Methods and compositions for producing triacetic acid lactone

Abstract

Method and compositions for producing malonyl-CoA and / or triacetic acid lactone within peroxisomes using genetically modified yeast cells to metabolize a substrate containing fatty acids, vegetable oil, or alkanes, and genetically modified organisms are provided. Also provided are methods to generate acetyl-CoA, which is retained in the peroxisomes due to genetic modifications that prevent its export therefrom. A modified enzyme with a peroxisomal targeting sequence catalyzes a reaction between acetyl-CoA and oxaloacetate, producing pyruvate and malonyl-CoA. The methods can include deleting carnitine acetyltransferase (CAT2) alleles to prevent acetyl-CoA export from peroxisomes, and using genes encoding methylmalonyl-CoA carboxyltransferase subunits with peroxisomal targeting sequences. Additionally, the method incorporates 2-pyrone synthase genes to further facilitate production processes.

Description

METHODS AND COMPOSITIONS FOR PRODUCING TRIACETIC ACID LACTONEINCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Ser. No. 63 / 658816, filedJune 11 , 2024 which is hereby incorporated by reference in its entirety.BACKGROUNDField

[0002] The present disclosure relates to methods and compositions of producing malonyl-coenzyme A and triacetic acid lactone. More particularly, the disclosure relates to methods of using, and compositions of, genetically modified yeast cells for producing malonylcoenzyme A and triacetic acid lactone in the peroxisomes thereof using a fatty acid, vegetable oil, and / or alkane feedstock.Description of the Related Art

[0003] Triacetic acid lactone (TAL) was recently described as a “bioprivileged” molecule, defined as a biologically derived chemical intermediate that can be efficiently converted to a diversity of chemical products including both novel molecules and drop-in replacements. Consequently, TAL is a useful precursor for a wide range of compounds and a much-needed biobased substitute for petrochemicals. However, it is prohibitively expensive to use natural sources of TAL, such as Gerbera daisies, to produce sufficient quantities of TAL to support commercial applications. Therefore, there is a need for synthetic means of producing TAL, in particular, for bioengineered microbial production of TAL.SUMMARY

[0004] For purposes of summarizing the disclosure and describing certain advantages that may be achieved, certain objects have been described. Of course, it is to be understood that not necessarily all such objects or advantages may be achieved in accordance with any particular embodiment of the disclosure. Thus, for example, those skilled in the art will recognize that theor group of advantages as taught herein without necessarily achieving other objects or advantages as may be taught or suggested herein.

[0005] Some embodiments provided herein relate to methods of producing malonylcoenzyme A and triacetic acid lactone, and compositions related to the same.

[0006] Some embodiments provided herein relate to methods of producing malonyl- CoA. In some embodiments, the methods include providing a substrate including a feedstock. In some embodiments, the methods include providing a genetically modified yeast cell to the substrate. In some embodiments, the methods include metabolizing the substrate in a peroxisome of the genetically modified yeast cell to produce acetyl-CoA. In some embodiments, the methods include introducing a modified enzyme to the peroxisome. In some embodiments, the methods include catalyzing a reaction in the peroxisome with the modified enzyme to produce malonyl- CoA. In some embodiments, the feedstock is a fatty acid, a vegetable oil, or an alkane. In some embodiments, the modified enzyme includes a peroxisomal targeting sequence that localizes the modified enzyme in the peroxisome. In some embodiments, the modified enzyme is an acyl-CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase. In some embodiments, the genetically modified yeast cell is engineered to prevent export of the acetyl- CoA from the peroxisome. In some embodiments, the genetically modified yeast cell is engineered to delete carnitine acetyltransferase (CAT2) alleles from the genome of the genetically modified yeast cell are deleted. In some embodiments, the genetically modified yeast cell includes a gene encoding methylmalonyl-CoA carboxyltransferase, methylmalonyl-CoA carboxyltransferase 12S subunit, methylmalonyl-CoA carboxyltransferase 5S subunit, or methylmalonyl-CoA carboxyltransferase 1.3S subunit. In some embodiments, the methylmalonyl-CoA carboxyltransferase 12S subunit is tagged with a peroxisomal targeting amino acid sequence. In some embodiments, the peroxisomal targeting amino acid sequence is added to a carboxyl terminus of the methylmalonyl- Co A carboxyltransferase 12S subunit. In some embodiments, the genetically modified yeast cell includes a gene encoding 2-pyrone synthase. In some embodiments, the gene encoding the 2-pyrone synthase is derived from Gerbera spp. In some embodiments, the 2-pyrone synthase is tagged with a peroxisomal targeting amino acid sequence. In some embodiments, the methods include upregulating expression of genes encoding pyruvatemalonyl-CoA hydrolase. In some embodiments, the genetically modified yeast cell is Candida spp. In some embodiments, the genetically modified yeast cell is Candida viswanathii. In some embodiments, the genetically modified yeast cell includes one or more diploid yeast species. In some embodiments, the substrate includes oleic acid. In some embodiments, the substrate includes a co-feed. In some embodiments, the co-feed includes glycerol or acetate. In some embodiments, the feedstock includes a by-product of vegetable oil production.

[0007] Some embodiments provided herein relate to methods of producing malonyl- CoA and pyruvate. In some embodiments, the methods include providing a substrate including a feedstock. In some embodiments, the methods include providing a genetically modified yeast cell to the substrate. In some embodiments, the methods include metabolizing the substrate in a peroxisome of the genetically modified yeast cell to produce acetyl-CoA. In some embodiments, the methods include introducing a modified enzyme to the peroxisome. In some embodiments, the methods include catalyzing a reaction in the peroxisome with the modified enzyme to produce pyruvate and malonyl-CoA. In some embodiments, the methods include directing polyketide synthases to the peroxisome to react with the pyruvate, thereby producing triacetic acid lactone. In some embodiments, the feedstock is fatty acid, vegetable oil, or an alkane. In some embodiments, the modified enzyme is an acyl-CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase. In some embodiments, the genetically modified yeast cell is engineered to prevent export of the acetyl-CoA from the peroxisome. In some embodiments, the methods include adding carboxylating the pyruvate to produce oxaloacetate. In some embodiments, the genetically modified yeast cell is engineered to delete carnitine acetyltransferase (CAT2) alleles from the genome of the genetically modified yeast cell are deleted. In some embodiments, the genetically modified yeast cell includes a gene encoding methylmalonyl-CoA carboxyltransferase, methylmalonyl-CoA carboxyltransferase 12S subunit, methylmalonyl-CoA carboxyltransferase 5S subunit, or methylmalonyl-CoA carboxyltransferase 1.3S subunit. In some embodiments, the methylmalonyl-CoA carboxyltransferase 12S subunit is tagged with a peroxisomal targeting amino acid sequence. In some embodiments, the peroxisomal targeting amino acid sequence is added to a carboxyl terminus of the methylmalonyl-CoA carboxyltransferase 12S subunit. In some embodiments, the genetically modified yeast cell includes a gene encoding 2-pyrone synthase. Insome embodiments, the 2-pyrone synthase is tagged with a peroxisomal targeting amino acid sequence. In some embodiments, the methods include upregulating expression of genes encoding pyruvate carboxylase in the genetically modified yeast cell. In some embodiments, the modified enzyme is malonyl-CoA hydrolase. In some embodiments, the genetically modified yeast cell is Candida spp. In some embodiments, the genetically modified yeast cell is Candida viswanathii. In some embodiments, the genetically modified yeast cells include one or more diploid yeast species. In some embodiments, the substrate includes oleic acid. In some embodiments, the substrate includes a co-feed. In some embodiments, the co-feed includes glycerol or acetate. In some embodiments, the feedstock includes a by-product of vegetable oil production. In some embodiments, the genetically modified yeast cell produces at least 2,000 pg of triacetic acid lactone per unit liter.

