Squalene hopaene cyclase variants for production of sclareolide

Abstract

Variants of squalene hopaene cyclase (SHC) are provided for the enzymatic conversion of homofarnesic acid to sclareolide, which can be non-enzymatically converted to ambergris, in particular to a squalene hopaene cyclase (SHC) for use in the enzymatic conversion of homofarnesic acid to sclareolide.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 352,352, filed on June 15, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file entitled "IFF652WOPCT_SequenceListing.xml", created on June 13, 2023, and is 95 bytes in size. The information in the electronic sequence listing is incorporated by reference in its entirety. Technical Field

[0005] Variants of squalene hopene cyclase (SHC) are provided for the enzymatic conversion of homofarnesoic acid to sclareolide, which can be non-enzymatically converted to ambroxan. Background Art

[0006] Compounds with a dodecahydronaphtho[2,1-b]furan skeleton are of great economic importance as aroma chemicals. Among them, (3aR,5aS,9aS,9bR)-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan), known as ambrox, is particularly important for providing the base note of fragrance compositions. Originally obtained from the ambergris of sperm whales, synthetic methods have now been developed to produce ambrox. In one route, sclareol, a component of clary sage (Salvia sclarea), is used as a starting material. Oxidative degradation of sclareol with, for example, chromic acid, permanganate, H2O2 or ozone provides sclareolide, which is subsequently reduced with, for example, LiAlH4 or NaBH4 to give ambrox-1,4-diol. Alternatively, sclareolide can be prepared from sclareol by biotransformation using Hyphozyma roseoniger (EP 0204009). Finally, ambra diol or tetraranor labdane diol is cyclized in a series of chemical processes to give the compound ambroxol ((-)-2). The preparation of the racemate rac-2 of ambroxol has been accomplished, among others, by homofarnesylic acid and 4-(2,6,6-trimethylcyclohex-1-enyl)butan-2-one.

[0007] In another route, ambroxan is prepared biocatalytically using squalene-hopeene cyclase (SHC; Scheme 1) (Neumann et al. (1986) Biol. Chem. Hoppe Seyler 367:723).

[0008] Solution 1

[0009] While SHC naturally catalyzes the cyclization of squalene to hompane, catalysis by ambroxol is a secondary reaction with a specific activity of 0.02 mU / mg protein. SHC from Alicyclobacillus acidocaldarius (formerly Bacillus acidocaldarius), Zymomonas mobilis, and Bradyrhizobium japonicum were purified and characterized for their natural (e.g., squalene) and non-natural substrates (e.g., homofarnesol and citral). See, e.g., WO 2010 / 139719, WO 2012 / 066059, JP 2009060799, and Seitz et al. (2012) J. Molecular Catalysis B: Enzymatic 84:72-77. Furthermore, WO 2016 / 170099 describes SHC variants with improved conversion of E,E-homofarnesol to ambroxol. US11091752 provides further SHC variants. Summary of the invention

[0010] Variant SHC molecules and methods of use thereof are described for producing sclareolide from homofarnesic acid, which can be non-enzymatically converted to ambroxan.

[0011] In one aspect, a variant squalene-hopaene cyclase (SHC) polypeptide is provided, which has at least 60% sequence identity to SEQ ID NO: 2 and comprises an amino acid substitution at one or more positions corresponding to positions V45, Q54, Q178, M184, V222, R249, I278, Y284, T326, R348, A574, A683 relative to SEQ ID NO: 2, wherein the variant exhibits increased sclareolide production compared to SEQ ID NO: 2. In some embodiments, the amino acid substitution at the one or more positions is V45L, Q54E, Q178S, M184T, V222Q, V222R, V222K, R249R, I278T, I278A, I278S, Y284W, T326S, R348R, A574A, A683E, or any combination thereof. In some embodiments, the variant SHC polypeptide comprises the following amino acid substitutions: (a) V45L, Q54E, I278T, T326S; (b) V45L, Q54E, M184T, R249R, I278T, T326S; (c) V45L, Q54E, V222Q, R249R, I278T, T326S; (d) V45L, Q54E, R249R, I278T, T326S; (e) V45L, Q54E, V222R, I278T, T326S; (f) V45L, Q54E, V222K, I278T, T326S; (g) V45L, Q54E, V222K, I278S, T32 6S; (h) V45L, Q54E, V222K, I278A, T326S; (i) V45L, Q54E, R249R, I278A, T326S; (j) V45L, Q54E, R249 R, I278T, T326S, A574A; (k) V45L, Q54E, V222K, R249R, I278T, T326S; (l) V45L, Q54E, Q178S, V222K, I278S, T326S; (m) V45L, Q54E, Q178S, V222R, I278T, T326S; (n) V45L, Q54E, V222Q, R249R, I278T, T3 26S, A683E; (o) V45L, Q54E, V222Q, Y284W, R249R, I278T, T326S; (p) V45L, Q54E, V222R, I278T, T326S, R348R; (q) V45L, Q54E, V222K, R249R, I278A, T326S; or (r) V45L, Q54E, Q178S, V222K, I278T, T326S.In some embodiments, the variant SHC polypeptide has at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.

[0012] In one aspect, a nucleic acid molecule is provided comprising a nucleic acid sequence encoding a variant SHC polypeptide described herein. In some embodiments, the nucleic acid sequence: i) encodes an amino acid sequence having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25; ii) encodes an amino acid sequence having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 NO:41 or SEQ ID NO:42 has at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity; or iii) hybridizes under stringent conditions to a nucleic acid sequence having a sequence complementary to the sequence shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42. In some embodiments, the nucleic acid sequence comprises a heterologous regulatory sequence, optionally a promoter sequence.

[0013] In one aspect, an expression vector is provided, comprising a nucleic acid molecule encoding a variant SHC described herein. In one aspect, an expression vector is provided, comprising a nucleic acid molecule described herein. In one aspect, a recombinant host cell is provided, comprising a nucleic acid molecule encoding a variant SHC described herein. In one aspect, a recombinant host cell is provided, comprising an expression vector described herein.

[0014] In one aspect, a method for producing sclareolide is provided, comprising contacting homofarnesic acid with a variant SHC polypeptide described herein or a recombinant host cell described herein. In some embodiments, the method comprises collecting the sclareolide. In some embodiments, the homofarnesic acid comprises (3E,7E) homofarnesic acid. In some embodiments, the method comprises non-enzymatically converting the sclareolide to ambroxol.

[0015] Various aspects and embodiments of these variant molecules and methods are described in the following independently numbered paragraphs.

[0016] 1. In one aspect, a non-naturally occurring variant of a wild-type squalene-hopaene cyclase (SHC) polypeptide is described, having at least 80% sequence identity to SEQ ID NO: 1 and comprising a first amino acid substitution at position V45, Q54, I278 and T326 relative to SEQ ID NO: 1 and a second amino acid substitution at position M184 and / or V222 relative to SEQ ID NO: 2, wherein the variant exhibits increased sclareolide production compared to a variant of wild-type SHC having only the amino acid substitutions V45L, Q54E, I278T and T326S relative to SEQ ID NO: 1 for numbering.

[0017] 2. In some embodiments of the variant SHC as described in paragraph 1, the first amino acid substitution is V45L, Q54E, I278T and T326S.

[0018] 3. In some embodiments of the variant SHC as described in paragraphs 1 or 2, the second amino acid substitution is selected from the group consisting of: M184T, V222Q, V222R and V222K, or a combination thereof.

[0019] 4. In some embodiments, the variant SHC as described in paragraph 1 has an amino acid substitution selected from the group consisting of: (i) V45L, Q54E, M184T, I278T and T326S, (ii) V45L, Q54E, V222Q, I278T and T326S, (iii) V45L, Q54E, I278T and T326S, (iv) V45L, Q54E, V222R, I278T and T326S, (v) V45L, Q54E, V222K, I278T and T326S, or (vi) V45L, Q54E, V222K, I278S and T326S, all relative to SEQ ID NO: 1 for numbering, wherein relative to SEQ ID NO: 2 for numbering, NO:2, the variant exhibits increased sclareolide production compared to the wild-type SHC variant having only the amino acid substitutions V45L, Q54E, I278T and T326S.

[0020] 5. In some embodiments, the variant SHC as described in any of paragraphs 1-4 has at least 90% amino acid sequence identity with SEQ ID NO:2.

[0021] 6. In another aspect, a recombinant vector encoding the variant SHC polypeptide of any one of paragraphs 1-5 is provided.

[0022] 7. In another aspect, a recombinant host cell comprising the recombinant vector as described in paragraph 6 is provided.

[0023] 8. In another aspect, a method for producing sclareolide is provided, comprising

[0024] (a) contacting homofarnesic acid with (i) a variant of wild-type SHC having only the amino acid substitutions V45L, Q54E, I278T, and T326S relative to SEQ ID NO: 1 or (ii) a variant of wild-type SHC as described in any of paragraphs 1-5; and (b) collecting sclareolide produced thereby.

[0025] 9. In another aspect, provided is a method for producing sclareolide, comprising (a) contacting homofarnesic acid with the recombinant host cell of paragraph 7, and (b) collecting sclareolide produced thereby.

[0026] 10. In some embodiments of the method of paragraph 8 or 9, the homofarnesic acid comprises (3E, 7E) homofarnesic acid.

