic acid, along with their respective times of retention. FIG. 3 provides the HPLC plots demonstrating that phloretin is transformed to phloroglucinol and phloretic acid. FIG.4 provides a schematic of enzymatically mediated conversion of naringenin chalcone to phloroglucinol and p-coumaric acid. FIG.5 provides a schematic of process of generation of phloretin. V. Detailed Description The present application provides compositions and methods for production of phloroglucinol in a cell-free medium, wherein one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of an organic material to phloroglucinol. The one or more enzymes may be engineered. The engineered enzyme may be non-naturally occurring. 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.” As used herein, “reaction solution” may refer to all components necessary for enzyme- based chemical transformation. This is typically, but not limited to, buffering agent, salts, cofactor, and substrate (starting material). 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 Patent Application “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. 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. 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. A host cell, organism, or microorganism engineered to express or overexpress a gene or nucleic acid sequence, or to overexpress an enzyme or polypeptide has been genetically engineered through recombinant DNA technology to include a gene or nucleic acid sequence that does not naturally encode the enzyme or polypeptide or to express an endogenous gene at a level that exceeds its level of expression in a non-altered cell. As nonlimiting examples, a host cell, organism, or microorganism engineered to express or overexpress a gene or a nucleic acid sequence, or to overexpress an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene. Overexpression of a gene can also be by increasing the copy number of a gene in the cell or organism. Similarly, a host cell, organism, or microorganism engineered to under-express or to have reduced expression of a gene, nucleic acid sequence, or to under-express an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene. Specifically included are gene disruptions, which include any insertions, deletions, or sequence mutations into or of the gene or a portion of the gene that affect its expression or the activity of the encoded polypeptide. Gene disruptions include “knockout” mutations that eliminate expression of the gene. Modifications to under-express a gene also include modifications to regulatory regions of the gene that can reduce its expression. 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 Patent Applicationas bacteria), isolated via chromatography and / or any other methods, and then added into abioreactor 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. Phloroglucinol Phloroglucinol is a polyphenolic compound that chemical structure includes an aromaticphenyl ring with three hydroxyl groups. Phloroglucinol (1,3,5-trihydroxybenzene) and itsderivatives are widely used in commerce. The structure of phloroglucinol (1,3,5-trihydroxybenzene) is provided below: Phloroglucinol and its derivatives, e.g., trimethylphloroglucinol, are used as pharmaceutical agents, e.g., as antispasmodics. Phloroglucinol is used as a starting material or intermediate in pharmaceutical, microbicide, and other organic syntheses. Phloroglucinol is used as a stain for microscopy samples that contain lignin (e.g., wood samples), and it is used in the manufacture of dyes, including leather, textile, and hair dyes. It is used in the manufacture of adhesives and as an epoxy resin curing agent, and in the preparation of explosives, e.g., thethermally- and shock-stable high explosive, 1,3,4-triamino-2,4,6-trinitrobenzene (TATB).Phloroglucinol also functions as an antioxidant, stabilizer, and corrosion resistance agent, and is utilized as a coupling agent for photosensitive duplicating paper, as a substitute for silver iodide in rain-making, as a bone sample decalcifying agent, and as a floral preservative. Phloroglucinol can also be converted to resorcinol by catalytic hydrogenation. Patent Application In certain embodiments, phloroglucinol is commonly used to alleviate pain. In certain embodiments, phloroglucinol is used as an gastrointestinal drug with antispasmodic effects, used in the following cases: symptomatic treatment of pain associated with dysfunction of the digestive system and biliary tract; treatment of acute pain caused by urinary tract spasm: Renal colic; symptomatic treatment of pain caused by spasms in gynecology; supportive treatment of contractions during pregnancy combined with convalescence. Preparation of phloroglucinol: Currently, phloroglucinol is commercially produced by chemical organic synthesis using caustics and high temperatures, beginning with petroleum-derived starting materials and creating much environmentally problematic waste. For example, a process for preparation involves synthesis of phloroglucinol by adding hydrochloric acid to 2,4,6-trinitrobenzoic acid. Org. Synth.1929, 9, 74. Phloroglucinols are secondary metabolites that occur naturally in certain plant species. It is also produced by brown algae and bacteria. Formylated phloroglucinol compounds (euglobals, macrocarpals and sideroxylonals) can be found in Eucalyptus species. Hyperforin and adhyperforin are two phloroglucinols found in St John's wort. Humulone is a phloroglucinol derivative with three isoprenoid side-chains. Two side-chains are prenyl groups and one is an isovaleryl group. Humulone is a bitter-tasting chemical compound found in the resin of mature hops (Humulus lupulus). Brown algae, such as Ecklonia stolonifera, Eisenia bicyclis or species in the genus Zonaria, produce phloroglucinol and phloroglucinol derivatives. Brown algae also produce a type of tannins known as phlorotannins. The bacterium Pseudomonas fluorescens produces phloroglucinol, phloroglucinol carboxylic acid and diacetylphloroglucinol. In Pseudomonas fluorescens, biosynthesis of phloroglucinol is performed with a type III polyketide synthase. The synthesis begins with the condensation of three malonyl-CoAs. Then decarboxylation followed by the cyclization of the activated 3,5-diketoheptanedioate product leads to the formation of phloroglucinol. The enzyme pyrogallol hydroxytransferase uses 1,2,3,5-tetrahydroxybenzene and 1,2,3-trihydroxybenzene (pyrogallol) to produce 1,3,5-trihydroxybenzene (phloroglucinol) and 1,2,3,5- Patent Application tetrahydroxybenzene. It is found in the bacterium species Pelobacter acidigallici. The enzyme phloroglucinol reductase uses dihydrophloroglucinol and NADP+ to produce phloroglucinol, NADPH, and H+. It is found in the bacterium species Eubacterium oxidoreducens. The legume- root modulating, microsymbiotic nitrogen-fixing bacterium species