PRIORITY CLAIM

[0001] This application claims the benefit of U.S. Provisional Application No. 63/171,418, filed April 06, 2021, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure generally relates to biological processes of producing beta hydroxyisovalerate, more particularly methods to create non-natural microorganisms comprising non-natural bH I V synthase enzymes and processes for using said microorganisms to produce beta hydroxyisovalerate, and more specifically to non-natural microorganisms that produce beta hydroxyisovalerate.

BACKGROUND

[0003] The beta hydroxyisovalerate (bHΐn) molecule (shown below), which is also known as 3- hydroxy-3-methylbutric acid, has potential applications ranging from liquid crystals to pharmaceutical ingredients and dietary supplements.

As such, a number of methods to produce b-hydroxyisovalerate are known in the art. They are mainly centered around chemical, organic synthesis starting with 4-hydroxy-4-methyl-2- pentanone. bHΐn can be synthesized by the oxidation of 4-hydroxy-4-methyl-2-pentanone. One suitable procedure is described by Coffman et al., J. Am. Chem. Soc. 80: 2882-2887 (1958). See also, for example, US6248922, US6090978 US1016471653, US6090918 and US2014025698. As described therein, bHΐn is synthesized by an alkaline sodium hypochlorite oxidation of diacetone alcohol. The product is recovered in free acid form, which can be converted to a salt. For example, bHΐn can be prepared as its calcium salt by a procedure similar to that of Coffman et al. (1958) in which the free acid of bHΐn is neutralized with calcium hydroxide and recovered by crystallization from an aqueous ethanol solution.

[0004] Biological methods to produce bHΐn are also known. For example, bHΐn can also be prepared by the conversion of 3 -methyl crotonate (3-methylbut-2-enoate) by cell-free extracts of Galactomyces reessii [Dhar and JPN Rosazza. Journal of Industrial Microbiology & Biotechnology 2002, 28, 81 - 87]. Cell free extracts of Galactomyces reessii contain an enoyl CoA hydratase that can catalyze the transformation of 3 -methyl crotonic acid to bHΐn. Resting cells of Galactomyces reessii could convert b-methylbutyrate into b-hydroxyisovalerate [Lee IY, Nissen SL, Rosazza JP. Applied and environmental microbiology 1997, 63(11):4191-4195; Lee IY, Rosazza JP. Arch. Microbiol., 1998 Mar;169(3):257-62] Using a two-step fed-batch fermentation process where biomass was first produced to sufficient density in the first step, followed by the addition of b-methylbutyrate to the washed biomass in the second step, Lee et al. reported producing 38 g/L of bHΐn. US10676765B2 describes an alternative enzymatic method to produce bHΐn through the conversion of 3 -methyl crotonyl-CoA into bHΐn via 3-hydroxy-3- methylbutyryl-CoA. The availability of 3 -methyl crotonic acid or b-methylbutyrate in economically viable quantities for in vitro or in vivo production of bHΐn is still a challenge that needs to be overcome before this process can become commercially viable.

[0005] Indeed, bHΐn is synthesized in humans through the metabolism of L-leucine (see for example Nutrient Metabolism, Martin Kohlmeier, Academic Press, 2015) as a result of the conversion of its keto acid, a-ketoisocaproate (KIC) by the promiscuous action of 4- hydroxyphenylpyruvate dioxygenase (HPPD). Dioxygenases are enzymes that incorporate diatomic oxygen to form oxo-intermediates. To reduce diatomic oxygen, these enzymes require a source of electrons as well as a cofactor capable of one-electron chemistry. The ferrous ion is the most common cofactor capable of localizing substrates by acting as a conduit to transfer the electrons from the substrates to oxygen. Common coordinated reductant for the ferrous ion is the a-keto acid moiety and a-keto acid dependent oxygenases are very versatile and play a key role in the secondary metabolism [Purpero and Moran, J. Biol. Inorg. Chem. 12 (2007) 587-601]. [0006] A majority of the a-keto acid dependent oxygenases have three substrates - oxygen, a- ketoglutarate (the source of the a-keto acid) and the substrate, whose transformation is the catalytic objective [Hausinger, Crit. Rev. Biochem. Mol. Biol. 39 (2004) 21-68] HPPD and hydroxymandelate synthase (HMS) are an exception to this general principal by having only two substrates. HPPD and HMS receive electrons from their common a-keto acid substrate, 4- hydroxyphenylpyruvate (HPP), and also transform it into their hydroxylated and decarboxylated products homogentisate and hydroxymandelate, respectively, without the need for a- ketoglutarate. These two enzymes are believed to have evolved from an entirely different lineage than all other a-keto acid oxygenases [Moran, G.M., Archives of Biochemistry and Biophysics 544 (2014) 58-68] although their core catalytic mechanism is consistent with the enzyme family. [0007] There is a large body of literature on HPPD, owing to its importance in agriculture and medicine. The primary product of HPPD reaction is homogentisate, which is the precursor to plastoquinone and tocopherols in plants and archaea. They are intimately involved in electron transport in the photosynthetic system, serve as antioxidants and plant hormones. Therefore, inhibiting the synthesis of homogentisate is commonly used to inhibit the growth of plants and weeds. A number of molecules such as leptospermone and usnic acid and their similars inhibit HPPD activity and are used as ingredients in herbicides [Beaudegnies et al., Bioorg. Med. Chem. 17 (2009) 4134-4152]. HPPD inhibitors such as NTBC (nitisinone) is used to treat Type 1 tyrosinemia. Inborn genetic errors leading to aberrant metabolic enzymes in the catabolism of homogentisate causes Type 1 tyrosinemia. NTBC has been used as a treatment by repressing the synthesis of homogentisate by inhibiting HPPD [Lindstedt et al., Lancet 340 (1992) 813-817]. [0008] Interestingly, HPPD was also shown to produce bHΐn as a result of its promiscuity towards a-ketoisocaproate, theketo acid of leucine [Crouch NP, E.Baldwin, M.-H. Lee, C.H. MacKinnon, Z.H. Zhang, Bioorg Med Chem Lett 1996, 6(13): 1503-1506] . In addition to its involvement in aromatic amino acid metabolism, HPPD is involved in the metabolism of leucine by converting excess a-ketoisocaproate into bHΐn [Crouch NP, Lee MH, Iturriagagoitia-Bueno T, MacKinnon CH. Methods in enzymology 2000, 324:342-355]. Prior to the elucidation of the promiscuity of HPPD, a dedicated dioxygenase to transfonn a-ketoisocaproate into bHΐn was alleged to exist [Sabourin PJ, Bieber LL: The Journal ofbiological chemistry 1982, 257(13):7468-7471 ; Sabourin PJ, Bieber LL: Methods in enzymology 1988, 166:288-297; Sabourin PJ, Bieber LL: Metabolism: clinical and experimental 1983, 32(2): 160-164; Xu et al., Biochemical and Biophysical Research Communications 276, (2000), 1080-1084] Baldwin et al., (1995) published early reports of HPPD having several fold higher activity with HPP than with a-ketoisocaproate [Baldwin et al., Bioorganic and Medicinal Chemistry Letters, 5(12) (1995), 1255-1260] Subsequently, sequence studies and further biochemical analyses by Crouch et al, (1996) and Crouch et al., (2000) confirmed that the alleged dioxygenase was HPPD which catalyzed the conversion of a- ketoisocaproate into bHΐn as a result of its promiscuity. Indeed, Crouch et al., 1996 suggested any further reference to HPPD as a-ketoisocaproate dioxygenase be discontinued. The promiscuity of HPPD is also evident by its transformation of 2-keto-4-(methylthio)butyric acid, the keto acid of methionine [Adlington, R.M., et al., Bioorganic & Medicinal Chemistry Letters, Volume 6, Issue 16, 20 August 1996, 2003-2006] [0009] Unlike some enzymes where substrate specificity is dictated only by active site conformation, the substrate specificity in HPPD extends beyond its active site pocket. The active site of HPPD is enclosed by a C-terminal a-helix which is believed to function as a gate-keeper for substrate access (Fritze et ah, Plant Physiol. Vol. 134, 2004). The rat HPPD was completely inactive when 14 residues were deleted from the C-terminus [Lee et ah, FEBS Letters, Vol. 393, Issues 2-3, 1996, 269-272]. Similarly, the human enzyme was inactive when 15 residues of the C-terminus were deleted [Lin et ah, PLoS ONE 8(8): e69733, 2013]. Other roles for the C- terminus are also possible. For example, homology modeling for a closed conformation for rat HPPD suggests that Q375 forms bifurcate hydrogen bonds with N380 and S250 while D384 may form a salt bridge pair with K249 in SEQ ID NO: 1. Therefore, the dynamic interactions of these sites (and others) might mediate the position of the C-terminal helix to ensure that the gate is opened during the catalytic cycle to allow binding of KIC and releasing HMB. These interactions may be necessary to maintain the integrity of the active site and ensure correct substrate orientation. Not only the presence of an intact C-terminal helix is critical, but its conformation also appears to play a significant role in substrate specificity.

[0010] There are several examples in the food, pharmaceutical, animal feed, biofuel, and biopolymer industries of producing ingredients through the use of metabolically engineered microorganisms and employing them in a fermentation process. Not all microorganisms are suited for the production of products. For example, bacteria are conventionally better suited for the production of amino acids, vitamins and enzymes while yeasts are better suited for the production of alcohols and organic acids. Therefore, selecting the appropriate microorganism to produce bHΐn is critical. This disclosure relates to methods of selecting a microorganism for bHΐn production.

[0011] Given that bHΐn is produced using chemical processes that are not only energy-intensive, but also result in toxic by-products, there is a clear and urgent need to develop environmentally benign processes that use renewable feedstocks. There is also a need for the production of high quality bHΐn that is cost-effective and efficiently produced.

SUMMARY

[0012] The subject of the present disclosure satisfies the need and provides related advantages as well. Provided herein are certain embodiments to create non-natural enzymes with increased bHIU synthase catalytic activity. Also provided herein are embodiments of using the non-natural bHΐn synthase enzymes in genetically modified microorganisms as well as in a cell-free manner. Also provided herein are methods to select and engineer microorganisms to produce beta hydroxyisovalerate (bHΐn) and uses of the engineered, non-natural microorganisms. This disclosure also provides methods of producing bHΐn by culturing the genetically modified microorganisms in the presence of at least one carbon source, then isolating bHΐn from the culture. In certain embodiments, the carbon source is one or more of glucose, xylose, arabinose, sucrose and lactose.

[0013] In some embodiments, the parent enzymes with basal bHΐn synthase activity are polypeptides that are at least 65% identical to a polypeptide selected from SEQ ID NOs: 1 - 3. In certain embodiments, the polypeptide with bHΐn synthase activity is derived from Rattus norvegicus. In some embodiments, the parent enzymes with bHΐn synthase activity are polypeptides that are at least 65% identical to a polypeptide selected from SEQ ID NOs: 4 - 148. [0014] In some embodiments, naturally occurring enzymes with basal bHΐn synthase activity are modified or mutated to increase their ability to catalyze a-ketoisocaproate into bHΐn. Examples of such non-natural enzymes with increased bHΐn activity compared with their corresponding wild-type parent are those having one or more modifications or mutations at positions corresponding to amino acids selected from A361, F336, F347, F364, F368, F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 1 or SEQ ID NO: 6.

[0015] In some embodiments, the non-natural enzymes with bHΐn synthase activity have at least one or more of the following modifications or mutations: leucine, isoleucine or methionine at position 361, leucine, isoleucine, methionine or tryptophan at position 336, tryptophan, tyrosine or isoleucine at position 347, alanine, leucine, isoleucine, methionine or tryptophan at position 364, tyrosine, tryptophan, leucine, isoleucine or methionine at position 368, leucine, isoleucine or methionine at position 371, leucine, isoleucine or methionine at position 362, leucine, valine or methionine at position 227, leucine, valine or methionine at position 252, phenylalanine, tryptophan or methionine at position 224, leucine, valine or methionine at position 289, tryptophan, tyrosine or isoleucine at position 323, leucine, isoleucine, tryptophan or methionine at position 367, phenylalanine, tryptophan or methionine at position 187, phenylalanine, tryptophan or methionine at position 241 , isoleucine, methionine or valine at position 363, leucine at position 239 of SEQ ID NO: 1, methionine, isoleucine or proline at position 251, methionine, isoleucine or proline at position 265, valine, methionine, isoleucine or leucine at position 226, phenylalanine, leucine, isoleucine or tryptophan at position 212, isoleucine, leucine or methionine at position 217, isoleucine, leucine or methionine at position 228, and/or leucine at position 210, wherein the modifications or mutations are of SEQ ID NO: 1 or SEQ ID NO: 6.

[0016] In some embodiments, at least one nucleic acid encoding a polypeptide with bHΐn synthase activity is introduced into a microorganism, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 1 - 3. In some embodiments, at least one nucleic acid encoding a polypeptide with bHΐn synthase activity is introduced into a microorganism, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 4 - 8.