[0027] 11. In some embodiments, the method of any of paragraphs 8-10 further comprises non-enzymatic conversion of the sclareolide to ambroxol.

[0028] Each of the aspects and embodiments described herein can be used together unless explicitly or clearly excluded from the context of an embodiment or aspect.

[0029] These and other aspects and embodiments of the variant molecules and methods are described below with reference to any of the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1Provided is an amino acid sequence comparison of Gluconobacter morbifer squalene-hopene cyclase (GmSHC) to related SHC enzymes from Z. mobilis (ZmSHC), Bradyrhizobium sp. (BspSHC), Rhodopseudomonas palustris (RpSHC), Streptomyces coelicolor (ScSHC), Burkholderia ambifaria (BamSHC), Bacillus anthracis (BanSHC), and A. acidocaldarius (AaSHC). The underlined residues represent the core sequence Gln-Xaa-Xaa-Xaa-Gly-Xaa-Trp (SEQ ID NO:3), and the bolded residues represent the Asp-Xaa-Asp-Asp-Thr-Ala (SEQ ID NO:4) active site motif. DETAILED DESCRIPTION

[0031] 1. Definitions and Abbreviations

[0032] Before describing these variations and methods in detail, the following terms are defined. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. The singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. This document is organized into several sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this manner, the titles used for the different sections of this disclosure should not be construed as limiting.

[0033] All publications (including patent documents, scientific articles and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. Nothing herein should be construed as an admission that such publications constitute prior art in the appended claims. If definitions set forth herein are contrary to or otherwise inconsistent with definitions set forth in patents, applications, published applications and other publications incorporated herein by reference, the definitions set forth herein take precedence over definitions incorporated herein by reference.

[0034] 1.1. Definition

[0035] As used herein, the term "ambroxan" refers to (3aR,5aS,9aS,9bR)-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan), which is commercially available under the names AMBROX (Firmenich), Ambroxan (Henkel), (Givaudan), (Quest), Laevo (Firmenich), (International Flavors & Fragrances Inc.), and and / or Norambrenolide Ether (Pacific). The desirable sensory benefits of Ambroxan come from the (-) stereoisomer, not the (+) enantiomer. The odor of the (-) stereoisomer is described as musky, woody, warm, or amber, while the (+) enantiomer has a relatively weaker note.

[0036] As used herein, "GmSHC" refers to a squalene-hopene cyclase isolated from Gluconobacter mossoni. In particular, when not modified by "variant", "GmSHC" refers to a wild-type protein having an amino acid sequence according to SEQ ID NO: 1. By comparison, a "variant GmSHC" or "GmSHC variant" refers to a GmSHC in which the amino acid sequence is altered compared to the amino acid sequence of the reference (or wild-type) GmSHC sequence of SEQ ID NO: 1. In some embodiments, when not modified by "variant", "GmSHC" refers to a wild-type protein having an amino acid sequence according to SEQ ID NO: 2. In some embodiments, a "variant GmSHC" or "GmSHC variant" refers to a GmSHC in which the amino acid sequence is altered compared to the amino acid sequence of the reference (or wild-type) GmSHC sequence of SEQ ID NO: 2. The terms variant SHC and SHC variant are used interchangeably herein to refer to variant GmSHC and GmSHC variant.

[0037] As used herein, the term "target yield" refers to the number of grams of product that can be recovered per gram of feedstock (which can be calculated as a molar conversion percentage).

[0038] As used herein, the term "activity" means the ability of an enzyme to react with a substrate to provide a target product. Activity can be determined in a so-called activity assay by the increase of the target product as a function of time, the decrease of the substrate (or starting material), or a combination of these parameters.

[0039] As used herein, the term "target productivity" refers to the amount of recoverable target product per hour of bioconversion time (ie, time after substrate addition) in grams per liter of fermentation capacity. In addition, GmSHC variants can exhibit improved target yield factors compared to reference GmSHC proteins.

[0040] As used herein, the term "target yield factor" refers to the ratio between the obtained product concentration and the concentration of the GmSHC variant in the reaction medium (eg, purified GmSHC enzyme or extract from recombinant host cells expressing the GmSHC enzyme).

[0041] As used herein, the term "recombinant host", also known as a "genetically modified host cell" or a "transgenic cell", refers to a host cell that contains a heterologous nucleic acid or whose genome has been augmented by at least one incorporated DNA sequence.

[0042] As used herein, the term "nucleic acid molecule" refers to a polynucleotide of the present disclosure, which may be DNA, cDNA, genomic DNA, synthetic DNA, or RNA, and may be double-stranded or single-stranded, sense strand and / or antisense strand.

[0043] As used herein, the term "isolated DNA", as used herein, refers to a nucleic acid or polynucleotide isolated from a natural source (e.g., Gluconobacter möglioni), or a nucleic acid or polynucleotide produced by recombinant DNA technology, such as a DNA construct comprising a polynucleotide heterologous to a host cell, which is optionally incorporated into a host cell.

[0044] "Hybridization" and its grammatical variants refer to the process by which a nucleic acid strand forms a duplex (i.e., base pairs) with a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by hybridization under the following conditions: 65°C and 0.1X SSC (where 1X SSC = 0.15M NaCl, 0.015M trisodium citrate (Na3 citrate), pH 7.0). Hybridized double-stranded nucleic acids are characterized by a melting temperature (Tm), wherein half of the hybridized nucleic acids are unpaired with the complementary strand. Mismatched nucleotides within the duplex lower the Tm.

[0045] As used herein, an "expression vector" includes a recombinant nucleic acid molecule encoding wild-type GmSHC or a SHC variant or homologue as described herein and necessary regulatory regions suitable for expressing a polypeptide. The choice of an expression vector (e.g., a plasmid, cosmid, viral or phage vector) will generally depend on the host cell into which it will be introduced. In some embodiments, the vector is a plasmid. In some embodiments, a recombinant nucleic acid molecule encoding wild-type GmSHC or a SHC variant or homologue as described herein is operably linked to various promoters and regulatory factors to drive expression when present, for example, in a host cell. An expression vector may also be referred to herein as a recombinant vector.

[0046] As used herein, the term "transformed" refers to the introduction of foreign or heterologous DNA into a cell. The transforming DNA may or may not be integrated, i.e., covalently linked, into the genome of the cell.

[0047] As used herein, the term "selective crystallization" refers to a process step in which (-)-sclareolide is crystallized from a solvent while the remaining isomers remain dissolved in the crystallization solvent.

[0048] In certain embodiments, the final product is isolated (-)-sclareolide. The term "isolated" used with respect to (-)-sclareolide refers to a biotransformation product that is separated from or purified from components that accompany it. An entity produced in a cell system from a source different from its natural origin is "isolated" because it is necessarily free of components that naturally accompany it. The degree of separation or purity can be measured by any appropriate method, such as gas chromatography (GC), HPLC or NMR analysis.

[0049] 1.2 Abbreviations and acronyms

[0050] Unless otherwise stated, the following abbreviations / acronyms have the following meanings:

[0051] ℃ Celsius

[0052] dH2O or DI deionized water

[0053] g or gm gram

[0054] FID Flame Ionization Detector

[0055] GC Gas Chromatography

[0056] hr hour

[0057] kg kilogram

[0058] LGA Lamarckian genetic algorithm

[0059] M Moore

[0060] mg milligram

[0061] min

[0062] mL and ml

[0063] mm Millimeters

[0064] mM millimole

[0065] MW Molecular weight

[0066] SDS Sodium dodecyl sulfate

[0067] PAGE polyacrylamide gel electrophoresis

[0068] sec seconds

[0069] U Unit

[0070] v / v volume / volume

[0071] w / v weight / volume

[0072] w / w weight / weight

[0073] wt% weight percentage

[0074] 2. Variant squalene-hopaene cyclase, an enzyme with an altered product spectrum

[0075] Variants of a parent squalenehopaene cyclase (SHC), exemplified by the homofarnesol-ambroxyl cyclase (HAC) isolated from Gluconobacter mossoni, are described that exhibit improved conversion of homofarnesic acid (HFA) to sclareolide compared to the relative parent enzyme. Direct enzymatic conversion of HFA to ambroxyl has been described, but is not an efficient process. Enzymatic conversion of HFA to sclareolide, followed by biochemical conversion of sclareolide to ambroxyl may be more efficient and less expensive than direct enzymatic conversion. Variants that exhibit improved conversion of HFA to sclareolide have not been described to date.

