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ENGINEERED ENONE REDUCTASE AND KETOREDUCTASE VARIANT ENZYMES

20230242946 · 2023-08-03

    Inventors

    Cpc classification

    International classification

    Abstract

    The present disclosure provides engineered enone reductase enzymes (EREDs), polypeptides having ERED activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing ERED enzymes are also provided. The present disclosure also provides engineered ketoreductase enzymes (KREDs), polypeptides having KRED activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing KRED enzymes are also provided. The present disclosure further provides compositions comprising the ERED and KRED enzymes and methods of using the engineered ERED and KRED enzymes. The present disclosure finds particular use in the production of pharmaceutical compounds.

    Claims

    1. An engineered enone reductase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 262, 10, 20, 162, 282, 294, 322, and/or 346, or a functional fragment thereof, wherein the polypeptide sequence of said engineered enone reductase comprises at least one substitution or substitution set and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NOs: 262, 10, 20, 162, 282, 294, 322, and/or 346.

    2. The engineered enone reductase of claim 1, wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10, and wherein the polypeptide sequence of said enone reductase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 32/127/250/261/297/384, 5, 10, 13, 18/103, 19/260/363/394, 30, 32/127/250/384, 44, 56, 92, 99, 100, 103, 103/154, 107, 109, 124, 149, 154, 156, 168, 169, 172, 183, 209, 250, 250/290/384, 250/309/384, 250/384, 279, 306, 307, 341, 359, 369, 394, 398, and 399, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 10.

    3. The engineered enone reductase of claim 1, wherein said polypeptide sequence of said engineered enone reductase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20, and wherein said polypeptide sequence of said engineered enone reductase comprises at least one substitution or substitution set at one or more positions selected from 32/44/103/107/124/127/150/250, 32/44/103/107/124/127/341/394, 32/44/107/124/127/150/183, 32/103/107/124, 32/103/107/124/127/150/250, 32/103/107/124/183/250, 32/103/107/124/209/250, 32/103/107/124/250/394, 32/103/109/154/168/183/341/398, 32/107/124/209/394, 32/124/127/250, 32/183, 32/183/341/399, 38, 40, 44/103/107/124/127, 44/103/107/124/127/183, 44/103/124/127/150/183/260, 44/103/124/250/394, 83, 103/107/124, 103/107/124/127/150, 103/107/124/209/394, 103/107/124/250, 114, 118, 124/150, 148, and 261, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 20.

    4. The engineered enone reductase of claim 1, wherein said polypeptide sequence of said engineered enone reductase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 162, and wherein said polypeptide sequence of said engineered enone reductase comprises at least one substitution or substitution set at one or more positions selected from 4, 7, 7/307, 56/378, 95, 100, 109, 127, 146/333, 161, 209, 209/378, 258, 297, 298, 299, 302, 306, 307, 333, 336, 341, 359, and 378, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 162.

    5. The engineered enone reductase of claim 1, wherein said polypeptide sequence of said engineered enone reductase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 262, and wherein said polypeptide sequence of said engineered enone reductase comprises at least one substitution or substitution set at one or more positions selected from 4/100/209/258/359/378, 4/151/307/378, 4/151/333, 4/151/359/378, 4/209/359, 4/209/359/378, 7/95/100, 95/100/326/333/378, 95/100/326/378, 95/258/378, 95/333, 100/146/151/258/359/378, 100/209/258/359, 100/378, 146/151/359/378, 209, 209/258, 209/298/359/378, 209/333/359, 258/378, 341, and 378, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 262.

    6. The engineered enone reductase of claim 1, wherein said polypeptide sequence of said engineered enone reductase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 282, and wherein said polypeptide sequence of said engineered enone reductase comprises at least one substitution or substitution set at one or more positions selected from 89, 243, and 283, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 282.

    7. The engineered enone reductase of claim 1, wherein said polypeptide sequence of said engineered enone reductase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 294, and wherein said polypeptide sequence of said engineered enone reductase comprises at least one substitution or substitution set at one or more positions selected from 47, 118, 148, 258/261, 314, 374/378, 377/378, and 378, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 294.

    8. The engineered enone reductase of claim 1, wherein said polypeptide sequence of said engineered enone reductase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 322, and wherein said polypeptide sequence of said engineered enone reductase comprises at least one substitution or substitution set at one or more positions selected from 47/89/95/148/258/261, 47/89/95/243/258/261/378, 47/89/258, 47/95, 89/95/243, 95/148, 95/148/243/258/261, 95/148/243/261, 95/148/258/261, 95/243, 100/243, 100/243/374, and 148/243, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 322.