[0017] In some embodiments, at least one nucleic acid encoding a polypeptide with bHI V synthase activity is introduced into a microorganism, wherein said polypeptide comprises at least or more of the mutations mentioned above. In some embodiments, the non-natural microorganism produces bHΐn or produces more bHΐn than its unmodified parent.

[0018] In some embodiments, a non-natural microorganism comprises a metabolic pathway relating to one or more steps of (i) pyruvate to acetolactate, (ii) acetolactate to 2,3- dihydroxyisovalerate, (iii) 2,3-dihydroxyisovalerate to a-ketoisovalerate, (iv) a-ketoisovalerate to a-isopropylmalate, (v) a-isopropylmalate to b-isopropylmalate, (vi) b-isopropylmalate to a- ketoisocaproate and (vii) a-ketoisocaproate to bHΐn. In some aspects, if the non-natural microorganism has multiple sub-cellular compartments, one or more genes for the one or more steps (i) to (vii) of the metabolic pathway encodes an enzyme that is localized to the cytosol. [0019] In some embodiments, a non-natural microorganism comprises a metabolic pathway relating to one or more steps of (i) pyruvate into acetolactate, (ii) acetolactate into 2,3- dihydroxyisovalerate, (iii) 2,3-dihydroxyisovalerate into a-ketoisovalerate, (iv) a-ketoisovalerate into 2-isopropylmalate, (v) 2-isopropylmalate into 2-isopropylmaleate, (vi) 2-isopropylmaleate into 3-isopropylmalate, (vii) 3-isopropylmalate into 2-isopropyl-3-oxosuccinate, (viii) 2- isopropyl-3-oxosuccinate into a-ketoisocaproate, and (ix) a-ketoisocaproate into bHΐn. In some aspects, if the non-natural microorganism has multiple sub-cellular compartments, one or more genes for the one or more steps (i) to (ix) of the metabolic pathway encodes an enzyme that is localized to the cytosol. [0020] In some embodiments, the non-natural microorganism expresses or overexpresses at least one of the genes encoding for acetolactate synthase (EC: 2.2.1.6), keto-acid reductoisomerase (EC: 1.1.1.86), dihydroxyacid dehydratase (EC: 4.2.1.9), 2-isopropylmalate synthase (EC: 2.3.3.13), isopropylmalate isomerase (EC: 4.2.1.33), 3-isopropylmalate dehydrogenase (EC: 1.1.1.85) and bHΐn synthase. In some aspects, the non-natural microorganism expresses or overexpresses two or more genes encoding for acetolactate synthase, keto-acid reductoisomerase, dihydroxyacid dehydratase, 2-isopropylmalate synthase, isopropylmalate isomerase, 3- isopropylmalate dehydrogenase and bHΐn synthase.

[0021] In certain embodiments, the non-natural microorganisms having multiple sub-cellular compartments, a bHΐn producing metabolic pathway according to Figure 1 or Figure 2 with at least one bHΐn pathway enzyme is localized in the cytosol. In an exemplary embodiment, the non-natural microorganisms comprise a bHΐn producing metabolic pathway with all the bHΐn pathway enzymes localized in the cytosol.

[0022] In some embodiments, the non-natural eukaryotic microorganism expresses or overexpresses at least one of the genes encoding for cytosolic acetolactate synthase, cytosolic keto-acid reductoisomerase, cytosolic dihydroxyacid dehydratase, cytosolic 2-isopropylmalate synthase, cytosolic isopropylmalate isomerase, cytosolic 3-isopropylmalate dehydrogenase and cytosolic bHΐn synthase. In some aspects, the non-natural eukaryotic microorganism expresses or overexpresses two or more genes encoding for cytosolic acetolactate synthase, cytosolic keto- acid reductoisomerase, cytosolic dihydroxyacid dehydratase, cytosolic 2-isopropylmalate synthase, cytosolic isopropylmalate isomerase, cytosolic 3-isopropylmalate dehydrogenase and cytosolic bHΐn synthase.

[0023] In some embodiments, the non-natural microorganism comprises at least one nucleic acid encoding a polypeptide with beta hydroxyisovalerate synthase activity wherein said polypeptide is at least about 65% identical to at least one polypeptide selected from SEQ ID NOs: 1 - 3. In certain embodiments, the polypeptide with bHΐn synthase activity is derived from Rattus norvegicus.

[0024] In certain embodiments, the non-natural microorganism comprises at least one nucleic acid encoding a polypeptide with bHΐn synthase activity wherein said polypeptide is at least about 65% identical to at least one polypeptide selected from SEQ ID NOs: 4 - 5. In certain embodiments, the polypeptide with bHIU synthase activity is derived from Yarrowia lipolytica. In certain embodiments, the non-natural microorganism comprises at least one nucleic acid encoding a polypeptide with bHΐn synthase activity wherein said polypeptide is at least about 65% identical to at least one polypeptide selected from SEQ ID NOs: 6 - 8. In certain embodiments, the polypeptide with bHΐn synthase activity is derived from Homo sapiens

[0025] In another embodiment, the non-natural microorganism comprises a dioxygenase enzyme which has been modified or mutated to increase the ability of the enzyme to preferentially utilize a-ketoisocaproate as its substrate. According to certain aspects of the present invention, the nonnatural enzyme comprises one or more dioxygenase enzymes having one or more modifications or mutations at substrate-specificity positions corresponding to amino acids selected from A361, F336, F347, F364, F368, F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 1 or SEQ ID NO: 6. [0026] In some aspects, at least one of the substrate-specificity positions corresponding to amino acids selected from the group consisting of A361, F336, F347, F364, F368, F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 1 or SEQ ID NO: 6 has been replaced with one of the corresponding disclosed amino acids to alter the respective substrate-specificity residue.

[0027] In some other aspects, two or more of the substrate-specificity positions corresponding to amino acids selected from the group consisting of A361, F336, F347, F364, F368, F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 1 or SEQ ID NO: 6 have been replaced with one of the corresponding disclosed amino acids to alter the respective substrate-specificity residue.

[0028] In yet some other aspects, at least 3 and up to 24 of the substrate-specificity positions corresponding to amino acids selected from the group consisting of A361, F336, F347, F364, F368, F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 1 or SEQ ID NO: 6 have been replaced with one of the corresponding disclosed amino acids to alter the respective substrate-specificity residue.

[0029] In certain embodiments, the non-natural microorganisms may be prokaryotic microorganisms. In some embodiments, the prokaryotic microorganisms may be Gram-positive selected from the group comprising of Corynebacterium, Lactobacillus, Lactococcus and Bacillus. In some embodiments, the non-natural prokaryotic microorganisms may be Gram- negative selected from the group comprising of Escherichia and Pseudomonas. In another embodiment, the non-natural microorganism may be non-natural eukaryotic microorganisms. In certain embodiments, the non-natural eukaryotic microorganisms may be non-natural yeast microorganisms. In some embodiments, the non-natural yeast microorganism may be selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Pfansenula, or Candida.

[0030] In another embodiment, the non-natural microorganism may be cultivated in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of bHΐn is produced and optionally, recovering the bHΐn. In certain embodiments, the non-natural microorganism produces bHΐn from a carbon source with a yield of at least about 0.1 percent of theoretical yield. In another aspect, the non-natural microorganism produces bHΐn from a carbon source with a yield of at least 1 percent of theoretical yield. In another aspect, the non-natural microorganism produces bHΐn from a carbon source with a yield of at least about 5 percent of theoretical yield. In another aspect, the non-natural microorganism produces bHΐn from a carbon source with a yield of at least 20 percent of theoretical yield. In another aspect, the non-natural microorganism produces bHΐn from a carbon source with a yield of at least 50 percent, at least about 75 percent, at least about 80 percent, or at least about 85 percent of the theoretical yield. [0031] In some aspects, the non-natural microorganism produces bHΐn from a carbon source with a yield of at least about 0.1 percent up to 100 percent of theoretical yield, in some aspects at least about 1 percent up to 99.9 percent of theoretical yield, in some aspects at least about 5 percent up to about 99.5 of theoretical yield, in some aspects at least 20 percent up to about 99.5 percent of theoretical yield, in some aspects at least 50 percent up to about 99.5 percent of theoretical yield, in some aspects at least about 75 percent up to about 99.5 percent of theoretical yield, in some aspects at least about 80 percent up to about 99.5 percent of theoretical yield, and in some aspects at least about 85 percent up to about 99.5 percent of theoretical yield.

[0032] In some embodiments, the non-natural polypeptide comprising bHΐn synthase activity may enable the “in vitro" conversion of a-ketoisocaproate into bHΐn in the presence of co-substrates and co-factors.

[0033] In some embodiments, the present invention is directed to a composition comprising bHΐn produced by a non-natural enzyme, wherein the bHΐn prior to any isolation or purification process has not been in substantial contact with any component comprising a halogen-containing component. In some aspects, the halogen-containing component is a chemical derivative produced by a typical chemical production process of bHΐn. In some aspects, the halogen- containing component comprises hydrochloric acid and/or chloroform.

[0034] In some embodiments, the present invention is directed to a composition comprising bII I V produced by a non-natural microorganism, wherein the bHΐn prior to any isolation or purification process has not been in substantial contact with any component comprising a halogen-containing component. In some aspects, the halogen-containing component is a chemical derivative produced by a typical chemical production process of bHΐn. In some aspects, the halogen- containing component comprises hydrochloric acid and/or chloroform.

[0035] The above summary is not intended to describe each illustrated embodiment or every implementation of the subject matter hereof. The figures and the detailed description that follow more particularly exemplify various embodiments.

BRIEF DESCRIPTION OF THE FIGURES

[0036] Subject matter hereof may be more completely understood in consideration of the following detailed description of various embodiments in connection with the accompanying figures, in which:

[0037] Figure 1 illustrates a bHΐn metabolic pathway, according to certain embodiments of the present invention. According to some aspects this disclosure, the metabolic pathway can also comprise an active transporter to transport bHΐn out of the non-natural microorganism.

[0038] Figure 2 illustrates another bHΐn metabolic pathway, according to certain embodiments of the present invention. According to some aspects of this disclosure, the metabolic pathway can also comprise an active transporter to transport bHΐn out of the non-natural microorganism. [0039] Figure 3 illustrates the growth of microorganisms as maximum specific growth rate in the presence of various concentrations of bHΐn in the media as an indicator of metabolic activity. [0040] Figure 4 is a bar graph illustrating the relative activity of exemplary bHΐn synthase variants with at least one mutation that have different activity with HPP (open bars) and KIC (closed bars), according to certain embodiments of the present invention.

[0041 ] Figure 5 is a bar graph illustrating the relative activity of exemplary bHΐn synthase variants with more than one mutation that have different activity with HPP (open bars) and KIC (closed bars) , according to certain embodiments of the present invention. [0042] Figure 6A-6E illustrates Python scripts used to calculate sequence entropy within dioxygenases described herein, according to certain embodiments of the present invention. [0043] Figure 7 illustrates the production of bHΐn using non-natural bacteria.

[0044] Figure 8 is a bar graph illustrating the accumulation of bHΐn by non-natural yeast microorganism harboring an unmodified dioxygenase enzyme (SB556) or an improved bHΐn synthase (SB557), according to certain embodiments of the present invention.

[0045] While various embodiments are amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the claimed inventions to the particular embodiments described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the subject matter as defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The following description of the present disclosure is merely intended to illustrate various embodiments. As such, the specific modifications discussed are not to be construed as limitations on the scope of the present disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein are incorporated by reference in their entirety.

[0047] The present disclosure relates to non-natural microorganisms and the use of said microorganisms in a fermentation process to produce higher value products such as organic acids. More specifically, the present disclosure relates to engineered microorganisms that produce b- hydroxyisovaleric acid (bHΐn). As a molecule with unique structure, bHΐn has potential applications ranging from liquid crystals to pharmaceutical ingredients and dietary supplements. [0048] As used herein, “b-hydroxyisovalerate” or “beta hydroxyisovalerate” or “bHΐn” or “b- hydroxy^-methylbutyrate” or “3 -hydroxy-3 -methylbutyric acid” refer to the same compound having the following molecular structures (free acid form on left and conjugate base on the right). Furthermore, these terms not only include the free acid form or conjugate base, but also the salt form with a cation and derivatives thereof, or any combination of these compounds. For instance, a calcium salt of bHIU includes calcium bHIU hydrate having the following molecular structure.

While the foregoing terms mean any form of [iHIV, the form of bHΐn used within the context of the present disclosure preferably is selected from the group comprising of a free acid, a calcium salt, an ester and a lactone.

[0049] As used herein, the temi “microorganism” refers to a prokaryote such as a bacterium or a eukaryote such as a yeast or a fungus. As used herein, the temi “non-natural microorganism” refers to a microorganism that has at least one genetic alteration not normally found in a naturally occurring strain of the species, including wild-type strains of the reference species. Genetic alterations include, for example, human-intervened modifications introducing expressible nucleic acids encoding polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microorganism’s genetic material. When a microorganism is genetically engineered to overexpress a given enzyme, it is manipulated such that the host cell has the capability to express, and preferably, overexpress an enzyme, thereby increasing the biocatalytic capability of the cell. When a microorganism is engineered to inactivate a gene, it is manipulated such that the host cell has decreased, and preferably, lost the capability to express an enzyme. As used herein, the tern “overexpress” refers to increasing the expression of an enzyme to a level greater than the cell normally produces. The term encompasses overexpression of endogenous as well as exogenous enzymes. As used herein, the terms “gene deletion” or “gene knockout” or “gene disruption” refer to the targeted disruption of the gene in vivo resulting in the removal of one or more nucleotides from the genome resulting in decreased or loss of function using genetic manipulation methods such as homologous recombination, directed mutagenesis or directed evolution.