Bradyrhizobium japonicum is able to degrade catechin with formation of phloroglucinol carboxylic acid, further decarboxylated to phloroglucinol, which is dehydroxylated to resorcinol and hydroxyquinol. As a result, it would be beneficial to provide more efficient and cleaner processes for the production of phloroglucinol. One possible solution might be to provide a biosynthetic route for production of phloroglucinol. Biosynthetic production of compounds related to phloroglucinol has been reported in plants, algae, and microbes, e.g.: acetyl phloroglucinols from Pseudomonas spp.; hyperforins, hyperfoliatins, hyperjovinols, and hyperatomarins from Hypericum spp.; pallidusol, dehydropallidusol, pallidol, mallopallidol, and homomallopallidol from Mallotus spp.; garcinielliptones from Garcinia spp.; flavaspidic acids from Dryopteris spp.; macrocarpals and sideroxylonals from Eucalyptus spp.; 1,3,5-trimethoxybenzene from Rosa spp.; as well as phloroglucinol-containing glycosides and phlorotannins. However, there are no known commercially viable methods for production of phloroglucinols from these sources. However, production of phloroglucinol is reported in such plants and microbes as merely a degradation product of more complex, and thus less abundant and / or more costly, starting materials. See, e.g.: L. Schoefer et al., Appl. Environ. Microbiol.70(10):6131-37 (2004); D. Baas & J. Rétey, Eur. J. Biochem.265:896-901 (1999). In addition, microbial biosynthetic production of di-acetyl phloroglucinols has been proposed as a means for improving the anti-fungal activity of recombinant bacteria to be released into the agricultural environment as biocontrol agents against phytopathogens. See U.S. Pat. No.6,051,383, Thomashow et al., issued Apr.18, 2000; and M. G. Bangera & L. S. Thomashow, J Bact.181(10):3155-63 (1999). Yet, a route of anabolic biosynthetic production of phloroglucinol, e.g., from inexpensive starting materials such as glucose, is not shown. Moreover, there is no known cell-free method for bioproduction of phloroglucinol. The methods provided in the current invention disclose cell-free production of phloroglucinol by converting a substrate to phloroglucinol. In certain embodiments, the substrate is phloretin. In certain other embodiments, the substrate is naringenin chalcone. The methods Patent Application provided in the invention related to cell-free production of phloroglucinol are economic and reliable as compared to other conventional methods of production of phloroglucinol. In addition, the methods provided in the invention provide higher titer values of phloroglucinol from these processes as compared to phloroglucinol produced from other conventional methods. Moreover, phloroglucinol is toxic to cells. Thus, the cell-free production methods of the current invention are beneficial because they are cell-free and thus there is no concern for toxicity of phloroglucinol to the cells. The methods of the invention are also environmentally friendly and they have a smaller manufacturing footprint as compared to the other conventional methods of production of phloroglucinol. In certain embodiments, the methods of the invention also result in production of phloretic acid and / or p-coumaric acid. Phloretic acid and p-coumaric acid may be purified from the reaction mixture. In certain aspects, the method of the present invention provides a method for bioproduction of phloroglucinol, the method comprising providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of a substrate to phloroglucinol. In certain embodiments, the substrate is phloretin. In certain embodiments, the substrate is naringenin chalcone. In certain embodiments, the method results in production of phloroglucinol and phloretic acid. In certain embodiments, the method results in production of phloroglucinol and p-coumaric acid. In certain embodiments, the one or more enzyme is a hydrolase enzyme. In certain embodiments, the hydrolase enzyme is phloretin hydrolase. In certain embodiments of the invention, phloretin is transformed to phloroglucinol and phloretic acid by phloretin hydrolase enzyme. The structure of phloretin is provided below: to hydrolase mediated transformation of phloretin to phloroglucinol and phloretic acid is provided in FIG. 1. This method is a cell-free method of bioproduction of phloroglucinol. FIG. 2 provides the analytical standards for phloretin, Patent Application phloroglucinol, and phloretic acid, along with their respective times of retention. FIG.3 provides the HPLC plots demonstrating that phloretin is transformed to phloroglucinol and phloretic acid. The plot on top demonstrates the composition of reaction mixture at 1 minute, and the bottom panel describes the reaction mixture after 60 minutes. As evident from the plot in the lower panel of FIG. 3, phloretin is transformed to phloroglucinol and phloretic acid after 60 minutes of the start of the reaction in presence of a phloretin hydrolase enzyme. In certain embodiments of the invention, naringenin chalcone is transformed to phloroglucinol and p-coumaric acid by phloretin hydrolase enzyme. The structure of naringenin chalcone is provided below: of naringenin chalcone to phloroglucinol and p- coumaric acid is provided in FIG. 4. This method is also a cell-free method of bioproduction of phloroglucinol. In certain embodiments, the methods of the invention involve additional steps to increase the availability of naringenin chalcone, which is the substrate for bioproduction of phloroglucinol. The additional steps may include any of the steps provided in PCT / US2022 / 024591, which is incorporated by reference in its entirety. For example, the additional steps may include overexpression of chalcone synthase (EC 2.3.1.74), which transforms malonyl-CoA and p-coumaroyl-CoA to naringenin chalcone. In certain embodiments, both chalcone synthase and phloretin hydrolase are expressed in the same host organism prior to lysis. In certain embodiments, chalcone synthase and phloretin hydrolase are expressed in different same host organism prior to lysis. In certain embodiments, the method further provides for removal of chalcone synthase from the reaction mixture. In certain embodiments, chalcone synthase is not removed from the reaction mixture after the lysing the cells. Patent Application In certain embodiments, chalcone synthase used for production of phloroglucinol is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in Table 3 of PCT / US2022 / 024591, reproduced below: Organism GenBank Accession Number Petunia hybrida AAF602971 Patent Application In certain embodiments, the cell-free medium 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. In certain embodiments, the host organism is selected from a group consisting of: bacteria, yeast, and / or mammalian cells. In certain embodiments, wherein 