[0076] The amino acid sequence of wild-type Gluconobacter mössleri SHC (ie, (GmSHC)) is available under GENBANK Accession Nos. WP_040507485 and EHH69691. The amino acid sequence of wild-type GmSHC is provided below as SEQ ID NO: 1:

[0077] MLPEAVSSAC DWLIDQQKPD GHWVGPVESN ACMEAQWCLA

[0078] LWFLGQEDHP LRPRLAQALL

[0079] EMQREDGSWG IYVGADHGDI NTTVEAYAAL RSMGYAADMP

[0080] IMAKSAAWIQ QKGGLRNVRV

[0081] FTRYWLALIG EWPWDKTPNL PPEIIWLPDN FIFSIYNFAQ

[0082] <h2 style=";text-align:left;direction:ltr">WARATMMPLT ILSARRPSRP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0083] <h2 style=";text-align:left;direction:ltr"> LLPENRLDGL FPEGRENFDY ELPVKGEEDL WGRFFRAADK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0084] <h2 style=";text-align:left;direction:ltr"> GLHSLQSFPV RRFVPREAAI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0085] <h2 style=";text-align:left;direction:ltr"> RHVIEWIIRH QDADGGWGGIQPPWIYGLMA LSVEGYPLHH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0086] <h2 style=";text-align:left;direction:ltr"> PVLAKAMDAL NDPGWRRDKG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0087] <h2 style=";text-align:left;direction:ltr"> DASWIQATNS PVWDTMLAVL ALHDAGAEDR YSPQMDKAIG WLLDRQVRVKGDWSIKLPDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0088] <h2 style=";text-align:left;direction:ltr"> EPGGWAFEYA NDKYPDTDDT AVALIALAGC RHRPEWRERD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0089] <h2 style=";text-align:left;direction:ltr"> IEGAISRGVN WLLAMQSSSG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0090] <h2 style=";text-align:left;direction:ltr"> GWGAFDKDNN RSILTKIPFC DFGEALDPPS VDVTAHVLEA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0091] <h2 style=";text-align:left;direction:ltr"> FGLLGISRNH PSVQKALAYI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0092] <h2 style=";text-align:left;direction:ltr"> RSEQERNGAW FGRWGVNYVY GTGAVLPALA AIGEDMTQPY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0093] <h2 style=";text-align:left;direction:ltr"> IVRACDWLMS VQQENGGWGE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0094] <h2 style=";text-align:left;direction:ltr"> SCASYMDINA VGHGVATASQ TAWALIGLLA AKRPKDREAI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0095] <h2 style=";text-align:left;direction:ltr"> ARGCQFLIER QEDGSWTEEE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0096] <h2 style=";text-align:left;direction:ltr"> YTGTGFPGYG VGQAIKLDDP SLPDRLLQGA ELSRAFMLRY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0097] <h2 style=";text-align:left;direction:ltr"> DLYRQYFPVM ALSRARRMMK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0098] <h2 style=";text-align:left;direction:ltr"> EDASAAA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0099] The amino acid sequence of wild-type GmSHC is also provided in SEQ ID NO:2:

[0100] MSPADISTKS SSFQRLDNML PEAVSSACDW LIDQQKPDGH

[0101] WVGPVESNAC MEAQWCLALW

[0102] FLGQEDHPLR PRLAQALLEM QREDGSWGIY VGADHGDINT

[0103] TVEAYAALRS MGYAADMPIM

[0104] AKSAAWIQQK GGLRNVRVFT RYWLALIGEW PWDKTPNLPP

[0105] EIIWLPDNFI FSIYNFAQWA

[0106] RATMMPLTIL SARRPSRPLL PENRLDGLFP EGRENFDYEL

[0107] PVKGEEDLWG RFFRAADKGL

[0108] HSLQSFPVRR FVPREAAIRH VIEWIIRHQD ADGGWGGIQP

[0109] PWIYGLMALS VEGYPLHHPV

[0110] LAKAMDALND PGWRRDKGDA SWIQATNSPV WDTMLAVLAL

[0111] HDAGAEDRYS PQMDKAIGWL

[0112] LDRQVRVKGD WSIKLPDTEP GGWAFEYAND KYPDTDDTAV

[0113] ALIALAGCRH RPEWRERDIE

[0114] GAISRGVNWL LAMQSSSGGW GAFDKDNNRS ILTKIPFCDF

[0115] GEALDPPSVD VTAHVLEAFG

[0116] LLGISRNHPS VQKALAYIRS EQERNGAWFG RWGVNYVYGT

[0117] GAVLPALAAI GEDMTQPYIV

[0118] RACDWLMSVQ QENGGWGESC ASYMDINAVG HGVATASQTA

[0119] WALIGLLAAK RPKDREAIAR

[0120] GCQFLIERQE DGSWTEEEYT GTGFPGYGVG QAIKLDDPSL

[0121] PDRLLQGAELSRAFMLRYDL

[0122] YRQYFPVMAL SRARRMMKED ASAAA

[0123] Alignment of the GmSHC amino acid sequence with similar SHC amino acid sequences from Zymomonas mobilis, Bradyrhizobium species, Rhodopseudomonas palustris, Streptomyces coelicolor, Burkholderia bifida, Bacillus anthracis, and Alicyclobacillus acidocaldarius indicated that the amino acid sequence identities ranged between 37% and 76% (Table 1). Table 1 shows the alignment with SEQ ID NO: 1.

[0124] Table 1. Alignment of various SHC amino acid sequences and identity with GmSHC.

[0125] Source organism Accession Number Identity (%) Zymomonas mobilis Q5NM88 76 Bradyrhizobium species A5EBP6 72 Burkholderia bifida Q0B5S3 37 Bacillus anthracis A0A0E0W268 48 Rhodopseudomonas palustris WP_011665849 66 Streptomyces coelicolor Q9X7V9 45 Alicyclobacillus acidocaldarius P33247 44

[0126] SHC contains the core sequence Gln-Xaa-Xaa-Xaa-Gly-Xaa-Trp (SEQ ID NO:3) (Reipen et al. (1995) Microbiology 141:155-61) and an Asp-Xaa-Asp-Asp-Thr-Ala (SEQ ID NO:4) motif associated with the SHC active site (Wendt et al. (1997) Science 277:1811-5). (See Figure 1 The data presented herein demonstrate that when expressed in a heterologous host cell such as E. coli, the variants or variants of the SHC enzyme can readily convert HFA to sclareolide, which can then be non-enzymatically converted to ambroxol.

[0127] As used herein, the term "amino acid change" refers to the insertion of one or more amino acid residues, the deletion of one or more amino acid residues, or the substitution of one or more amino acid residues (which may be conservative, non-conservative or synonymous) relative to the amino acid sequence of a reference amino acid sequence (e.g., such as the wild-type amino acid sequence of SEQ ID NO: 2). Amino acid changes can be easily identified by comparing the amino acid sequence of a GmSHC derivative with the amino acid sequence of a reference or wild-type GmSHC.

[0128] For example, conservative amino acid substitutions can be made based on similarities in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into the following six standard amino acid groups: (1) hydrophobic - Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic - Cys, Ser, Thr, Asn, Gln; (3) acidic - Asp, Glu; (4) basic - His, Lys, Arg; (5) residues affecting chain orientation - Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Thus, as used herein, the term "conservative substitution" means that an amino acid is exchanged for another amino acid listed in the same group of the six standard amino acid groups shown above. For example, the exchange of Glu for Asp retains a negative charge in the modified polypeptide. In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices. Some preferred conservative substitutions in the above six groups are exchanges in the following subgroups: (i) Ala, Val, Leu and Ile; (ii) Ser and Thr; (iii) Asn and Gln: (iv) Lys and Arg; and (v) Tyr and Phe. Given the known genetic code and recombinant and synthetic DNA technology, scientists in the field can easily construct DNA encoding conservative amino acid variants. Although synonymous or silent mutations do not directly change the amino acid sequence of the encoded protein, they still affect the accuracy or efficiency of splicing. Silent or synonymous mutations can be referred to as substitutions in this article.

[0129] As used herein, "non-conservative substitution" or "non-conservative amino acid exchange" is defined as an amino acid exchanged by another amino acid listed in different groups of the six standard amino acid groups (1) to (6) shown above. Typically, non-conservative substitutions that alter the biological function of wild-type GmSHC are used to prepare the GmSHC derivatives of the present disclosure.

[0130] Amino acid substitutions can be introduced using known recombinant gene technology protocols including PCR, gene cloning, cDNA site-directed mutagenesis, host cell transformation and in vitro transcription, which can be used to introduce such changes into the SHC sequence to generate SHC variant enzymes. The variants can then be screened for SHC functional activity.

[0131] The SHC variants may have about 1 to about 45 amino acid substitutions, about 1 to about 40 amino acid substitutions, about 1 to about 35 amino acid substitutions, about 1 to about 30 amino acid substitutions, about 1 to about 25 amino acid substitutions, about 1 to about 20 amino acid substitutions, about 1 to about 15 amino acid substitutions, about 1 to about 10 amino acid substitutions or about 1 to about 5 amino acid substitutions relative to the amino acid sequence of a reference (or wild-type) SHC sequence according to SEQ ID NO: 1.

[0132] The SHC variants may have about 1 to about 45 amino acid substitutions, about 1 to about 40 amino acid substitutions, about 1 to about 35 amino acid substitutions, about 1 to about 30 amino acid substitutions, about 1 to about 25 amino acid substitutions, about 1 to about 20 amino acid substitutions, about 1 to about 15 amino acid substitutions, about 1 to about 10 amino acid substitutions or about 1 to about 5 amino acid substitutions relative to the amino acid sequence of a reference (or wild-type) SHC sequence according to SEQ ID NO: 2.

[0133] Alternatively, the SHC variant may have at least 5, at least 10 amino acids or at least 15 amino acid substitutions, but ideally no more than about 30 or 40 amino acid substitutions relative to the amino acid sequence of the reference (i.e., wild-type) SHC sequence according to SEQ ID NO: 1. In various embodiments, the SHC variant may have about 1 amino acid substitution, about 2 amino acid substitutions, about 3 amino acid substitutions, about 4 amino acid substitutions, about 5 amino acid substitutions, about 6 amino acid substitutions, about 7 amino acid substitutions, about 8 amino acid substitutions, about 9 amino acid substitutions, about 10 amino acid substitutions, about 11 amino acid substitutions, about 12 amino acid substitutions, about 15 amino acid substitutions, about 20 amino acid substitutions, about 25 amino acid substitutions, about 30 amino acid substitutions, about 35 amino acid substitutions, about 40 amino acid substitutions, about 45 amino acid substitutions, or about 50 amino acid substitutions relative to the reference SHC.