[0050] As used herein, the tenn “gene” refers to a nucleic acid sequence that can be transcribed into messenger RNA and further translated into protein. [0051] The term “nucleic acid” as used herein, includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.

[0052] Usually, the nucleotide sequence encoding an enzyme is operably linked to a promoter that causes sufficient expression of the corresponding nucleotide sequence in the host microorganism according to the present disclosure to confer to the cell the ability to produce b-hydroxyisovaleric acid. As used herein, the term “operably linked” refers to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. In order to increase the likelihood that an exogenous gene is translated into an enzyme that is in active form, the corresponding nucleotide sequence may be adapted to optimize its codon usage to that of the chosen host microorganism. Several methods for codon optimization are known in the art and are embedded in computer programs such as Codon W, GenSmart, CodonOpt, etc.

[0053] As used herein, the term “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for a nucleic acid polymerase, transcription initiation sites and any other DNA sequences known to one of skill in the art. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation.

[0054] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, the protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms “polypeptide”, “peptide” and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma- carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0055] The tenn “enzyme” as used herein is defined as a protein which catalyzes a (bio)chemical reaction in a cell. The interaction of an enzyme with other molecules such as the substrate can be quantified by the Michaelis constant (K _M ), which indicates the affinity of the substrate to the active site of the enzyme. K can be quantified using prior art (see for example, Stryer, Biochemistry, 4th edition, W.H. Freeman, Nelson and Cox, Lehninger Principles of Biochemistry, 6th edition, W.H. Freeman). The rate of biocatalysis or enzymatic activity is defined by k _cat , which is the enzyme turnover number. Therefore, the ratio of the rate of enzymatic activity to the substrate affinity is widely considered to be representative of an enzyme’s catalytic efficiency. As defined herein, the efficiency of an enzyme to act on a specific substrate is quantified by the ratio ofk _cai /KM. Therefore, an enzyme with higher value of k _cat / w for a certain substrate can catalyze the reaction more efficiently than another enzyme with a lower value of k _cat /K _M for the same substrate. A non-natural enzyme refers to an enzyme that comprises at least one amino acid alteration at the desired position that is not normally found in nature. Amino acid alternations include, for example, human-intervened modifications introducing replacing one naturally occurring amino acid with another, addition or deletion of amino acids such that the modified enzyme has the capability of enhanced catalytic activity.

[0056] As used herein, b-hydroxyisovalerate synthase refers to an enzyme that can catalyze the conversion of a-ketoisocaproate into bΐΐ IV. One Unit (U) of bHΐn synthase activity is defined here as the amount of enzyme needed to convert one micromole of a-ketoisocaproate into bHΐn in one minute under the reaction conditions. Accordingly, a variant of bHΐn synthase that can convert more a-ketoisocaproate into bHΐn than the same amount of another variant is preferred. [0057] The term "biosynthetic pathway", also referred to as "metabolic pathway", refers to a set of anabolic or catabolic biochemical reactions for converting one chemical species into another. Gene products belong to the same "metabolic pathway" if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product. As used herein, the term "bHΐn metabolic pathway" or “bHΐn pathway” refers to an enzyme pathway which produces bHΐn from pyruvate, as illustrated in Figure 1 or Figure 2.

[0058] FIPPD is found in most aerobic forms of life (Gunsior et al, Biochemistry 43, 2004, 663- 674). HPPD has been shown to have reasonably broad substrate specificity, recognizing a range of polar and non-polar a-keto acids as substrates. For example, rat HPPD can decarboxylase and oxygenate KIC into bHΐn and a-keto-5-thiahexanoic acid into 4-thiapentanoic acid-4-oxide (Baldwin et ah, Bioorg. Med. Chem. Lett. 5, 1995, 1255-1260; Adlington et ah, Bioorg. Med. Chem. Lett. 6, 1996, 2003-2006). However, HPPD from Streptomyces has been shown to be entirely devoid of either decarboxylase or dioxygenase activity in the presence of KIC (Johnson- Winters, Biochemistry, 2003, 42: 2072-2080). Therefore, not all HPPDs have the catalytic ability to convert KIC into bHΐn. This disclosure relates to identifying HPPDs that have the basal promiscuous activity to produce bHΐn.

[0059] Several HPPD crystal structures from different organisms have been resolved and are available in the Protein Data Bank (PDB, www.rcsb.org). HPPD is observed to form oligomers. The monomer is folded into structural domains that are arranged as an N-terminal and C-terminal open b-barrel of eight b-strands each. The active site is located inside the b-barrel of the C- terminal domain and contains a 2-His-l-carboxylate motif that non-covalently binds a Fe ²⁺ ion (Fritze et ah, 2004, Plant Physiol 134:1388-1400). An examination of 19 crystal structures (17 HPPDs from rat, human, bacterial, and plant and 2 bacterial hydroxymandelate synthases) in the PDB revealed two conformations for HPPD - an “open” and a “closed”. The C-terminal helix forms a “lid” at the active site, with the N-terminal portion of the C-terminal helix in close proximity to the catalytic iron site. It is possible that the C-terminal helix serves as a gatekeeper for substrate docking and/or product release. It is also possible that C-terminal helix structure changes in response to substrate docking and could be allosterically coupled to enzyme catalysis. Given the position and the apparent dynamic structure of the C-terminal helix, it is likely to play a critical role in substrate specificity. Lability of the C-tenninal helix may also be related to the ability of these enzymes to catalyze reactions with multiple substrates (“enzyme promiscuity”). Presumably, another source of enzyme promiscuity is the capacity of the catalytic iron to activate molecule oxygen without strict requirements as to the chemical identity of the distal portion of the keto acid substrate. [0060] The naturally occurring parent enzymes identified herein have low activity using a- ketoisocaproate, thereby not enabling efficient production of bHΐn. The present disclosure describes methods of increasing bHΐn production through the use of non-natural enzymes. Accordingly, the present disclosure is directed to an isolated nucleic acid encoding a polypeptide with bHΐn synthase activity, wherein the polypeptide sequence is at least 65% identical to at least one polypeptide selected from SEQ ID Nos: 1 - 148. Methods to determine identity and similarity are codified in publicly available computer programs. Example computer program methods to determine identity and similarity between two sequences include BLASTP and BLASTN, publicly available fromNCBI and other sources (BLAST Manual, Altschul, S., et ah, NCBI NLM NIH Bethesda, Md. 20894). Example parameters for amino acid sequences comparison using BLASTP are gap open 11.0, gap extend 1 , Blosum 62 matrix.

[0061] In certain embodiments, the polypeptide with bHΐn synthase activity is derived from the genus Rattus. In an example embodiment, the polypeptide with bHΐn synthase activity is derived from Rattus norvegicus, F alloantigen Rattus norvegicus, Rattus rattus or Rattus losea. In another example embodiment, the polypeptide with bHΐn synthase activity is selected from at least one of SEQ ID NOS: 1 - 3.

[0062] In some embodiments, the polypeptide with bHΐn synthase activity has at least 65% identity to at least one polypeptide selected from SEQ ID NOS: 1 - 148. Further within the scope of the present application are polypeptides with bHΐn synthase activity which are at least about 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 98%, 97%, 98%, 99%, or 99.5% identical to at least one polypeptide selected from SEQ ID NOS: 1 - 148.

[0063] The promiscuous activity of HPPD with a-ketoisocaproate is indicative of a basal level recognition of the desired substrate and the present disclosure discloses methods to increase a- ketoisocaproate activity relative to 4-hydroxyphenyl pyruvate activity (KIC/HPP) by modifying certain amino acids at specific positions in the sequence. Modifying amino acids that play a role in the catalysis can lead to alterations in the enzyme activity. One skilled in the art can recognize the position of these amino acids in homologous protein sequences by aligning the sequences. Two sequences are said to be “optimally aligned” when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences. Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art. The BLOSUM82 matrix is often used as a default scoring substitution matrix in sequence alignment protocols such as Gapped BLAST 2.0. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acids positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences, so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer- implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402, and made available to the public at the National Center for Biotechnology Information Website. Optimal alignments, including multiple alignments, can be prepared using, e.g., PSI-BLAST with no compositional adjustments.

[0064] As described herein, the present inventors identified polypeptides with bHΐn synthase activity. One desirable feature of a polypeptide with bHΐn synthase activity is the ability to exhibit high activity for the conversion of a-ketoisocaproate into bHΐn. Another desirable property of a polypeptide with bHΐn synthase activity is low activity with the native substrate, HPP, thereby reducing the impact on other substrates. The present disclosure identifies several beneficial modifications or mutations which can be made to an existing dioxygenase enzyme to improve the dioxygenase enzyme's ability to catalyze the conversion of KIC to bHΐn with higher activity. In some embodiments, the non-natural enzyme is a polypeptide with increased KIC/HPP activity, wherein the sequence of the polypeptide has at least one modification.

[0065] According to certain aspects of the present invention, the non-natural enzyme comprises one or more modifications at substrate-specificity positions corresponding to amino acids selected from A361, F336, F347, F364, F368, F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 1 or SEQ ID NO: 6.

[0066] In some embodiments, the dioxygenase enzyme has been modified or mutated to alter one or more one of the substrate-specificity residues. In certain embodiments, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 361 is replaced with a residue selected from methionine, leucine and isoleucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 336 is replaced with leucine, methionine, isoleucine and tryptophan. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 347 is replaced with tryptophan, tyrosine and isoleucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 364 is replaced with methionine, alanine, isoleucine, leucine and tryptophan. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 368 is replaced with tyrosine, tryptophan, leucine, isoleucine and methionine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 371 is replaced with methionine, leucine and isoleucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 362 is replaced with methionine, leucine and isoleucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 227 is replaced with methionine, leucine and isoleucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 252 is replaced with methionine, leucine and valine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 361 is replaced with threonine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 224 is replaced with methionine, phenylalanine and tryptophan. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 289 is replaced with methionine, leucine and isoleucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 323 is replaced with tryptophan, tyrosine and isoleucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 367 is replaced with methionine, leucine, isoleucine and tryptophan. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 187 is replaced with methionine, phenylalanine and tryptophan. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 241 is replaced with methionine, phenylalanine and tryptophan. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 363 is replaced with methionine, isoleucine and valine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 239 is replaced with leucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 251 is replaced with methionine, isoleucine and proline. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 265 is replaced with methionine, isoleucine and proline. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 226 is replaced with methionine, valine, isoleucine and leucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 212 is replaced with phenylalanine, leucine, isoleucine or tryptophan. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 217 is replaced with methionine, isoleucine or leucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 228 is replaced with methionine, isoleucine or leucine. In another embodiment, the dioxygenase enzyme of SEQ ID NO: 1 or SEQ ID NO: 6 is modified, wherein the residue corresponding to position 210 is replaced with leucine.

[0067] In some aspects, at least one of the substrate-specificity positions corresponding to amino acids selected from the group consisting of A361, F336, F347, F364, F368, F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 1 or SEQ ID NO: 6 has been replaced with one of the corresponding disclosed amino acids to alter the respective substrate-specificity residue.

[0068] In some other aspects, two or more of the substrate-specificity positions corresponding to amino acids selected from the group consisting of A361, F336, F347, F364, F368, F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 1 or SEQ ID NO: 6 have been replaced with one of the corresponding disclosed amino acids to alter the respective substrate-specificity residue.

[0069] In yet some other aspects, at least 3 and up to 24 of the substrate-specificity positions corresponding to amino acids selected from the group consisting of A361, F336, F347, F364, F368, F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 1 or SEQ ID NO: 6 have been replaced with one of the corresponding disclosed amino acids to alter the respective substrate-specificity residue.

[0070] In an exemplary embodiment, the modified dioxygenase enzyme is derived from a corresponding unmodified dioxygenase that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a polypeptide selected from any of SEQ ID NOS: 1 - 8.

[0071 ] In some embodiments, the present disclosure relates to a polypeptide with increased bHΐn synthase activity, wherein the polypeptide sequence is derived from Yarrowia lipolytica and is at least 65% identical to a polypeptide selected from either of SEQ ID NOs: 4 - 5 and has been modified or mutated to alter one or more of the substrate-specificity residues. In certain embodiments, the polypeptide is modified at one or more positions corresponding to amino acids selected from A374, F349, F360, F377, F381, 1384, G375, V240, 1265, A374, L237, 1302, L336, L380, N200, N254, N377, P252, Q264, Q278, S239, V225, 1230, V241 and W223. In an exemplary embodiment, the modified decarboxylase enzyme is derived from a corresponding unmodified decarboxylase that is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a polypeptide selected from either of SEQ ID NOs: 4 - 5.