    9. The engineered enone reductase of claim 1, wherein said polypeptide sequence of said engineered enone reductase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 346, and wherein said polypeptide sequence of said engineered enone reductase comprises at least one substitution or substitution set at one or more positions selected from 10, 11, 13, 20, 21, 29, 64, 99, 99/398, 108, 175, 235, 243, 320, 333, 388, 392, 393, and 397, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 346.

    10. An engineered ketoreductase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 432 and/or 476, or a functional fragment thereof, wherein the polypeptide sequence of said engineered ketoreductase comprises at least one substitution or substitution set and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NOs: 432 and/or 476.

    11. The engineered ketoreductase of claim 10, wherein said polypeptide sequence of said engineered ketoreductase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 432, and wherein the polypeptide sequence of said ketoreductase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 21/103, 2/72, 2/72/172, 21, 21/65/72/73/103, 21/65/72/131/147/181, 21/65/72/147, 21/65/103/152, 21/65/131/152, 21/65/147/152, 21/65/152, 21/72/73/103, 21/72/103, 21/72/103/131/152/226, 21/72/103/147, 21/72/103/147/152/181, 21/72/131/181/197, 21/72/152/181, 21/73/103, 21/73/131/147, 21/73/147, 21/73/181, 21/103, 21/103/147, 21/103/147/152, 21/147, and 26/173/221, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 432.

    12. The engineered ketoreductase of claim 10, wherein said polypeptide sequence of said engineered ketoreductase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 476, and wherein said polypeptide sequence of said engineered ketoreductase comprises at least one substitution or substitution set at one or more positions selected from 17, 43, 45, 54, 71, 96, 190, 194, 195, 198, 205, and 250, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 476.

    13. The engineered enone reductase of claim 1, wherein said engineered enone reductase comprises a variant engineered enone reductase set forth in SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, and/or 346.

    14. The engineered ketoreductase of claim 10, wherein said engineered ketoreductase comprises a variant engineered ketoreductase set forth in SEQ ID NOs: 432, and/or 476.

    15. The engineered enone reductase of claim 1, wherein said engineered enone reductase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered enone reductase variant set forth in the even numbered sequences of SEQ ID NOs: 12-430.

    16. The engineered ketoreductase of claim 10, wherein said engineered ketoreductase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered ketoreductase variant set forth in the even numbered sequences of SEQ ID NOs: 434-524.

    17. The engineered enone reductase of claim 1, wherein said engineered enone reductase comprises a polypeptide sequence set forth in at least one of the even numbered sequences of SEQ ID NOs: 12-430.

    18. The engineered ketoreductase of claim 10, wherein said engineered ketoreductase comprises a polypeptide sequence set forth in at least one of the even numbered sequences of SEQ ID NOs: 434-524.

    19. The engineered enone reductase of claim 1, wherein said engineered enone reductase comprises at least one improved property compared to a wild-type enone reductase or another engineered enone reductase.

    20. The engineered ketoreductase of claim 10, wherein said engineered ketoreductase comprises at least one improved property compared to a wild-type ketoreductase or another engineered ketoreductase.

    21. The engineered enone reductase of claim 19, wherein said improved property comprises improved activity on a substrate.

    22. The engineered ketoreductase of claim 20, wherein said improved property comprises improved activity on a substrate.

    23. The engineered enone reductase of claim 21, wherein said substrate comprises compound (1).

    24. The engineered ketoreductase of claim 22, wherein said substrate comprises compound (2).

    25. The engineered enone reductase of claim 1, wherein said engineered enone reductase is purified.

    26. The engineered ketoreductase of claim 10, wherein said engineered ketoreductase is purified.

    27. A polynucleotide sequence encoding at least one engineered enone reductase of claim 1.

    28. A polynucleotide sequence encoding at least one engineered enone reductase comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 9, 19, 161, 261, 281, 293, 321, and/or 345, or a functional fragment thereof.

    29. The polynucleotide sequence of claim 27, wherein said polynucleotide sequence is operably linked to a control sequence.

    30. The polynucleotide sequence of claim 27, wherein said polynucleotide sequence is codon optimized.

    31. The polynucleotide sequence of claim 27, wherein said polynucleotide sequence comprises a polynucleotide sequence set forth in the odd numbered sequences of SEQ ID NOs: 11-429.

