Methods and systems for preparing panthenol

Abstract

Methods and systems for bioproduction of panthenol in microbial hosts and cell-free are provided, which include a pantothenate synthetase (PanC) enzyme that is engineered for improved activity and increased panthenol production.

Description

[0001] Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0002] METHODS AND SYSTEMS FOR PREPARING PANTHENOL

[0003] SEQUENCE LISTING

[0004] This application incorporates by reference a sequence listing submitted as a text file entitled “DEBU-036-01WO-Seq-Listing.xml” created October 29, 2025 and having a size of 23 kilobytes.

[0005] BACKGROUND

[0006] Panthenol is the alcohol analog of pantothenic acid, and exists in two optically active forms that can be present individually or as a racemic mixture (DL-panthenol, CAS Registry Number® 16485-10-2). The dextrorotatory form of panthenol (D-panthenol, CAS Registry Number® 81-13-0; synonyms: (A)-(+)-2,4-dihydroxy-N-(3-hydroxypropyl)-3,3- dimethylbutyramide, ( )-2,4-dihydroxy -3, 3 -dimethylbutyric 3-hydroxypropylamide, D- pantothenyl alcohol, dexpanthenol, provitamin B5) is oxidized to produce D-pantothenic acid / pantothentate (Vitamin B5), which is one of the water-soluble B-complex vitamins important for protein metabolism and synthesis of red blood cells. Panthenol (D-panthenol or DL-panthenol) is useful in a variety of pharmaceutical and cosmetic products, including but not limited to topical preparations (ointments, creams, lotions, etc.) with moisturizing, wound healing, anti-aging, anti-inflammatory, and / or anti-oxidant effects.

[0007] Panthenol is typically manufactured via chemical synthesis. However, improved methods of panthenol production are needed in the art.

[0008] SUMMARY

[0009] The present invention provides, in various embodiments, methods and systems for bioproduction of panthenol in microbial hosts or cell-free.

[0010] In some embodiments, the invention provides a composition for preparing panthenol, comprising: a variant of pantothenate synthetase (PanC), wherein the variant of PanC is engineered for increased activity for panthenol production as compared to wild-type PanC; ( )- pantoate; and 3-amino-l -propanol, wherein the engineered variant of PanC contains one or more mutations, each mutation at a position corresponding to E66, V109, R121, H124, T175, K184, R187, R196, 1255, K269, or R271 in SEQ ID NOV. Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0011] In some embodiments, the engineered variant of PanC comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10.

[0012] In some embodiments, the engineered variant of PanC comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10.

[0013] In some embodiments, the engineered variant of PanC comprises an amino acid sequence having at least 99% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 10.

[0014] In some embodiments, the engineered variant of PanC comprises an amino acid sequencing having at least 99% identity to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 10.

[0015] In some embodiments, the one or more mutations are selected from the group consisting of E66D, V109T, V109C, V109F, V109L, R121W, R121D, H124V, H124I, H124L, H124M, T175I, T175V, K184F, K184S, R187A, R187C, R187D, R187E, R187F, R187G, R187H, R187L, R187M, R187P, R187S, R187T, R187V, R187W, R187Y, R196Y, K269Y, I255T, I255D, I255R, I255K, K269Y, R271A, R271F, R271G, R271P, and R271T.

[0016] In some embodiments, the engineered variant of PanC contains a combination of two or more mutations, wherein the combination is one of the combinations listed in Table 3.

[0017] In some embodiments, the composition further comprises a microbial host strain overexpressing the engineered variant of PanC.

[0018] In some embodiments, the microbial host strain comprises bacteria, yeast, and / or fungal cells.

[0019] In some embodiments, the microbial host strain is engineered to include modifications that enhance production of (7? -pantoate, including but not limited to overexpression of panB, panE, and ALS.

[0020] In some embodiments, the microbial host strain is modified to overexpress (i) one or more variants of ketopantoate hydroxymethyltransferase; (ii) an acetolactate synthase; (iii) 2- dehydropantoate 2-reductase; or (v) any combination thereof.

[0021] In some embodiments, the host strain is in a defined M9 minimal media. Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0022] In some embodiments, the (7?)-pantoate concentration is about 0.1 to about 1 mM.

[0023] In some embodiments, the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM.