[0008] Some embodiments provided herein relate to genetically modified yeast cells. In some embodiments, the cells include a mutation adapted to prevent export of the acetyl-CoA from a peroxisome and a modified enzyme targeted to the peroxisome. In some embodiments, the cells are adapted to produce malonyl-CoA and triacetic acid lactone. In some embodiments, the mutation includes deletion of carnitine acetyltransferase (CAT2) alleles. In some embodiments, the modified enzyme is acyl-CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase, or malonyl-CoA hydrolase. In some embodiments, the genetically modified yeast cell is adapted to grow on a substrate including a feedstock, the feedstock including at least one of a fatty acid, a vegetable oil, and an alkane. In some embodiments, the genetically modified yeast cell includes a gene encoding methylmalonyl-CoA carboxyltransferase, methylmalonyl-CoA carboxyltransferase 12S subunit, methylmalonyl-CoA carboxyltransferase 5S subunit, or methylmalonyl-CoA carboxyltransferase 1.3S subunit, and including the gene includes a tag with a peroxisomal targeting amino acid sequence.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The features and advantages of the methods and compositions described herein will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. These drawings depict only several embodimentsdrawings, similar reference numbers or symbols typically identify similar components, unless context dictates otherwise. In some instances, the drawings may not be drawn to scale.

[0010] FIG. 1 illustrates an example embodiment of acetyl-CoA transport out of the peroxisome and to the mitochondria via acetyl-carnitine as an intermediate.

[0011] FIG. 2 illustrates an example embodiment of malonyl-CoA production from acetyl-CoA and oxaloacetate in the peroxisome.

[0012] FIG. 3 illustrates an example embodiment of quantification of TAL in shake flask fermentations.DETAILED DESCRIPTION

[0013] The figures and the following description relate to various example embodiments by way of illustration only. It should be noted that from the following discussion, example embodiments of the structures, methods, and associated models disclosed herein will be readily recognized as viable alternatives that may be employed without departing from the principles of what is claimed. Reference will now be made in detail to several embodiments, examples of which are illustrated in the accompanying figures. It is noted that wherever practicable, similar or like reference numbers may be used in the figures and may indicate similar or like functionality. The figures depict embodiments of the disclosed methods and one skilled in the art will readily appreciate from the following description that various example embodiments of the methods and equipment described herein may be employed without departing from the principles described herein.

[0014] Disclosed herein are methods of producing malonyl- coenzyme A (malonyl- CoA) and triacetic acid lactone (TAL), and compositions related to the same. More particularly, the disclosure relates to methods of using genetically modified yeast cells for producing malonyl- CoA and TAL in the peroxisomes thereof, and using a fatty acid, vegetable oil, and / or alkane feedstock, and compositions related to the same.

[0015] Some embodiments provided herein relate to modified microorganisms, including, but not limited to, genetically modified yeast cells. In some embodiments, the genetically modified yeast cells are used to produce TALs. Some embodiments provided herein relate to the use of fatty acids as a substrate for growing the yeast cells. In some embodiments,and are an underutilized waste stream from a range of industries. In some embodiments, yeast cells are efficient at consuming fatty acids by beta-oxidation to produce acetyl-CoA, which occurs in the peroxisome of the yeast cells. Thus, provided herein are various embodiments including novel methods for 1) using acetyl-CoA derived from beta-oxidation to produce malonyl-CoA, 2) compartmentalizing production of malonyl-CoA to the peroxisome, 3) facilitating strain development using adaptive laboratory evolution and 4) improving carbon dioxide fixation by the yeast cells.

[0016] In some embodiments, the methods disclosed herein harness the native production of acetyl-CoA produced by beta-oxidation of fatty acids in the peroxisome. The carnitine shuttle is responsible for moving acetyl-CoA out of the peroxisome and into the cytosol and mitochondria. By controlling how the acetyl-CoA is channeled out of the peroxisome, acetyl- CoA can efficiently be directed towards biosynthesis of compounds such as TAL. Deletion of the gene encoding carnitine acetyltransferase (CAT2) prevents acetyl-CoA generated by betaoxidation from leaving the peroxisome, which produces a strain that is unable to grow by consuming fatty acids and like substances. Accordingly, herein disclosed are methods for transferring acetyl-CoA out of the peroxisome while simultaneously producing malonyl-CoA and pyruvate, the pyruvate consumable by mitochondria to support yeast cell growth.

[0017] In some embodiments, methylmalonyl-CoA carboxyltransferase (MMC) enzyme complex is introduced into the peroxisome. MMC catalyzes the interconversion of acetyl- CoA and oxaloacetate to pyruvate and malonyl-CoA. In some embodiments, one or more subunit of MMC can be engineered to target the peroxisome. In some embodiments, the reactants and products of MMC, e.g., acetyl-CoA and malonyl-CoA, can be confined to the peroxisome by virtue of being esters of coenzyme A, while pyruvate and oxaloacetate can freely cross the peroxisomal membrane. In some further embodiments, a polyketide synthase to produce TAL, including, but not limited to, 2-pyrone synthase, can also be inserted into the peroxisome to utilize peroxisomal acetyl-CoA and malonyl-CoA. Accordingly, disclosed herein are embodiments of simultaneously maximizing yeast cell growth and polyketide production dependent on malonyl-CoA.

[0018] Accordingly, it is an object of the present disclosure to provide methods of producing malonyl-CoA that can include providing a substrate that can include a feedstock, providing a genetically modified yeast cell to the substrate, metabolizing the substrate inperoxisomes, and catalyzing a reaction in the peroxisomes between the acetyl-CoA and an oxaloacetate with the modified enzyme to produce a pyruvate and the malonyl-CoA. In some embodiments, the feedstock can include at least one fatty acid, vegetable oil and / or alkane. In some embodiments, metabolizing by the genetically modified yeast cell can produce acetyl-CoA. In some embodiments, each of the genetically modified yeast cells can be engineered to be unable to export the acetyl-CoA from the peroxisomes. In some embodiments, the modified enzyme can include a peroxisomal targeting sequence that can localize the modified enzyme in each of the peroxisomes. In some embodiments, the modified enzyme can include one or more of an acyl- CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase. In some further embodiments, the modified enzyme can include malonyl-CoA hydrolase.