[0072] Corresponding amino acids in other dioxygenases are easily identified by visual inspection of the amino acid sequences or by using commercially available homology software programs. Thus, given the defined regions for changes and the assays described in the present application, one with skill in the art can make one or a number of modifications which would result in an increased ability to specifically catalyze the conversion of KIC to bHΐn, in any homologous dioxygenase enzyme of interest.

[0073] The modified polypeptides can be optimally aligned with the corresponding unmodified, wild-type dioxygenase enzymes to generate a similarity score which is at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, or most preferably at least about 95% of the score for the reference sequence using the BLOSUM82 matrix, with a gap existence penalty of 11 and a gap extension penalty of 1. [0074] In some embodiments, the non-natural bHΐn synthase enzyme comprises fragments of the disclosed polypeptides, which comprises at least 25, 30, 40, 50, 100, 150, 200, 250, 300 or 375 amino acids and retain bHΐn synthase activity. Such fragments may be obtained by deletion mutation, by recombinant techniques that are routine and well-known in the art, or by enzymatic digestion of the polypeptides of interest using any of a number of well-known proteolytic enzymes.

[0075] In addition, as is understood by the skilled artisan, not all positions within an enzyme are created equal. Certain "permissive sites" are more likely to accommodate mutations without affecting activity or stability. In a sequence family such as the 4-hydroxyphenylpyruvate dioxygenase (HPPD), there are hundreds of sites that are more permissive to mutation than other sites. One method to identify permissive sites is by quantifying the extent to which each site has variable amino acids among a collection of homologs. A standard calculation to quantify this variability is to compute the sequence entropy for each site.

[0076] In an example calculation, selection of sequences was initiated by performing a blastp (version 2.7.1+, default parameters) search against the NCBI nr database using the SEQ ID NO: 1 as the query. Of the 5000 sequences retrieved, the top 246 BLAST hits were selected for further analysis as this group was largely made up of mammalian sequences, whereas lower ranked sequenced were predominately non mammalian. Identical sequences were grouped using CD- HIT2 (c flag = 1.0) and a single sequence was chosen from each group. The remaining 219 unique sequences were then aligned using clustal-w2 (version 2.0.12, default parameters) and the alignment visually inspected using Jalview (version 2.11.1-3). Sequences with poorly aligned regions, likely resulting from miscalled coding regions, were eliminated. This resulted in a final curated set of 140 sequences. These sequences, corresponding to SEQ ID NOs: 9 - 148, were aligned using multiple sequence alignment. Accordingly, the multiple sequence alignment has a number of gaps. Typically, sequence identity is calculated by counting the number of matching amino acids after aligning two sequences, ignoring gaps in the alignment. To proceed, the analysis was limited to positions in the multiple sequence alignment where at least half of the sequences (>= 70) have an amino acid rather than a gap. Furthermore, for numbering simplicity, only sites for which a reference sequence (SEQ ID NO: 1) has an amino acid rather than a gap were considered. This resulted in 393 aligned positions. For each of these aligned positions, the sequence entropy was calculated as shown in Figure 6. First, the probability P, of observing each amino acid variant found at this site was calculated. Then the sum of -P * ln(P) over all amino acid variants at this site was calculated. If the site is completely conserved (common to all 140 aligned sequences), the sequence entropy would be 0. In contrast, if all 20 amino acids were found with equal probability, the sequence entropy would be 3.0.

[0077] Several positions within the multiple sequence aligmnent are diverse, with significant sequence entropy. Of the 393 positions, this example shows that 193 have sequence entropy exceeding a threshold of 0.05, 177 also exceed 0.10, 131 also exceed 0.20, and 84 also exceed 0.40. For example, the site for SERI 75 from SEQ ID NO: 1 has sequence entropy = 1.822. At this site 9 amino acid variants are represented, with the most common variants being SER (42 / 140), LYS (30 / 140), ASN (28 / 140), ARG (15 / 140), GLN (8 / 140), THR (7 / 140), ASP (5 / 140), HIS (4 / 140), and ALA (1 / 140).

[0078] As used herein, a permissive site exceeds a specified sequence entropy threshold using the code illustrated in Figure 6 Using a threshold level of 0.05 for permissive sites, the following positions corresponding to residues in SEQ ID NO: 1 residues are relatively pemiissive sites within the multiple sequence alignment: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 15, 17, 22, 33, 34, 35, 38, 40, 43, 44, 45, 48, 53, 56, 57, 58, 61, 62, 63, 64, 65, 66, 68, 70, 71, 72, 74, 78, 79, 80, 81, 82, 84, 87, 92, 96, 97, 101, 104, 105, 106, 107, 111, 112, 113, 116, 117, 118, 119, 121, 123, 125, 127, 128, 130, 133, 135, 136, 139, 147, 149, 150, 151, 153, 155, 160, 161, 162, 163, 164, 165, 166,

168, 169, 171, 172, 175, 176, 177, 179, 180, 181, 184, 188, 189, 191, 192, 194, 195, 196, 197,

198, 201, 202, 203, 205, 215, 217, 220, 221, 223, 227, 229, 230, 232, 235, 245, 247, 251, 256,

257, 259, 262, 267, 268, 269, 270, 272, 275, 276, 277, 278, 279, 280, 282, 283, 286, 287, 289,

290, 291, 293, 294, 297, 298, 300, 301, 302, 304, 305, 306, 308, 309, 311, 313, 314, 315, 316,

318, 321, 322, 324, 326, 328, 329, 332, 340, 346, 350, 352, 354, 365, 366, 369, 371, 373, 374,

376, 377, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392 and 393.

[0079] In contrast, sites below a specified sequence entropy threshold can be used to identify relatively non-permissive sites. Accordingly, as used herein, a non-permissive site falls below a specified threshold using the code illustrated in Figure 6. Using a threshold level of < 0.05 for non-permissive sites, the following positions corresponding to residues in SEQ ID NO: 1 are relatively non-permissive sites within the multiple sequence alignment: 11, 14, 16, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 36, 37, 39, 41, 42, 46, 47, 49, 50, 51, 52, 54, 55, 59, 60, 67, 69, 73, 75, 76, 77, 83, 85, 86, 88, 89, 90, 91, 93, 94, 95, 98, 99, 100, 102, 103, 108, 109, 110, 114, 115, 120, 122, 124, 126, 129, 131, 132, 134, 137, 138, 140, 141, 142, 143, 144, 145, 146, 148,

152, 154, 156, 157, 158, 159, 167, 170, 173, 174, 178, 182, 183, 185, 186, 187, 190, 193, 199,

200, 204, 206, 207, 208, 209, 210, 211, 212, 213, 214, 216, 218, 219, 222, 224, 225, 226, 228,

231, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 246, 248, 249, 250, 252, 253, 254,

255, 258, 260, 261, 263, 264, 265, 266, 271, 273, 274, 281, 284, 285, 288, 292, 295, 296, 299,

303, 307, 310, 312, 317, 319, 320, 323, 325, 327, 330, 331, 333, 334, 335, 336, 337, 338, 339,

341, 342, 343, 344, 345, 347, 348, 349, 351, 353, 355, 356, 357, 358, 359, 360, 361, 362, 363,

364, 367, 368, 370, 372, 375, 378, 379, 380 and 381.

[0080] In certain embodiments, the threshold level may be set at 0.10. Using a threshold level of > 0.10 for permissive sites, the following positions corresponding to residues in SEQ ID NO: 1 residues are relatively permissive sites within the multiple sequence alignment: 1, 2, 3, 4, 5, 6, 7, 9, 10, 12, 13, 15, 17, 22, 33, 34, 35, 38, 40, 43, 44, 45, 48, 53, 56, 57, 58, 61, 62, 63, 65, 66, 68,

70, 71, 72, 74, 78, 79, 80, 84, 87, 92, 96, 97, 104, 105, 106, 111, 112, 113, 116, 117, 118, 119, 123, 125, 127, 128, 130, 133, 136, 139, 147, 149, 150, 151, 153, 155, 160, 161, 162, 163, 164,

165, 166, 168, 169, 171, 172, 175, 176, 177, 179, 180, 181, 184, 188, 189, 191, 192, 194, 195,

196, 197, 198, 201, 202, 205, 215, 217, 220, 221, 223, 227, 229, 230, 232, 235, 245, 247, 251,

257, 259, 262, 267, 268, 269, 270, 272, 276, 277, 278, 279, 280, 282, 283, 286, 289, 290, 291,

293, 294, 297, 298, 300, 301, 302, 305, 306, 308, 309, 313, 314, 315, 316, 321, 324, 326, 328,

329, 332, 340, 346, 350, 352, 354, 365, 366, 369, 371, 373, 374, 376, 377, 382, 383, 384, 385,

386, 387, 388, 389, 390, 391, 392, 393. Likewise, using a threshold level of < 0.10 for non- permissive sites, the following positions corresponding to SEQ ID NO: 1 residues are relatively non-permissive sites within the multiple sequence alignment: 8, 11, 14, 16, 18, 19, 20, 21, 23, 24,

25, 26, 27, 28, 29, 30, 31, 32, 36, 37, 39, 41, 42, 46, 47, 49, 50, 51, 52, 54, 55, 59, 60, 64, 67, 69,

73, 75, 76, 77, 81, 82, 83, 85, 86, 88, 89, 90, 91, 93, 94, 95, 98, 99, 100, 101, 102, 103, 107, 108, 109, 110, 114, 115, 120, 121, 122, 124, 126, 129, 131, 132, 134, 135, 137, 138, 140, 141, 142,

143, 144, 145, 146, 148, 152, 154, 156, 157, 158, 159, 167, 170, 173, 174, 178, 182, 183, 185,

186, 187, 190, 193, 199, 200, 203, 204, 206, 207, 208, 209, 210, 211, 212, 213, 214, 216, 218,

219, 222, 224, 225, 226, 228, 231, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 246,

248, 249, 250, 252, 253, 254, 255, 256, 258, 260, 261, 263, 264, 265, 266, 271, 273, 274, 275,

281, 284, 285, 287, 288, 292, 295, 296, 299, 303, 304, 307, 310, 311, 312, 317, 318, 319, 320,

322, 323, 325, 327, 330, 331, 333, 334, 335, 336, 337, 338, 339, 341, 342, 343, 344, 345, 347, 348, 349, 351, 353, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 367, 368, 370, 372, 375, 378, 379, 380, 381.

[0081] In certain embodiments, the threshold level may be set at 0.20. Using a threshold level of > 0.20 for permissive sites, the following positions corresponding to SEQ ID NO: 1 residues are relatively permissive sites within the multiple sequence alignment: 1, 3, 5, 6, 7, 9, 12, 13, 15, 22,

35, 38, 40, 43, 44, 45, 48, 57, 58, 62, 65, 66, 68, 70, 71, 72, 78, 80, 84, 96, 97, 104, 105, 111, 113, 116, 117, 118, 119, 123, 125, 128, 130, 133, 150, 151, 153, 155, 160, 161, 162, 163, 164, 165,

166, 168, 169, 171, 172, 175, 176, 177, 179, 180, 184, 191, 194, 195, 196, 197, 198, 201, 202,

205, 215, 217, 221, 223, 227, 230, 235, 245, 247, 257, 259, 262, 270, 272, 276, 277, 279, 280,

282, 283, 286, 289, 290, 293, 294, 297, 298, 302, 305, 309, 313, 314, 316, 321, 326, 340, 346,

354, 365, 366, 369, 371, 373, 374, 376, 377, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,

393. Likewise, using a threshold level of < 0.20 for non-permissive sites, the following positions corresponding to SEQ ID NO: 1 residues are relatively non-permissive sites within the multiple sequence alignment: 2, 4, 8, 10, 11, 14, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31,

32, 33, 34, 36, 37, 39, 41, 42, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 59, 60, 61, 63, 64, 67, 69, 73,

74, 75, 76, 77, 79, 81, 82, 83, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 98, 99, 100, 101, 102, 103, 106, 107, 108, 109, 110, 112, 114, 115, 120, 121, 122, 124, 126, 127, 129, 131, 132, 134, 135,

136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 152, 154, 156, 157, 158,

159, 167, 170, 173, 174, 178, 181, 182, 183, 185, 186, 187, 188, 189, 190, 192, 193, 199, 200,

203, 204, 206, 207, 208, 209, 210, 211, 212, 213, 214, 216, 218, 219, 220, 222, 224, 225, 226,

228, 229, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 246, 248, 249, 250,

251, 252, 253, 254, 255, 256, 258, 260, 261, 263, 264, 265, 266, 267, 268, 269, 271, 273, 274,

275, 278, 281, 284, 285, 287, 288, 291, 292, 295, 296, 299, 300, 301, 303, 304, 306, 307, 308,

310, 311, 312, 315, 317, 318, 319, 320, 322, 323, 324, 325, 327, 328, 329, 330, 331, 332, 333,

334, 335, 336, 337, 338, 339, 341, 342, 343, 344, 345, 347, 348, 349, 350, 351, 352, 353, 355,

356, 357, 358, 359, 360, 361, 362, 363, 364, 367, 368, 370, 372, 375, 378, 379, 380, 381, 382.