[0024] In some embodiments, the composition is cell-free.

[0025] In some embodiments, the engineered variant of PanC is in lysate or purified form.

[0026] In some embodiments, the engineered variant of PanC is a lysate and has a concentration of about 0.1 to about 20 vol%.

[0027] In some embodiments, the (A)-pantoate concentration is about 1 to about 5 mM.

[0028] In some embodiments, the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM.

[0029] In some embodiments, the composition further comprises a buffer at a concentration of about 20 to about 250 mM.

[0030] In some embodiments, the buffer is Tris pH 7.5.

[0031] In some embodiments, the composition further comprises MgCh at a concentration of about 1 to about 20 mM.

[0032] In some embodiments, the composition further comprises KC1 at a concentration of about 1 to about 20 mM.

[0033] In some embodiments, the composition further comprises ATP at a concentration of about 1 to about 5 mM.

[0034] In some embodiments, the composition further comprises the engineered variant of PanC provides at least about a 1.5-fold improvement in panthenol generation as compared to an unmodified PanC control.

[0035] In some embodiments, the composition further comprises the engineered variant of PanC provides at least about a 2-fold improvement in panthenol generation as compared to an unmodified PanC control.

[0036] In some embodiments, the composition further comprises the engineered variant of PanC provides at least about a 2.5-fold improvement in panthenol generation as compared to an unmodified PanC control.

[0037] In some embodiments, the composition further comprises the engineered variant of PanC provides at least about a 3-fold improvement in panthenol generation as compared to an unmodified PanC control. Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0038] In some embodiments, the composition further comprises the engineered variant of PanC provides at least about a 4-fold improvement in panthenol generation as compared to an unmodified PanC control.

[0039] In some embodiments, the invention provides a method of preparing panthenol, comprising: providing a reaction mixture comprising a variant of pantothenate synthetase (PanC) engineered for increased activity for panthenol production as compared to wild-type PanC, (R)- pantoate, and 3 -amino- 1 -propanol; and incubating the reaction mixture containing the engineered variant of PanC, the (AJ)-pantoate, and the 3 -amino- 1 -propanol at a temperature of about 20 to about 40 degrees Celsius for at least about 1 to about 24 hours, wherein the engineered variant of PanC contains one or more mutations, each mutation at a position corresponding to E66, VI 09, R121, H124, T175, K184, R187, R196, 1255, K269, or R271 in SEQ ID NOV.

[0040] In some embodiments, the method is performed in a microbial host, and the step of providing the reaction mixture comprises: culturing a microbial host strain overexpressing the engineered variant of PanC in a growth media having 3-amino-l -propanol added thereto, wherein microbial host strain produces the R)-pantoate, or wherein the (7?)-pantoate is provided in the media.

[0041] In some embodiments, the microbial host strain comprises bacteria, yeast, and / or fungal cells.

[0042] In some embodiments, the microbial host strain is engineered to include modifications that enhance production of (R)-pantoate, including but not limited to overexpression of panB, panE, and ALS.

[0043] In some embodiments, the microbial host strain is modified to overexpress (i) one or more variants of ketopantoate hydroxymethyltransferase; (ii) an acetolactate synthase; (iii) 2- dehydropantoate 2-reductase; or (v) any combination thereof.

[0044] In some embodiments, the (A)-pantoate concentration is about 0.1 to about 1 mM.

[0045] In some embodiments, the 3-amino-l -propanol concentration is about 1 to about 200 mM.

[0046] In some embodiments, the method is performed cell-free, and the step of providing the reaction mixture comprises: providing a cell-free reaction mixture comprising the engineered variant of PanC in lysate or purified form, the (A)-pantoate and the 3-amino-l -propanol. Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0047] In some embodiments, the engineered variant of PanC is a lysate and has a concentration of about 0.1 to about 20 vol%.

[0048] In some embodiments, the (A)-pantoate concentration is about 1 to about 5 mM.

[0049] In some embodiments, the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM.

[0050] In some embodiments, wherein the reaction mixture further comprises Tris pH 7.5 at a concentration of about 20 to about 250 mM.

[0051] In some embodiments, wherein the reaction mixture further comprises MgCh at a concentration of about 1 to about 20 mM.

[0052] In some embodiments, the reaction mixture further comprises KC1 at a concentration of about 1 to about 20 mM.