[0019] It is another object of the present disclosure to provide methods of producing malonyl-CoA and triacetic acid lactone, including providing a substrate including a feedstock, the feedstock including at least one fatty acid, vegetable oil and / or alkane, providing a genetically modified yeast cell to the substrate, metabolizing the substrate in peroxisomes of each of the genetically modified yeast cells, introducing a modified enzyme to the peroxisomes, catalyzing a reaction in the peroxisomes between the acetyl-CoA and an oxaloacetate with the modified enzyme to produce a pyruvate and the malonyl-CoA, and directing polyketide synthases to the peroxisomes to react with the pyruvate, thereby producing triacetic acid lactone. In some embodiments, the feedstock can include at least one fatty acid, vegetable oil and / or alkane. In some embodiments, metabolizing by the genetically modified yeast cell can produce acetyl-CoA. In some embodiments, each of the genetically modified yeast cells can be engineered to be unable to export the acetyl-CoA from the peroxisomes. In some embodiments, the modified enzyme can include a peroxisomal targeting sequence that can localize the modified enzyme in each of the peroxisomes. In some embodiments, the modified enzyme can include one or more of an acyl-CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase. In some further embodiments, the modified enzyme includes malonyl-CoA hydrolase.

[0020] In some embodiments, the method can further include preventing the export of acetyl-CoA from the peroxisomes by deleting carnitine acetyltransferase (CAT2) alleles from thecell can include genes encoding the methylmalonyl-CoA carboxyltransferase. For example, the genes encoding the methylmalonyl-CoA carboxyltransferase can include genes encoding a methylmalonyl-CoA carboxyltransferase 12S subunit, a methylmalonyl-CoA carboxyltransferase 5S subunit and a methylmalonyl-CoA carboxyltransferase 1.3S subunit. In some further embodiments, a subunit of the methylmalonyl-CoA carboxyltransferase can be tagged with an amino acid sequence to localize the methylmalonyl-CoA carboxyltransferase to the peroxisomes. For example, the methylmalonyl-CoA carboxyltransferase 12S subunit can be tagged with a peroxisomal targeting amino acid sequence. In some embodiments, amino acid sequences from enzymes found in Propionibacterium freudenreichii subsp. shermanii can be used to localize the methylmalonyl-CoA carboxyltransferase in the peroxisomes. For example, the 12S subunit can be tagged with a peroxisomal targeting sequence to place the targeting sequence at the C-terminus or N-terminal sequence of the 12S subunit. By way of further example, the peroxisomal targeting sequence can include an amino acid sequence of -GRRAKL or MDRLNQLSGQL-, binding to the carboxyl terminus (C-terminal) and amino terminus (N-terminal) of the 12S subunit, respectively. In some embodiments, the peroxisomal targeting sequence can be N-termini of peroxisomal thiolases such as POTI or FOX3. In some embodiments, the peroxisomal targeting amino acid sequence can be added to the C-terminus of the methylmalonyl-CoA carboxyltransferase 12S subunit. For example, placement of a peroxisomal targeting sequence at the C-terminus of the 12S subunit may not affect function of the 12S subunit.

[0021] In some embodiments, the genetically modified yeast cell can be engineered to include a gene encoding 2-pyrone synthase. In some embodiments, 2-pyrone synthase can catalyze the biosynthesis of 6-methyl-4-hydroxy-2-pyrone from malonyl-CoA. In some embodiments, the gene encoding the 2-pyrone synthase is derived from Gerbera spp., including, but not limited to, Gebera x hybrida. In some embodiments, the 2-pyrone synthase can be tagged with a peroxisomal targeting amino acid sequence.

[0022] In some embodiments, the method can include upregulating expression of genes encoding pyruvate carboxylase in each of the genetically modified yeast cell. For example, the pyruvate carboxylase can catalyze the carboxylation of pyruvate to form oxaloacetate.

[0023] In some embodiments, the genetically modified yeast cell can include unconventional yeast species or strains. In some embodiments, the genetically modified yeast cellinclude Candida viswanathii. In some embodiments, the genetically modified yeast cell can include one or more diploid yeast species.

[0024] In some embodiments, the substrate can include a feedstock. For example, the feedstock can include an oleic acid, a plant-derived oil, or like substances. In some further embodiments, the substrate can include a co-feed. For example, the co-feed can include a glycerol and / or acetate. In some embodiments, the feedstock can include by-products of plant-derived oil production, including, but not limited to, vegetable oil production.

[0025] In some embodiments, the method can include carboxylating the pyruvate to produce oxaloacetate, thereby perpetuating malonyl-CoA production in the peroxisomes. In some embodiments, the pyruvate can freely cross membranes of the peroxisomes into mitochondria, thereby supporting growth of the genetically modified yeast cell.

[0026] In some embodiments, the genetically modified yeast cells can be engineered to produce LAC at a higher concentration than non-genetically modified yeast cells. In some embodiments, the genetically modified yeast cells can produce 250 pg, 500 pg, 750 pg, 1,000 pg, 1,250 pg, 1,500 pg, 1,750 pg, 2,000 pg, 2,250 pg, 2,500 pg, 2,750 pg, 3,000 pg, 3,250 pg, 3,500 pg, or more LAC per unit liter.

[0027] Also disclosed herein is a genetically modified yeast cell adapted to produce malonyl-coenzyme A (malonyl-CoA) and triacetic acid lactone (TAL). More particularly, the disclosure relates to a genetically modified yeast cell adapted to produce malonyl-CoA and TAL in the peroxisomes thereof, and adapted to grow on a substrate including a feedstock, the feedstock including at least one of a fatty acid, a vegetable oil, and an alkane feedstock.

[0028] Some embodiments provided herein relate to modified microorganisms, including, but not limited to, a genetically modified yeast cell. In some embodiments, the genetically modified yeast cells are adapted to produce TALs. Some embodiments provided herein relate to the genetically modified yeast cell adapted to grow on a substrate including a feedstock including fatty acids. In some embodiments, fatty acids represent a more direct source of a polyketide metabolic precursor, such as acetyl-CoA, and are an underutilized waste stream from a range of industries. In some embodiments, the genetically modified yeast cells are adapted to consume fatty acids by beta-oxidation to produce acetyl-CoA. In some embodiments, consumption of the fatty acids takes place in the peroxisome of the yeast cells. Thus, provided herein are variousCoA derived from beta-oxidation to produce malonyl-CoA, 2) compartmentalize production of malonyl-CoA to the peroxisome, 3) facilitate strain development using adaptive laboratory evolution and 4) improve carbon dioxide fixation by the yeast cells.