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 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 methods of the invention further comprise lysing of cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of phloroglucinol. In certain embodiments, the one or more enzymes are purified and / or separated from the cell lysate. The purified enzyme may be used for the cell-free reaction. In certain embodiments, the one or more enzymes are not purified from the cell lysate. In these embodiments, the cell lysate comprising the one or more enzymes would be used for conducting the cell-free reaction for the production of phloroglucinol. In certain embodiments, phloretin hydrolase is expressed in a host organism, wherein the host organism is selected from a group consisting of: bacteria, yeast, and / or mammalian cells. In certain embodiments, wherein the phloretin hydrolase enzyme introduced in the host organism by integration into genome of the host organism or on a plasmid. In certain embodiments, the host organisms expressing phloretin hydrolase enzyme are cultured until a pre-determined biomass is achieved to produce the requisite quantity of phloretin hydrolase enzyme. In certain embodiments, the methods of the invention further comprise lysing of cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of phloroglucinol. In certain embodiments, phloretin hydrolase enzyme are purified and / or separated from the cell lysate. The purified phloretin hydrolase enzyme may be used for the cell-free reaction. In certain embodiments, phloretin hydrolase enzyme is not purified from the cell lysate. In these embodiments, the cell lysate comprising phloretin hydrolase enzyme would be used for conducting the cell-free reaction for the production of phloroglucinol. In certain embodiments, the one or more enzymes to produce phloroglucinol is phloretin hydrolase. Phloretin hydrolase is an enzyme that is used to catalyze the conversion of phloretin to phloretic acid and phloroglucinol. Phloretin hydrolase (EC 3.7.1.4) is also known to hydrolyses Patent Application other C-acylated phenols related to phloretin. In certain embodiments, phloretin hydrolase used for cell-free production of phloroglucinol is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in Table 1 below. In certain embodiments, phloretin hydrolase used for cell-free production of phloroglucinol is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in Table 1 below. Table 1: UniProt ID Organism A0A4D7YTB6 Rhi bi di b t In certain embodiments, the invention provides engineered phloretin hydrolase enzyme for the cell-free production of phloroglucinol. In certain embodiments, the engineered phloretin hydrolase enzyme is optimized for cell-free production of phloroglucinol. In certain embodiments, engineered phloretin hydrolase enzyme includes genetic modifications. In certain embodiments, the genetic modifications may be selected from a group consisting of: point mutations, insertions, deletions, and / or any other modifications such that those enzymes result in efficient and optimal cell-free production of phloroglucinol. In certain embodiments, the phloretin hydrolase enzyme used in the cell-free production of phloroglucinol is any enzymes disclosed in this application. In certain embodiments, the cell-free medium may further comprise any other additional ingredients required for the cell-free production of phloroglucinol. For example, in certain Patent Application embodiments, the cell-free medium comprises buffer, phloretin, organic solvent, ascorbic acid / ascorbate, cell lysate, and / or water. In certain embodiments, the buffer used in the cell-free reaction medium is any buffer suitable for the enzymatic reaction. In certain embodiments, the buffer maintains the pH of about 6 to about 12 in the reaction mixture. In certain embodiments, the buffer maintains the pH of about 7 to about 11 in the reaction mixture. In certain embodiments, the buffer maintains the pH of about 8 to about 10 in the reaction mixture. In certain preferred embodiments, the buffer is a phosphate buffer. In certain embodiments, the buffer is present at a concentration of about 1 mM to about 500 mM. In certain embodiments, the buffer is present at a concentration of about 10 mM to about 400 mM. In certain embodiments, the buffer is present in the reaction mixture at a concentration of about 50 mM to about 300 mM. In certain embodiments, the buffer is present in the reaction mixture at a concentration of about 100 mM to about 200 mM. In certain embodiments, ethanol is present in the reaction mixture in a concentration of about 1% to about 60% (v / v). In certain embodiments, ethanol is present in the reaction mixture in a concentration of about 1% to about 50% (v / v). In certain embodiments, ethanol is present in the reaction mixture in a concentration of about 5% to about 30% (v / v). In certain embodiments, the concentration of ascorbic acid / ascorbate in the reaction mixture is from about 0.5 mM to about 30 mM. In certain embodiments, the concentration of ascorbic acid / ascorbate in the reaction mixture is from about 1 mM to about 20 mM. In certain embodiments, phloretin is present in the in the reaction mixture at a concentration of from about 1 mM to about 1000 mM. In certain embodiments, phloretin is present in the in the reaction mixture at a concentration of from about 1 mM to about 500 mM. In certain embodiments, phloretin is present in the in the reaction mixture at a concentration of from about 100 mM to about 400 mM. In certain embodiments, wherein the cell lysate is present in the reaction mixture at a concentration of about including the one or more enzymes is present at a concentration of from about 0.1% (v / v) to about 50% (v / v). In certain embodiments, wherein the cell lysate is present in the reaction mixture at a concentration of about including the one or more enzymes is present at a concentration of from about 1% (v / v) to about 40% (v / v). In certain embodiments, purified isolated Patent Application phloretin hydrolase enzyme is present in the reaction mixture at a concentration of from about 0.1% (v / v) to about 50% (v / v). In certain embodiments, purified isolated phloretin hydrolase enzyme is present in the reaction mixture at a concentration of from about 1% (v / v) to about 40% (v / v). In certain embodiments, the reaction for production of phloroglucinol is conducted for a duration till the desired quantity of phloroglucinol is obtained. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 10 minutes to about 48 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 10 minutes to about 36 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 20 minutes to about 24 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 0.5 hours to about 20 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 30 minutes to about 20 hours. In certain embodiments, the reaction for cell-free production of phloroglucinol is carried out from about 1 hour to about 15 hours. In certain embodiments, the temperature of the reaction mixture for cell-free production of phloroglucinol is varied to obtain optimal results for the production of phloroglucinol. In certain embodiments, the temperature of the reaction mixture for cell-free production of phloroglucinol is from about 15 ℃ to about 45 ℃. In certain embodiments, the temperature of the reaction mixture for cell-free production of phloroglucinol is from about 20 ℃ to about 40 ℃. In certain embodiments, the temperature of the reaction mixture for cell-free production of phloroglucinol is from about 22.5 ℃ to about 37.5 ℃. The temperature of the reaction mixture for cell-free production of phloroglucinol may influence the rate of production of phloroglucinol. Consequently, the duration of reaction may be adjusted according to the temperature of the reaction mixture to obtain optimal yield of phloroglucinol in cell-free production of phloroglucinol. 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 phloroglucinol. In certain embodiments, the reaction for cell-free production of phloroglucinol 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 phloroglucinol are in a solution. In certain embodiments, the reaction for cell-free production of phloroglucinol is conducted in a bubble Patent Application 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 phloroglucinol 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 phloroglucinol were utilized. In certain embodiments, the reaction for cell-free production of phloroglucinol 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 phloroglucinol. 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. The methods provided in the invention are advantageous over other conventional methods of production of phloroglucinol. In certain embodiments, the methods of the invention provide for cell-free production of phloroglucinol. Because the methods of the invention are conducted in cell- free medium, they provide significant economic efficiency by reducing the cost of production of phloroglucinol in other conventional methods. In certain embodiments, because the reaction for production of phloroglucinol 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 particular, 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. In certain aspects, the invention provides that the phloretin used as a substrate for production of phloroglucinol is produced in a cell. In certain embodiments, phloretin used as a substrate for production of phloroglucinol is produced in an engineered host cell. In certain embodiments, the engineered host cell comprises one or more genetic modifications to increase the production of phloretin through transformation by one or more enzymes of a carbon source by the engineered host cell. In certain embodiments, the carbon source is selected from a group Patent Application consisting of naringenin or p-coumaric acid. In certain embodiments, the carbon source is provided to the engineered cells of the invention by external supplementation. In certain embodiments, the carbon source may be added to the medium in which the cells are grown. In certain embodiments, the carbon source may be produced by the engineered cells, wherein the engineered cells include one or more genetic modifications designed to increase the production of the carbon source. The methods of the invention provide biosynthetic routes for conversion of naringenin or p-coumaric acid to phloretin through one or more intermediates. The methods of the invention also provide for engineered host cells wherein the host cells include one or more genetic modifications to optimize the production of phloretin and / or a precursor or ingredient used in the production of phloretin. The methods of the invention recognize that it would be beneficial to optimize the production of precursor and / or ingredient in the pathway for production of phloretin because it would increase the overall yield of phloretin. For example, in certain embodiments, the one or more genetic modifications in the engineered host cell may be genetic modifications targeted to increasing the production of naringenin or p-coumaric acid in the engineered host cell. In other embodiments, one or more genetic modifications in the engineered host cell may be genetic modifications targeted to decrease the degradation of naringenin or p-coumaric acid in the engineered host cell. The invention further recognizes that one or more the steps involved in the conversion of naringenin or p-coumaric acid to phloretin may be conducted in a cell-free medium. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified flavonone / flavonol-cleaving reductase (FCR) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified enoate reductase (ER) or a homolog thereof, and (iii) any combinations thereof. In certain embodiments, the flavonone / flavonol-cleaving reductase (FCR) enzyme is an enzyme with an amino acid sequence at least 85%, 90%, 95%, or 100% identical to any one of enzymes selected from the group consisting of: Accession # Organism Patent Application MBN1191749 Dehalococcoidales bacterium AGS82961 Eubacterium ramulus In certain embodiments, the eonate reductase (ER) enzyme with an amino acid sequence is at least 85%, 90%, 95%, or 100% identical to any one of enzymes selected from the group consisting of: Accession # Organism Patent Application A0A4R1R8M8 Hydrogenispora ethanolica OX A0A1G4Q083 Lachnospiraceae bacterium C10 OX In certain embodiments, the engineered host cell further comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified 4-coumarate-CoA ligase (4CL) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified chalcone synthase (CHS) or a homolog thereof, (iii) nucleic acid sequences encoding native or modified chalcone isomerase (CHI) or a homolog thereof, (iv) nucleic acid sequences encoding native or tyrosine ammonia lyase (TAL) or a homolog thereof and (v) any combinations thereof. Tyrosine ammonia-lyase (TAL) can be, for example, a member of the aromatic amino acid deaminase family that catalyzes the elimination of ammonia from L-tyrosine to yield p-coumaric acid. An exemplary tyrosine ammonia lyase is the Saccharothrix espanaensis tyrosine ammonia lyase (TAL). Also considered for use in the engineered cells provided herein are TALs with TALs listed in the table below. TAL homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID:1 that have the activity of a tyrosine ammonia lyase that produces p-coumaric acid from tyrosine. Table: Tyrosine ammonia-lyase Organism GenBank Accession Number Patent Application Similar to tyrosine ammonia-lyase, phenylalanine ammonia-lyase (PAL) can be a member of the aromatic amino acid deaminase family that catalyzes the non-oxidative deamination of L- phenylalanine to form trans-cinnamic acid. An exemplary phenylalanine ammonia-lyase is the Brevibacillus laterosporus phenylalanine ammonia-lyase (PAL). 