[0053] In some embodiments, wherein the reaction mixture further comprises ATP at a concentration of about 1 to about 5 mM.

[0054] In some embodiments, the invention provides a microbial host strain expressing a variant of pantothenate synthetase (PanC), wherein the variant of PanC is engineered for increased activity for panthenol production as compared to wild-type PanC, and wherein the engineered variant of PanC contains one or more mutations, each mutation at a position corresponding to E66, V109, R121, H124, T175, K184, R187, R196, 1255, K269, or R271 in SEQ ID NO:9.

[0055] In some embodiments, the one or more mutations are selected from the group consisting of E66D, V109T, V109C, V109F, V109L, R121W, R121D, H124V, H124I, H124L, H124M, T175I, T175V, K184F, K184S, R187A, R187C, R187D, R187E, R187F, R187G, R187H, R187L, R187M, R187P, R187S, R187T, R187V, R187W, R187Y, R196Y, K269Y, I255T, I255D, I255R, I255K, K269Y, R271A, R271F, R271G, R271P, and R271T.

[0056] In some embodiments, the engineered variant of PanC contains a combination of two or more mutations, wherein the combination is one of the combinations listed in Table 3.

[0057] In some embodiments, the microbial host strain comprises bacteria, yeast, and / or fungal cells.

[0058] In some embodiments, the microbial host strain is modified to overexpress (i) one or more variants of ketopantoate hydroxymethyltransferase; (ii) an acetolactate synthase; (iii) 2- dehydropantoate 2-reductase; or (v) any combination thereof. Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0059] Additional features and advantages of embodiments of the present invention are described further below. This summary section is meant merely to illustrate certain features of embodiments of the invention, and is not meant to limit the scope of the invention in any way. The failure to discuss a specific feature or embodiment of the invention, or the inclusion of one or more features in this summary section, should not be construed to limit the invention as claimed.

[0060] BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The foregoing summary, as well as the following detailed description of certain embodiments of the invention, will be better understood when read in conjunction with the appended drawings. For the purposes of illustrating the systems and methods of the present application, there are shown in the drawings preferred embodiments. It should be understood, however, that the application is not limited to the precise arrangements and instrumentalities shown.

[0062] FIG. 1 shows an overview of the panthenol production pathway according to embodiments of the present invention.

[0063] FIG. 2 shows chromatograms of a panthenol analytical standard and the results of a whole-cell assay demonstrating panthenol production in a host strain engineered according to embodiments of the present invention.

[0064] FIG. 3 shows chromatograms of a panthenol analytical standard and the results of cell- free reactions with PanC in E.coli lysate or as a purified enzyme according to embodiments of the present invention.

[0065] FIG. 4 shows improvement of panthenol generation using pantothenate synthetase (PanC) variants engineered according to embodiments of the present invention. Mutations listed are relative to SEQ ID NO:9; experiments performed as described in Example 1.

[0066] FIG. 5 shows improvement of panthenol generation using pantothenate synthetase (PanC) variants engineered according to embodiments of the present invention. Mutations listed are relative to SEQ ID NO:9; experiments performed as described in Example 1.

[0067] FIG. 6 shows a protein sequence alignment for various pantothenate synthetase (PanC) homologs that may be engineered according to embodiments of the present invention. Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0068] FIG. 7 shows the activity of two purified PanC homologs for panthenol production according to embodiments of the present invention. Experiments performed as described in Example 3.

[0069] FIG. 8 shows improvement of panthenol generation using pantothenate synthetase (PanC) variants engineered according to embodiments of the present invention. Mutations listed are relative to SEQ ID NO:3; experiments performed as described in Example 1..

[0070] FIG. 9 shows kinetic parameters of variants of PanC009 (SEQ ID NO:9) demonstrating improved performance for utilizing 3 -amino- 1 -propane (3A1P). Mutations listed are relative to SEQ ID NO:9.

[0071] FIG. 10 shows improvement of panthenol generation using pantothenate synthetase (PanC) variants engineered according to embodiments of the present invention. Mutations listed are relative to SEQ ID NO:9; experiments performed as described in Example 2.

[0072] FIG. 11 shows strain modifications leading to improved panthenol production. Experiments performed as described in Example 4.