[0029] In some embodiments, the genetically modified yeast cell can include a methylmalonyl-CoA carboxyltransferase (MMC) enzyme complex that is introduced into the peroxisome. MMC catalyzes the interconversion of acetyl-CoA and oxaloacetate to pyruvate and malonyl-CoA. In some embodiments, one or more subunit of MMC can be engineered to target the peroxisome. In some embodiments, the reactants and products of MMC, e.g., acetyl-CoA and malonyl-CoA, can be confined to the peroxisome by virtue of being esters of coenzyme A, while pyruvate and oxaloacetate can freely cross the peroxisomal membrane. In some further embodiments, the genetically modified yeast cell can include a polyketide synthase to produce TAL, including, but not limited to, 2-pyrone synthase. For example, the polyketide synthase can be inserted into the peroxisome to utilize peroxisomal acetyl-CoA and malonyl-CoA. Accordingly, disclosed herein are embodiments of the genetically modified yeast cell that is adapted to simultaneously maximize growth and polyketide production dependent on malonyl-CoA.

[0030] It is a further object of the present disclosure to provide the genetically modified yeast cell adapted to produce malonyl-CoA on a substrate that can include a feedstock. For example, the genetically modified yeast cell can be adapted to grow on the substrate and to metabolize the substrate in peroxisomes. In some embodiments, the genetically modified yeast cell can include an introduced modified enzyme to the peroxisomes, the modified enzyme adapted to catalyze a reaction in the peroxisome between the acetyl-CoA and an oxaloacetate to produce a pyruvate and the malonyl-CoA. In some embodiments, the feedstock can include at least one fatty acid, vegetable oil and / or alkane. In some embodiments, the genetically modified yeast cell is adapted to metabolize one or more feedstock such as to produce acetyl-CoA. In some embodiments, the genetically modified yeast cell can be unable to export the acetyl-CoA from the peroxisome. In some embodiments, the modified enzyme can include a peroxisomal targeting sequence that can localize the modified enzyme in each of the peroxisomes. In some embodiments, the modified enzyme can include one or more of an acyl-CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or ainclude malonyl-CoA hydrolase.

[0031] It is another object of the present disclosure to provide a substrate that supports growth of the genetically modified yeast cell. For example, the substrate is configured to maximize malonyl-CoA and triacetic acid lactone by the genetically modified yeast cell. In some embodiments, the substrate can include a feedstock, the feedstock including at least one fatty acid, vegetable oil and / or alkane. In some embodiments, the genetically modified yeast cell can be adapted to metabolize the substrate in peroxisomes of each of the genetically modified yeast cells. In some embodiments, the modified enzyme can be adapted to locate in the peroxisomes, therein adapted to catalyze a reaction between the acetyl-CoA and an oxaloacetate to produce a pyruvate and the malonyl-CoA. In some embodiments, a polyketide synthase can be adapted to locate in the peroxisomes to react with the pyruvate, thereby producing triacetic acid lactone. In some embodiments, the feedstock can include at least one fatty acid, vegetable oil and / or alkane. In some embodiments, the genetically modified yeast cells are adapted to produce acetyl-CoA. In some embodiments, the genetically modified yeast cells can be unable to export the acetyl-CoA from the peroxisomes. In some embodiments, the modified enzyme can include a peroxisomal targeting sequence that can be adapted to localize the modified enzyme in each of the peroxisomes. In some embodiments, the modified enzyme can include one or more of an acyl-CoA oxidase, an enoyl- CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase. In some further embodiments, the modified enzyme can include malonyl-CoA hydrolase.

[0032] In some embodiments, the genetically modified yeast cell is adapted to inhibit export of acetyl-CoA from the peroxisomes by deleting carnitine acetyltransferase (CAT2) alleles from the genome of the modified yeast cells. In some embodiments, the genetically modified yeast cells can include genes encoding the methylmalonyl-CoA carboxyltransferase. For example, the genes encoding the methylmalonyl-CoA carboxyltransferase can include genes encoding a methylmalonyl-CoA carboxyltransferase 12S subunit, a methylmalonyl-CoA carboxyltransferase 5S subunit and a methylmalonyl-CoA carboxyltransferase 1.3S subunit. In some further embodiments, a subunit of the methylmalonyl-CoA carboxyltransferase can be tagged with an amino acid sequence to localize the methylmalonyl-CoA carboxyltransferase to the peroxisomes. For example, the methylmalonyl-CoA carboxyltransferase 12S subunit can be tagged with aenzymes found in Propionibacterium freudenreichii subsp. shermanii can be used to localize the methylmalonyl-CoA carboxyltransferase in the peroxisomes. For example, the 12S subunit can be tagged with a peroxisomal targeting sequence to place the targeting sequence at the C-terminus or N-terminal sequence of the 12S subunit. By way of further example, the peroxisomal targeting sequence can include an amino acid sequence of -GRRAKL or MDRLNQLSGQL-, binding to the carboxyl terminus (C-terminal) and amino terminus (N-terminal) of the 12S subunit, respectively. In some embodiments, the peroxisomal targeting sequence can be N-termini of peroxisomal thiolases such as POTI or F0X3. In some embodiments, the peroxisomal targeting amino acid sequence can be added to the C-terminus of the methylmalonyl-CoA carboxyltransferase 12S subunit. For example, placement of a peroxisomal targeting sequence at the C-terminus of the 12S subunit may not affect function of the 12S subunit.

[0033] In some embodiments, the genetically modified yeast cell can be engineered to include a gene encoding 2-pyrone synthase. In some embodiments, 2-pyrone synthase can catalyze the biosynthesis of 6-methyl-4-hydroxy-2-pyrone from malonyl-CoA. In some embodiments, the gene encoding the 2-pyrone synthase is derived from Gerbera spp., including, but not limited to, Gebera x hybrida. In some embodiments, the 2-pyrone synthase can be tagged with a peroxisomal targeting amino acid sequence.

[0034] In some embodiments, the genetically modified yeast cell can be adapted to upregulate expression of genes encoding pyruvate carboxylase. For example, the pyruvate carboxylase can catalyze the carboxylation of pyruvate to form oxaloacetate.

[0035] In some embodiments, the genetically modified yeast cell can be configured to carboxylate the pyruvate to produce oxaloacetate, thereby perpetuating malonyl-CoA production in the peroxisomes. In some embodiments, the pyruvate can freely cross membranes of the peroxisomes into mitochondria, thereby supporting growth of each of the genetically modified yeast cell.

[0036] FIG. 1 illustrates an example embodiment of acetyl-CoA transport out of the peroxisome 110 and to the mitochondria 120 via acetyl- carnitine 160 as an intermediate. This process is vital for cellular energy production, as it allows for the efficient utilization of fatty acids as a nutrient source. In wild type populations of some species and / or strains of yeast, particularly those possessing a fully functional CAT2 gene, the encoded CAT2 protein plays a central role inCoA 140, generated within the peroxisome, into acetyl-carnitine. This conversion is crucial because acetyl-CoA cannot directly cross the peroxisomal membrane.