[0082] In certain embodiments, the threshold level may be set at 0.40. Using a threshold level of > 0.40 for permissive sites, the following positions corresponding to SEQ ID NO: 1 residues are relatively pennissive sites within the multiple sequence alignment: 3, 5, 6, 9, 12, 35, 38, 48, 62, 66, 71, 72, 104, 105, 116, 118, 119, 123, 125, 128, 133, 150, 151, 153, 155, 160, 162, 163, 164, 165, 166, 168, 169, 171, 175, 177, 179, 180, 184, 194, 198, 201, 202, 230, 235, 245, 247, 270, 272, 277, 279, 280, 282, 283, 286, 290, 293, 294, 297, 298, 302, 305, 309, 313, 314, 316, 321,

340, 354, 366, 373, 376, 377, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393. Likewise, using a threshold level of < 0.40 for non-pemiissive sites, the following positions corresponding to SEQ ID NO: 1 residues are relatively non-permissive sites within the multiple sequence alignment: 1, 2, 4, 7, 8, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 65, 67, 68, 69, 70, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 117, 120, 121, 122, 124, 126, 127, 129, 130, 131, 132, 134, 135, 136, 137, 138,

139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 152, 154, 156, 157, 158, 159, 161, 167,

170, 172, 173, 174, 176, 178, 181, 182, 183, 185, 186, 187, 188, 189, 190, 191, 192, 193, 195,

196, 197, 199, 200, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,

218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 231, 232, 233, 234, 236, 237, 238,

239, 240, 241, 242, 243, 244, 246, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,

260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 271, 273, 274, 275, 276, 278, 281, 284, 285,

287, 288, 289, 291, 292, 295, 296, 299, 300, 301, 303, 304, 306, 307, 308, 310, 311, 312, 315,

317, 318, 319, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,

337, 338, 339, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 355, 356, 357,

358, 359, 360, 361, 362, 363, 364, 365, 367, 368, 369, 370, 371, 372, 374, 375, 378, 379, 380,

381, 382.

[0083] Directed evolution can be considered as an orthogonal method for enzyme improvement that can be used either by itself or in conjunction with rational design (see Arnold, F., Acc. Chem. Res. 1998, 31, 125-131 and Johannes and Zhao, Curr Opinion in Microbiol 2006, 9:261-267 for more information). Unlike rational design that depends on structural modeling and scoring functions to identify specific residues to mutate, directed evolution relies on simple yet powerful principles of mutation. Starting from a single target gene that encodes for the parent enzyme, a library of mutant genes is created through mutagenesis and/or gene recombination. Iterative accumulation of small improvements identified using multiple rounds of diversification and screening can lead to substantial net enzyme improvements. Methods to subject enzymes through directed evolution are well-characterized in the literature (see for example, Bloom et al, Current Opinion in Structural Biology 2005, 15:447M52). In some embodiments, the non-natural bHIU synthase is subjected to directed evolution and variants with higher bHΐn synthase activity are selected.

[0084] Accordingly, in some embodiments, the present disclosure provides a nucleic acid molecule encoding a modified dioxygenase, wherein said modified dioxygenase is derived from a corresponding wild-type, unmodified dioxygenase, wherein the sequence of non-permissive sites within said modified dioxygenase is at least about 60%, at least about 70%, at least about 80%, or at least about 90% identical to the sequence of non-permissive sites within the corresponding wild-type, unmodified dioxygenase. In one embodiment, the threshold level for distinguishing between permissive and non-permissive sites using the code illustrated in Figure 5 is 0.05. In certain other embodiments, the threshold level for distinguishing between permissive and non-permissive sites using the code illustrated in Figure 6 is selected from 0.1, 0.2 and 0.4. In an exemplary embodiment, the modified dioxygenase enzyme is derived from a corresponding wild-type, unmodified dioxygenase selected from any of SEQ ID NOs: 1 - 148. In some embodiments, the corresponding wild-type, unmodified dioxygenase is obtained from Rattus species. In a further exemplary embodiment, the corresponding wild-type, unmodified dioxygenase is obtained from Rattus species classified into a genera selected from the group consisting of Rattus norvegicus, Rattus rattus , R. xanthurus , R. leucopus and R. fuscipes. In another further exemplary embodiment, the corresponding wild-type, unmodified dioxygenase is obtained from Homo sapiens. In another further embodiment, the corresponding wild-type, unmodified dioxygenase is obtained from Arabidopsis. In another further exemplary embodiment, the corresponding wild-type, unmodified dioxygenase is obtained from Yarrowia. In another further exemplary embodiment, the corresponding wild-type, unmodified dioxygenase is obtained from Pseudomonas . In another further exemplary embodiment, the corresponding wild-type, unmodified dioxygenase is obtained from Streptomyces. In an exemplary embodiment, the corresponding wild-type, unmodified dioxygenase is obtained from Rattus norvegicus. In another exemplary embodiment, the corresponding wild-type, unmodified dioxygenase is 4- hydroxyphenyl pyruvate dioxygenase (SEQ ID NO: 1 - 3).

[0085] In accordance with the present disclosure, any number of mutations can be made to the dioxygenase enzymes, and in certain embodiments, multiple mutations can be made to result in an increased ability to catalyze the conversion of KIC to [ill IV with high catalytic efficiency. Such mutations can include point mutations, frame shift mutations, deletions, and insertions. In certain embodiments, one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more, etc.) point mutations may be preferred.

[0086] In some embodiments, the bHΐn synthase will have an intact C-terminus. As defined herein, the C-terminus of bHΐn synthase is the stretch of residues that include the C-terminal a- helix that shields the active site. For example, in SEQ ID NO: 1, the stretch of amino acids from 361 to 393 are considered the C-terminus. In some embodiments, the residues comprising the C- terminus are modified to allow increased activity with KIC. In some embodiments, the C-terminus of the bHΐn synthase is engineered to favor a conformation with high specificity for KIC.

[0087] In some embodiments, the non-natural microorganism comprises at least one nucleic acid molecule encoding a polypeptide with bHΐn synthase activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOS: 1 - 149. Further within the scope of present disclosure are recombinant microorganisms comprising at least one nucleic acid molecule encoding a polypeptide with bHΐn synthase activity, wherein said polypeptide is at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a polypeptide selected from SEQ ID NOS: 1 - 149.

[0088] The present disclosure relates to a non-natural microorganism for producing bHΐn. Tolerance to high concentrations of bHΐn is an important trait of a suitable microorganism. An ideal microorganism to enable bHΐn production is capable of conducting fermentation at low pH levels to decrease downstream recovery costs, resulting in more economical production. Additional characteristics of a suitable microorganism include rapid growth and exhibit overall process robustness. In some embodiments, the subject of the present disclosure relates to a non natural microorganism having an active bHΐn metabolic pathway from pyruvate to bHΐn.

[0089] A bHΐn metabolic pathway is shown in Figure 1. In some embodiments, bHΐn metabolic pathway comprises of the conversion of pyruvate into 2-acetolactate, 2-acetolactate into 2,3- dihydroxy-isovalerate, 2,3-dihydroxy-isovalerate into a-ketoisovalerate, a-ketoisovalerate into 2- isopropylmalate, 2-isopropylmalate into 3-isopropylmalate, 3-isopropylmalate into KIC and KIC into bHΐn.

[0090] Another bHΐn metabolic pathway is shown in Figure 2. In some embodiments, bHΐn metabolic pathway comprises of the conversion of pyruvate into 2-acetolactate, 2-acetolactate into 2,3 -dihydroxy-isovalerate, 2,3-dihydroxy-isovalerate into a-ketoisovalerate, a- ketoisovalerate into 2-isopropylmalate, 2-isopropylmalate into 2-isopropylmaleate, 2- isopropylmaleate into 3-isopropylmalate, 3-isopropylmalate into 2-isopropyl-3-oxosuccinate, 2- isopropyl-3-oxosuccinate into KIC, KIC into bHΐn.

[0091] In some embodiments, the bHΐn pathway also comprises a hydroxy acid transporter to facilitate the export of bHΐn formed inside the microorganism to extracellular environment. [0092] As used herein, the “theoretical yield” of bHΐn refers to the molar ratio of bHΐn that is produced extracellularly to the carbon source that is used. The theoretical yield can be calculated based on the biochemical conversion of glucose to pyruvate and the subsequent conversion of pyruvate into bHΐn in the bHΐn metabolic pathway, taking into consideration of the microorganism’s native redox constraints to enable the conversion. For example, in yeast, the theoretical yield of bHΐn from glucose is 0.667.

[0093] In a first example embodiment, the non-natural microorganism of the present disclosure (a) expresses or overexpresses at least one gene encoding for bHΐn synthase, (b) expresses or overexpresses at least one gene encoding for acetolactate synthase, (c) expresses or overexpresses at least one gene encoding for acetohydroxy acid reductoisomerase, (d) expresses or overexpresses at least one gene encoding for 2,3 -dihydroxy isovalerate dehydratase, (e) expresses or overexpresses at least one gene encoding for 2-isopropylmalate synthase (f) expresses or overexpresses at least one gene encoding for isopropylmalate isomerase, or (g) expresses or overexpresses at least one gene encoding for 3-isopropylmalate dehydrogenase. In some aspects, the non-natural microorganism of the present disclosure comprises a combination of two or more of (a), (b), (c) (d), (e), (f) and (g).

[0094] In a second example embodiment, the non-natural microorganism of the present disclosure (a) expresses or overexpresses at least one gene encoding for bHΐn synthase, (b) expresses or overexpresses at least one gene encoding for acetolactate synthase, (c) expresses or overexpresses at least one gene encoding for 2,3-keto-acid reductoisomerase, (d) expresses or overexpresses at least one gene encoding for dihydroxy isovalerate dehydratase, (e) expresses or overexpresses at least one gene encoding for 2-isopropylmalate synthase, (i) expresses or overexpresses at least one gene encoding for 2-isopropylmalate hydrolyase (2-isopropylmaleate-forming), (j) expresses or overexpresses at least one gene encoding for 2-isopropylmalate hydrolyase (3-isopropylmalate- forming), (g) expresses or overexpresses at least one gene encoding for 3-isopropylmalate dehydrogenase. In some aspects, the non-natural organism of the present disclosure comprises a combination of two or more of (a), (b), (c) (d), (e), (i), (j) and (g). [0095] In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide with acetolactate synthase (EC: 2.2.1.6) activity. For example, acetolactate synthases capable of converting pyruvate to acetolactate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including B. subtilis (GenBank Accession No. Q04789.3), L. lactis (GenBank Accession No. NP_267340.1), S. mutans (GenBank Accession No. NP_721805.1), K. pneumoniae (GenBank Accession No. PTD93137.1), C. glutamicum (GenBank Accession No. 1238373540), E, cloacae (GenBank Accession No. WP 013097652.1), M. maripaiudis (GenBank Accession No. ABX01060.1), P. grisea (GenBank Accession No. AAB81248.1), T. stipitatus (GenBank Accession No. XP 002485976.1), or S. cerevisiae ILV2 (GenBank Accession No. 1789111829). Additional acetolactate synthases capable of converting pyruvate to acetolactate are described in WO2013016724, which incorporated herein by reference in its entirety. A review article characterizing the biosynthesis of acetolactate from pyruvate via the activity of acetolactate synthases is provided by Chipman et al., 1998, Biochimica et Biophysica Acta 1385: 401 -19. Chipman et al, provide an alignment and consensus for the sequences of a representative number of acetolactate synthases. Motifs shared in common between the majority of acetolactate synthases include: SGPG(A/C/V)(T/S)N, GX(P/A)GX(V7A/T),

GX(Q/G)(T/A)(IJM)G(Y/F/W)(A/G)X(P/G)(W/A)AX(G/T)(A/V) and GD(G/A)(G/S/C)F, at amino acid positions corresponding to the 163-169, 240-245, 521 -535, and 549-553 residues, respectively, of the S. cerevisiae ILV2. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit acetolactate synthase activity. In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide that is at least about 65% identical to at least one polypeptide selected from SEQ ID NOs: 297 - 300.

[0096] In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide with acetohydroxy acid reductoisomerase activity (EC: 1.1.1.86). Acetohydroxy acid reductoisomerases capable of converting acetolactate to 2,3- dihydroxyisovaierate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including E. coli (GenBank Accession No. EGB30597.1), L. lactis (GenBank Accession No. WP_012897822.1), Shewanella sp, (GenBank Accession No. WP_01 1621167.1), A. fischeri (GenBank Accession No. WP_005421503.1), M. maripaiudis (GenBank Accession No. AB035228.1), B. subtilis (GenBank Accession No. CAB14789), S. pombe (GenBank Accession No. NP_001018845) or S. cerevisiae ILV 5 (GenBank Accession No. NP_013459.1). Additional ketol-acid reductoisomerases capable of converting acetolactate to 2,3- dihydroxyisovalerate are described in WO2013016724, incorporated herein by reference in its entirety. Motifs shared between a majority of acetohydroxy acid reductoisomerases include G(Y/C/W)GXQ(G/A), (F/Y/L)(S/A)HG(F/L), V(V/I/F)(M/L/A)(A/C)PK,

D(L/I)XGE(Q/R)XXLXG and S (D/N AT)T A(E/Q/R)XG at amino acid positions corresponding to the 89-94, 175-179, 194-200, 282- 272, and 459-465 residues, respectively, of the E. coli acetohydroxy acid reductoisomerase encoded by ilvC. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit acetohydroxy acid reductoisomerase activity. The naturally existing acetohydroxy acid reductoisomerases preferentially use NADPH as a cofactor. Cofactor specificity can be switched to preferentially use NADH as a cofactor by means of modifying specific residues. Examples of such acetohydroxy acid reductoisomerases with increased preference for using NADH as a co factor are described in US Publication No. 2010/0143997. In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide that is at least about 65% identical to at least one polypeptide selected from SEQ ID NOs: 301 - 303.