[0073] DETAILED DESCRIPTION

[0074] Methods and systems are described herein for bioproduction of panthenol in microbial hosts or cell-free. In various embodiments, engineered variants of pantothenate synthetase are used, which improve (i.e., increase) the enzyme’s activity for panthenol production.

[0075] FIG. 1 shows an overview of panthenol production according to embodiments of the present invention. Host strain engineering is used to convert feedstock sugars to A-pantoate. Enzyme engineering of a pantothenate synthetase (PanC) enzyme (EC 6.3.2.1) is used to increase the enzyme activity for panthenol production using 3 -amino- 1 -propanol as a substrate. In each embodiment, 3 -amino- 1 -propanol is added for the final step.

[0076] FIG. 2 shows chromatograms of a panthenol analytical standard and the results of a whole-cell assay demonstrating panthenol production in a host strain engineered according to embodiments of the present invention.

[0077] FIG. 3 shows chromatograms of a panthenol analytical standard and the results of cell- free reactions with PanC in E.coli lysate or as a purified enzyme according to embodiments of the present invention. Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0078] The enzyme used for FIGS. 2 and 3 was unmodified PanC009 (SEQ ID NO:9; see below) or a variant of PanC009.

[0079] Sequences for various homologs of the enzyme pantothenate synthetase (PanC), which are suitable for engineering to produce panthenol as described above, are provided below in Table 1, with the sequence identifier, NCBI accession number, and origin followed by the sequence. Sequences with 90% or greater identity to the sequences identified below may also be engineered to produce panthenol as described above.

[0080] Table 1 Attorney Docket No. DEBU-036 / 01WO 37396 / 184 Attorney Docket No. DEBU-036 / 01WO 37396 / 184 Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0081] In some embodiments, the pantothenate synthetase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10.

[0082] In some embodiments, the pantothenate synthetase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NON, or SEQ ID NO: 10.

[0083] In some embodiments, the pantothenate synthetase comprises an amino acid sequence having at least 98% identity to SEQ ID NON, SEQ ID NO:2, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NON, or SEQ ID NO: 10.

[0084] In some embodiments, the pantothenate synthetase comprises an amino acid sequence having at least 99% identity to SEQ ID NON, SEQ ID NO:2, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, or SEQ ID NONO.

[0085] In some embodiments, the pantothenate synthetase comprises an amino acid sequence identical to SEQ ID NO: 1, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, or SEQ ID NO: 10.

[0086] Table 2 and Table 3 show lists of mutations introduced in an exemplary PanC homolog (SEQ ID NON). Engineered variants of SEQ ID NON as listed in Table 2 and Table 3 improved the activity of the enzyme towards the non-native substrate (3 -amino- 1 -propanol) as compared to the unmodified enzyme. FIG. 4 and FIG. 5 show panthenol generation by the wild-type enzyme (control; unmodified SEQ ID NON) and some of the engineered variants listed in Table 2 and Table 3, respectively. The Y-axis shows improvement as fold over internal control (FOIC). The X-axis shows variant sequence number (internal identification number of the engineered variant) ordered by FOIC (ascending).

[0087] Table 2 Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0088] Table 3

[0089] The amino acid changes listed in Table 2 and Table 3 were made in one example PanC homolog (SEQ ID NO:9), but it is specifically contemplated that equivalent changes (e.g., at positions aligning with / corresponding to E66, V109, R121, H124, T175, K184, R187, R196, 1255, K269, and / or R271 in SEQ ID NO:9) could be made in other PanC homologs such as, but not limited to, those identified above (SEQ ID NOs: 1-8, 10). FIG. 6 shows a protein sequence alignment of SEQ ID NO:9 with SEQ ID NOs: 4, 6, 5, 10, 7, 2, and 3, respectively. In FIG. 6, R187 and R271 of SEQ ID NO:9, and equivalents in the other PanC homologs, are marked by boxes with asterisk. Any of the R187 and / or R271 mutations listed in Table 2, for example, could be made at the corresponding residues in the other PanC homolog sequences (see, e.g., FIG. 8, which shows data for SEQ ID NO:3 as well as R187F and R87F, R121N variants thereof). In addition, although the amino acid changes listed in Table 2 were evaluated Attorney Docket No. DEBU-036 / 01WO 37396 / 184 individually, it is specifically contemplated that an engineered PanC variant according to embodiments of the present invention could include a combination of more than one of the listed mutations (such as, but not limited to, the combinations listed in Table 3), and / or could include a mutation that is not listed in Table 2, but is a homologous replacement (a different amino acid with biochemical properties similar to the one listed; e.g., R187I instead of R187L).