[0037] The process begins with the introduction of a fatty acid 150, or similar substances, into the peroxisome 110. Within the peroxisome, fatty acyl-CoA oxidase (FAA) initiates the beta-oxidation pathway 130, breaking down the fatty acid into smaller acetyl-CoA molecules. These acetyl-CoA molecules serve as the substrate for the CAT2 protein, which catalyzes their transformation into acetyl-carnitine. Once converted, the acetyl-carnitine molecules are transported out of the peroxisome and into the cytoplasm. From there, they traverse the mitochondrial membrane, facilitated by specific transport proteins. Upon entering the mitochondria, the acetyl-carnitine molecules are reconverted back into acetyl-CoA 170. This final step is essential as it allows the acetyl-CoA molecules to enter the Kreb’s cycle, where they undergo further oxidation to generate ATP, the primary energy currency of the cell. By utilizing acetyl-carnitine as an intermediate, cells are able to overcome the limitations of membrane permeability and ensure the continuous supply of acetyl-CoA to the mitochondria for maximizing energy generation.

[0038] FIG. 2 illustrates an example embodiment of malonyl-CoA production from acetyl-CoA and oxaloacetate in the peroxisome of a genetically modified yeast cell. In some embodiments, fatty acids 250, or other like substances, are provided to a peroxisome 210 of a genetically modified yeast cell. In some embodiments, the fatty acids 250 are reacted by the betaoxidation pathway 230 to yield acetyl-CoA 240. In some embodiments, the genetically modified yeast cell includes a AG4T2 allele if haploid, or AG4T2 / AG4T2 alleles if diploid. For example, a diploid CAT2I I.CAT2 transgenic yeast cell can include Candida sp., including, but not limited to Candida viswanathii. Because the CAT2 gene is nonfunctional of deleted in the genetically modified yeast cells, acetyl-CoA 240 remains inside the peroxisome 210. In some embodiments, the peroxisome-bound acetyl-CoA 240 can react with oxaloacetate 295 to produce pyruvate 260 and malonyl-CoA 265. In some embodiments, the pyruvate 260 is reacted with a carboxyl group 280 to regenerate the oxaloacetate 295. In some embodiments, the pyruvate 260 is transported 290 to a mitochondrion 220 to provide promote yeast cell growth. In some embodiments, the malonyl- CoA 265 is reacted an MMC to synthesize polyketides 270, including TAL.or similar substances, into the peroxisome 210 of a genetically modified yeast cell. These fatty acids serve as the raw material for the subsequent biochemical reactions. The peroxisome 210, a specialized organelle within the yeast cell, acts as the primary site for fatty acid metabolism. Inside the peroxisome 210, the beta-oxidation pathway 230 breaks down the fatty acids into acetyl-CoA 240 molecules. This pathway is a series of enzymatic reactions that sequentially shorten the fatty acid chains, generating acetyl-CoA 240 units as a byproduct. Acetyl-CoA 240 is a crucial metabolic intermediate, serving as a precursor for various biosynthetic pathways. A key feature of the genetically modified yeast cell is the deletion or inactivation of the CAT2 gene. In wild-type yeast cells, the CAT2 protein facilitates the transport of acetyl-CoA 240out of the peroxisome 210. However, in the engineered yeast strains in some embodiments and as depicted in FIG. 2, the CAT2 gene is nonfunctional or deleted, leading to the accumulation of acetyl-CoA within the peroxisome 210. This accumulation is a critical factor in enabling the subsequent reactions.

[0040] With acetyl-CoA 240 confined to the peroxisome, it can react with oxaloacetate 295, a four-carbon compound derived from the citric acid cycle. This reaction, catalyzed by a specific enzyme, leads to the formation of pyruvate 260 and malonyl-CoA 265. Pyruvate is a three- carbon molecule that plays a central role in cellular metabolism, while malonyl-CoA is a key building block for fatty acid synthesis. To ensure a continuous supply of oxaloacetate 295 for the reaction with acetyl-CoA 240, a recycling mechanism is employed. The pyruvate generated in the previous step is transported 290 out of the peroxisome and into the cytoplasm. From there, it enters the mitochondria 220, where it undergoes carboxylation, a reaction that adds a carboxyl group 280 to the pyruvate molecule, effectively converting it back into oxaloacetate 295. This regenerated oxaloacetate 295 is then transported back into the peroxisome 210, replenishing the pool for further reactions with acetyl-CoA. The malonyl-CoA produced in the peroxisome serves as a substrate for polyketide synthases (PKSs). PKSs are large multi-enzyme complexes that catalyze the synthesis of polyketides 270, a diverse class of natural products with various biological activities. Among the polyketides that can be synthesized by the engineered yeast cells is triacetic acid lactone, a precursor for the production of large hydrocarbon molecules.

[0041] FIG. 3 illustrates an example embodiment of quantification of TAL from fatty acids 300 in shake flask fermentations. Strain 33 320 contains MMC and 2-pyrone synthaseproduces 30-35x more TAL than control wild-type yeast cells.

[0042] Further, non-limiting embodiments provided herein are further described in the following enumerated arrangements.

[0043] 1. A method of producing malonyl-CoA, comprising: providing a substrate comprising a feedstock; providing a genetically modified yeast cell to the substrate; metabolizing the substrate in a peroxisome of the genetically modified yeast cell to produce acetyl-CoA; introducing a modified enzyme to the peroxisome; and catalyzing a reaction in the peroxisome with the modified enzyme to produce malonyl-CoA.

[0044] 2. The method of arrangement 1, wherein the feedstock is a fatty acid, a vegetable oil, or an alkane.

[0045] 3. The method of any one of arrangements 1 -2, wherein the modified enzyme comprises a peroxisomal targeting sequence that localizes the modified enzyme in the peroxisome.

[0046] 4. The method of any one of arrangements 1-3, wherein the modified enzyme is an acyl-CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a betaketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase.

[0047] 5. The method of any one of arrangements 1-4, wherein the genetically modified yeast cell is engineered to prevent export of the acetyl-CoA from the peroxisome.

[0048] 6. The method of arrangement 5, wherein the carnitine acetyltransferase (CAT 2) alleles from the genome of the genetically modified yeast cell are deleted.

[0049] 7. The method of any one of arrangements 1-6, wherein the genetically modified yeast cell comprises a gene encoding methylmalonyl-CoA carboxyltransferase, methylmalonyl-CoA carboxyltransferase 12S subunit, methylmalonyl-CoA carboxyltransferase 5S subunit, or methylmalonyl-CoA carboxyltransferase 1.3S subunit.