    32. An expression vector comprising at least one polynucleotide sequence of claim 27.

    33. A host cell comprising at least one polynucleotide sequence of claim 27.

    34. A method of producing an engineered enone reductase in a host cell, comprising culturing the host cell of claim 33, under suitable conditions, such that at least one engineered enone reductase is produced.

    35. A polynucleotide sequence encoding at least one engineered ketoreductase of claim 10.

    36. A polynucleotide sequence encoding at least one engineered ketoreductase comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 432 and/or 476, or a functional fragment thereof.

    37. The polynucleotide sequence of claim 35, wherein said polynucleotide sequence is operably linked to a control sequence.

    38. The polynucleotide sequence of claim 35, wherein said polynucleotide sequence is codon optimized.

    39. The polynucleotide sequence of claim 35, wherein said polynucleotide sequence comprises a polynucleotide sequence set forth in the odd numbered sequences of SEQ ID NOs: 433-523.

    40. An expression vector comprising at least one polynucleotide sequence of claim 35.

    41. A host cell comprising at least one polynucleotide sequence of claim 35.

    42. A method of producing an engineered ketoreductase in a host cell, comprising culturing the host cell of claim 41, under suitable conditions, such that at least one engineered ketoreductase is produced.

    Description

    DETAILED DESCRIPTION OF THE INVENTION

    [0150] The present disclosure provides engineered enone reductase enzymes (EREDs), polypeptides having ERED activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing ERED enzymes are also provided. The present disclosure also provides engineered ketoreductase enzymes (KREDs), polypeptides having KRED activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing KRED enzymes are also provided. The present disclosure further provides compositions comprising the ERED and KRED enzymes and methods of using the engineered ERED and KRED enzymes. The present disclosure finds particular use in the production of pharmaceutical compounds.

    [0151] In some embodiments, the present invention provides enzymes suitable for reduction of certain enone compounds, as depicted in Scheme 1.

    ##STR00001##

    [0152] The ERED catalyzes the reduction of the enone substrate of compound (1) using NADPH as a cofactor, to the intermediate acid of compound (2). The intermediate compound (2) is then converted to the alcohol product of compound (3) by a KRED enzyme. The KRED enzyme also recycles oxidized NADP.sup.+ to NADPH, using the reversible conversion of isopropanol to acetone.

    [0153] Glucose dehydrogenase (GDH) can also be used to recycle NADP to NADPH, using the conversion glucose to gluconate, according to Scheme 2.

    ##STR00002##

    The coupled reaction of Schemes 1 and 2 are comprised of the ERED and KRED reactions depicted in Scheme 3 and Scheme 4, respectively.

    ##STR00003##

    [0154] Various stereoisomers of compound (2) and compound (3) are possible from the coupled reaction. The ERED half reaction (Scheme 3) may produce the R acid (undesired) or S acid (compound (2)). KRED enzymes also create chiral S and R alcohol products. In Examples 3-9 of the present disclosure, KRED P2-G03 (Codexis, Inc.) was used to select for the S product (>99% e.e.). Thus, the stereoselectivity of an ERED in the coupled reaction can be measured through the proportion of cis-3 or trans-3 in the final alcohol product, according the Scheme 5, below. This allowed selection for desired trans-3/S-2 selectivity during the ERED evolution to achieve the desired alcohol product of compound (3).

    ##STR00004##

    [0155] The coupled reaction may also lead to an undesired side reaction if the KRED acts directly on enone compound (1), rather than intermediate acid compound (2). This side reaction, depicted below in Scheme 6, produces the allylic alcohol of compound (4).

    ##STR00005##

    [0156] The present invention was developed in order to address the potential use of ERED and KRED enzymes to produce compound (3) with increased substrate conversion, reduced side reactivity, and increased stereoselectivity. In some embodiments, the present disclosure provides ERED and KRED enzymes that are useful in optimizing production of compound (2) and/or compound (3).