[0090] Engineered variants according to embodiments of the present invention are not limited to those comprising the amino acid changes listed in Table 2 (and equivalents as described above), and are specifically contemplated to include any PanC mutations that increase the activity of the enzyme with 3 -amino- 1 -propanol. Such mutations may be made at positions other than the identified positions (E66, V109, R121, H124, T175, K184, R187, R196, 1255, K269, and / or R271 in SEQ ID NO: 9 and equivalents in other PanC homologs), and may be present instead of, or in addition to, the mutations described above.

[0091] EXAMPLES

[0092] Example 1 : Screening of PanC Variants

[0093] Production of panthenol in microbial hosts was performed in two stages. In Stage 1 (seed), a microbial culture of the engineered E. coli host strain was grown for a time period (e.g., about 16 hours) at 32.5 °C in LB with 1% glycerol. In Stage 2 (production), the aforementioned culture was inoculated 1 :10 into M9 minimal media containing 0.5 mM (A -pantoate and 3- amino-1 -propanol (3A1P, 10 or 50 mM) and grown for an additional time period (e.g., about 22 hours) at 32.5 °C. After about 22 hours, the cultures were lOx diluted with water and analyzed by LCMS and / or HPLC. Variants of PanC enzymes were generated by standard mutagenesis protocols of the PanC gene in a plasmid. Libraries of PanC variants were expressed and tested as described above, and top-performing variants were subjected to further rounds of mutagenesis.

[0094] FIG. 4 shows the fold change increase from engineered PanC009 variants having single amino acid substitutions as compared to wild-type PanC009 (SEQ ID NOV).

[0095] FIG. 5 shows the fold change increase from engineered PanC009 variants having one or multiple amino acid substitutions as compared to wild-type PanC009 (SEQ ID NOV).

[0096] FIG. 8 shows the fold change increase from engineered PanC003 variants having one or two amino acid substitutions as compared to wild-type PanC009 (SEQ ID NO:3). Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0097] Example 2: PanC Screening Under Low 3A1P Conditions

[0098] PanC libraries were additionally screened under conditions employing low concentrations of 3A1P. Variants of PanC were generated by standard mutagenesis protocols of the PanC gene in a plasmid. E. coli cells were transformed with the libraries, and PanC variant libraries were expressed in 96-well plates in 200 piL LB broth with kanamycin at 32.5° C, 1000 rpm. After 20 hours, cultures were centrifuged, supernatant was discarded, and cells were resuspended in 100 .L of the reaction mixture: 50 mM sodium phosphate pH 7.5, 10 mM MgCh, 0.5 mM ( / ?)- pantoate, and 2.5 mM 3 -amino- 1 -propanol. Whole cell reactions were incubated at 1000 rpm at 32.5° C for 4 hours. The reactions were quenched by adding 900 pL of 60% ethanol, followed by filtration to remove precipitates. Analysis was performed via LCMS.

[0099] FIG. 9 shows kinetic parameters of wild-type PanC009 (SEQ ID NO:9) and engineered variants thereof, including variants identified by screening under low 3A1P conditions.

[0100] FIG. 10 shows the fold change increase from engineered PanC009 variants having multiple amino acid substitutions as compared to wild-type PanC009 (SEQ ID NOV).

[0101] Example 3: Panthenol Production Cell-Free

[0102] Cell-free production of panthenol is performed using a culture of E. coli expressing the engineered PanC enzyme. The cells are lysed with lysozyme. The resulting lysate can be used directly in cell-free reactions, or the enzyme can be further purified (see, e.g., FIG. 3). In some embodiments, cell-free production conditions may be as shown in Table 4. Cell-free reactions contain PanC enzyme either in lysate or purified, Tris pH 7.5, MgCh, KC1, (A)-pantoate, 3- amino-1 -propanol, ATP, and proceed for up to about 24 hours. The reaction may be quenched by filtering through a 10 kDa filter, or by heating to 50° C. Analysis may be performed via HPLC. ATP regeneration using various methods may also be employed when performing the reaction cell-free.