[0134] In some embodiments, the SHC variant may have at least 5, at least 10 amino acids, or at least 15 amino acid substitutions, but ideally no more than about 30 or 40 amino acid substitutions relative to the amino acid sequence of the reference (i.e., wild-type) SHC sequence according to SEQ ID NO: 2. In various embodiments, the SHC variant may have about 1 amino acid substitution, about 2 amino acid substitutions, about 3 amino acid substitutions, about 4 amino acid substitutions, about 5 amino acid substitutions, about 6 amino acid substitutions, about 7 amino acid substitutions, about 8 amino acid substitutions, about 9 amino acid substitutions, about 10 amino acid substitutions, about 11 amino acid substitutions, about 12 amino acid substitutions, about 15 amino acid substitutions, about 20 amino acid substitutions, about 25 amino acid substitutions, about 30 amino acid substitutions, about 35 amino acid substitutions, about 40 amino acid substitutions, about 45 amino acid substitutions, or about 50 amino acid substitutions relative to the reference SHC.

[0135] In some embodiments, the amino acid substitutions are silent mutations.Any one or more of the substitutions described herein may be silent mutations.

[0136] In these or other aspects, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the reference SHC of SEQ ID NO: 1.

[0137] In some aspects, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the reference SHC of SEQ ID NO:2.

[0138] In some embodiments, the variant SHC polypeptide comprises an amino acid substitution at one or more positions corresponding to positions V45, Q54, Q178, M184, V222, R249, 1278, Y284, T326, R348, A574, A683 relative to SEQ ID NO: 2. In some embodiments, the variant SHC polypeptide comprises an amino acid substitution at two or more positions corresponding to positions V45, Q54, Q178, M184, V222, R249, 1278, Y284, T326, R348, A574, A683 relative to SEQ ID NO: 2. In some embodiments, the variant SHC polypeptide comprises an amino acid substitution at three or more positions corresponding to positions V45, Q54, Q178, M184, V222, R249, 1278, Y284, T326, R348, A574, A683 relative to SEQ ID NO: 2. In some embodiments, the variant SHC polypeptide comprises an amino acid substitution at 4 or more positions corresponding to positions V45, Q54, Q178, M184, V222, R249, 1278, Y284, T326, R348, A574, A683 relative to SEQ ID NO: 2. In some embodiments, the variant SHC polypeptide comprises an amino acid substitution at 5 or more positions corresponding to positions V45, Q54, Q178, M184, V222, R249, 1278, Y284, T326, R348, A574, A683 relative to SEQ ID NO: 2. In some embodiments, the variant SHC polypeptide comprises an amino acid substitution at 6 or more positions corresponding to positions V45, Q54, Q178, M184, V222, R249, 1278, Y284, T326, R348, A574, A683 relative to SEQ ID NO: 2. In some embodiments, the variant SHC polypeptide comprises an amino acid substitution at 7 or more positions corresponding to positions V45, Q54, Q178, M184, V222, R249, 1278, Y284, T326, R348, A574, A683 relative to SEQ ID NO: 2. In some embodiments, the amino acid substitution is V45L, Q54E, Q178S, M184T, V222Q, V222R, V222K, R249R, I278T, I278S, I278A, Y284W, T326S, R348R, A574A, A683E, or any combination thereof. In some embodiments, the variant SHC further has at least 60%, 70%, 80%, 90%, 95% but less than 100% sequence identity to SEQ ID NO:2.In some embodiments, the amino acid substitution is V45L, Q54E, Q178S, M184T, V222Q, V222R, V222K, R249R, I278T, I278S, I278A, Y284W, T326S, R348R, A574A, A683E, or any combination thereof. In some embodiments, the variant SHC further has at least 70% but less than 100% sequence identity with SEQ ID NO: 2. In some embodiments, the variant SHC further has at least 80% but less than 100% sequence identity with SEQ ID NO: 2. In some embodiments, the variant SHC further has at least 90% but less than 100% sequence identity with SEQ ID NO: 2. In some embodiments, the variant SHC further has at least 95% but less than 100% sequence identity with SEQ ID NO: 2. In some embodiments, the variant SHC further has at least 60%, 70%, 80%, 90%, 95% but less than 100% sequence identity with SEQ ID NO: 1. In some embodiments, the amino acid substitution is V45L, Q54E, Q178S, M184T, V222Q, V222R, V222K, R249R, I278T, I278S, I278A, Y284W, T326S, R348R, A574A, A683E, or any combination thereof. In some embodiments, the variant SHC further has at least 70% but less than 100% sequence identity with SEQ ID NO: 1. In some embodiments, the variant SHC further has at least 80% but less than 100% sequence identity with SEQ ID NO: 1. In some embodiments, the variant SHC further has at least 90% but less than 100% sequence identity with SEQ ID NO: 1. In some embodiments, the variant SHC further has at least 95% but less than 100% sequence identity to SEQ ID NO: 1. In some embodiments, the variant SHC exhibits increased production of sclareolide compared to SEQ ID NO: 2. In some embodiments, the variant SHC exhibits increased production of sclareolide compared to SEQ ID NO: 1.

[0139] In some embodiments, the SHC variant comprises an amino acid substitution at position V45, Q54, I278 and / or T326. Such variants exhibit increased sclareolide production compared to the wild-type enzyme. Particular substitutions include, but are not limited to, V45L, Q54E, I278T and T326S. In some embodiments, substitutions include V45L, Q54E, I278T, I278S, I278A and T326S. In some embodiments, the SHC variant comprises an amino acid substitution at one or more (or all) positions of position V45, Q54, I278 and / or T326 relative to SEQ ID NO: 2. In some embodiments, the SHC variant comprises an amino acid substitution at one or more (or all) positions of position V45, Q54, I278 and / or T326 relative to SEQ ID NO: 1. In some embodiments, determining the position relative to SEQ ID NO: 1 comprises aligning SEQ ID NO: 1 with SEQ ID NO: 2. In some embodiments, the position of the substitution in SEQ ID NO: 1 corresponds to the position of the substitution in SEQ ID NO: 2. For example, if position 19 of SEQ ID NO: 2 is altered (e.g., substituted), then the corresponding position (position 1) in SEQ ID NO: 1 is altered (e.g., substituted) after alignment with SEQ ID NO: 2. In some embodiments, to determine the position relative to SEQ ID NO: 2, SEQ ID NO: 2 is aligned with a sequence that is or will undergo an amino acid change.

[0140] In particular embodiments, the SHC variant comprises additional amino acid substitutions at positions M184 and V222. In particular embodiments, the SHC variant comprises additional amino acid substitutions at positions Q178, M184, V222, Y284, A683. Such variants show further increased clareolide production compared to the wild-type enzyme and those having substitutions at positions V45, Q54, I278 and T326. Particular substitutions include, but are not limited to, M184T, V222Q, V222R and V222K, or a combination thereof. In some embodiments, substitutions include, but are not limited to, Q178S, M184T, V222Q, V222R and V222K, Y284W, A683E, or a combination thereof. In some embodiments, the SHC variant comprises one or more substitutions that are silent mutations. In some embodiments, the SHC variant comprises 1, 2, 3, 4, 5 or more silent mutations. In some embodiments, the SHC variant comprises 1, 2, 3, 4 or 5 silent mutations. In some embodiments, the SHC variant comprises 1, 2, 3 or 4 silent mutations. In some embodiments, the SHC variant comprises 1, 2 or 3 silent mutations. In some embodiments, the SHC variant comprises a silent mutation at position 249, 348, 574, or at any one position or combination thereof. In some embodiments, the silent mutation is R249R, R348R, A574A, or any combination thereof. In some embodiments, the position of the silent mutation is relative to SEQ ID NO: 2. In some embodiments, after alignment of SEQ ID NO: 1 with SEQ ID NO: 2, the silent mutation is located at a position in SEQ ID NO: 1 that corresponds to a position in SEQ ID NO: 2.

[0141] In some embodiments, the SHC variant has the following amino acid substitutions:

[0142] V45, Q54, I278, T326;

[0143] V45, Q54, M184, R249, I278, T326;

[0144] V45, Q54, V222, R249, I278, T326;

[0145] V45, Q54, R249, I278, T326;

[0146] V45, Q54, V222, I278, T326;

[0147] V45, Q54, V222, I278, T326;

[0148] V45, Q54, V222, I278, T326;

[0149] V45, Q54, V222, I278, T326;

[0150] V45, Q54, R249, I278, T326;

[0151] V45, Q54, R249, I278, T326, A574;

[0152] V45, Q54, V222, R249, I278, T326;

[0153] V45, Q54, Q178, V222, I278, T326;

[0154] V45, Q54, Q178, V222, I278, T326;

[0155] V45, Q54, V222, R249, I278, T326, A683;

[0156] V45, Q54, V222, R249, I278, Y284, T326;

[0157] V45, Q54, V222, I278, T326, R348;

[0158] V45, Q54, V222, R249, I278, T326; or

[0159] V45, Q54, Q178, V222, I278, T326.