[0050] 8. The method of arrangement 7, wherein the methylmalonyl-CoA carboxyltransferase 12S subunit is tagged with a peroxisomal targeting amino acid sequence.

[0051] 9. The method of arrangement 8, wherein the peroxisomal targeting amino acid sequence is added to a carboxyl terminus of the methylmalonyl-CoA carboxyltransferase 12S subunit.

[0052] 10. The method of any one of arrangements 1-9, wherein the genetically modified yeast cell comprises a gene encoding 2-pyrone synthase.synthase is derived from Gerbera spp.

[0054] 12. The method of any one of arrangements 10-11, wherein the 2-pyrone synthase is tagged with a peroxisomal targeting amino acid sequence.

[0055] 13. The method of any one of arrangements 1-12, further comprising upregulating expression of genes encoding pyruvate carboxylase in the genetically modified yeast cell.

[0056] 14. The method of any one of arrangements 1-13, wherein the modified enzyme is malonyl-CoA hydrolase.

[0057] 15. The method of any one of arrangements 1-14, wherein the genetically modified yeast cell is Candida spp.

[0058] 16. The method of any one of arrangements 1-14, wherein the genetically modified yeast cell is Candida viswanathii.

[0059] 17. The method of any one of arrangements 1-16, wherein the genetically modified yeast cell comprises one or more diploid yeast species.

[0060] 18. The method of any one of arrangements 1-17, wherein the substrate comprises oleic acid.

[0061] 19. The method of any one of arrangements 1-18, wherein the substrate comprises a co-feed.

[0062] 20. The method of arrangement 19, wherein the co-feed comprises glycerol or acetate.

[0063] 21. The method of any one of arrangements 1-20, wherein the feedstock comprises a by-product of vegetable oil production.

[0064] 22. A method of producing malonyl-CoA and pyruvate, comprising: providing a substrate comprising a feedstock; providing a genetically modified yeast cell to the substrate; metabolizing the substrate in a peroxisome of the genetically modified yeast cell to produce acetyl- CoA; introducing a modified enzyme to the peroxisome; catalyzing a reaction in the peroxisome with the modified enzyme to produce pyruvate and malonyl-CoA; and directing polyketide synthases to the peroxisome to react with the pyruvate, thereby producing triacetic acid lactone.

[0065] 23. The method of arrangement 22, wherein the feedstock is fatty acid, vegetable oil, or an alkane.enzyme is an acyl-CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase.

[0067] 25. The method of any one of arrangements 22-24, wherein the genetically modified yeast cell is engineered to prevent export of the acetyl-CoA from the peroxisome.

[0068] 26. The method of any one of arrangements 22-25, further comprising carboxylating the pyruvate to produce oxaloacetate.

[0069] 27. The method of any one of arrangements 22-26, wherein carnitine acetyltransferase (CAT2) alleles from the genome of the genetically modified yeast cell are deleted.

[0070] 28. The method of any one of arrangements 22-27, wherein the genetically modified yeast cell comprises a gene encoding methylmalonyl-CoA carboxyltransferase, methylmalonyl-CoA carboxyltransferase 12S subunit, methylmalonyl-CoA carboxyltransferase 5S subunit, or methylmalonyl-CoA carboxyltransferase 1.3S subunit.

[0071] 29. The method of arrangement 28, wherein the methylmalonyl-CoA carboxyltransferase 12S subunit is tagged with a peroxisomal targeting amino acid sequence.

[0072] 30. The method of arrangement 29, wherein the peroxisomal targeting amino acid sequence is added to a carboxyl terminus of the methylmalonyl-CoA carboxyltransferase 12S subunit.

[0073] 31. The method of any one of arrangements 22-30, wherein the genetically modified yeast cell comprises a gene encoding 2-pyrone synthase.

[0074] 32. The method of arrangement 31, wherein the gene encoding the 2-pyrone synthase is derived from Gerbera spp.

[0075] 33. The method of any one of arrangements 31-32, wherein the 2-pyrone synthase is tagged with a peroxisomal targeting amino acid sequence.

[0076] 34. The method of any one of arrangements 22-33, further comprising upregulating expression of genes encoding pyruvate carboxylase in the genetically modified yeast cell.

[0077] 35. The method of any one of arrangements 22-34, wherein the modified enzyme is malonyl-CoA hydrolase.modified yeast cell is Candida spp.

[0079] 37. The method of any one of arrangements 22-35, wherein the genetically modified yeast cell is Candida viswanathii.

[0080] 38. The method of any one of arrangements 22-37, wherein the genetically modified yeast cell comprises one or more diploid yeast species.

[0081] 39. The method of any one of arrangements 22-38, wherein the substrate comprises oleic acid.

[0082] 40. The method of any one of arrangements 22-38, wherein the substrate comprises a co-feed.

[0083] 41. The method of arrangement 40, wherein the co-feed comprises glycerol or acetate.

[0084] 42. The method of any one of arrangements 22-41, wherein the feedstock comprises a by-product of vegetable oil production.

[0085] 43. The method of any one of arrangements 22-42, wherein the genetically modified yeast cell produces at least 2,000 pg of triacetic acid lactone per unit liter.

[0086] 44. A genetically modified yeast cell, comprising: a mutation adapted to prevent export of the acetyl-CoA from a peroxisome, and a modified enzyme targeted to the peroxisome in the genetically modified yeast cell, wherein the genetically modified yeast cell is adapted to produce malonyl-CoA and triacetic acid lactone.

[0087] 45. The genetically modified yeast cell of arrangement 44, wherein the mutation comprises deletion of carnitine acetyltransferase (G4T2) alleles.

[0088] 46. The genetically modified yeast cell of any one of arrangements 44-45, wherein the modified enzyme is acyl-CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl- CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl- CoA carboxyltransferase, or malonyl-CoA hydrolase.

[0089] 47. The genetically modified yeast cell of any one of arrangements 44-46, wherein the genetically modified yeast cell is Candida viswanathii.

[0090] 48. The genetically modified yeast cell of any one of arrangements 44-47, wherein the genetically modified yeast cell is adapted to grow on a substrate comprising a feedstock, the feedstock comprising at least one of a fatty acid, a vegetable oil, and an alkane.wherein the genetically modified yeast cell comprises a gene encoding methylmalonyl-CoA carboxyltransferase, methylmalonyl-CoA carboxyltransferase 12S subunit, methylmalonyl-CoA carboxyltransferase 5S subunit, or methylmalonyl-CoA carboxyltransferase 1.3S subunit, and wherein the gene comprises a tag with a peroxisomal targeting amino acid sequence.EXAMPLES

[0092] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the invention, as it is described herein above and in the claims.Example 1 - Generating Genetically Modified Yeast Cells

[0093] A population of diploid strains of Candida viswanathii are prepared in which alleles of CAT2 are deleted. Once deleted, the resulting strain cannot grow on fatty acids as a substrate. The strain can then be transformed with the following genes:

[0094] Genes encoding the three subunits of MMC. Specifically, subunits 12S, 5S and 1.3S derived from Propionibacterium freudenreichii subsp. shermanii. The 12S subunit is tagged with a peroxisomal targeting sequence, or PTS. The use of a PTS 1 places the peroxisomal targeting sequence at the C-terminus and use of a PTS2 places the peroxisomal targeting sequence at the N- terminal sequence. For example, PTS1 can include the peroxisomal targeting sequence -GRRAKL and PTS2 can include the peroxisomal targeting sequence MDRLNQLSGQL-, which is found in the N-termini of peroxisomal thiolases such as POTI or FOX3. The C-terminus of the 12S subunit of MMC is not essential for proper functioning of MMC.