    Engineered ERED and KRED Polypeptides

    [0157] The present invention provides engineered ERED and KRED polypeptides, polynucleotides encoding the polypeptides, methods of preparing the polypeptides, and methods for using the polypeptides. Where the description relates to polypeptides, it is to be understood that it also describes the polynucleotides encoding the polypeptides. In some embodiments, the present invention provides engineered, non-naturally occurring ERED and KRED enzymes with improved properties as compared to wild-type ERED and KRED enzymes or other engineered enzymes or reference enzymes. Any suitable reaction conditions find use in the present invention. In some embodiments, methods are used to analyze the improved properties of the engineered polypeptides to carry out the ERED reaction. In some embodiments, methods are used to analyze the improved properties of the engineered polypeptides to carry out the KRED reaction. In some embodiments, the reaction conditions are modified with regard to concentrations or amounts of engineered EREDs, engineered KREDs, substrate(s), buffer(s), solvent(s), pH, conditions including temperature and reaction time, and/or conditions with the engineered ERED and/or KRED polypeptide immobilized on a solid support, as further described below and in the Examples.

    [0158] As will be appreciated by the skilled artisan, in some embodiments, one or a combination of residue differences above (Summary of Invention) that is selected can be kept constant (i.e., maintained) in the engineered ERED and/or KRED as a core feature, and additional residue differences at other residue positions incorporated into the sequence to generate additional engineered ERED and/or KRED polypeptides with improved properties. Accordingly, it is to be understood for any engineered ERED and/or KRED containing one or a subset of the residue differences above, the present invention contemplates other engineered ERED and/or KRED that comprise the one or subset of the residue differences, and additionally one or more residue differences at the other residue positions disclosed herein.

    [0159] As noted above, the engineered ERED and/or KRED polypeptides are also capable of converting substrates (e.g., compound (1) to compound (2) and compound (2) to compound (3)). In some embodiments, the engineered ERED and/or KRED polypeptide is capable of converting the substrate compounds to the product compound with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, or more activity relative to the activity of the reference polypeptide of SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, 346, 432, and/or 476.

    [0160] In some embodiments, the engineered ERED polypeptide capable of converting the substrate compounds to the product compounds with at least 2 fold the activity relative to SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, and/or 346, comprises an amino acid sequence selected from the even-numbered sequences in SEQ ID NOs: 12-430.

    [0161] In some embodiments, the engineered KRED polypeptide capable of converting the substrate compounds to the product compounds with at least 2 fold the activity relative to SEQ ID NOs: 432 and/or 476, comprises an amino acid sequence selected from the even-numbered sequences in SEQ ID NOs: 434-524.

    [0162] In some embodiments, the engineered ERED has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, and/or 346, increases expression of the engineered ERED activity in a bacterial host cell, particularly in E. coli.

    [0163] In some embodiments, the engineered KRED has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NOs: 432 and/or 476, increases expression of the engineered KRED activity in a bacterial host cell, particularly in E. coli.

    [0164] In some embodiments, the engineered ERED and/or KRED polypeptide with improved properties has an amino acid sequence comprising a sequence selected from the even-numbered sequences in the range of SEQ ID NOs: 12-430 and 434-524.

    [0165] In some embodiments, the engineered ERED, comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one of the even-numbered sequences in the range of SEQ ID NOs: 12-430, and the amino acid residue differences as compared to SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, and/or 346, present in any one of the even-numbered sequences in the range of SEQ ID NOs: 12-430, as provided in the Examples.

    [0166] In some embodiments, the engineered KRED, comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one of the even-numbered sequences in the range of SEQ ID NOs: 434-524, and the amino acid residue differences as compared to NOs: 432 and/or 476, present in any one of the even-numbered sequences in the range of SEQ ID NOs: 434-524, as provided in the Examples.

    [0167] In addition to the residue positions specified above, any of the engineered ERED and/or KRED polypeptides disclosed herein can further comprise other residue differences relative to SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, 346, 432, and/or 476, at other residue positions (i.e., residue positions other than those included herein). Residue differences at these other residue positions can provide for additional variations in the amino acid sequence without adversely affecting the ability of the polypeptide to carry out the conversion of substrate to product. Accordingly, in some embodiments, in addition to the amino acid residue differences present in any one of the engineered ERED and/or KRED polypeptides selected from the even-numbered sequences in the range of SEQ ID NOs: 12-430 and 434-524, the sequence can further comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, or 1-50 residue differences at other amino acid residue positions as compared to the SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, 346, 432, and/or 476. In some embodiments, the number of amino acid residue differences as compared to the reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45 or 50 residue positions. In some embodiments, the number of amino acid residue differences as compared to the reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 residue positions. The residue differences at these other positions can be conservative changes or non-conservative changes. In some embodiments, the residue differences can comprise conservative substitutions and non-conservative substitutions as compared to the ERED and/or KRED polypeptide of SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, 346, 432, and/or 476.