4-coumarate-CoA ligase (4CL) catalyzes the activation of 4-coumarate to its CoA ester. Considered for use in the engineered cells provided herein are 4CLs of Petroselinum crispum, 4CLs in the Table below, and 4CL homologs of other species, as well as variants of naturally occurring 4CLs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to 4CL listed in the table below. Table: 4-coumarate-CoA ligases Organism GenBank Accession Number The chalcone synthase (CHS) can be, for example, a type III polyketide synthase that sequentially condenses three molecules of malonyl-CoA with one molecule of p-coumaryol-CoA Patent Application to produce the naringenin precursor naringenin chalcone or naringenin. An exemplary chalcone synthase is the chalcone synthase of Petunia x hybrida (CHS). Also considered for use in the engineered cells provided herein are the genes listed in the table below, and CHS homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the enzymes listed in the table below. Table: Chalcone synthases Organism GenBank Accession Number Patent Application Panax notoginseng QKV26463.1 Ranunculus asiaticus AYV994761 Chalcone isomerase (CHI, also referred to as chalcone flavonone isomerase) catalyzes the stereospecific and intramolecular isomerization of naringenin chalcone into its corresponding (2S)-flavanones. Considered for use in the engineered cells provided herein are CHI of Medicago sativa (SEQ ID NO: 6), CHI of Table 4, CHIs with SEQ ID NOS: 41-44, and CHI homologs of other species, as well as variants of naturally occurring CHI having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 6 (CHI, Medicago sativa) that have the activity of a chalcone isomerase. Table 4. Chalcone Isomerases Organism GenBank Accession Number Patent Application Handroanthus impetiginosus PIN05040.1 Lotus japonicus CAD690221 A nucleic acid sequence encoding a CHI can in some embodiments be fused to a nucleic acid sequence encoding a CHS in an engineered cell as provided herein, such that the CHI activity is fused to the chalcone synthase activity, i.e., a fusion protein is produced in the engineered cell that has both condensing and cyclization activities. The engineered host cell in the methods of the current invention is any host organism that is suitable for expression of the enzymes involved in the conversion of one or more substrates to phloretin. In certain preferred embodiments, the engineered host cell is E. coli or yeast. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the engineered host cell is yeast. In certain embodiments naringenin and / or naringenin chalcone are converted to phloretin in an engineered host cell through one or more chemical intermediates. In certain embodiments, the one or more enzymes is flavonone / flavonol-cleaving reductase (FCR), which plays a role in transformation of naringenin and / or naringenin chalcone to phloretin through one or more chemical intermediates. The engineered host cells used in the methods of the invention may include one or more genetic modifications listed in WO 2022 / 221392, which is incorporated herein by reference in its entirety. In certain embodiments, p-coumaric acid is converted to phloretin in an engineered host cell through one or more chemical intermediates. In certain embodiments, the one or more enzymes playing a role in transformation of p-coumaric acid to phloretin are: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and / or flavonone / flavonol-cleaving Patent Application reductase (FCR). In certain embodiments, the one or more chemical intermediates formed in the transformation of p-coumaric acid to phloretin are p-coumaroyl-CoA, naringenin chalcone, and naringenin. In certain embodiments, 4-coumarate-CoA ligase (4CL) transforms p-coumaric acid to p-coumaroyl-CoA, which is subsequently transformed by chalcone synthase (CHS) and / or chalcone isomerase (CHI) to naringenin. Naringenin is subsequently transformed to phloretin by flavonone / flavonol-cleaving reductase (FCR). In certain embodiments, p-coumaric acid is converted to phloretin in an engineered host cell through one or more chemical intermediates. In certain embodiments, the one or more enzymes playing a role in transformation of p-coumaric acid to phloretin is eonate reductase (ER). The enzyme eonate reductase transforms p-coumaric acid to phloretic acid. The phloretic acid is subsequently converted to phloretin through one or more intermediates. In certain embodiments, 4-coumarate-CoA ligase (4CL), and / or chalcone synthase (CHS) also play a role in transformation of phloretic acid to phloretin through one or more chemical intermediates. In certain embodiments, the processes for production of phloretin are provided in PCT / US2024 / 039825, which is incorporated by herein by reference in its entirety. In certain aspects, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of phloroglucinol through transformation by one or more enzymes of a carbon source by the engineered host cell. In certain embodiments, the carbon source is phloretin. In certain embodiments, the carbon source is transformed to phloroglucinol through one or more intermediates. In certain embodiments, one more intermediate is phloretin. In certain embodiments, the carbon source is selected from a group consisting of naringenin, naringenin chalcone, tyrosine, or p-coumaric acid. In certain embodiments, the carbon source is generated by the engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to enhance availability of the carbon source. In certain embodiments, one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under- expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof. Patent Application In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified flavonone / flavonol-cleaving reductase (FCR) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified enoate reductase (ER) or a homolog thereof, (iii) nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof, and (iv) any combinations thereof. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified 4-coumarate-CoA ligase (4CL) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified chalcone synthase (CHS) or a homolog thereof, (iii) nucleic acid sequences encoding native or modified chalcone isomerase (CHI) or a homolog thereof, (iv) nucleic acid sequences encoding native or tyrosine ammonia lyase (TAL) or a homolog thereof, (v) nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof, and (vi) any combinations thereof. In certain embodiments, the engineered host cell comprises nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof. In certain embodiments, the engineered host cell is E. coli or yeast. In certain aspects, the invention provides a method for production of phloroglucinol in an engineered host cell. In certain embodiments, the engineered host cell comprises one or more genetic modifications to increase the production of phloroglucinol through transformation by one or more enzymes of a substrate by the engineered host cell. In certain embodiments, the substrate is phloretin. In certain embodiments, the substrate is naringenin chalcone. In certain embodiments, the engineered host cell expresses one or more enzymes for conversion of the substrate to phloroglucinol. In certain embodiments, the one or more enzymes expressed in the engineered host cell for conversion of a substrate to phloroglucinol is phloretin hydrolase. In certain embodiments, phloretin hydrolase expressed in the engineered host cell for production of phloroglucinol is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in Table 1 below. In certain embodiments, phloretin hydrolase expressed in the engineered host cells for production of phloroglucinol is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in Table 1 below. Table 1: Patent Application UniProt ID Organism A0A4D7YTB6 Rhizobium radiobacter In certain aspects, the invention provides a method for production of phloroglucinol in an engineered host cell wherein the engineered host cell comprises one or more genetic modifications to increase the production of phloretin through transformation by one or more enzymes of a carbon source by the engineered host cell. Thus, in certain embodiments, the engineered host cell comprises one or more genetic modifications to produce phloretin and phloroglucinol. This approach is advantageous because it would provide efficiency for conversion of a carbon source to phloroglucinol, wherein phloretin is an intermediate in the production of phloroglucinol. This approach provides cost benefits in the production of phloroglucinol because there would be no requirement for separation and / or purification of phloretin. In certain embodiments, the processes for production of phloroglucinol in an engineered host cell comprises one more genetic modification listed in PCT / US2024 / 039825, which is incorporated by herein by reference in its entirety. In addition to these genetic modifications, the engineered host cell may additionally include one or more genetic modifications for conversion of phloretin to phloroglucinol in the engineered host cell. In certain embodiments, the engineered host cell expresses one or more enzymes for conversion of the substrate to phloroglucinol. In certain embodiments, the one or more enzymes expressed in the engineered host cell for conversion of a substrate to phloroglucinol is phloretin hydrolase. In certain embodiments, Patent Application phloretin hydrolase expressed in the engineered host cell for production of phloroglucinol is selected from the enzymes having at least 80%, 90%, or 95% amino acid sequence identity from the enzymes provided in Table 1 above. A host cell as provided herein can be a prokaryotic cell or a eukaryotic cell. Eukaryotic cells may be microbial eukaryotic cells, such as, for example, fungal cells or yeast cells. Prokaryotic cells that can be engineered as provided herein include bacterial cells and cyanobacterial cells. Host can be selected based on their ability to take up and utilize particular carbon sources, nitrogen sources, or precursor molecules or may be engineered to take up and utilize molecules that may be added to the culture medium. Nonlimiting examples of suitable microbial hosts for the bio-production of a flavonoid include, but are not limited to, any gram-negative organisms, more particularly a member of the family Enterobacteriaceae, such as E. coli, any gram-positive microorganism, for example Bacillus subtilis, Lactobacillus sp. or Lactococcus sp.; a yeast, for example Saccharomyces cerevisiae, Pichia pastoris or Pichia stipitis; and other groups or microbial species. More particularly, suitable microbial hosts for the bio-production of a flavonoid generally include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, and Saccharomyces. 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 producing phloretin and / or phloroglucinol include aerobic or microaerobic fermentation conditions. The culture conditions can be scaled up and grown continuously for producing phloretin and / or phloroglucinol. 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 ºC, depending on Patent Application 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. To purify phloretin and / or phloroglucinol produced in engineered host cells, the solution containing phloretin and / or phloroglucinol was concentrated and the product was purified via ion exchange or silica-based chromatography. Advantageously, the cell-free production of phloroglucinol provided herein provides a significantly higher titer value for phloroglucinol as compared to the conventional methods. The higher titer values of phloroglucinol provide additional cost advantages for production of phloroglucinol because the higher titer phloroglucinol would provide efficiency in purifying and / or concentrating phloroglucinol from the reaction mixture. In certain embodiments, the methods of the invention provide phloroglucinol titer value of from at least 5-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer value of from at least 10-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer value of from at least 50-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer value of from at least 100-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer value of from at least 500- fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer value of from at least 1000-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide phloroglucinol titer value of from at least 5000-fold higher than the conventional methods. In certain aspects, the invention provides compositions for cell-free production of phloroglucinol. The compositions of the invention are utilized for cell-free production of phloroglucinol in accordance with the methods described above. Patent Application In some embodiments, the isolated phloroglucinol 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%. In other embodiments, the isolated phloroglucinol 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%. VI. Examples Example 1: Bioproduction of phloroglucinol: The reaction conditions for the cell-free production of phloroglucinol from phlortein are provided below: Component Working Range Buffer pH 6-12 50-300 mM FIG. 3 provides the HPLC chromatograms for the formation of phloroglucinol and phloretic acid. Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, publicly accessible databases, have been Patent Application made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Equivalents 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