[0097] In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide with 2,3 -dihydroxy isovalerate dehydratase activity (EC: 4.2.1.9). Dihydroxy acid dehydratases capable of converting 2,3- dihydroxyisovalerate to a-ketoisovalerate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including M. tuberculosis (GenBank Accession No. CLR57443), L. lactis (GenBank Accession No. WP_010905837.1), S. mutans (GenBank Accession No. WP_002262431.1), M. stadtmanae (GenBank Accession No. WP_011407142.1), M. tractuosa (GenBank Accession No. WP_013453775.1), Eubacterium SCB49 (GenBank Accession No. WP_1 18518751.1), Y. lipolytica (GenBank Accession No. QNP96049.1), N. crassa (GenBank Accession No. XP_963045.1), or S. cerevissae ILV3 (GenBank Accession No. NP_012550.1). Additional dihydroxy acid dehydratases capable of 2,3 -dihydro xyisovaierate to a-ketoisovalerate are described in WO2013016724, incorporated herein by reference in its entirety. Motifs shared in common between the majority of 2,3-dihydroxy isovalerate dehydratases include: SLXSRXXIA, CDKXXPG, GXCXGXXTAN, GGSTN, GPXGXP GMRXE, ALXTDGRXSG, and GHXXPEA motifs at amino acid positions corresponding to the 93-101, 122-128, 193-202, 276- 280, 482-491, 509-518, and 526-532 residues, respectively, of the E. coli 2,3-dihydroxy isovalerate dehydratase. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 2,3-dihydroxy isovalerate dehydratase activity. In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide that is at least about 65% identical to at least one polypeptide selected from SEQ ID NOs: 304 - 307.

[0098] In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide with 2-isopropylmalate synthase activity (EC: 2.3.3.13). 2-isopropylmalate synthases capable of converting 3-methyl-2- oxobutanoate to (2S)-2-isopropylmalate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including C. glutamicum (GenBank Accession No. WP 015439406), E. coli (GenBank Accession No. WP 300082850.1), S. cerevisiae (GenBank Accession No. NP_0 14295.1 (Leu4) and NP_014751.1 (Leu9), M. maripaludis (GenBank Accession No. WP_011171007.1) or N. crassa (GenBank Accession No. XP_964875.1). Motifs shared in common between the majority of the 2-isopropylmalate synthases include: LRDGXQ, IEVXFPXXSXXD, ISXHXHNDXGXXV, AGAXXVEG, GXGERXGN XXL at amino acid positions corresponding to the 12-17, 43-54, 199-211, 220-227, 231-241 residues, respectively, of the E. coli 2-isopropylmalate synthase. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 2-isopropylmalate synthase activity. In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide that is at least about 65% identical to at least one polypeptide selected from SEQ ID NOs: 308 - 313.

[0099] In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide with 2-isopropylmalate isomerase activity (EC: 4.2.1.33). In some embodiments, the isomerization of 2-isopropylmalate into 3- isopropylmalate is catalyzed by an enzyme that is expressed by one gene. Such 2-isopropylmalate isomerases capable of converting 2-isopropylmalate into 3-isopropylmalate may be derived from a variety of sources, including S. cerevisiae (GenBank Accession NP 011506.1), P. kudriavzevii (GenBank Accession No. XP_029320833.1) or C. albicans (GenBank Accession No. XP_718655.1). In some embodiments, the isomerization of 2-isopropylmalate into 3- isopropylmalate is catalyzed by an enzyme that is expressed by two genes, each gene encoding for a different subunit. Such 2-isopropylmalate isomerases capable of converting 2- isopropylmalate into 3 -isopropylmalate may be derived from a variety of sources (e.g., bacterial, Archaea, etc.), including M. tuberculosis (GenBank Accession No. NP_217504.1), L. lactis (GenBank Accession No. WP_095586897.1), S. mutans (GenBank Accession No. WP_002262706.1), C. glutamicum (GenBank Accession No. WP_003858858.1), M. maripaludis (GenBank Accession No. WP_011171424.1) and E. coli. MG1655 (GenBank Accession No. NP 414614.1). In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide with 2-isopropylmalate isomerase activity (EC: 4.2.1.33), containing a subunit with 3 -isopropylmalate dehydratase activity. Motifs shared in common between the majority of the enzymes include: HEVTSPQAF, DSHTXTHGAFG, AFGIGTSEVEHVXATQT, CNMXIEXGA, VFXGSCTNXRXXDL, EXCASTSNRNFEGRQG, and GHXXPEA motifs at amino acid positions corresponding to the 33-41, 128-138, 141-157, 220-228, 342-355, and 422-437, residues, respectively, oftheis. coli 3- isopropylmalate dehydratase. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 3 -isopropylmalate dehydratase activity. In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide that is at least about 65% identical to at least one polypeptide selected from SEQ ID NOs: 314 - 315.

[0100] In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide with 3 -isopropylmalate dehydrogenase activity (EC: 1.1.1.85). 3 -isopropylmalate dehydrogenase capable of converting (2R,3S)-3- isopropylmalate to 4-Methyl-2-oxopentanoate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, plant, etc.), including A. thaliana (GenBank Accession No. NP_001322636.1), L. lactis (GenBank Accession No. WP_095586896.1), S. mutans (GenBank Accession No. WP 302262707.1), C. glutamicum (GenBank Accession No. WP_011014258.1), M. maripaludis (GenBank Accession No. WP_011170483.1), E. coli. MG 1655 (GenBank Accession No. NP 414615.4), P. kudriavzevii (GenBank Accession No. XP_029322355.1), C. albicans (GenBank Accession No. XP_720371.1), or S. cerevisiae S288C (GenBank Accession No. NP_009911.2). Motifs shared in common between the majority of 3 -isopropylmalate hydratases include: D AXLLG AXGXP , VRELXGGIYFG, DKXNVL, TXNXFGDILSDEA, LXEPXHGSAPD, and NPXAXILSXAMXL motifs at amino acid positions corresponding to the 69-79, 137-147, 260-265, 245-257, 279-289, and 297-309 residues, respectively, of the E. coli 3- isopropylmalate dehydrogenase. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 3-isopropylmalate dehydrogenase activity. In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide that is at least about 65% identical to at least one polypeptide selected from SEQ ID NOs: 316 - 320.

[0101] In some embodiments, the non-natural microorganism of the present disclosure expresses or overexpresses at least one gene encoding a polypeptide with bHΐn synthase activity. The non natural enzymes disclosed herein have low activity using 4-hydroxyphenylpyruvate, thereby not introducing any undesirable alterations in the metabolism. The present disclosure describes methods of increasing bEIΐn production through the use of non-natural microorganisms. Accordingly, the present disclosure is directed to an isolated nucleic acid encoding a polypeptide with bHΐn synthase activity, wherein the polypeptide sequence is at least 65% identical to at least one polypeptide selected from any of SEQ ID Nos: 1 - 148. In some embodiments, the bHΐn synthase will have an intact C-tenninus. As defined herein, the C-terminus of HPPD is the stretch of residues that include the C-tenninal a-helix that shields the active site. For example, in SEQ ID NO: 1, the stretch of amino acids from 361 to 393 are considered the C-terminus. In some embodiments, the residues comprising the C-terminus are modified to allow increased activity with KIC. In some embodiments, the C-tenninus of the HPPD is in a conformation to have the highest specificity for KIC.

[0102] In some embodiments, the non-natural microorganism expresses or overexpresses nucleotides that encode for polypeptides with bHΐn synthase activity has at least 65% identity to at least one polypeptide selected from any of SEQ ID NOS: 1 - 148. Further within the scope of the present application are polypeptides with bHΐn synthase activity which are at least about 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 98%, 97%, 98%, 99%, or 99.5% identical to at least one polypeptide selected from any of SEQ ID NOS: 1 - 148.

[0103] According to certain aspects of the present invention, the non-natural microorganism expresses or overexpresses nucleotides that encode for a bHΐn synthase enzyme comprising one or more modifications at substrate-specificity positions corresponding to amino acids selected from A361, F336, F347, F364, F368, F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 1 or SEQ ID NO: 6.

[0104] Substrate-specificity positions corresponding to those identified in SEQ ID NO: 1 can be similarly identified in other related enzymes by visual inspection of sequences that are aligned with SEQ ID NO: 1. In accordance with the present disclosure, any number of mutations can be made to the bHΐn synthase enzymes, and in certain embodiments, multiple mutations can be made to result in an increased ability to catalyze the conversion of KIC to bHΐn with high catalytic efficiency. Such mutations can include point mutations, frame shift mutations, deletions, and insertions. In certain embodiments, one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more, etc.) point mutations may be preferred.

[0105] In some embodiments, the non-natural microorganism expresses or overexpresses a nucleic acid encoding fragments of the disclosed polypeptides which comprises at least 25, 30, 40, 50, 100, 150, 200, 250, 300 or 375 amino acids and retain bHΐn synthase activity. Such fragments may be obtained by deletion mutation, by recombinant techniques that are routine and well-known in the art, or by enzymatic digestion of the polypeptides of interest using any of a number of well- known proteolytic enzymes.

[0106] In one embodiment, the bHΐn metabolic pathway is localized to the cytosol of the non natural microorganism with multiple sub-cellular compartments. In one embodiment, the non natural microorganism with multiple sub-cellular components comprises a bHΐn metabolic pathway with at least one pathway enzyme localized in the cytosol.

[0107] In some embodiments, the non-natural microorganism belongs to a genus selected from the group consisting of Escherichia, Corynebacterium, Lactobacillus, Lactococcus and Bacillus. In some embodiments, the non-natural microorganism belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Issatchenkia, Galactomyces, Pichia and Candida. [0108] In some embodiments where the non-natural microorganism is a eukaryote, the bHΐn metabolic pathway is expressed or overexpressed in its cytosol.

[0109] In certain embodiments, the non-natural microorganism comes in contact with a carbon source in a fermenter to produce bHΐn and introducing into the fermenter sufficient nutrients where the final concentration of b-hydroxyisovalerate concentration in the fennentation broth is greater than about 10 mg/L (for example, greater than about 100 mg/L, for example, greater than about 1 g/L, greater than about 5 g/L, greater than about 10 g/L, greater than about 20 g/L, greater than about 40 g/L, greater than 50 g/L), but usually below 150 g/L. In certain embodiments, the carbon source is selected from the group consisting of glucose, xylose, arabinose, sucrose, fructose, lactose, glycerol, and mixtures thereof.

[0110] In some embodiments, bHΐn thus produced is optionally recovered from the fermentation broth by first removing the cells, followed by separating the aqueous phase from the clarified fermentation broth along with the other by-products of the fermentation. In some embodiments, the bHΐn is co-purified with other fermentation-derived products, wherein the composition comprises at least one fennentation-derived impurity. In some embodiments, fermentation- derived products are selected from the group consisting of organic acids and amino acids. In some embodiments, bHΐn synthesized according to the present disclosure is substantially devoid of chlorofonn or hydrochloric acid.

[0111] The object of the present disclosure is further illustrated by the following examples that should not be construed as limiting. Examples are provided for clarity of understanding. While the object of the present disclosure has been described in connection with embodiments thereof, it will be understood that it is capable of further modifications and this disclosure is intended to cover variations, user or adaptations of the present disclosure following, in general, the principles of the present disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the present disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures and the Sequence Listings, are incorporated herein by reference for all purposes.

EXAMPLES

[0112] Example 1: Selection of microorganisms

[0113] This example illustrates a method to select microorganisms that are a priori suited to produce bHΐn. Several bacteria and yeasts were tested for their ability to grow in the presence of bHΐn. Bacterial strains - Corynebacterium glutamicum NRRL B-2784, Escherichia coli MG1655 and yeast strains - Saccharomyces cerevisiae CENPK.2, Kluyveromyces marxianus NRRL Y- 318, NRRL Y-6373, Pichia kudriavzevii NRRL Y-7551, NRRL Y-5396 were selected as exemplary microorganism to evaluate their ability to grow in the presence of bHΐn. Growth is considered an indicator of overall metabolic activity. The bacterial strains were started in LB medium and the yeast strains were started in YPD medium at conditions that are reportedly ideal for optimal growth. For example, E. coli strain was started from cryo-vials in LB medium at 37°C and S. cerevisiae was started from cryo-vials in YPD medium at 30°C.