    [0168] In some embodiments, the present invention also provides engineered polypeptides that comprise a fragment of any of the engineered ERED and/or KRED polypeptides described herein that retains the functional activity and/or improved property of that engineered ERED and/or KRED. Accordingly, in some embodiments, the present invention provides a polypeptide fragment capable of converting substrate to product under suitable reaction conditions, wherein the fragment comprises at least about 90%, 95%, 96%, 97%, 98%, or 99% of a full-length amino acid sequence of an engineered ERED and/or KRED of the present invention, such as an exemplary engineered ERED and/or KRED polypeptide selected from the even-numbered sequences in the range of SEQ ID NOs: 12-430 and 434-524. In some embodiments, the engineered ERED and/or KRED can have an amino acid sequence comprising a deletion in any one of the ERED and/or KRED polypeptide sequences described herein, such as the exemplary engineered polypeptides of the even-numbered sequences in the range of SEQ ID NOs: 12-430 and 434-524.

    [0169] Thus, for each and every embodiment of the engineered ERED and/or KRED polypeptides of the invention, the amino acid sequence can comprise deletions of one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the ERED and/or KRED polypeptides, where the associated functional activity and/or improved properties of the engineered ERED and/or KRED described herein are maintained. In some embodiments, the deletions can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues. In some embodiments, the number of deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues. In some embodiments, the deletions can comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues.

    [0170] In some embodiments, the engineered ERED and/or KRED polypeptide described herein can have an amino acid sequence comprising an insertion as compared to any one of the engineered ERED and/or KRED polypeptides described herein, such as the exemplary engineered polypeptides of the even-numbered sequences in the range of SEQ ID NOs: 12-430 and 434-524. Thus, for each and every embodiment of the ERED and/or KRED polypeptides of the disclosure, the insertions can comprise one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, or 50 or more amino acids, where the associated functional activity and/or improved properties of the engineered ERED and/or KRED described herein is maintained. The insertions can be to amino or carboxy terminus, or internal portions of the t ERED and/or KRED polypeptide.

    [0171] In some embodiments, the engineered ERED and/or KRED herein can have an amino acid sequence comprising a sequence selected from the even-numbered sequences in the range of SEQ ID NOs: 12-430 and 434-524, and optionally one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions.

    [0172] In the above embodiments, the suitable reaction conditions for the engineered polypeptides are provided as described in the Examples herein.

    [0173] In some embodiments, the polypeptides of the present invention are fusion polypeptides in which the engineered polypeptides are fused to other polypeptides, such as, by way of example and not limitation, antibody tags (e.g., myc epitope), purification sequences (e.g., His tags for binding to metals), and cell localization signals (e.g., secretion signals). Thus, the engineered polypeptides described herein can be used with or without fusions to other polypeptides.

    [0174] It is to be understood that the polypeptides described herein are not restricted to the genetically encoded amino acids. In addition to the genetically encoded amino acids, the polypeptides described herein may be comprised, either in whole or in part, of naturally occurring and/or synthetic non-encoded amino acids. Certain commonly encountered non-encoded amino acids of which the polypeptides described herein may be comprised include, but are not limited to: the D-stereomers of the genetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine (Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf); 2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff); 4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf); 3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf); 2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf); 4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf); 3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf); 2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf); 4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf); 3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine (Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif); 4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef); 3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff); 3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla); pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine (1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla); benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla); homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp); pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine (aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso); N(w)-nitroarginine (nArg); homolysine (hLys); phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer); phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid (hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid (PA), azetidine-3-carboxylic acid (ACA); 1-aminocyclopentane-3-carboxylic acid; allylglycine (aGly); propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal); homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle); homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal); homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) and homoproline (hPro). Additional non-encoded amino acids of which the polypeptides described herein may be comprised will be apparent to those of skill in the art (See e.g., the various amino acids provided in Fasman, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, Fla., pp. 3-70 [1989], and the references cited therein, all of which are incorporated by reference). These amino acids may be in either the L- or D-configuration.