Patent Application CLAIMS:

1. A method for bioproduction of phloroglucinol, the method comprising providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of a substrate to phloroglucinol.

2. The method of claim 1, wherein the substrate is phloretin.

3. The method of claim 1, wherein the substrate is naringenin chalcone.

4. The method of claim 2, wherein the method results in production of phloroglucinol and phloretic acid.

5. The method of claim 3, wherein the method results in production of phloroglucinol and p-coumaric acid.

6. The method of claims 2 or 3, wherein the one or more enzyme is a hydrolase enzyme.

7. The method of claim 6, wherein the hydrolase enzyme is phloretin hydrolase.

8. The method of claim 1, wherein the cell-free medium is a cell lysate.

9. The method of claim 8, wherein the cell lysate is a cell lysate from cells from a host organism expressing the one or more enzymes.

10. The method of claim 9, wherein the host organism is selected from a group consisting of: bacteria, yeast, and / or mammalian cells.

11. The method of claim 10, wherein the one or more enzymes are introduced in the host organism by integration into genome of the host organism or on a plasmid.Patent Application 12. The method of claim 11, 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.

13. The method of claim 12, further comprising lysing of cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of phloroglucinol.

14. The method of any of claims 8-13, wherein the enzyme is phloretin hydrolase.

15. The method of any of claims 8 – 13, wherein the one or more enzymes are purified and / or separated from the cell lysate.

16. The method of any of claims 8 – 13, wherein the one or more enzymes are not purified from the cell lysate.

17. The method of any of claims 1-16, wherein the cell-free medium further comprises one or more ingredients selected from the group consisting of: buffer, phloretin, organic solvent, ascorbic acid / ascorbate, cell lysate, and / or water.

18. The method of claim 17, wherein the buffer is a phosphate buffer.

19. The method of claim 17, wherein the organic solvent is ethanol.

20. The method of claim 17, wherein the buffer is present in the concentration of from about 10 mM to about 500 mM.

21. The method of claim 17, wherein the buffer is present in the concentration of from about 50 mM to about 300 mM.

22. The method of claim 17, wherein phloretin is present in the concentration of from about 1 mM to about 1000 mM.Patent Application 23. The method of claim 17, wherein phloretin is present in the concentration of from about 1 mM to about 500 mM.

24. The method of claim 17, wherein the concentration of organic solvent is from about 1% to about 50%.

25. The method of claim 17, wherein the concentration of organic solvent is from about 5% to about 40%.

26. The method of claim 17, wherein the concentration of ascorbic acid / ascorbate is from about 0.5 mM to about 30 mM.

27. The method of claim 17, wherein the concentration of ascorbic acid / ascorbate is from about 1 mM to about 20 mM.

28. The method of claim 17, wherein cell lysate including the one or more enzymes is present at a concentration of about 0.1% (v / v) to about 50% (v / v).

29. The method of claim 17, wherein cell lysate including the one or more enzymes is present at a concentration of about 1% (v / v) to about 40% (v / v).

30. The method of claim 17, wherein the one or more enzymes is present at a concentration of about 0.1% (v / v) to about 50% (v / v).

31. The method of claim 17, wherein reaction for cell-free production is carried out for a duration of about 0.5 hours to about 48 hours.

32. The method of claim 17, wherein reaction for cell-free production is carried out for a duration of about 0.5 hours to about 20 hours.

33. The method of claim 17, wherein temperature of the cell-free medium is from about 20 ℃ to about 40 ℃.Patent Application 34. The method of claim 17, wherein temperature of the cell-free medium is from about 25 ℃ to about 35 ℃.

35. The method of claim 1, wherein reaction is conducted in a bubble column reactor, wherein the one or more enzymes are in a solution.

36. The method of claim 1, wherein reaction is conducted in a packed bed reactor, wherein the one or more enzymes are immobilized.

37. The method of claim 7, wherein the phloretin hydrolase is an enzyme with an amino acid sequence at least 95% identical to any one of enzymes selected from the group consisting of: UniProt ID Organism38. The method of claim 5, wherein the p-coumaric acid is further used as a starting material to manufacture p-hydroxy-styrene.Patent Application 39. A composition for the cell-free production of phloroglucinol, wherein the composition comprises: one or more enzymes in a cell-free medium, wherein the one or more results in transformation of a substrate to phloroglucinol.

40. The composition of claim 39, wherein the substrate is phloretin.

41. The composition of claim 39, wherein the substrate is naringenin chalcone.

42. The composition of claim 40, wherein the transformation results in production of phloroglucinol and phloretic acid.

43. The composition of claim 41, wherein the method results in production of phloroglucinol and p-coumaric acid.

44. The composition of claims 40 or 41, wherein the one or more enzyme is a hydrolase enzyme.

45. The composition of claim 44, wherein the hydrolase enzyme is phloretin hydrolase 46. The composition of claim 39, wherein the cell-free medium is a cell lysate.

47. The composition of claim 46, wherein the cell lysate is a cell lysate from cells from a host organism expressing the one or more enzymes.

48. The composition of claim 47, wherein the host organism is selected from a group consisting of: bacteria, yeast, and / or mammalian cells.

49. The composition of claim 48, wherein the one or more enzymes are introduced in the host organism by integration into genome of the host organism or on a plasmid.

50. The composition of claim 49, 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.Patent Application 51. The composition of claim 50, further comprising lysing of cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of phloroglucinol.

52. The composition of any of claims 46-51, wherein the enzyme is phloretin hydrolase.

53. The composition of any of claims 46-51, wherein the one or more enzymes are purified and / or separated from the cell lysate.

54. The composition of any of claims 46-51, wherein the one or more enzymes are not purified from the cell lysate.

55. The composition of any of claims 39-54, wherein the cell-free medium further comprises one or more ingredients selected from the group consisting of: buffer, phloretin, organic solvent, ascorbic acid / ascorbate, cell lysate, and / or water.

