Patent Publication Number: US-11021724-B2

Title: Materials and methods for alkene reduction of levoglucosenone by an alkene reductase

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is the 35 U.S.C. § 371 national stage application of PCT Application No. PCT/US2018/025184, filed Mar. 29, 2018, which application claims the benefit of U.S. Provisional Application Ser. No. 62/478,430, filed Mar. 29, 2017, both of which are hereby incorporated by reference herein in their entireties, including any figures, tables, nucleic acid sequence, amino acid sequences, or drawings. 
    
    
     GOVERNMENT SUPPORT 
     This invention was made with government support under 1111791 awarded by the National Science Foundation. The government has certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Alkene reductase enzymes have become increasingly popular biocatalysts for converting prochiral substrates into optically pure building blocks [1-14]. Flavoproteins of the Old Yellow Enzyme superfamily are particularly useful for this purpose and a variety of homologs have been studied since the seminal work by Massey on  Saccharomyces pastorianus  OYE 1 [15,16]. These enzymes require no metal ions for catalytic activity and the reductions can be conducted under aqueous conditions. 
     Levoglucosenone (LGO) is a chiral platform chemical that can be produced from the catalytic fast pyrolysis of cellulose (see U.S. Publication No. 2012/0111714). LGO has the structure: 
                         
A variety of molecules can be synthesized from LGO, and from the alkene reduction product, 2,3-dihydro-levoglucosenone (2H-LGO), which is a molecule with a high added value (e.g., green solvent). 2,3-dihydro-levoglucosenone has value, for example, as a dipolar aprotic solvent.
 
                         
It is possible to reduce the double bond of LGO by a classic reduction using Pd/C under H 2  atmosphere; however, this process is not generally considered “green chemistry” since it requires organic solvents and the introduction of heavy metal ions in the reaction which must later be removed. Thus, there remains a need in the art for an enzymatic pathway to try to overcome this issue.
 
     BRIEF SUMMARY OF THE INVENTION 
     The subject invention concerns materials and methods for alkene reduction of compounds, such as levoglucosenone (LGO) and (S)-γ-hydroxymethyl-α,β-butenolide (HBO), using an alkene reductase enzyme. In one embodiment, a method of the invention comprises alkene reduction of a target compound by reacting the compound with an Old Yellow Enzyme (OYE) that reduces alkene bonds. In specific embodiments, LGO is contacted with an OYE to produce 2,3-dihydro-levoglucosenone (2H-LGO). In one embodiment, the OYE is OYE 2.6 from  Pichia sapitis  (GenBank Accession Nos. 3UPW_A and 3TJL_A) and comprises the amino acid sequence of SEQ ID NO:1. In a specific embodiment, the enzyme is an Old Yellow Enzyme (OYE) 2.6 mutant having an amino acid substitution at position 78 in the sequence, wherein the tyrosine at position 78 is substituted with a tryptophan amino acid (Y78W) and is designated as OYE 2.6 Y78W (SEQ ID NO:2). 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A-1 and 1A-2  show GC/MS spectrum of 2,3-dihydro-levoglucosenone/Quick extraction. 
         FIGS. 1B-1 and 1B-2  show GC/MS spectrum of 2,3-dihydro-levoglucosenone/Continuous extraction. 
         FIG. 2  shows  1 H NMR spectrum of 2,3-dihydro-levoglucosenone (2H-LGO). 
         FIG. 3  shows  13 C NMR spectrum of 2,3-dihydro-levoglucosenone (2H-LGO). 
         FIG. 4  shows  1 H NMR spectrum of control reaction with (S)-γ-hydroxymethyl-α,β-butenolide (HBO). 
         FIG. 5  shows  1 H NMR spectrum of crude reaction: (S)-γ-hydroxymethyl-α,β-butyrolactone (2H-HBO). 
     
    
    