[0160] In some embodiments, the SHC variant has the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1:

[0161] V45, Q54, I278, T326;

[0162] V45, Q54, M184, R249, I278, T326;

[0163] V45, Q54, V222, R249, I278, T326;

[0164] V45, Q54, R249, I278, T326;

[0165] V45, Q54, V222, I278, T326;

[0166] V45, Q54, V222, I278, T326;

[0167] V45, Q54, V222, I278, T326;

[0168] V45, Q54, V222, I278, T326;

[0169] V45, Q54, R249, I278, T326;

[0170] V45, Q54, R249, I278, T326, A574;

[0171] V45, Q54, V222, R249, I278, T326;

[0172] V45, Q54, Q178, V222, I278, T326;

[0173] V45, Q54, Q178, V222, I278, T326;

[0174] V45, Q54, V222, R249, I278, T326, A683;

[0175] V45, Q54, V222, R249, I278, Y284, T326;

[0176] V45, Q54, V222, I278, T326, R348;

[0177] V45, Q54, V222, R249, I278, T326; or

[0178] V45, Q54, Q178, V222, I278, T326.

[0179] In some embodiments, the variant has the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2:

[0180] V45, Q54, I278, T326;

[0181] V45, Q54, M184, R249, I278, T326;

[0182] V45, Q54, V222, R249, I278, T326;

[0183] V45, Q54, R249, I278, T326;

[0184] V45, Q54, V222, I278, T326;

[0185] V45, Q54, V222, I278, T326;

[0186] V45, Q54, V222, I278, T326;

[0187] V45, Q54, V222, I278, T326;

[0188] V45, Q54, R249, I278, T326;

[0189] V45, Q54, R249, I278, T326, A574;

[0190] V45, Q54, V222, R249, I278, T326;

[0191] V45, Q54, Q178, V222, I278, T326;

[0192] V45, Q54, Q178, V222, I278, T326;

[0193] V45, Q54, V222, R249, I278, T326, A683;

[0194] V45, Q54, V222, R249, I278, Y284, T326;

[0195] V45, Q54, V222, I278, T326, R348;

[0196] V45, Q54, V222, R249, I278, T326; or

[0197] V45, Q54, Q178, V222, I278, T326.

[0198] In a further specific embodiment, the variant has the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1:

[0199] (i) V45L, Q54E, M184T, I278T and T326S,

[0200] (ii) V45L, Q54E, V222Q, I278T and T326S,

[0201] (iii) V45L, Q54E, I278T and T326S,

[0202] (iv) V45L, Q54E, V222R, I278T and T326S,

[0203] (v) V45L, Q54E, V222K, I278T and T326S, or

[0204] (vi) V45L, Q54E, V222K, I278S and T326S.

[0205] In some embodiments, the variant has the following amino acid substitutions:

[0206] V45L, Q54E, I278T, T326S;

[0207] V45L, Q54E, M184T, R249R, I278T, T326S;

[0208] V45L, Q54E, V222Q, R249R, I278T, T326S;

[0209] V45L, Q54E, R249R, I278T, T326S;

[0210] V45L, Q54E, V222R, I278T, T326S;

[0211] V45L, Q54E, V222K, I278T, T326S;

[0212] V45L, Q54E, V222K, I278S, T326S;

[0213] V45L, Q54E, V222K, I278A, T326S;

[0214] V45L, Q54E, R249R, I278A, T326S;

[0215] V45L, Q54E, R249R, I278T, T326S, A574A;

[0216] V45L, Q54E, V222K, R249R, I278T, T326S;

[0217] V45L, Q54E, Q178S, V222K, I278S, T326S;

[0218] V45L, Q54E, Q178S, V222R, I278T, T326S;

[0219] V45L, Q54E, V222Q, R249R, I278T, T326S, A683E;

[0220] V45L, Q54E, V222Q, R249R, I278T, Y284W, T326S;

[0221] V45L, Q54E, V222R, I278T, T326S, R348R;

[0222] V45L, Q54E, V222K, R249R, I278A, T326S; or

[0223] V45L, Q54E, Q178S, V222K, I278T, T326.

[0224] In a further specific embodiment, the variant has the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1:

[0225] V45L, Q54E, I278T, T326S;

[0226] V45L, Q54E, M184T, R249R, I278T, T326S;

[0227] V45L, Q54E, V222Q, R249R, I278T, T326S;

[0228] V45L, Q54E, R249R, I278T, T326S;

[0229] V45L, Q54E, V222R, I278T, T326S;

[0230] V45L, Q54E, V222K, I278T, T326S;

[0231] V45L, Q54E, V222K, I278S, T326S;

[0232] V45L, Q54E, V222K, I278A, T326S;

[0233] V45L, Q54E, R249R, I278A, T326S;

[0234] V45L, Q54E, R249R, I278T, T326S, A574A;

[0235] V45L, Q54E, V222K, R249R, I278T, T326S;

[0236] V45L, Q54E, Q178S, V222K, I278S, T326S;

[0237] V45L, Q54E, Q178S, V222R, I278T, T326S;

[0238] V45L, Q54E, V222Q, R249R, I278T, T326S, A683E;

[0239] V45L, Q54E, V222Q, R249R, I278T, Y284W, T326S;

[0240] V45L, Q54E, V222R, I278T, T326S, R348R;

[0241] V45L, Q54E, V222K, R249R, I278A, T326S; or

[0242] V45L, Q54E, Q178S, V222K, I278T, T326.

[0243] In some embodiments, the position refers to the position in SEQ ID NO:2, and is the corresponding position in SEQ ID NO:1 after alignment of SEQ ID NO:1 with SEQ ID NO:2.

[0244] In some embodiments, the variant has the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 2:

[0245] (a) V45L, Q54E, I278T, T326S;

[0246] (b) V45L, Q54E, M184T, R249R, I278T, T326S;

[0247] (c)V45L, Q54E, V222Q, R249R, I278T, T326S;

[0248] (d)V45L, Q54E, R249R, I278T, T326S;

[0249] (e) V45L, Q54E, V222R, I278T, T326S;

[0250] (f)V45L, Q54E, V222K, I278T, T326S;

[0251] (g)V45L, Q54E, V222K, I278S, T326S;

[0252] (h)V45L, Q54E, V222K, I278A, T326S;

[0253] (i)V45L, Q54E, R249R, I278A, T326S;

[0254] (j)V45L, Q54E, R249R, I278T, T326S, A574A;

[0255] (k)V45L, Q54E, V222K, R249R, I278T, T326S;

[0256] (l)V45L, Q54E, Q178S, V222K, I278S, T326S;

[0257] (m)V45L, Q54E, Q178S, V222R, I278T, T326S;

[0258] (n)V45L, Q54E, V222Q, R249R, I278T, T326S, A683E;

[0259] (o)V45L, Q54E, V222Q, R249R, I278T, Y284W, T326S;

[0260] (p)V45L, Q54E, V222R, I278T, T326S, R348R;

[0261] (q) V45L, Q54E, V222K, R249R, I278A, T326S; or

[0262] (r)V45L, Q54E, Q178S, V222K, I278T, T326.

[0263] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO: 12.

[0264] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO: 13.

[0265] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO: 14.

[0266] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO: 15.

[0267] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO: 16.

[0268] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO: 17.

[0269] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO: 18.

[0270] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO: 19.

[0271] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO:20.

[0272] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO: 21.

[0273] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO:22.

[0274] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO:23.

[0275] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO:24.

[0276] In some embodiments, the SHC variant shares at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99%, or 100% sequence identity with SEQ ID NO:25.

[0277] In another aspect, nucleic acid molecules are provided which are or contain nucleic acid sequences encoding SHC variants. The nucleic acid sequences may encode a particular SHC variant described herein, or an SHC variant having a specified degree of amino acid sequence identity to a particular SHC variant.

[0278] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42. In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 30. In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 36. In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 37. In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 42.

[0279] In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid encoding a SHC variant (or a nucleic acid complementary to a SHC variant encoding a SHC variant) having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42. In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid encoding a SHC variant (or a nucleic acid complementary to a nucleic acid encoding a SHC variant) having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 30. In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid encoding a SHC variant (or a nucleic acid complementary to a nucleic acid encoding a SHC variant) having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 36. In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid encoding a SHC variant (or a nucleic acid complementary to a nucleic acid encoding a SHC variant) having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 37. In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid encoding a SHC variant (or a nucleic acid complementary to a nucleic acid encoding a SHC variant) having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:42.

[0280] Preferably, the SHC variants exhibit a better target yield of sclareolide compared to a reference SHC protein. In addition, the SHC variants may exhibit an improved (e.g., increased) target productivity relative to a reference SHC protein. In certain embodiments, the SHC variants exhibit at least a 2-fold, 3-fold, 4-fold, 6-fold, 8-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold increase in enzyme activity (e.g., conversion of HFA to sclareolide) relative to a reference SHC protein.