[0103] FIG. 7 shows the activity of two purified PanC homologs for panthenol production according to embodiments of the present invention. In FIG. 7, “PanC003” (lower plot) is SEQ ID NO:3 and “PanC009” (upper plot) is SEQ ID NOV.

[0104] Table 4 Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0105] Example 4: Panthenol production in strains

[0106] Panthenol production was also demonstrated in engineered strains that contain modifications to improve flux towards the precursor molecule (A)-pantoate. The engineered strains contain an improved PanC009 variant containing one or more mutations from Table 2, or additional mutations not listed in Table 2, and one or some combination of the following genome modifications: a) Overexpression of one or more ketopantoate hydroxymethyltransferase PanB variants from Table 5 with a constitutive promoter b) Overexpression of acetolactate synthase ALS variants from Table 6 with a constitutive promoter c) Overexpression of 2-dehydropantoate 2-reductase panE variants from Table 7 with a constitutive promoter

[0107] Production of panthenol in microbial hosts is performed in two stages. In Stage 1 (seed), a microbial culture of the engineered E. coli host strain is grown for a time period (e.g., about 16 hours) in a first medium, such as LB with 1% glycerol. In Stage 2 (production), the aforementioned culture is grown for an additional time period (e.g., about 22 hours) in a second medium, such as defined M9 minimal media containing 3 -amino- 1 -propanol (50 mM). After a given time period, the cultures are diluted with water and analyzed by LCMS and / or HPLC.

[0108] FIG. 11 shows the improvements in panthenol production achieved by incorporating the abovementioned modifications.

[0109] Table 5 provides exemplary sequences for ketopantoate hydroxymethyltransferase panB enzyme in accordance with the methods of invention. Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0110] Table 5 Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0111] In some embodiments, the ketopantoate hydroxymethyltransferase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

[0112] In some embodiments, the ketopantoate hydroxymethyltransferase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

[0113] In some embodiments, the ketopantoate hydroxymethyltransferase comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. In some embodiments, the ketopantoate hydroxymethyltransferase comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

[0114] In some embodiments, the ketopantoate hydroxymethyltransferase comprises an amino acid sequence identical to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

[0115] Table 6 provides exemplary sequences for acetolactate synthase ALS enzyme in accordance with the methods of invention.

[0116] Table 6 Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0117] In some embodiments, the acetolactate synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 15.

[0118] In some embodiments, the acetolactate synthase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 15.

[0119] In some embodiments, the acetolactate synthase comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 15.

[0120] In some embodiments, the acetolactate synthase comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 15.

[0121] In some embodiments, the acetolactate synthase comprises an amino acid sequence identical to SEQ ID NO: 15.

[0122] Table 7 provides exemplary sequences for 2-dehydropantoate 2-reductase panE enzyme in accordance with the methods of invention.

[0123] Table 7

[0124] In some embodiments, the 2-dehydropantoate 2-reductase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 16.

[0125] In some embodiments, the 2-dehydropantoate 2-reductase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 16.

[0126] In some embodiments, the 2-dehydropantoate 2-reductase comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 16.

[0127] In some embodiments, the 2-dehydropantoate 2-reductase comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 16. Attorney Docket No. DEBU-036 / 01WO 37396 / 184

[0128] In some embodiments, the 2-dehydropantoate 2-reductase comprises an amino acid sequence identical to SEQ ID NO: 16.

[0129] While there have been shown and described fundamental novel features of the invention as applied to the preferred and illustrative embodiments thereof, it will be understood that omissions and substitutions and changes in the form and details of the disclosed invention may be made by those skilled in the art without departing from the spirit of the invention. Moreover, as is readily apparent, numerous modifications and changes may readily occur to those skilled in the art. For example, various features and structures of the different embodiments discussed herein may be combined and interchanged. Hence, it is not desired to limit the invention to the exact construction and operation shown and described and, accordingly, all suitable modification equivalents may be resorted to falling within the scope of the invention as claimed. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.