     BRIEF DESCRIPTION OF THE SEQUENCES 
     SEQ ID NO:1 is an amino acid sequence of Old Yellow Enzyme 2.6. 
     SEQ ID NO:2 is an amino acid sequence of Old Yellow Enzyme 2.6 Y78W. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The subject invention concerns materials and methods for alkene reduction of compounds, such as levoglucosenone (LGO), using an alkene reductase enzyme. The methods of the invention can be used to cleanly and efficiently produce chemicals, drugs, herbicides, surfactants, etc. In one embodiment, a method of the invention comprises alkene reduction of a target compound by reacting the compound with an Old Yellow Enzyme (OYE), or an enzymatically active fragment or variant thereof, that reduces alkene bonds. In some embodiments, the OYE is from  Saccharomyces  (such as  S. pastorianus, S. cerevisiae , and  S. carlsbergensis ) or  Pichia . In specific embodiments of the methods, LGO is contacted with an OYE to produce 2,3-dihydro-levoglucosenone (2H-LGO). In a specific embodiment, the OYE is OYE 2.6 from  Pichia stipitis  (GenBank Accession Nos. 3UPW_A and 3TJL_A) and comprises the amino acid sequence of SEQ ID NO:1, or an enzymatically active fragment or variant thereof. In one embodiment, the enzyme is an OYE mutant having an amino acid substitution at position 78 in the sequence. In a more specific embodiment, the enzyme is an OYE 2.6 mutant having an amino acid substitution at position 78 in the sequence, wherein the tyrosine at position 78 is substituted with a tryptophan amino acid (Y78W) and is designated as OYE 2.6 Y78W (SEQ ID NO:2), or an enzymatically active fragment or variant thereof. 
     In one embodiment of the methods, levoglucosenone is contacted with an effective amount of OYE 2.6 Y78W in a suitable solvent, such as an aqueous solvent. The LGO can be dissolved in an alcohol, such as ethanol. In a specific embodiment, an LGO solution is added dropwise to the reaction mixture over the course of several hours. In an exemplified embodiment, the LGO solution is added dropwise to the reaction mixture over about six hours. In specific embodiments, the reaction mixture can comprise one or more of glucose, potassium phosphate, NADP +  and glucose dehydrogenase. The reaction can be allowed to proceed for a suitable period of time, e.g., several hours. Completion of the reaction can be performed by testing for the presence/amount of LGO remaining in the reaction mixture. The alkene reduction product can be extracted from the solution following reaction completion using, for example, ethyl acetate and evaporation of the organic phase. The methods of the invention can also be used for alkene reduction of other molecules, such as (S)-γ-hydroxymethyl-α,β-butenolide (HBO). 
     Methods of the present invention allow for the preparation of desired molecules through a safer (no organic solvent nor hydrogen needed), greener (no harmful reagents) and less toxic (no residual metal) synthetic process. This is particularly useful in the case of fine chemicals or end products used in the pharmaceutic, cosmetic and food/feed sectors. Moreover, compared to commonly used metal-catalyzed hydrogenation of levoglucosenone, the methods of the present invention provide better yield and do not require a purification step to remove a Pd catalyst. 
     Substitution of amino acids other than those specifically exemplified or naturally present in a wild type or mutant enzyme of the invention are also contemplated within the scope of the present invention. For example, non-natural amino acids can be substituted for the amino acids of a polypeptide, so long as the polypeptide having the substituted amino acids retains substantially the same biological or functional activity (e.g., enzymatic) as the polypeptide in which amino acids have not been substituted. Examples of non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid, ε-amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogues in general. Non-natural amino acids also include amino acids having derivatized side groups. Furthermore, any of the amino acids in the protein can be of the D (dextrorotary) form or L (levorotary) form. Allelic variants of a protein sequence of a wild type enzyme of the present invention are also encompassed within the scope of the invention. 
     Amino acids can be generally categorized in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an enzyme of the present invention having an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the polypeptide having the substitution still retains substantially the same biological or functional activity (e.g., enzymatic) as the polypeptide that does not have the substitution. Table 1 provides a listing of examples of amino acids belonging to each class. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Class of Amino Acid 
                 Examples of Amino Acids 
               
               
                   
                   
               
             
            
               
                   
                 Nonpolar 
                 Ala, Val, Leu, Ile, Pro, Met, Phe, Trp 
               
               
                   
                 Uncharged Polar 
                 Gly, Ser, Thr, Cys, Tyr, Asn, Gln 
               
               
                   
                 Acidic 
                 Asp, Glu 
               
               
                   
                 Basic 
                 Lys, Arg, His 
               
               
                   
                   
               
            
           
         
       
     
     Fragments and variants of an enzyme are contemplated within the scope of the present invention and can be generated and tested for the presence of enzymatic function using standard techniques known in the art. Thus, an ordinarily skilled artisan can readily prepare and test fragments and variants of an enzyme of the invention and determine whether the fragment or variant retains enzymatic activity relative to full-length or a non-variant enzyme. 
     Enzymes contemplated within the scope of the subject invention can also be defined in terms of more particular identity and/or similarity ranges with those sequences of the invention specifically exemplified herein or known in the art. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein. Unless otherwise specified, as used herein percent sequence identity and/or similarity of two sequences can be determined using the algorithm of Karlin and Altschul [21], modified as in Karlin and Altschul [22]. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. [19]. BLAST searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. [20]. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and)(BLAST) can be used. See NCBI/NIH website. 
     Single letter amino acid abbreviations are defined in Table 2. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Letter Symbol 
                 Amino Acid 
               
               
                   
                   
               
             
            
               
                   
                 A 
                 Alanine 
               
               
                   
                 B 
                 Asparagine or 
               
               
                   
                   
                 aspartic acid 
               
               
                   
                 C 
                 Cysteine 
               
               
                   
                 D 
                 Aspartic Acid 
               
               
                   
                 E 
                 Glutamic Acid 
               
               
                   
                 F 
                 Phenylalanine 
               
               
                   
                 G 
                 Glycine 
               
               
                   
                 H 
                 Histidine 
               
               
                   
                 I 
                 Isoleucine 
               
               
                   
                 K 
                 Lysine 
               
               
                   
                 L 
                 Leucine 
               
               
                   