[0095] The gene encoding 2-pyrone synthase. The 2-pyrone synthase gene can be targeted to the peroxisome using a PTS1 or PTS2. For example, a 2-pyrone synthase from a Gerbera spp, including, but not limited to Gerbera x hybrida.

[0096] Genes encoding a pyruvate carboxylase. Alternatively, PEP carboxylase via pyruvate phosphate dikinase to convert pyruvate to PEP.

[0097] Genes encoding malonyl-CoA hydrolase, such as EHD3 El 24V from Saccharomyces cerevisiae.Example 2 - Controlling Pests

[0098] The following example demonstrates use of the genetically modified yeast to maximize efficiency and output of malonyl-CoA and / or LAC.

[0099] Adaptive evolution relies on 1) efficient interconversion of acetyl-CoA and oxaloacetate to malonyl-CoA and pyruvate by MMC, 2) utilization of pyruvate for growth and as an intermediate for carboxylation to regenerate oxaloacetate and 3) utilization of malonyl-CoA in the peroxisome for biosynthesis of TAL. For example, oleic acid can serve as feedstock for yeast cells, which can be supplemented with a co-feed of glycerol or acetate. The appropriate polyketide synthase is important for adaptive evolution as accumulation of malonyl-CoA is detrimental to C. viswanathii. Several strains can be developed in parallel for adaptive evolution assays to account for different challenges in producing a functional strain. Strains are selected for their high growth rate and cell density on oleic acid as a sole carbon source that also produce higher titers of the target polyketide. Both TAL and flaviolin are colorful and can be used as a colorimetric readout of product titers.

[0100] Various modifications to the implementations described in this disclosure may be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other implementations without departing from the spirit or scope of this disclosure. Thus, the claims are not intended to be limited to the implementations shown herein, but are to be accorded the widest scope consistent with this disclosure, the principles and the novel features disclosed herein. Additionally, a person having ordinary skill in the art will readily appreciate, the terms “upper” and “lower” are sometimes used for ease of describing the figures, and indicate relative positions corresponding to the orientation of the figure on a properly oriented page, and may not reflect the proper orientation of a feature as implemented.

[0101] While certain embodiments have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the disclosure. Indeed, the novel methods and systems described herein may be embodied in a variety of other forms. Furthermore, various omissions, substitutions and changes in the systems and methods described herein may be made without departing from the spirit of the disclosure. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the disclosure.particular aspect, embodiment, or example are to be understood to be applicable to any other aspect, embodiment or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The protection is not restricted to the details of any foregoing embodiments. The protection extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0103] The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any aspect or embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or embodiments. Various aspects of the novel systems and methods are described more fully hereinafter with reference to the accompanying drawings. This disclosure may, however, be embodied in many different forms and should not be construed as limited to any specific structure or function presented throughout this disclosure. Rather, these aspects are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Based on the teachings herein one skilled in the art should appreciate that the scope of the disclosure is intended to cover any aspect of the novel systems and methods disclosed herein, whether implemented independently of, or combined with, any other aspect described. For example, a method may be practiced using any number of the aspects set forth herein. In addition, the scope of the disclosure is intended to cover such a method which is practiced using other structure, functionality, or structure and functionality in addition to or other than the various aspects of the disclosures set forth herein. It should be understood that any aspect disclosed herein may be embodied by one or more elements of a claim.

[0104] Furthermore, certain features that are described in this disclosure in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination.from a claimed combination can, in some cases, be excised from the combination, and the combination may be claimed as a subcombination or variation of a subcombination.

[0105] The features and attributes of the specific embodiments disclosed above may be combined in different ways to form additional embodiments, all of which fall within the scope of the present disclosure. Also, the separation of various system components in the implementations described above should not be understood as requiring such separation in all implementations, and it should be understood that the described components and systems can generally be integrated together in a single product or packaged into multiple products.

[0106] Moreover, while operations may be depicted in the drawings or described in the specification in a particular order, such operations need not be performed in the particular order shown or in sequential order, or that all operations be performed, to achieve desirable results. Other operations that are not depicted or described can be incorporated in the example methods and processes. For example, one or more additional operations can be performed before, after, simultaneously, or between any of the described operations. Further, the operations may be rearranged or reordered in other implementations. Those skilled in the art will appreciate that in some embodiments, the actual steps taken in the processes illustrated and / or disclosed may differ from those shown in the figures. Depending on the embodiment, certain of the steps described above may be removed, others may be added. Furthermore, the features and attributes of the specific embodiments disclosed above may be combined in different ways to form additional embodiments, all of which fall within the scope of the present disclosure.

[0107] For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. Not necessarily all such advantages may be achieved in accordance with any particular embodiment. Thus, for example, those skilled in the art will recognize that the disclosure may be embodied or carried out in a manner that achieves one advantage or a group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

[0108] Conditional language, such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements, and / or steps. Thus, such conditional language is not generally intendedor that one or more embodiments necessarily include logic for deciding, with or without user input or prompting, whether these features, elements, and / or steps are included or are to be performed in any particular embodiment.

[0109] Conjunctive language such as the phrase “at least one of X, Y, and Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be either X, Y, or Z. Thus, such conjunctive language is not generally intended to imply that certain embodiments require the presence of at least one of X, at least one of Y, and at least one of Z. Thus, as used herein, a phrase referring to “at least one of X, Y, and Z” is intended to cover: X; Y; Z; X and Y; X and Z; Y and Z; and X, Y, and Z.

[0110] The headings provided herein, if any, are for convenience only and do not necessarily affect the scope or meaning of the devices and methods disclosed herein.

[0111] Language of degree used herein, such as the terms “approximately,” “about,” “generally,” and “substantially” as used herein represent a value, amount, or characteristic close to the stated value, amount, or characteristic that still performs a desired function or achieves a desired result. For example, the terms “approximately,” “about,” “generally,” and “substantially” may refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, and within less than 0.01% of the stated amount.

[0112] The scope of the present disclosure is not intended to be limited by the specific disclosures of embodiments in this section or elsewhere in this specification and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive.