    [0175] Those of skill in the art will recognize that amino acids or residues bearing side chain protecting groups may also comprise the polypeptides described herein. Non-limiting examples of such protected amino acids, which in this case belong to the aromatic category, include (protecting groups listed in parentheses), but are not limited to: Arg(tos), Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester), Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

    [0176] Non-encoding amino acids that are conformationally constrained of which the polypeptides described herein may be composed include, but are not limited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine carboxylic acid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

    [0177] In some embodiments, the engineered polypeptides can be in various forms, for example, such as an isolated preparation, as a substantially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, and/or as cell extracts and/or lysates of such cells. The enzymes can be lyophilized, spray-dried, precipitated or be in the form of a crude paste, as further discussed below.

    [0178] In some embodiments, the engineered polypeptides can be in the form of a biocatalytic composition. In some embodiments, the biocatalytic composition comprises (a) a means for conversion of a ketone compound to a chiral alcohol via an acid intermediate by contact with an ERED polypeptide and a KRED polypeptide and (b) a suitable cofactor. In some embodiments, the biocatalytic composition comprises an ERED having activity on a enone substrate. In some embodiments, the biocatalytic composition comprises a KRED having activity on a ketone. In some further embodiments, the biocatalytic composition comprises an ERED and a KRED that catalyze a multistep reaction pathway in a single pot. In some embodiments, the biocatalytic composition comprises a NADPH cofactor.

    [0179] In some embodiments, additional reaction components or additional techniques are utilized to supplement the reaction conditions. In some embodiments, these include taking measures to stabilize or prevent inactivation of the enzyme, reduce product inhibition, shift reaction equilibrium to desired product formation.

    [0180] In some further embodiments, any of the above described process for the conversion of substrate compound to product compound can further comprise one or more steps selected from: extraction, isolation, purification, crystallization, filtration, and/or lyophilization of product compound(s). Methods, techniques, and protocols for extracting, isolating, purifying, and/or crystallizing the product(s) from biocatalytic reaction mixtures produced by the processes provided herein are known to the ordinary artisan and/or accessed through routine experimentation. Additionally, illustrative methods are provided in the Examples below.

    Engineered ERED and KRED Polynucleotides Encoding Engineered Polypeptides, Expression Vectors and Host Cells

    [0181] The present invention provides polynucleotides encoding the engineered enzyme polypeptides described herein. In some embodiments, the polynucleotides are operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. In some embodiments, expression constructs containing at least one heterologous polynucleotide encoding the engineered enzyme polypeptide(s) is introduced into appropriate host cells to express the corresponding enzyme polypeptide(s).

    [0182] As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode an engineered enzyme (e.g., ERED or KRED) polypeptide. Thus, the present invention provides methods and compositions for the production of each and every possible variation of enzyme polynucleotides that could be made that encode the enzyme polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., in the various Tables).

    [0183] In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria. Consequently, codon optimized polynucleotides encoding the engineered enzyme polypeptides contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full length coding region.

    [0184] In some embodiments, the enzyme polynucleotide encodes an engineered polypeptide having enzyme activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from the SEQ ID NOs provided herein, or the amino acid sequence of any variant (e.g., those provided in the Examples), and one or more residue differences as compared to the reference polynucleotide(s), or the amino acid sequence of any variant as disclosed in the Examples (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions). In some embodiments, the reference polypeptide sequence is selected from SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, 346, 432, and/or 476.

    [0185] In some embodiments, the polynucleotides are capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from any polynucleotide sequence provided herein, or a complement thereof, or a polynucleotide sequence encoding any of the variant enzyme polypeptides provided herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an enzyme polypeptide comprising an amino acid sequence that has one or more residue differences as compared to a reference sequence.

    [0186] In some embodiments, an isolated polynucleotide encoding any of the engineered enzyme polypeptides herein is manipulated in a variety of ways to facilitate expression of the enzyme polypeptide. In some embodiments, the polynucleotides encoding the enzyme polypeptides comprise expression vectors where one or more control sequences is present to regulate the expression of the enzyme polynucleotides and/or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art. In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, suitable promoters are selected based on the host cells selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25 [1983]). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992]).

    [0187] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et al., supra).

    [0188] In some embodiments, the control sequence is also a suitable leader sequence (i.e., a non-translated region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable leader sequence that is functional in the host cell of choice finds use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

    [0189] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA). Any suitable polyadenylation sequence which is functional in the host cell of choice finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (See e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990 [1995]).

    [0190] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway). In some embodiments, the 5′ end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5′ end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva, Microbiol. Rev., 57:109-137 [1993]). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.