Claims

1. Attorney Docket No. DEBU-036 / 01WO 37396 / 184CLAIMS1. A composition for preparing panthenol, comprising: a variant of pantothenate synthetase (PanC), wherein the variant of PanC is engineered for increased activity for panthenol production as compared to wild-type PanC;(7?)-pantoate; and3 -amino- 1 -propanol, wherein the engineered variant of PanC contains one or more mutations, each mutation at a position corresponding to E66, V109, R121, H124, T175, K184, R187, R196, 1255, K269, or R271 in SEQ ID NO:9.

2. The composition of claim 1, wherein the engineered variant of PanC comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 1, SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID N0:9, or SEQ ID NO: 10.

3. The composition of claim 1, wherein the engineered variant of PanC comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOT, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NOTO.

4. The composition of claim 1, wherein the engineered variant of PanC comprises an amino acid sequence having at least 99% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NOT, SEQ ID NOT, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 10.

5. The composition of claim 1, wherein the engineered variant of PanC comprises an amino acid sequencing having at least 99% identity to SEQ ID NOT, SEQ ID NOT, SEQ ID NO:9, or SEQ ID NO: 10.

6. The composition of claim 1, wherein the one or more mutations are selected from the group consisting of E66D, V109T, V109C, V109F, V109L, R121W, R121D, H124V, H124I, H124L,Attorney Docket No. DEBU-036 / 01WO 37396 / 184H124M, T175I, T175V, K184F, K184S, R187A, R187C, R187D, R187E, R187F, R187G, R187H, R187L, R187M, R187P, R187S, R187T, R187V, R187W, R187Y, R196Y, K269Y, I255T, I255D, I255R, I255K, K269Y, R271A, R271F, R271G, R271P, and R271T.

7. The composition of claim 1, wherein the engineered variant of PanC contains a combination of two or more mutations, wherein the combination is one of the combinations listed in Table 3.Table 38. The composition of any one of claims 1-7, wherein the composition further comprises a microbial host strain overexpressing the engineered variant of PanC.

9. The composition of claim 8, wherein the microbial host strain comprises bacteria, yeast, and / or fungal cells.

10. The composition of claim 9, wherein the microbial host strain is modified to overexpress (i) one or more variants of ketopantoate hydroxymethyltransferase; (ii) an acetolactate synthase; (iii) 2-dehydropantoate 2-reductase; or (v) any combination thereof.

11. The composition of claim 8, wherein the host strain is in a defined M9 minimal media.Attorney Docket No. DEBU-036 / 01WO 37396 / 18412. The composition of claim 8, wherein the (7?)-pantoate concentration is about 0.1 to about 1 mM.

13. The composition of claim 8, wherein the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM.

14. The composition of claim 1, wherein the composition is cell-free.

15. The composition of claim 14, wherein the engineered variant of PanC is in lysate or purified form.

16. The composition of claim 15, wherein the engineered variant of PanC is a lysate and has a concentration of about 0.1 to about 20 vol%.

17. The composition of claim 14, wherein the (A)-pantoate concentration is about 1 to about 5 mM.

18. The composition of claim 14, wherein the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM.

19. The composition of claim 14, further comprising a buffer at a concentration of about 20 to about 250 mM.

20. The composition of claim 10, wherein the buffer is Tris pH 7.5.

21. The composition of claim 14, further comprising MgCh at a concentration of about 1 to about 20 mM.

22. The composition of claim 14, further comprising KC1 at a concentration of about 1 to aboutAttorney Docket No. DEBU-036 / 01WO 37396 / 18423. The composition of claim 14, further comprising ATP at a concentration of about 1 to about5 mM.

24. The composition of any one of claims 1-23, wherein the engineered variant of PanC provides at least about a 1.5-fold improvement in panthenol generation as compared to an unmodified PanC control.

25. The composition of any one of claims 1-23, wherein the engineered variant of PanC provides at least about a 2-fold improvement in panthenol generation as compared to an unmodified PanC control.

26. The composition of any one of claims 1-23, wherein the engineered variant of PanC provides at least about a 2.5-fold improvement in panthenol generation as compared to an unmodified PanC control.

27. The composition of any one of claims 1-23, wherein the engineered variant of PanC provides at least about a 3-fold improvement in panthenol generation as compared to an unmodified PanC control.

28. The composition of any one of claims 1-23, wherein the engineered variant of PanC provides at least about a 4-fold improvement in panthenol generation as compared to an unmodified PanC control.