56. The composition of claim 55, wherein the buffer is a phosphate buffer.

57. The composition of claim 55, wherein the organic solvent is ethanol.

58. The composition of claim 55, wherein the buffer is present in the concentration of from about 10 mM to about 500 mM.

59. The composition of claim 55, wherein the buffer is present in the concentration of from about 50 mM to about 300 mM.

60. The composition of claim 55, wherein phloretin is present in the concentration of from about 1 mM to about 1000 mM.

61. The composition of claim 55, wherein phloretin is present in the concentration of from about 1 mM to about 500 mM.

62. The composition of claim 55, wherein the concentration of organic solvent is from about 1% to about 50%.Patent Application 63. The composition of claim 55, wherein the concentration of organic solvent is from about 5% to about 40%.

64. The composition of claim 55, wherein the concentration of ascorbic acid / ascorbate is from about 0.5 mM to about 30 mM.

65. The composition of claim 55, wherein the concentration of ascorbic acid / ascorbate is from about 1 mM to about 20 mM.

66. The composition of claim 55, wherein cell lysate including the one or more enzymes is present at a concentration of about 0.1% (v / v) to about 50% (v / v).

67. The composition of claim 55, wherein cell lysate including the one or more enzymes is present at a concentration of about 1% (v / v) to about 40% (v / v).

68. The composition of claim 55, wherein the one or more enzymes is present at a concentration of about 0.1% (v / v) to about 50% (v / v).

69. The composition of claim 55, wherein reaction for cell-free production is carried out for a duration of about 0.5 hours to about 48 hours.

70. The composition of claim 55, wherein reaction for cell-free production is carried out for a duration of about 0.5 hours to about 20 hours.

71. The composition of claim 55, wherein temperature of the cell-free medium is from about 20 ℃ to about 40 ℃.

72. The composition of claim 55, wherein temperature of the cell-free medium is from about 25 ℃ to about 35 ℃.

73. The composition of claim 39, wherein reaction is conducted in a bubble column reactor, wherein the one or more enzymes are in a solution.Patent Application 74. The composition of claim 39, wherein reaction is conducted in a packed bed reactor, wherein the one or more enzymes are immobilized.

75. The composition of claim 45, wherein the phloretin hydrolase is an enzyme with an amino acid sequence at least 95% identical to any one of enzymes selected from the group consisting of: UniProt ID Organism A A4D7YTB Rhi i i76. The method of claim 44, wherein the p-coumaric acid is further used as a starting material to manufacture p-hydroxy-styrene.

77. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of phloroglucinol through transformation by one or more enzymes of a carbon source by the engineered host cell.

78. The engineered host cell of claim 77, wherein the carbon source is phloretin.

79. The engineered host cell of claim 77, wherein the carbon source is transformed to phloroglucinol through one or more intermediates.Patent Application 80. The engineered host cell of claim 79, wherein the one more intermediate is phloretin.

81. The engineered host cell of claim 80, wherein the carbon source is selected from a group consisting of naringenin, naringenin chalcone, tyrosine, or p-coumaric acid.

82. The engineered host cell of claim 77, wherein the carbon source is generated by the engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to enhance availability of the carbon source.

83. The engineered host cell of claim 77, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof.

84. The engineered host cell of claim 77, wherein the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified flavonone / flavonol-cleaving reductase (FCR) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified enoate reductase (ER) or a homolog thereof, (iii) nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof, and (iv) any combinations thereof.

85. The engineered host cell of claim 77, wherein the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified 4-coumarate-CoA ligase (4CL) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified chalcone synthase (CHS) or a homolog thereof, (iii) nucleic acid sequences encoding native or modified chalcone isomerase (CHI) or a homolog thereof, (iv) nucleic acid sequences encoding native or tyrosine ammonia lyase (TAL) or a homolog thereof, (v) nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof, and (vi) any combinations thereof.Patent Application 86. The engineered host cell of claim 77, wherein the engineered host cell comprises nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof.

87. The engineered host cell of claim 77, wherein the engineered host cell is E. coli or yeast.

88. A method for bioproduction of phloroglucinol, wherein the method comprises an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of phloroglucinol through transformation by one or more enzymes of a carbon source by the engineered host cell.

89. The method of claim 88, wherein the carbon source is phloretin.

90. The method of claim 88, wherein the carbon source is transformed to phloroglucinol through one or more intermediates.

91. The method of claim 90, wherein the one more intermediate is phloretin.

92. The method of claim 91, wherein the carbon source is selected from a group consisting of naringenin, naringenin chalcone, tyrosine, or p-coumaric acid.

93. The method of claim 88, wherein the carbon source is generated by the engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to enhance availability of the carbon source.

94. The method of claim 88, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof.

95. The method of claim 88, wherein the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequencesPatent Application encoding native or modified flavonone / flavonol-cleaving reductase (FCR) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified enoate reductase (ER) or a homolog thereof, (iii) nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof, and (iv) any combinations thereof.

96. The method of claim 88, wherein the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding native or modified 4-coumarate-CoA ligase (4CL) enzyme or a homolog thereof, (ii) nucleic acid sequences encoding native or modified chalcone synthase (CHS) or a homolog thereof, (iii) nucleic acid sequences encoding native or modified chalcone isomerase (CHI) or a homolog thereof, (iv) nucleic acid sequences encoding native or tyrosine ammonia lyase (TAL) or a homolog thereof, (v) nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof, and (vi) any combinations thereof.

97. The method of claim 88, wherein the engineered host cell comprises nucleic acid sequences encoding native or modified phloretin hydrolase or a homolog thereof.

98. The method of claim 88, wherein the engineered host cell is E. coli or yeast.

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