    [0191] In some embodiments, the control sequence is also a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from any suitable source, including, but not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (See e.g., WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

    [0192] In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

    [0193] In another aspect, the present invention is directed to a recombinant expression vector comprising a polynucleotide encoding an engineered enzyme polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some embodiments, the various nucleic acid and control sequences described herein are joined together to produce recombinant expression vectors which include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the enzyme polypeptide at such sites. Alternatively, in some embodiments, the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In some embodiments involving the creation of the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

    [0194] The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the enzyme polynucleotide sequence. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

    [0195] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector is one in which, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon is utilized.

    [0196] In some embodiments, the expression vector contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.

    [0197] In another aspect, the present invention provides a host cell comprising at least one polynucleotide encoding at least one engineered enzyme polypeptide of the present invention, the polynucleotide(s) being operatively linked to one or more control sequences for expression of the engineered enzyme enzyme(s) in the host cell. Host cells suitable for use in expressing the polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E. coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (AfhuA) and BL21). Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, and or tetracycline resistance.

    [0198] In some embodiments, the expression vectors of the present invention contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. In some embodiments involving integration into the host cell genome, the vectors rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.

    [0199] In some alternative embodiments, the expression vectors contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements preferably contain a sufficient number of nucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

    [0200] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are P15A ori or the origins of replication of plasmids pBR322, pUC19, pACYC177 (which plasmid has the P15A ori), or pACYC184 permitting replication in E. coli, and pUB110, pE194, or pTA1060 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes it's functioning temperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433 [1978]).

    [0201] In some embodiments, more than one copy of a nucleic acid sequence of the present invention is inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

    [0202] Many of the expression vectors for use in the present invention are commercially available. Suitable commercial expression vectors include, but are not limited to the p3×FLAG™ expression vectors (Sigma-Aldrich Chemicals), which include a CMV promoter and hGH polyadenylation site for expression in mammalian host cells and a pBR322 origin of replication and ampicillin resistance markers for amplification in E. coli. Other suitable expression vectors include, but are not limited to pBluescriptII SK(-) and pBK-CMV (Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (See e.g., Lathe et al., Gene 57:193-201 [1987]).

    [0203] Thus, in some embodiments, a vector comprising a sequence encoding at least one variant ERED or KRED is transformed into a host cell in order to allow propagation of the vector and expression of the variant KRED(s) or ERED(s). In some embodiments, the variant EREDs or KREDs are post-translationally modified to remove the signal peptide and in some cases may be cleaved after secretion. In some embodiments, the transformed host cell described above is cultured in a suitable nutrient medium under conditions permitting the expression of the variant ERED(s) or KRED(s). Any suitable medium useful for culturing the host cells finds use in the present invention, including, but not limited to minimal or complex media containing appropriate supplements. In some embodiments, host cells are grown in HTP media. Suitable media are available from various commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection).

    [0204] In another aspect, the present invention provides host cells comprising a polynucleotide encoding an improved ERED or KRED polypeptide provided herein, the polynucleotide being operatively linked to one or more control sequences for expression of the ERED or KRED enzyme in the host cell. Host cells for use in expressing the ERED or KRED polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E. coli, Bacillus megaterium, Lactobacillus kefir, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Appropriate culture media and growth conditions for the above-described host cells are well known in the art.

    [0205] Polynucleotides for expression of the ERED or KRED may be introduced into cells by various methods known in the art. Techniques include among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells are known to those skilled in the art.

    [0206] In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, the fungal host cells are yeast cells and filamentous fungal cells. The filamentous fungal host cells of the present invention include all filamentous forms of the subdivision Eumycotina and Oomycota. Filamentous fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose and other complex polysaccharides. The filamentous fungal host cells of the present invention are morphologically distinct from yeast.

    [0207] In some embodiments of the present invention, the filamentous fungal host cells are of any suitable genus and species, including, but not limited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, and/or Volvariella, and/or teleomorphs, or anamorphs, and synonyms, basionyms, or taxonomic equivalents thereof.

    [0208] In some embodiments of the present invention, the host cell is a yeast cell, including but not limited to cells of Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the present invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

    [0209] In some embodiments of the invention, the host cell is an algal cell such as Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp. ATCC29409).