[0114] Seed cultures of all the strains were transferred into minimal mineral salts medium, supplemented with 2% glucose. To the same media bHΐn was added to a final concentration of 20 g/L, 40 g/L or 50 g/L. Growth of the microorganisms in the presence of bHΐn was monitored in BioLector II (m2p Labs, Aachen, Germany) in triplicate. The maximum specific growth rate of these microorganisms in the presence of various concentrations of bHΐn was calculated and shown in Figure 3. Typically yeasts grew faster than bacteria at higher concentrations of bHΐn, suggesting faster glucose uptake. Specifically, Y-6373, Y-7551 and Y-5396 exhibited the fastest growth and are potential candidates to host the bHΐn metabolic pathway. This example illustrates a method to screen and select appropriate host microorganisms for producing bHΐn.

[0115] Example 2: Identification of dioxygenase enzymes with high bHΐn synthase activity [0116] Example 2 demonstrates the promiscuity of 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzymes and identification of preferred enzymes with higher activity with KIC. Exemplary HPPD enzymes were selected from human (P32754), rat (P32755), A. thaliana (P93836), P. aeruginosa (Q9I576), Y. lipolytica (Q6CDR5) and S. avermitilis (Q53586). Hydro xymandelate synthase from A. orientalis (052791) was also selected. Codon-optimized genes encoding these enzymes were synthesized and cloned into pET28 vector for expression in E. coli BL21 Rosetta cells. The genes also contained a hexa-histidine tag at the N-terminus to allow easy purification. Cells from overnight cultures were harvested and lysed. The enzymes were purified with Qiagen Ni-NTA columns and the concentration of the purified enzyme was quantified with Qubit 4 (ThermoFisher).

[0117] Enzyme activity was measured by monitoring the dissolved oxygen concentration in the reaction mixture. The reaction was performed in 0.2 M Tris-Maleate buffer (pH 6.5) containing 1 lnM FeSCfi, 0.5 mM ascorbic acid and 1 mM dithiothreitol. Purified enzyme was added to the reaction mixture and incubated at 30°C. The reaction was started by adding 1 mM KIC and the decrease in the concentration of dissolved oxygen was monitored using a Clarke electrode in Oxytherm (Hansatech Instruments). Enzyme activity was calculated from the rate of decrease in the dissolved oxygen after correcting for the slope prior to the substrate addition. One unit of volumetric enzyme activity is defined as the amount of enzyme required to consume 1 nmol of dissolved oxygen per minute under the assay conditions. Activity is expressed as specific activity, which is calculated as volumetric enzyme activity normalized by the total enzyme in the reaction mixture.

[0118] Table 1 summarizes the specific activity of exemplary dioxygenases using HPP or KIC as substrates. The rat HPPD exhibited high activity using HPP or KIC as the substrate. The rat HPPD exhibited the highest KIC activity among the enzymes studied, followed by the human HPPD and Y. lipolytica HPPD. The human enzyme had the highest KIC/HPP ratio for the specific activity. Hydroxymandelate synthase from A. orientalis exhibited even higher activity with HPP than the rat enzyme and converts HPP into hydroxymandelate using a mechanism that is believed to have evolved differently than that in HPPD.

Table 1. Specific dioxygenase activity of exemplary enzymes using KIC or HPP as substrates. The sequence identity with the rat HPPD is also shown.

Specific Activity

Enzyme ID% _ KIC/HPP

HPP KIC

Human (P32754) 89.8% 0.52 0.28 0.54

Rat (P32755) 100.0% 17.98 0.70 0.04

A. thaliana (P93836) 27.3% 0.65 0.17 0.26

Y. lipolytica (Q6CDR5) 49.5% 6.02 0.23 0.04 P. aeruginosa (Q9I576) 24.1% 0.80 0.19 0.24 S. avermitilis (Q53586) 43.2% 0.41 0.11 0.27 A. orientalis (052791) 26.2% 26.50 0.20 0.01

[0119] As a result of the high sequence identity between the human and the rat dioxygenases, there are relatively few amino acid differences that are close to the catalytic iron site. Species differences in activity with HPP and KIC may be due to different structure or dynamics of the C- terminal helix. Information is scare on the conformation of the C-terminal a-helix and its relationship with rate and specificity. Factors independent of sequence identity may also contribute to [1HIV synthase activity. Accordingly, the present disclosure discloses methods to select dioxygenase enzymes that have high bHIU synthase activity. [0120] Example 3: Role of C-terminal helix in catalysis

[0121] The purpose of Example 3 is to illustrate the importance of the C-tenninal helix of the dioxygenase enzyme in substrate specificity as well as the rate of catalysis. As shown in Table 1, HPPD from rat and human are -90% identical and yet, differ substantially in their catalytic ability using HPP or KIC as substrates. Human HPPD has lower activity with either substrate than the rat enzyme. To determine the role of C-terminus, the region corresponding to residues 361 to 393 (corresponding to the C-tenninal domain) was swapped between the human and rat enzymes. Gene fragments corresponding to these residues were PCR-amplified and fused to create the swap. The resulting chimeric enzyme is the human HPPD with rat C-tenninus (human-rC). The gene encoding the chimeric enzyme was cloned into pET28a expression vector, which was transfonned into E. coli Rosetta cells. The cells were grown and activity of the purified enzyme assayed as described in Example 2.

[0122] Upon swapping the C-tenninal domain of the human HPPD with that from rat, the activity of the chimeric enzyme decreased using HPP as the substrate but remained unchanged with KIC. The results shown in Table 2 are indicative of the importance of the role of the C-terminal domain in substrate specificity.

Table 2. Enzyme activity

Specific Activity

Enzyme _

HPP KIC

Human (P32754) 5.36 0.49 Human-rC 1.11 0.49

[0123] Example 4: Engineering dioxygenase for improved bHΐn synthase activity

[0124] Example 4 demonstrates methods to mutate or modify certain pennissive sites as identified in the description to identify mutated or modified dioxygenase variants that have improved activity with KIC. Exemplary pennissive amino acid residues, as identified in this disclosure, in SEQ ID NO: 1 were mutated one at a time to evaluate their impact on KIC activity. The residues selected for illustrative purpose were F371, V212, F364 and Q251. These native residues were mutated using Q5© Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol. The integrity of the mutated variants was confinned by Sanger sequencing and transformed into E. coli Rosetta BL21 cells for expression. The activity of these variants was assayed using the procedure described in Example 2.

[0125] The relative activity of the non-natural enzyme variants is shown in Figure 4. As exemplified in Figure 4, not all mutations have a similar impact on the relative activity using KIC or HPP as the substrate. Some enzyme variants exhibited increased activity with KIC and others exhibited increased activity with FIPP, relative to the corresponding activity of the unmutated natural wild-type enzyme. Accordingly, Example 3 demonstrates methods to make amino acid changes in pennissive sites and identify enzyme variants that have increased activity with KIC. [0126] Example 5: Improved bHΐn synthase enzymes

[0127] Example 5 further builds on the Example 4 to demonstrate how mutation or modification of residues at multiple pennissive sites simultaneously can result in improved activity with KIC that may not be possible with individual changes alone. Rather than being constrained into using nucleotide degenerate codons, variant spread-out low diversity libraries were synthesized. The enzyme variants cloned into pET28a plasmid were transformed into E. coli BL21 Rosetta cells to obtain individual colonies, which were grown overnight. The cells were collected, lysed in lysis buffer (200 mM NaCl, 50 mM Tris-HCl pH 8.0, 200 pg/mL Lysozyme, 5 U/mL DNase, Sigma P8849 protease inhibitor cocktail) and the lysate was used to assay the dioxygenase activity using HPP or KIC as the substrates.

[0128] Figure 5 illustrates the relative activity of the non-natural bHΐn synthase enzyme variants compared with that of unmodified SEQ ID NO: 1. Mutations to multiple pennissive sites increased the relative activity with KIC. For example, the combination of mutations at residues at V212 and L224 appeared to increase the enzyme activity significantly with KIC and reduced the enzyme activity with HPP. Since these two residues are in close proximity to the C-tenninal helix, these mutations are likely to affect the structure, dynamics, or both for the C-tenninal helix. Additional sites, either within the C-terminal helix itself or sites that directly influence its conformation, could also be reasonably targeted for mutations. Accordingly, through Example 5, the present disclosure discloses methods to identify residues that could be modified or mutated to alter the substrate specificity of unmodified dioxygenase enzymes to create improved bHΐn synthase enzymes.

[0129] Example 6: Screening and identification of improved bHIU enzymes [0130] This example illustrates a methodology for identifying improved bHΐn enzymes from a large number of variants. Starting with a Swissmodel homology model (https://swissmodel.expasy.org/repository/uniprot/P32755) which uses chain B of the PDB entry 3isq (human HPPD) as the template for rat HPPD, SHARPEN/CHOMP software was used (Loksha et al., Journal of Computational Chemistry, Volume 30(6), 2009: 999-1005). The script uses a Python module “smallmol” that extends or wraps OpenBabel, pybel, and the semi- empirical optimization software MOP AC and generated a 3D confonnation of KIC from the SMILES format description of the molecule. The most promising structures obtained from MopacSuperScan , a customized code, were re-optimized without dihedral constraints. KIC and HMB were docked onto the catalytic iron by alignment of the keto acid to comparable ligand atoms found in multiple HPPD crystal structures. Each of these small molecule poses, along with the rat HPPD homology model, were used as input for a Rosetta protein design calculation. The amino acids selected for the enzyme active site were collated in Table 3.

Table 3. Permissive amino acid palette design calculations

[0131] Using the same approach, several additional rounds of Rosetta design calculations were performed. The library was gradually focused on a smaller number of residues that could more feasibly be sampled using medium throughput experimental assays. These were used, in combination with manual inspection of the enzyme models and biophysical intuition, to select combinatorial libraries for experimental synthesis. Rosetta suggested: avoiding mutations to Val 212, considering mutating Leu224 to Met, strongly recommended mutating Gin 251 to Pro, strongly recommended mutating Phe 364 to Ala, avoiding mutations to Leu 367, considering mutating Phe368 (to Tyr, His, or Leu), and strongly recommended mutating Phe371 to Leu. Given the Rosetta calculation results, combinatorial libraries were devised that could efficiently explore the space of mutations recommended by Rosetta, as well as by biophysical intuition about locations that could tolerate mutations and locations suitable to directly impact substrate specificity.

Table 4: Exemplary combinatorial library design

[0132] DNA fragments comprising the degenerate codons were synthesized as Spread Out Low Diversity libraries by Twist Bioscience to intentionally retain the native residue at each selected locus and cloned into pET28a vector. The resulting ligation mixture was transformed into E. coli Rosetta cells and the colonies that appeared on selection plates were cultured in 96-well plates. After growth for 8 h and overnight induction, the cells were collected by centrifugation and lysed to obtain the cell-free extract. The cell-free extract comprising the active enzyme variant was used in an assay mixture (Johnson-Winters, Biochemistry, 2003, 42: 2072-2080) in OxoPlates OP96U (Presens, Regensburg, Germany) to follow the reduction in dissolved oxygen. The calibrations and calculations were performed according to the manufacturer's instructions. Approximately 5400 variants were evaluated, and those with at least 90% of the wild-type activity with KIC and less than 50% of the wild-type activity with HPP were identified. Improved pHIV activity in the variants that were thus identified in the first screen was validated in triplicate assays and those that confirmed the improvement were sequenced to identify the mutations. Specific changes to five residues (V212L, L224M, Q251P, F368Y and F371L) emerged to be critical to increasing the enzyme activity with KIC while concomitantly reducing the activity with HPP.

[0133] Example 7: Evaluating the catalytic efficiency of the pHIV synthase variants [0134] This example illustrates a method to quantify the catalytic efficiency of bHΐn synthase enzymes and select improved variants. Exemplary bHΐn synthase enzyme variants identified in Example 6 were produced in E. coli Rosetta cells and were purified from the lysates using Ni- NTA columns (Qiagen, Valencia, CA). The purified enzyme was assayed for activity using Oxythem oxygen electrode (Hansatech Instruments, Norfolk, UK) as described in Example 2. After initial observation of the nonenzymatic rate of oxygen consumption due to Fenton chemistry, the enzymatic reaction was initiated by the addition of different amounts of KIC or HPP. The kinetic constants were calculated by fitting the reaction velocity and the substrate concentration to Michaelis-Menten equation.

[0135] Some enzyme variants exhibited improved bHΐn synthase activity, as shown in Table 5. The kinetic constants confirmed improved activity with KIC and concomitant reduction in activity with HPP.