                 M 
                 Methionine 
               
               
                   
                 N 
                 Asparagine 
               
               
                   
                 P 
                 Proline 
               
               
                   
                 Q 
                 Glutamine 
               
               
                   
                 R 
                 Arginine 
               
               
                   
                 S 
                 Serine 
               
               
                   
                 T 
                 Threonine 
               
               
                   
                 V 
                 Valine 
               
               
                   
                 W 
                 Tryptophan 
               
               
                   
                 Y 
                 Tyrosine 
               
               
                   
                 Z 
                 Glutamine or 
               
               
                   
                   
                 glutamic acid 
               
               
                   
                   
               
            
           
         
       
     
     Materials and Methods 
     2,3-dihydro-levoglucosenone (2H-LGO). To a reaction mixture containing 200 mM glucose, 100 mM KP i , pH 7.5 and 0.3 mM nicotinamide adenine dinucleotide phosphate (NADP + ) in a final volume of 50 mL, was added 125 Units of glucose dehydrogenase (GDH, Codexis CDX-901) and 375 μg of purified Y78W Old Yellow Enzyme 2.6 (Y78W OYE 2.6). The solution was gently stirred at room temperature and 120 μL (1 mmol) of levoglucosenone (LGO) dissolved in 1.5 mL of ethanol was added dropwise over 6 hours, reaching a final concentration of 20 mM. Completion of the reaction was verified by removing 500 μL of the reaction mixture, adding 500 μL of ethyl acetate+0.01% methyl benzoate, and vortex mixing for 30 seconds. The organic phase was analyzed by GC-MS using a 0.25 mm×30 m DB-17 column. No GC peak corresponding to LGO was observed, indicating that the reaction was complete. The 50 mL reaction mixture was subjected to continuous extraction with 75 mL of ethyl acetate overnight, then the organic phase was evaporated under vacuum to give pure 2H-LGO with ˜99% conversion and in quantitative yield. 
     
       
         
         
             
             
         
       
     
     GC-MS: m/z 128 (2.6%), 100 (52%), 82 (40%), 70 (23%), 57 (37%), 54 (100%) (see  FIGS. 1A-1, 1A-2, 1B-1, and 1B-2 ). 
       1 H NMR (300 MHz, CDCl 3 ): δ 5.03 (s, 1H, H 6 ), 4.66 (m, 1H 5b ), 3.98 (m, 2H, H 5a,4 ), 2.58 (m, 1H, H 2a ), 2.28 (m, 2H, H 2b,3a ), 2.00 (m, 1H, H 3b ) (see  FIG. 2 ). 
       13 C NMR (100 MHz, CDCl 3 ): δ 200.2 (s, C 1 ), 101.4 (d, C 6 ), 73.0 (d, C 4 ), 67.4 (t, C 5 ), 31.0 (t, C 2 ), 29.8 (t, C 3 ) (see  FIG. 3 ). 
     (S)-γ-hydroxymethyl-α,β-butyrolactone (2H-HBO). To a reaction mixture containing 50 mM KP i , pH 7.5, and 10 mM NADPH, with a final volume of 1 mL, was added 75 μg of Y78W OYE 2.6 and 10 mM (1.20 μL, mmol) of (S)-γ-hydroxymethyl-α,β-butenolide (HBO). The reaction mixture was stirred at room temperature overnight, and then evaporated using a Speed-Vac. The resulting crude oil was then dissolved in D 2 O and analyzed by  1 H NMR. The absence of the peaks corresponding to the alkene bond suggest that the reaction went to completion. A control reaction with all components except Y78W OYE 2.6 gave no evidence of reduction by  1 H NMR. 
                         
Control reaction:  1 H NMR (300 MHz, CDCl 3 ): δ 7.69 (1H, H 3 ), 6.25 (1H, H 2 ) (see  FIG. 4 ).
 
Crude reaction:  1 H NMR (300 MHz, CDCl 3 ): δ no peak for either H 2  or H 3  (see  FIG. 5 ).
 
     All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. 
     Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. 
     Example 1 
       Pichia stipitis  OYE 2.6 was chosen for reducing levoglucosenone since the enzyme shows higher stability under process conditions than the  S. pastorianus  OYE 1 [17]. A variety of site-directed mutants of  P. stipitis  OYE 2.6 were created with the aim of altering its stereoselectivity [18]. One particular variant, Tyr 78 Trp (Y78W), showed especially interesting properties and its x-ray crystal structure was solved [18]. While the overall size of a Trp side-chain is larger than that of Tyr, the former&#39;s shape actually creates substrate binding volume in the active site. Because levoglucosenone is a relatively large substrate, we initially tested the Y78W mutant of  P. stipitis  OYE 2.6 for its reduction. When this proved successful, we also tested wild-type  P. stipitis  OYE 2.6 as a catalyst for levoglucosenone alkene reduction. The wild-type  P. stipites  OYE 2.6 also proved successful in reduction of levoglucosenone (data not shown). 
     It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto. 
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         U.S. Publication No. 2012/0111714 
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