[0281] To facilitate the expression of SHC and the production and isolation of sclareolide, SHC or SHC variants are expressed in recombinant host cells. Host cells useful for the purposes of the present disclosure include, but are not limited to, prokaryotic cells such as bacteria (e.g., E. coli and B. subtilis), which can be transformed with, for example, expression vectors containing nucleic acid molecules encoding GmSHC or variant SHC described herein, recombinant phage DNA, plasmid DNA, bacterial artificial chromosomes, or cosmid DNA expression vectors; simple eukaryotic cells, like yeast (e.g., Saccharomyces and Pichia), which can be transformed with, for example, recombinant yeast expression vectors containing polynucleotide molecules of the present disclosure; insect cells (e.g., baculovirus insect cell expression systems); human cells (e.g., HeLa, CHO, and Jurkat) and plant cells (Arabidopsis and tobacco). Depending on the host cell and the corresponding vector used to introduce the nucleic acid molecule encoding GmSHC or a variant SHC described herein, the nucleic acid molecule may be integrated into, for example, the chromosome or mitochondrial DNA, or may be maintained extrachromosomally (e.g., as an episome), or may be carried only transiently by the cell. In embodiments involving eukaryotic cells, preferably the cell is a fungal, mammalian, or plant cell. Suitable eukaryotic cells include, for example, but are not limited to, mammalian cells, yeast cells (e.g., Saccharomyces, Candida, Kluyveromyces, Schizosaccharomyces, Yarrowia, Pichia, and Aspergillus), or insect cells (including Sf9), amphibian cells (including melanocytes), or worm cells including Caenorhabditis (including Caenorhabditis elegans) cells. Suitable mammalian cells include, for example, but are not limited to, COS cells (including Cos-1 and Cos-7), CHO cells, HEK293 cells, HEK293T cells, or other transfectable eukaryotic cell lines. In embodiments involving prokaryotes, preferably the cell is Escherichia coli, Bacillus sp., or Streptomyces sp. In some embodiments, the host cell comprises a nucleic acid molecule or expression vector containing a nucleic acid molecule encoding a variant SHC described herein. In some embodiments, the host cell is a yeast, a bacterium, a mammalian cell, or a plant cell. In some embodiments, the host cell is Escherichia coli. In some embodiments, the nucleic acid molecule encoding the variant SHC is codon optimized.While some embodiments include the use of intact whole cells or cell extracts, other embodiments include the use of free, optionally purified or partially purified SHC enzymes or immobilized SHC enzymes for the bioconversion of HFAs to sclareolide. In this regard, when soluble wild-type SHC or SHC variants are used as the biocatalyst, this is considered a two-phase system.

[0282] Sclareolide produced by the present two-phase system can be collected, for example, by steam extraction / distillation or organic solvent extraction using a water-immiscible solvent (to separate the reaction products and unreacted substrate from the biocatalyst remaining in the aqueous phase), followed by subsequent solvent evaporation to obtain the crude reaction product as determined by gas chromatography (GC) analysis.

[0283] In some embodiments, sclareolide is produced by contacting a variant SHC described herein or a host cell comprising an expression vector comprising a nucleic acid molecule encoding a variant SHC described herein with an HFA. In some embodiments, the HFA comprises (3E, 7E) HFA. In some embodiments, the contacting occurs in the absence or absence of added metal ions.

[0284] Sclarea lactone can be further selectively crystallized to remove unreacted HFA substrate from the final product. In some embodiments, the isolated crystalline material contains only (-)-sclarea lactone product. In other embodiments, the isolated crystalline material contains other isomers, wherein the isomers are present only in an olfactory acceptable amount.

[0285] Examples of suitable water-miscible and water-immiscible organic solvents suitable for the extraction and / or selective crystallization of (-)-sclareolide include, but are not limited to, aliphatic hydrocarbons, preferably those having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; halogenated aliphatic hydrocarbons, preferably those having one or two carbon atoms, such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane or tetrachloroethane; aromatic hydrocarbons, such as benzene, toluene, xylene, chlorobenzene or dichlorobenzene; aliphatic acyclic and cyclic ethers or alcohols, preferably those having 4 to 8 carbon atoms, such as ethanol, isopropanol, diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran; or esters, such as ethyl acetate or n-butyl acetate, or ketones, such as methyl isobutyl ketone or dioxane or mixtures of these. The solvent that is particularly preferably used is the above-mentioned heptane, methyl tert-butyl ether (also referred to as MTBE, tert-butyl methyl ether and iBME), diisopropyl ether, tetrahydrofuran, ethyl acetate and / or its mixture. Preferably, a water-miscible solvent such as ethanol is used to extract (-)-sclareolide from the solid phase of the reaction mixture. The use of ethanol is advantageous because it is easy to handle, nontoxic and environmentally friendly.

[0286] Ideally, the amount of (-)-sclareolide produced is in the range of about 1 mg / L to about 20,000 mg / L (20 g / L) or higher, such as about 20 g / L to about 200 g / L or 100 g / L to 200 g / L, preferably about 125 g / L or 150 g / L.

[0287] Various applications of (-)-sclareolide include, but are not limited to, the non-enzymatic production of ambrox for use in fine fragrances or consumer products such as fabric care, toiletry, beauty care and cleaning products, including substantially all products in which the currently available ambrox ingredients are used commercially, including, but not limited to, AMBROX (Henkel), AMBERLYN (Quest) and ambroxolide (Pacific), as well as those marketed under the trademarks (Givaudan), Laevo (Firmenich) and / or (Aromor). In some embodiments, enzymatically produced sclareolide can be used in fragrances such as fine fragrances, consumer fragrances, full fragrances, sketches and / or accords via non-enzymatically produced ambroxan. The selective crystallization of (-)-ambroxan may be affected by the presence of unreacted HFA substrate and the ratio of (-)-ambroxan to other detectable isomers. In some embodiments, the selective crystallization of (-)-ambroxan may be affected by the presence of unreacted HFA and / or its byproducts and the ratio of (-)-ambroxan to other detectable isomers. Selective crystallization of (-)-ambroxan is still possible even if only a 10% conversion of the HFA substrate to the enzymatically produced (-)-sclareolide to the non-enzymatically produced (-)-ambroxan is obtained.

[0288] Non-enzymatic methods for converting sclareolide to ambroxol (e.g., (-)-ambroxol) include, but are not limited to, methods for reducing sclareolide. In some embodiments, sclareolide can undergo a two-step reaction process for conversion. In some embodiments, the first reaction comprises reacting sclareolide with a reducing agent (e.g., but not limited to, lithium aluminum hydride) in a solvent (e.g., but not limited to, diethyl ether). In some embodiments, the second reaction comprises reacting the product of the first step with a reagent (e.g., but not limited to, tosyl chloride) in a solvent (e.g., but not limited to, pyridine). A method for converting ambroxan to sclareolide is described in Functionalization of C-12 in labdanic diterpenes: synthesis of the natural diterpenic lactones isolated from Cistus ladaniferus L, Teresa, J. de Pascual; Urones, JG; Montana Pedrero, A.; Basabe Barcala, P., Tetrahedron Letters (1985), 26(46), 5717-20, which is incorporated by reference in its entirety.

[0289] The following non-limiting examples are provided to further illustrate the present invention.

[0290] Examples

[0291] Example 1: Generation of SHC variants

[0292] Three different approaches were used to generate SHC variants: rational mutagenesis (site-directed mutagenesis), semi-rational mutagenesis (by site-saturation library), and random mutagenesis (error-prone PCR). Variants were expressed in heterologous systems and screened by GC.

[0293] Homology modeling. Homology modeling was used to build the three-dimensional structure of GmSHC. The templates used were crystal 1GSZ (Lenhart et al. (2002) Chem. Biol. 9:639-45) and 3SQC (Wendt et al. (1999) J. Mol. Biol. 286:175-87), which share 44% and 43% sequence identity with 95% of the GmSHC sequence.

[0294] Molecular docking. The ground state representation of HFA was then docked to the active center of the GmSHC structure. This was achieved by defining a three-dimensional grid box centered on the protonated oxygen atom of the first proton donor. The grid box defines the active center pocket region where substrate conformations will be sampled during the molecular docking run. Molecular docking was then performed using the Lamarckian genetic algorithm (LGA; Morris et al. (1998) J. Comput. Chem. 19:1639-62; Morris et al. (2009) J. Comput. Chem. 30:2785-91). A total of 1000 LGA runs were performed for each system. The population was 300, GA elitism = 1, the maximum number of generations was 27,000, and the maximum number of energy evaluations was 2,500,000. Therefore, for each LGA run, the first generation started with a population of 300 random substrate conformations. The best substrate conformation in the current population automatically survives to the next generation (GA elite = 1). Therefore, the next generation population starts with the most suitable substrate conformation of the previous generation plus another 299 conformations. When the maximum number of generations or energy evaluations is reached, the LGA run stops. For each LGA run, a substrate conformation is obtained. The substrate conformations are then classified according to energy and root mean square deviation. The highest ranked structure corresponds to the lowest binding energy structure with the lowest average binding energy of the densest population.