Table 5: Exemplary bHΐn synthase variants

KTC H P P kcat KM kcat/Klvi kcat KM kcat/KM

Mutant Mutation

[s ¹ ] [mM] [s ¹ M ¹ ] [s ¹ ] [mM] [s ¹ M ¹ ]

WT 0.013 0.55 24 065 003 23214

L F371L 0.008 0.14 53 0.10 0.04 2546

PL Q251P/F371L 0.013 0.26 52 0.00 0.01 126

LM V212L/L224M 0.014 0.13 114 0.05 0.03 1670

[0136] Example 8: Production of bHΐn in bacteria

[0137] This example illustrates the production of bHΐn in bacteria and yeast. A strain of Corynebacterium glutamicum MV-KICFl (Applied Microbiol, Microb Biotech ,8, 351-360) that was metabolically modified to produce a-ketoisocaproate was used to introduce a gene that encodes for a polypeptide encoding SEQ ID NO: 1. The codons in the nucleic acid sequence of the gene were optimized according to the codon usage of the bacterium and the DNA was cloned into pZ8-ptac vector (Cleto et ah, ACS Synth Biol. 2016 May 20; 5(5): 375-385) and transformed into MV-KICFl by electroporation. The non-natural bacterial strain endowed with the capability to produce bHΐn was propagated in Brain-Heart Infusion medium and cultivated in CGXII medium (Hoffman et ah, J Appl Microbiol., 2014, 117: 663—678) using glucose as the main carbon source to evaluate the bHΐn production. Substrate consumption and product formation was evaluated on Agilent 1200 HPLC using 5mM H2S04 as the mobile phase with Aminex HPX- 87c column (BioRad, Hercules, CA). As illustrated in Figure 7, the non-natural bacterium comprising bHΐn synthase produced more bHΐn than its parent wild-type.

[0138] Example 9: Assembling pHIV metabolic pathway in yeast cytosol

[0139] This example provides methods to assemble bHΐn metabolic pathway shown in Figure 1 in Y-5396 yeast. This strain of yeast was specifically selected based on high metabolic activity as illustrated in Example 1. The example also provides methods for discriminating between enzymes (genes) that perform the same function. All DNA modifications were performed based on a reference genome that is available publicly (Douglass et ah, 2018, PLoS Pathog, 14(7):el007138). To facilitate use of URA3 as a selection marker, both alleles of URA3 in the diploid yeast Y-5396 were deleted. The first URA3 allele was replaced by a KanMX cassette conferring Geneticin resistance and flanked by loxP sites by transforming a KanMX cassette flanked by -500 bases of homology upstream and downstream of URA3 into Y-5396. Transfonnants were selected on media containing Geneticin and insertion of the cassette in the URA3 locus confirmed by PCR, creating strain SB500. To delete the second URA3 allele -500 bases upstream and -500 bases downstream of URA3 were joined via overlap extension PCR to create a clean deletion construct and transformed into SB500. Transformants were plated on 5-fluorootic acid to select for mutants that had lost both alleles of URA3. The clean deletion of the second allele was confirmed by PCR and the KanMX cassette insertion was re-confirmed, to create strain SB501. Finally, the KanMX cassette was removed by transient expression of Cre recombinase from an unstable plasmid that conferred resistance to Hygromycin. Colonies were then screened for loss of Geneticin resistance (loss of KanMX), loss of Hygromycin resistance (plasmid marker), and ability to grow on 5-FOA (double deletion of URA3). Genomic DNA at the LIRA 3 locus was PCR-amplified and sequenced to confirm the deletions had occurred as expected. The resulting strain called SB502, which has the two copies of the URA3 gene deleted to facilitate the use of uracil for selection. [0140] Genes encoding for enzymes in the bHΐn metabolic pathway were inserted in the chromosome at intergenic loci using homologous recombination. Chromosomal integration of heterologous genes to assemble the bHΐn metabolic pathway is illustrated for a-isopropylmalate synthase (IPMS) as an example. One plasmid (pi) was constructed containing the integration site 5’ homology ann, IPMS expression cassette, and the 3’ two-thirds of the URA3 cassette. A second plasmid (p2) was constructed with the 5’ two-thirds of the URA3 cassette (such that there was -500 bases overlap with pi) and the integration site 3’ homology arm. This strategy decreases the rate of unwanted recombination events because only when both halves integrate into the same site will a functional URA3 cassette be fonned. All PCR reactions were performed using NEB Q5 high fidelity polymerase according to manufacturer’s instructions. Plasmids were assembled using NEBuilder HiFi assembly mix according to manufacturer’s instructions, routinely using 30 base pair overlaps to facilitate assembly. The URA3 cassette is flanked by loxP sites to facilitate removal of the marker by expression of Cre recombinase. Genes encoding for enzymes with SEQ ID NOS: 308 - 312 were evaluated for their IPMS activity.

[0141] Plasmids shown in Table 6 were assembled using NEBuilder HiFi assembly mix according to manufacturer’s instructions, routinely using 30 base pair overlaps to facilitate assembly. IPMS genes were inserted into two separate intergenic loci on chromosome A (NC_042506). Intergenic locus A2193833 (aka igA2.2) and A1207782 (aka igA1.2).

[0142] Table 6. Relevant plasmids used to modify strains in this example

[0143] To construct pSBOl 1-15 the 5’ homology arm and ioTDH3 promoter were PCR amplified with primers shown in SEQ ID NO: 321 and SEQ ID NO: 322 and SEQ ID NO: 323 and SEQ ID NO: 324 respectively, using SB502 genomic DNA as template. The IPMS genes were codon optimized for Issatchenkia orientalis and synthesized as gene fragments by Twist Biosciences (San Francisco, CA). The genes needed to be split into two fragments because of their length and complexity. The ioTKL terminator & 3’ portion of the URA3 cassette and vector backbone (pTwist-Kan high copy), were PCR amplified from a plasmid synthesized by Twist Biosciences. To construct pSB017 the 3’ homology arm was amplified using primers SEQ ID NO: 325 and SEQ ID NO: 326 and SB502 genomic DNA as template. The 5’ portion of the URA3 cassette and vector backbone was amplified using primers ig2.2p2 gib vec F+R and plasmid igl.6p2 as template. Clones were screened by PCR and/or restriction digest for proper assembly and sequences were confinued via Sanger sequencing. The pi and p2 inserts were liberated from their vector backbones via restriction digest and inserts purified via gel extraction (NEB Monarch gel purification kit) to be transformed into suitable yeast strain.

[0144] All transformations were performed using the lithium acetate method as described in Geitz & Schiestl, 2007, Nature Protocols, 31-34. Individual transformants were screened using colony PCR to confirm correct integration of the 5’ flank (using primers SEQ ID NO: 327 and SEQ ID NO: 328) and 3’ flank (using primers SEQ ID NO: 329 and SEQ ID NO: 330) and to confirm correct assembly of the ura3 marker (using primers SEQ ID NO: 331 and SEQ ID NO: 332) and presence of the gene of interest (SEQ ID NO: 333 and SEQ ID NO: 334). Primers amplifying the native integration site (SEQ ID NO: 327 and SEQ ID NO: 329) were also used to identify any heterozygosity. The resulting strains containing a single copy of a gene encoding enzymes with SED ID NOs: 308 - 312, designated SB507 - SB511, respectively, were assayed for IPMS activity. The activity was detenuined by measuring the amount of free CoA liberated as described in Kohlhaw and Leary, 1969, Vol. 244, No. 8 pp. 2218-2225. Total protein concentration in cell lysates was measured using Bradford assay. The recorded activity from these strains is expressed in nmol/mg prot/min and shown in Table 7.

[0145] Table 7. Enzyme activity in yeast strains

Strain Sequence Activity

SB507 SEQ ID NO: 308 86.5

SB508 SEQ ID NO: 309 56.0

SB509 SEQ ID NO: 310 25.5

SB510 SEQ ID NO: 311 89.3

SB511 SEQ ID NO: 312 5.4

SB512 Control 5.5

[0146] As illustrated in Table 7, even a single copy of the gene could significantly enhance enzyme activity. Strains SB507 and SB510 were selected for inserting a second copy of the gene to make the locus homozygous. These strains were transformed with lpg of pECOlO plasmid containing a Cre expression cassette and KanMX cassette conferring resistance to Geneticin. Transformants were plated on YPD + 500ug/mL G418-sulfate. A single colony was used to inoculate YPD broth + 500ug/mL G418-sulfate and grown overnight. The G418 culture was then used to inoculate SC+lg/L 5-FOA which selected for clones that had lost the ura3 marker. The 5-FOA culture was grown for 24-48 hours until visible growth was observed then cells were streaked for isolation onto a YPD plate. Single colonies were replica plated onto YPD, YPD+G418500 and SC-Ura plates. Clones that did not grow in the presence of G418 (had lost pECOlO) or SC-Ura (lacked URA3) were screened via colony PCR to confirm URA3 loop-out. The second copy of the gene was integrated in a second round of transfonnation using the same pi and p2 constructs. Successful integration and homozygosity were confirmed using colony PCR. The resulting strain was rendered auxotrophic for uracil by repeating the method described above, to facilitate further modification. In the manner described above, the other genes in the bHIU metabolic pathway shown in Figure 1 were inserted subsequently. Sequences of exemplary enzymes that catalyze various steps of the bHΐn metabolic pathway are step (b): SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300 step (c): SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, step (d): SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, step (f): SEQ ID NO: 314, SEQ ID NO: 315 and step (g): SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320. Therefore, the resulting yeast strains have the multiple variations of the bHΐn metabolic pathway leading up to KIC.

[0147] A strain, designated SB553, comprising homozygous integration of genes that encode for SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 304, SEQ ID NO: 308, SEQ ID NO: 315 and SEQ ID NO: 319, was grown in 50 mL of yeast minimal salts medium supplemented with 40 mg/L of uracil, trace metals, vitamins and glucose as the carbon source (Verduyn, et al. Yeast 8, 7: 501 -517, 1992). After 44 h of growth, the yeast culture was harvested and centrifuged to remove the yeast cells. The clarified supernatants were analyzed for residual glucose and bHΐn synthesis via HPLC as described in Example 5. The strain SB502 was also grown under identical conditions to serve as a control. SB502 produced only 0.32 g/L of KIC while SB553 produced 1.84 g/L of KIC. Increased KIC production is clearly illustrative of the increased activity of all the enzymes expressed or overexpressed in the yeast cytosol.

[0148] Example 10: Production of bHΐn in yeast

[0149] This example will illustrate the integration of the complete bHΐn metabolic pathway that will result in bHΐn production by a non-natural yeast. All genetic manipulations were carried out using the methods described in Example 9. Step (a) of the bHΐn metabolic pathway shown in Figure 1, encoding for bHΐn synthase, was assembled in strain SB553. Codon-optimized sequences of genes that encode for bHΐn synthase variants corresponding to SEQ ID NO: 1 and SEQ ID NO: 335 were chromosomally integrated in SB553 to result in strains SB556 and SB557. The three strains were grown under identical conditions using minimal salts medium described in Example 9. After 42 h of growth, the culture was harvested, and cells separated by centrifugation. The clarified supernatant was analyzed for bHΐn using an HPLC as described in Example 3. As illustrated in Figure 8, supernatant from SB553 did not have any detectable bHΐn. However, SB556 supernatant had 0.04 g/L of bHΐn and SB557 supernatant had 0.12 g/L of bHΐn. The result confirms the conversion of glucose into bHΐn in SB556 and SB557 yeast using the metabolic pathway shown in Figure 1 and the ability of the yeast cell to export bHΐn into the medium using a transporter. Furthermore, the result also illustrates increased bHΐn production using different variants of bHΐn.

[0150] Various embodiments of systems, devices, and methods have been described herein. These embodiments are given only by way of example and are not intended to limit the scope of the claimed inventions. It should be appreciated, moreover, that the various features of the embodiments that have been described may be combined in various ways to produce numerous additional embodiments. Moreover, while various materials, dimensions, shapes, configurations and locations, etc. have been described for use with disclosed embodiments, others besides those disclosed may be utilized without exceeding the scope of the claimed inventions.

[0151] Persons of ordinary skill in the relevant arts will recognize that the subject matter hereof may comprise fewer features than illustrated in any individual embodiment described above. The embodiments described herein are not meant to be an exhaustive presentation of the ways in which the various features of the subject matter hereof may be combined. Accordingly, the embodiments are not mutually exclusive combinations of features; rather, the various embodiments can comprise a combination of different individual features selected from different individual embodiments, as understood by persons of ordinary skill in the art. Moreover, elements described with respect to one embodiment can be implemented in other embodiments even when not described in such embodiments unless otherwise noted.

[0152] Although a dependent claim may refer in the claims to a specific combination with one or more other claims, other embodiments can also include a combination of the dependent claim with the subject matter of each other dependent claim or a combination of one or more features with other dependent or independent claims. Such combinations are proposed herein unless it is stated that a specific combination is not intended.

[0153] Any incorporation by reference of documents above is limited such that no subject matter is incorporated that is contrary to the explicit disclosure herein. Any incorporation by reference of documents above is further limited such that no claims included in the documents are incoiporated by reference herein. Any incorporation by reference of documents above is yet further limited such that any definitions provided in the documents are not incorporated by reference herein unless expressly included herein.

[0154] For purposes of interpreting the claims, it is expressly intended that the provisions of 35 U.S.C. § 112(f) are not to be invoked unless the specific terns “means for” or “step for” are recited in a claim.