29. A method of preparing panthenol, comprising: providing a reaction mixture comprising a variant of pantothenate synthetase (PanC) engineered for increased activity for panthenol production as compared to wild-type PanC, (R)- pantoate, and 3 -amino- 1 -propanol; and incubating the reaction mixture containing the engineered variant of PanC, the (R)- pantoate, and the 3-amino-l -propanol at a temperature of about 20 to about 40 degrees Celsius for at least about 1 to about 24 hours,Attorney Docket No. DEBU-036 / 01WO 37396 / 184 wherein the engineered variant of PanC contains one or more mutations, each mutation at a position corresponding to E66, V109, R121, H124, T175, K184, R187, R196, 1255, K269, or R271 in SEQ ID NO:9.

30. The method of claim 29, wherein the method is performed in a microbial host, and wherein the step of providing the reaction mixture comprises: culturing a microbial host strain overexpressing the engineered variant of PanC in a growth media having 3 -amino- 1 -propanol added thereto, wherein microbial host strain produces the (R)-pantoate, or wherein the (R)-pantoate is provided in the media.

31. The method of claim 30, wherein the microbial host strain comprises bacteria, yeast, and / or fungal cells.

32. The method of claim 30, wherein the microbial host strain is modified to overexpress (i) one or more variants of ketopantoate hydroxymethyltransferase; (ii) an acetolactate synthase; (iii) 2- dehydropantoate 2-reductase; or (v) any combination thereof.

33. The method of claim 30, wherein the media is a defined M9 minimal media.

34. The method of claim 30, wherein the (7?)-pantoate concentration is about 0.1 to about 1 mM.

35. The method of claim 30, wherein the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM.

36. The method of claim 29, wherein the method is performed cell-free, and wherein the step of providing the reaction mixture comprises: providing a cell-free reaction mixture comprising the engineered variant of PanC in lysate or purified form, the (7?)-pantoate and the 3 -amino- 1 -propanol.

37. The method of claim 36, wherein the engineered variant of PanC is a lysate and has a concentration of about 0.1 to about 20 vol%.Attorney Docket No. DEBU-036 / 01WO 37396 / 18438. The method of claim 36, wherein the (A)-pantoate concentration is about 1 to about 5 mM.

39. The method of claim 36, wherein the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM.

40. The method of claim 36, wherein the reaction mixture further comprises Tris pH 7.5 at a concentration of about 20 to about 250 mM.

41. The method of claim 36, wherein the reaction mixture further comprises MgCh at a concentration of about 1 to about 20 mM.

42. The method of claim 36, wherein the reaction mixture further comprises KC1 at a concentration of about 1 to about 20 mM.

43. The method of claim 36, wherein the reaction mixture further comprises ATP at a concentration of about 1 to about 5 mM.

44. A microbial host strain expressing a variant of pantothenate synthetase (PanC), wherein the variant of PanC is engineered for increased activity for panthenol production as compared to wild-type PanC, and wherein the engineered variant of PanC contains one or more mutations, each mutation at a position corresponding to E66, V109, R121, H124, T175, K184, R187, R196, 1255, K269, or R271 in SEQ ID NO:9.

45. The microbial host strain of claim 44, wherein the one or more mutations are selected from the group consisting of E66D, V109T, V109C, V109F, V109L, R121W, R121D, H124V, H124I, H124L, H124M, T175I, T175V, K184F, K184S, R187A, R187C, R187D, R187E, R187F, R187G, R187H, R187L, R187M, R187P, R187S, R187T, R187V, R187W, R187Y, R196Y, K269Y, I255T, I255D, I255R, I255K, K269Y, R271A, R271F, R271G, R271P, and R271T.Attorney Docket No. DEBU-036 / 01WO 37396 / 18446. The microbial host strain of claim 44, wherein the engineered variant of PanC contains a combination of two or more mutations, wherein the combination is one of the combinations listed in Table 3.Table 347. The microbial host strain of claim 44, wherein the microbial host strain comprises bacteria, yeast, and / or fungal cells.

48. The microbial host strain of claim 44, wherein the microbial host strain is modified to overexpress (i) one or more variants of ketopantoate hydroxymethyltransferase; (ii) an acetolactate synthase; (iii) 2-dehydropantoate 2-reductase; or (v) any combination thereof.

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