[0295] SHC structural analysis and catalytic mechanism. SHC is an intact single-sided membrane protein with a dimer 3D arrangement. Each monomer is characterized by eight QW motifs (Sato et al. (1998) Biosci. Biotechnol. Biochem. [Bioscience, Biotechnology and Biochemistry] 62: 407-11), which tightly connect many α-helices to form two highly stable α / α-barrel domains (Wendt et al. (1999) J. Mol. Biol. [Journal of Molecular Biology] 286: 175-87). The active center cavity is buried in the two α / α-barrel domains and can be accessed through an internal hydrophobic channel. For AaSHC, the channel and the active center cavity are separated by a narrow constriction composed of residues F166, V174, F434 and C435, which is responsible for substrate recognition (Lenhart et al. (2002) Chem. Biol. [Chemical Biology] 9: 639-45). For GmSHC, these residues correspond to F176, M184, F457, and C458. Unless otherwise indicated, the positions of amino acid residues provided for GmSHC refer to SEQ ID NO:2. Residues that form the conserved DXDD motif are observed at the top of the active center cavity (Wendt et al. (1999) J. Mol. Biol. 286:175-87). One of those residues is the first proton donor D396, which initiates cyclization by donating a proton to the double bonds 2 and 3 (Scheme 2). In GmSHC, the oxygen atom of D396 is 2.5 Å away from the carbon 3 of the double bond 2,3.

[0296]

[0297] The DXDD motif is followed by tryptophan and phenylalanine residues, which are responsible for stabilizing the cationic intermediate through strong cation-π interactions (Dougherty (1996) Science 271:163-168). At the bottom of the cavity is a glutamate residue, the final proton acceptor, which can receive a proton from the hydroxyl group and lead to the closure of the third ring and the formation of the product sclareolide. Structural analysis of GmSHC suggests that this enzyme has two possible final proton acceptors. However, based on the docking results, E386 of GmSHC is most likely the final proton acceptor. The distance between the sclareolide hydroxyl oxygen and E386 is only Apparently, this arrangement of the final proton acceptor plays an important role in the catalytic efficacy of this enzyme.

[0298] Using molecular modeling, the GmSHC residues and other key catalytic residues that establish the cation-π interactions responsible for stabilizing the cationic intermediate were identified. Notably, most of the important catalytic residues are conserved for other RpSHC enzymes (WO 2010 / 139719). The main differences include: (a) the GmSHC active center is residue 45; (b) GmSHC residue 184 together with residues 176, 457 and 458 is responsible for the narrow constriction between the hydrophobic channel and the active center cavity, which is related to substrate selectivity; and (c) the pattern of the QW motif is somewhat different.

[0299] Structural hot spots. Based on molecular modeling and molecular docking results, the following active center structural hot spots were identified that, when mutated, could improve the chemical steps catalyzed by the enzyme: residues V45, E46, Q54, F176, M184, F457, C458, W179, I278, Q279, T326, F385, E386, D397, F443, F460, F624, F654, and L656. Position V222 was also identified.

[0300] Specificity determining positions and conserved residues. Specificity determining positions indicate which residues have co-evolved within a subset of proteins of a family sharing a given catalytic specificity. It thus allows tracking evolutionary processes associated with the acquisition of multiple biological functions within the same protein family. Specificity determining positions were calculated from a multiple sequence alignment containing 1000 homologous sequences using the Xdet algorithm.

[0301] Evolution of GmSHC. To improve the catalytic conversion of HFA to sclareolide, GmSHC was modified to (1) improve the Michaelis-Menten complex; (2) introduce mutations that increase the cation-π stability of the carbocationic intermediate based on structure and coevolutionary hotspots; (3) open the catalytic cavity by mutating residues that are critical only for catalysis of the 5-ring natural substrate squalene; (4) mutate residues that assist the final proton acceptor to promote product formation; (5) alter the active center; (6) mutate residues responsible for the narrow constriction between the hydrophobic channel and the active center cavity; and (7) add a QW motif.

[0302] Using molecular docking, GmSHC variants designed to improve the Michaelis-Menten complex, increase the cation-π stability of the carbocation intermediate, open the catalytic cavity by mutating residues that are critical only for catalysis of the 5-ring natural substrate squalene, mutate residues that assist the final proton acceptor to promote product formation, and alter the active center were tested in silico. The results of this analysis are presented in Table 2.

[0303] Table 2. GmSHC modified to improve the Michaelis-Menten complex

[0304]

[0305]

[0306] Table 3 lists additional mutations for each of the above modifications.

[0307] Table 3. Additional GmSHCs modified to improve the Michaelis-Menten complex

[0308]

[0309]

[0310]

[0311]

[0312]

[0313] *From literature.

[0314] SHC variant enzyme expression.The wild type and GmSHC variants of Table 5 are cloned into pET28a (+) one by one.These DNA constructs are transformed into Lemo21 (DE3) Escherichia coli and plated on agar plates containing kanamycin.These are incubated overnight at 37°C.Pick a single bacterial colony for inoculating 10mL LB+ kanamycin in a 50mL Falcon tube.The primary culture is incubated overnight with stirring at 37°C.These cultures (10mL) are used to inoculate 1L LB+ kanamycin in a shake flask, and then incubated for about 4 hours at 37°C, 180rpm.Then 0.4mM IPTG and 0.2mM L-rhamnose are added to induce protein expression.The incubator temperature is reduced to 25°C, and the culture is further incubated overnight at 180rpm.The next day, the culture was centrifuged at 4000rpm for 10 minutes, and the supernatant was discarded. The cell pellets were resuspended in 0.1 M potassium phosphate buffer pH 7.4 and exposed to 1 cycle of freeze / thaw before lyophilization and then used in the reaction assays.

[0315] The cell pellet (1 μL) was spotted on a nitrocellulose membrane and air-dried for 30 minutes. The membrane was placed in 5% milk powder for 1 hour at room temperature with gentle stirring. The membrane was then rinsed 3×5 minutes with phosphate buffered saline (PBS). Anti-histidine antibody solution (1 / 10,000 dilution) was added and incubated at room temperature with shaking for 1 hour. The blot was then washed 3×5 minutes in PBS. A developing solution (6 mg diaminobenzidine (DAB) and 5 μL 30% H2O2 (in 10 mL PBS)) was added to the blot. Once developed, the developing solution was immediately removed and the blot was rinsed with water.

[0316] The results of this analysis showed that all constructs were expressed in E. coli at 25°C after the addition of 1 mM IPTG. Following enzyme expression and processing, dot blots were performed to assess whether the introduction of specific mutations altered protein expression. Notably, most GmSHC variants showed similar expression levels to the wild-type GmSHC construct.

[0317] SHC variant screening reaction. 0.2M, pH 5 sodium citrate buffer was prepared, and 500 μL of this buffer was added to the freeze-dried whole cells of each variant (from 1L shake flask fermentation of Escherichia coli transformed with the desired variant plasmid). These results were obtained when the enzyme loading was 50% (w / w). Subsequently, HFA (20 mg / mL) was added to the buffer / enzyme mixture. The reaction was incubated with stirring for 72 hours at 25°C. In order to stop the reaction and extract the product, 2× volumes of 3:2 heptane:isopropanol were added to each reaction. These were then incubated with stirring for 30 minutes at 37°C to fully mix. The reaction was centrifuged at 4000rpm for 10 minutes to precipitate any cell material. The upper organic layer was then taken out and put into a clean gas chromatography (GC) vial.

[0318] GC Analysis Method. A GC analysis method was used to detect each starting material and product used in the screening reactions. Due to the large amount of sample generated, a rapid method was developed with a run time of only 4.5 minutes. The GC analysis conditions are presented in Table 4.

[0319] Table 4. Starting materials and products used in screening reactions

[0320]

[0321]

[0322] Analysis of SHC variant reaction samples after addition of 10 mg / mL HFA with 100% enzyme loading showed that many SHC variants exhibited improved activity (based on peak area %) compared to the wild-type enzyme (Table 5). The wild-type enzyme did not show any conversion of HFA to sclareolide, so all fold changes were calculated based on the first mutant enzyme showing sclareolide formation, the V45L+Q54E+I278T+T326S variant. No metal ions were included in the reactions.

[0323] Table 5. SHC variants with improved sclareolide production

[0324] mutation Multiple improvements V45L+Q54E+I278T+T326S -0- V45L+Q54E+M184T+R249R+I278T+T326S 3.3 V45L+Q54E+V222Q+R249R+I278T+T326S 3.9 V45L+Q54E+R249R+I278T+T326S 4.2 V45L+Q54E+V222R+I278T+T326S 4.2 V45L+Q54E+V222K+I278T+T326S 5.4 V45L+Q54E+V222K+I278S+T326S 7.4

[0325] Further analysis was performed to identify one or more optimal SHC enzymes. Specifically, the substrate loading was increased to 20 mg / mL and incubated at a reduced enzyme loading (50%). As shown in Table 6, several variant enzymes exhibited higher activity than the wild-type and original mutant SHC enzymes.

[0326] Table 6. SHC variants with improved sclareolide production (50% enzyme loading).