Claims

1. A method of producing malonyl-CoA, comprising: providing a substrate comprising a feedstock; providing a genetically modified yeast cell to the substrate; metabolizing the substrate in a peroxisome of the genetically modified yeast cell to produce acetyl-CoA; introducing a modified enzyme to the peroxisome; and catalyzing a reaction in the peroxisome with the modified enzyme to produce malonyl-CoA.

2. The method of claim 1, wherein the feedstock is a fatty acid, a vegetable oil, or an alkane.

3. The method of claim 1, wherein the modified enzyme comprises a peroxisomal targeting sequence that localizes the modified enzyme in the peroxisome.

4. The method of claim 1, wherein the modified enzyme is an acyl-CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase.

5. The method of claim 1, wherein the genetically modified yeast cell is engineered to prevent export of the acetyl-CoA from the peroxisome.

6. The method of claim 5, wherein the carnitine acetyltransferase (CAT 2) alleles from the genome of the genetically modified yeast cell are deleted.

7. The method of claim 1, wherein the genetically modified yeast cell comprises a gene encoding methylmalonyl-CoA carboxyltransferase, methylmalonyl-CoA carboxyltransferase 12S subunit, methylmalonyl-CoA carboxyltransferase 5S subunit, or methylmalonyl-CoA carboxyltransferase 1.3S subunit.

8. The method of claim 7, wherein the methylmalonyl-CoA carboxyltransferase 12S subunit is tagged with a peroxisomal targeting amino acid sequence.

9. The method of claim 8, wherein the peroxisomal targeting amino acid sequence is added to a carboxyl terminus of the methylmalonyl-CoA carboxyltransferase 12S subunit.

10. The method of claim 1, wherein the genetically modified yeast cell comprises a gene encoding 2-pyrone synthase.derived from Gerbera spp.

12. The method of claim 10, wherein the 2-pyrone synthase is tagged with a peroxisomal targeting amino acid sequence.

13. The method of claim 1, further comprising upregulating expression of genes encoding pyruvate carboxylase in the genetically modified yeast cell.

14. The method of claim 1 , wherein the modified enzyme is malonyl-CoA hydrolase.

15. The method of claim 1 , wherein the genetically modified yeast cell is Candida spp.

16. The method of claim 15, wherein the genetically modified yeast cell is Candida viswanathii.

17. The method of claim 1 , wherein the genetically modified yeast cell comprises one or more diploid yeast species.

18. The method of claim 1, wherein the substrate comprises oleic acid.

19. The method of claim 1, wherein the substrate comprises a co-feed.

20. The method of claim 19, wherein the co-feed comprises glycerol or acetate.

21. The method of claim 1, wherein the feedstock comprises a by-product of vegetable oil production.

22. A method of producing malonyl-CoA and pyruvate, comprising: providing a substrate comprising a feedstock; providing a genetically modified yeast cell to the substrate; metabolizing the substrate in a peroxisome of the genetically modified yeast cell to produce acetyl-CoA; introducing a modified enzyme to the peroxisome; catalyzing a reaction in the peroxisome with the modified enzyme to produce pyruvate and malonyl-CoA; and directing polyketide synthases to the peroxisome to react with the pyruvate, thereby producing triacetic acid lactone.

23. The method of claim 22, wherein the feedstock is fatty acid, vegetable oil, or an alkane.enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase.

25. The method of claim 22, wherein the genetically modified yeast cell is engineered to prevent export of the acetyl-CoA from the peroxisome.

26. The method of claim 22, further comprising carboxylating the pyruvate to produce oxaloacetate.

27. The method of claim 22, wherein carnitine acetyltransferase (CAT 2) alleles from the genome of the genetically modified yeast cell are deleted.

28. The method of claim 22, wherein the genetically modified yeast cell comprises a gene encoding methylmalonyl-CoA carboxyltransferase, methylmalonyl-CoA carboxyltransferase 12S subunit, methylmalonyl-CoA carboxyltransferase 5S subunit, or methylmalonyl-CoA carboxyltransferase 1.3S subunit.

29. The method of claim 28, wherein the methylmalonyl-CoA carboxyltransferase 12S subunit is tagged with a peroxisomal targeting amino acid sequence.

30. The method of claim 29, wherein the peroxisomal targeting amino acid sequence is added to a carboxyl terminus of the methylmalonyl-CoA carboxyltransferase 12S subunit.

31. The method of claim 22, wherein the genetically modified yeast cell comprises a gene encoding 2-pyrone synthase.

32. The method of claim 31, wherein the gene encoding the 2-pyrone synthase is derived from Gerber a spp.

33. The method of claim 31, wherein the 2-pyrone synthase is tagged with a peroxisomal targeting amino acid sequence.

34. The method of claim 22, further comprising upregulating expression of genes encoding pyruvate carboxylase in the genetically modified yeast cell.

35. The method of claim 22, wherein the modified enzyme is malonyl-CoA hydrolase.

36. The method of claim 22, wherein the genetically modified yeast cell is Candida spp.

37. The method of claim 36, wherein the genetically modified yeast cell is Candida viswanathii.or more diploid yeast species.

39. The method of claim 22, wherein the substrate comprises oleic acid.

40. The method of claim 22, wherein the substrate comprises a co-feed.

41. The method of claim 40, wherein the co-feed comprises glycerol or acetate.

42. The method of claim 22, wherein the feedstock comprises a by-product of vegetable oil production.

43. The method of claim 22, wherein the genetically modified yeast cell produces at least 2,000 pg of triacetic acid lactone per unit liter.

44. A genetically modified yeast cell, comprising: a mutation adapted to prevent export of the acetyl-CoA from a peroxisome, and a modified enzyme targeted to the peroxisome in the genetically modified yeast cell, wherein the genetically modified yeast cell is adapted to produce malonyl- CoA and triacetic acid lactone.

45. The genetically modified yeast cell of claim 44, wherein the mutation comprises deletion of carnitine acetyltransferase (CAT2) alleles.

46. The genetically modified yeast cell of claim 44, wherein the modified enzyme is acyl-CoA oxidase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a betaketothiolase, a thiolase, an acyl-CoA synthase, or a methylmalonyl-CoA carboxyltransferase, or malonyl-CoA hydrolase.

47. The genetically modified yeast cell of claim 44, wherein the genetically modified yeast cell is Candida viswanathii.

48. The genetically modified yeast cell of claim 44, wherein the genetically modified yeast cell is adapted to grow on a substrate comprising a feedstock, the feedstock comprising at least one of a fatty acid, a vegetable oil, and an alkane.

49. The genetically modified yeast cell of claim 44, wherein the genetically modified yeast cell comprises a gene encoding methylmalonyl-CoA carboxyltransferase, methylmalonyl- CoA carboxyltransferase 12S subunit, methylmalonyl-CoA carboxyltransferase 5S subunit, or methylmalonyl-CoA carboxyltransferase 1.3S subunit, and wherein the gene comprises a tag with a peroxisomal targeting amino acid sequence.

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