[0327] mutation Multiple improvements V45L+Q54E+I278T+T326S 0 V45L+Q54E+R249R+I278T+T326S 7 V45L+Q54E+V222K+I278T+T326S 12 V45L+Q54E+V222K+I278A+T326S 20.4 V45L+Q54E+R249R+I278A+T326S 25.6 V45L+Q54E+V222R+I278T+T326S 27 V45L+Q54E+R249R+I278T+T326S+A574A 27 V45L+Q54E+V222K+I278S+T326S 28.4 V45L+Q54E+V222Q+R249R+I278T+T326S 30.3 V45L+Q54E+V222K+R249R+I278T+T326S 33.2 V45L+Q54E+Q178S+V222K+I278S+T326S 37.3 V45L+Q54E+Q178S+V222R+I278T+T326S 43.8 V45L+Q54E+V222Q+R249R+I278T+T326S+A683E 44.6 V45L+Q54E+V222Q+Y284W+R249R+I278T+T326S 46.2 V45L+Q54E+V222R+I278T+T326S+R348R 60

[0328] Example 2: Summary of GmSHC variants

[0329] SHC is an integral single-sided membrane protein that adopts a three-dimensional arrangement of dimers. Each monomer is characterized by a QW motif that tightly connects many α-helices to form two highly stable α / α-barrel domains (Wendt et al. (1999) J. Mol. Biol. [Journal of Molecular Biology] 286: 175-87). The active center cavity is buried within the two α / α-barrel domains and is accessible through an internal hydrophobic channel that is considered to be the membrane-immersed region of the enzyme (Lenhart et al. (2002) Chem. Biol. [Chemical Biology] 9: 639-45). The channel and the active center cavity are separated by a narrow constriction that is responsible for substrate recognition (Lenhart et al. (2002) Chem. Biol. [Chemical Biology] 9: 639-45). For GmSHC, those residues correspond to Phe176, Met184, Phe457, and Cys458. Residues constituting the conserved DXDD motif are found at the top of the active center cavity (Wendt et al. (1999) J. Mol. Biol. 286:175-87). One of those residues is the first proton donor, Asp396, which initiates the cyclization of homofarnesol by donating a proton to the double bond C2=C3. The DXDD motif is followed by tryptophan, tyrosine, and phenylalanine residues, which are responsible for stabilizing the cationic intermediate through strong cation-π interactions (Dougherty (1996) Science 271:163-8). At the bottom of the cavity is a negatively charged residue, the final proton acceptor, which receives a proton from the hydroxyl group, leading to the closure of the third ring and the formation of ambroxol.

[0330] To improve the conversion of HFA to sclareolide, GmSHC was mutated at one or more residues at positions 45, 54, 178, 184, 222, 278, 284, 326, 348, 574, and 683 of SEQ ID NO: 2. Silent mutations at positions 249, 348, and 574 identified in the error-prone library were also introduced into GmSHC of SEQ ID NO: 2.

[0331] Positions 45 and 326. According to the GmSHC homology model and molecular docking calculations, residues V45 and T326 are located near the substrate hydroxyl group. GmSHC position 45 is mutated to glutamine, leucine or isoleucine, and position 326 is mutated to serine to increase the intermolecular interaction with the substrate. In some cases, position 45 is mutated to leucine. In some cases, position 326 is mutated to serine.

[0332] Structural alignment between position 54 AaSHC (Reinhert et al. (2004) Chem. Biol. 11: 121-6) and the GmSHC homology model indicated that Q54 of GmSHC overlaps with residue E45 of AaSHC, which is the final proton acceptor of the enzyme (Dang & Prestwich (2000) Chem. Biol. 7: 643-9). Therefore, residue 54 of GmSHC was mutated to glutamic acid to incorporate the final proton acceptor at this position without negatively affecting the charge network associated with the conserved DXDD motif.

[0333] Position 178. Structural alignment between the homology model of GmSHC and the homologous human lanosterol synthase (Thoma et al. (2004) Nature 432: 118-22) indicated that Q178 of GmSHC overlaps with residue H232 of human lanosterol synthase, which is the final proton acceptor of the enzyme. Therefore, residue 178 of GmSHC was mutated to glutamic acid to incorporate the final proton acceptor at this position. Q178S was selected by site saturation mutagenesis to improve activity.

[0334] Position 184. Residue M184 is located in a narrow constriction responsible for substrate recognition (Lenhart et al. (2002) Chem. Biol. 9:639-45). Therefore, in order to alter substrate recognition, M184 of GmSHC was mutated to a nonpolar amino acid, i.e., Leu, Ile, Val and Ala. By mutating this position, any methionine oxidation phenomena, which could have a negative impact on substrate recognition, were also prevented. By site saturation mutagenesis, it was observed that the mutant M184T resulted in increased activity.

[0335] Position 278. According to the GmSHC homology model and molecular docking calculations, residue I278 is located just below the substrate hydroxyl group. When residue I278 is mutated to valine, molecular docking calculations indicate that the substrate arrangement within the active center is improved by positioning the substrate C2=C3 double bond closer to the first proton donor D396. Mutant I278A opens the catalytic cavity near the final proton acceptor. Mutant I278T interacts with the substrate and assists the final proton acceptor in the final product formation. When residue I278 is mutated to serine, molecular docking calculations indicate that it not only interacts with the final proton acceptor but also promotes the substrate arrangement within the active center, bringing it closer to the final proton donor.

[0336] Position 222. Each SHC monomer is characterized by a QW motif that tightly connects many α-helices to form two highly stable α / α-barrel domains (Wendt et al. (1999) J. Mol. Biol. 286: 175-87). The variant V222Q aims to establish a new QW motif together with W229, which further increases the structural stability of the enzyme. In a sequence alignment of 1,000 homologous enzymes of Gluconobacter mori SHC (GmSHC), the consensus residue at position 222 is arginine. Consensus residues, like V222R, are typically associated with higher structural stability (Steipe et al. (1994) J. Mol. Biol. 240 (3): 188-92). Therefore, mutating V222 to Q or R is expected to improve activity. It was subsequently discovered that K is in fact most beneficial at this position.

[0337] Position 249. R249R was identified in the error-prone library. This residue interacts with the cell membrane. This residue is located in the enzyme: membrane interface.

[0338] Position 284. Like F176, M184, F457 and C458, Y284 is also located in a narrow constriction that separates the channel from the active center pocket and is responsible for substrate recognition. Variant Y284W was observed to increase activity by site saturation mutagenesis, most likely by being able to increase the enzyme's specificity for HFA.

[0339] Position 348.R348R was identified in the error-prone library and is located in a flexible loop of the enzyme.

[0340] Position 574.A574A was identified in the error-prone library. This silent mutation is located in a flexible loop.

[0341] Position 683.A683E is located in the C-terminal domain of the enzyme and introduces a negatively charged residue in this region that has a positive impact on activity.

Claims

1. A variant squalene-hopaene cyclase (SHC) polypeptide having at least 60% sequence identity to SEQ ID NO:2 and comprising an amino acid substitution at one or more positions corresponding to position V45, Q54, Q178, M184, V222, R249, 1278, Y284, T326, R348, A574 or A683 relative to SEQ ID NO:2, wherein the variant exhibits increased sclareolide production compared to SEQ ID NO:

2.

2. The variant SHC of claim 1, wherein the amino acid substitution at the one or more positions is V45L, Q54E, Q178S, M184T, V222Q, V222R, V222K, R249R, I278T, I278S, I278A, Y284W, T326S, R348R, A574A, A683E, or any combination thereof.

3. The variant SHC of claim 1 or claim 2, wherein the variant SHC polypeptide comprises the following amino acid substitutions: (a) V45L, Q54E, I278T, T326S; (b)V45L, Q54E, M184T, R249R, I278T, T326S; (c)V45L, Q54E, V222Q, R249R, I278T, T326S; (d)V45L, Q54E, R249R, I278T, T326S; (e) V45L, Q54E, V222R, I278T, T326S; (f)V45L, Q54E, V222K, I278T, T326S; (g)V45L, Q54E, V222K, I278S, T326S; (h)V45L, Q54E, V222K, I278A, T326S; (i)V45L, Q54E, R249R, I278A, T326S; (j)V45L, Q54E, R249R, I278T, T326S, A574A; (k)V45L, Q54E, V222K, R249R, I278T, T326S; (l)V45L, Q54E, Q178S, V222K, I278S, T326S; (m)V45L, Q54E, Q178S, V222R, I278T, T326S; (n)V45L, Q54E, V222Q, R249R, I278T, T326S, A683E; (o)V45L, Q54E, V222Q, R249R, I278T, Y284W, T326S; (p)V45L, Q54E, V222R, I278T, T326S, R348R; (q) V45L, Q54E, V222K, R249R, I278A, T326S; or (r)V45L, Q54E, Q178S, V222K, I278T, T326S.

4. The variant SHC of any one of claims 1 to 3, wherein the variant SHC polypeptide has at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO:

25.

5. A nucleic acid molecule comprising a nucleic acid sequence encoding the variant SHC polypeptide of any one of claims 1 to 4.

6. The nucleic acid molecule of claim 5, comprising the following nucleic acid sequence: i) encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25; ii) has at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42; or iii) hybridizes under stringent conditions to a nucleic acid sequence having a sequence complementary to the sequence shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:

42.

7. A nucleic acid molecule as claimed in claim 5 or claim 6, comprising a heterologous regulatory sequence, optionally a promoter sequence.

8. An expression vector comprising a nucleic acid molecule encoding a variant SHC polypeptide according to any one of claims 1 to 4 or a nucleic acid molecule according to any one of claims 5 to 7.

9. A recombinant host cell comprising the nucleic acid molecule according to any one of claims 5 to 7 or the expression vector according to claim 8.

10. A method for producing sclareolide, comprising contacting homofarnesic acid with the variant SHC polypeptide of any one of claims 1-4 or the recombinant host cell of claim 9.

11. The method of claim 10, comprising collecting the sclareolide.

12. The method of claim 10 or claim 11, wherein the homofarnesic acid comprises (3E,7E) homofarnesic acid.

13. The method of any one of claims 10-12, comprising non-enzymatically converting the sclareolide to ambroxan.

Images