    [0210] In some other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include, but are not limited to Gram-positive, Gram-negative and Gram-variable bacterial cells. Any suitable bacterial organism finds use in the present invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia and Zymomonas. In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces, or Zymomonas. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments the bacterial host strain is an industrial strain. Numerous bacterial industrial strains are known and suitable in the present invention. In some embodiments of the present invention, the bacterial host cell is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A. rubi). In some embodiments of the present invention, the bacterial host cell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus, and A. ureafaciens). In some embodiments of the present invention, the bacterial host cell is a Bacillus species (e.g., B. thuringensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefaciens). In some embodiments, the host cell is an industrial Bacillus strain including but not limited to B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausii, B. stearothermophilus, or B. amyloliquefaciens. In some embodiments, the Bacillus host cells are B. subtilis, B. licheniformis, B. megaterium, B. stearothermophilus, and/or B. amyloliquefaciens. In some embodiments, the bacterial host cell is a Clostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, and C. beijerinckii). In some embodiments, the bacterial host cell is a Corynebacterium species (e.g., C. glutamicum and C. acetoacidophilum). In some embodiments the bacterial host cell is an Escherichia species (e.g., E. coli). In some embodiments, the host cell is Escherichia coli W3110. In some embodiments, the bacterial host cell is an Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, and E. terreus). In some embodiments, the bacterial host cell is a Pantoea species (e.g., P. citrea, and P. agglomerans). In some embodiments the bacterial host cell is a Pseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, and S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (e.g., Z. mobilis, and Z. lipolytica).

    [0211] Many prokaryotic and eukaryotic strains that find use in the present invention are readily available to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

    [0212] In some embodiments, host cells are genetically modified to have characteristics that improve protein secretion, protein stability and/or other properties desirable for expression and/or secretion of a protein. Genetic modification can be achieved by genetic engineering techniques and/or classical microbiological techniques (e.g., chemical or UV mutagenesis and subsequent selection). Indeed, in some embodiments, combinations of recombinant modification and classical selection techniques are used to produce the host cells. Using recombinant technology, nucleic acid molecules can be introduced, deleted, inhibited or modified, in a manner that results in increased yields of ERED(s) or KRED(s) within the host cell and/or in the culture medium. For example, knockout of Alp1 function results in a cell that is protease deficient, and knockout of pyr5 function results in a cell with a pyrimidine deficient phenotype. In one genetic engineering approach, homologous recombination is used to induce targeted gene modifications by specifically targeting a gene in vivo to suppress expression of the encoded protein. In alternative approaches, siRNA, antisense and/or ribozyme technology find use in inhibiting gene expression. A variety of methods are known in the art for reducing expression of protein in cells, including, but not limited to deletion of all or part of the gene encoding the protein and site-specific mutagenesis to disrupt expression or activity of the gene product. (See e.g., Chaveroche et al., Nucl. Acids Res., 28:22 e97 [2000]; Cho et al., Molec. Plant Microbe Interact., 19:7-15 [2006]; Maruyama and Kitamoto, Biotechnol Lett., 30:1811-1817 [2008]; Takahashi et al., Mol. Gen. Genom., 272: 344-352 [2004]; and You et al., Arch. Microbiol., 191:615-622 [2009], all of which are incorporated by reference herein). Random mutagenesis, followed by screening for desired mutations also finds use (See e.g., Combier et al., FEMS Microbiol. Lett., 220:141-8 [2003]; and Firon et al., Eukary. Cell 2:247-55 [2003], both of which are incorporated by reference).

    [0213] Introduction of a vector or DNA construct into a host cell can be accomplished using any suitable method known in the art, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, PEG-mediated transformation, electroporation, or other common techniques known in the art. In some embodiments, the Escherichia coli expression vector pCK100900i (See, U.S. Pat. No. 9,714,437, which is hereby incorporated by reference) finds use.

    [0214] In some embodiments, the engineered host cells (i.e., “recombinant host cells”) of the present invention are cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the ERED or KRED polynucleotide. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and are well-known to those skilled in the art. As noted, many standard references and texts are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archebacterial origin.

    [0215] In some embodiments, cells expressing the variant ERED or KRED polypeptides of the invention are grown under batch or continuous fermentations conditions. Classical “batch fermentation” is a closed system, wherein the compositions of the medium is set at the beginning of the fermentation and is not subject to artificial alternations during the fermentation. A variation of the batch system is a “fed-batch fermentation” which also finds use in the present invention. In this variation, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Batch and fed-batch fermentations are common and well known in the art. “Continuous fermentation” is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

    [0216] In some embodiments of the present invention, cell-free transcription/translation systems find use in producing variant ERED(s) or KRED (s). Several systems are commercially available and the methods are well-known to those skilled in the art.

    [0217] The present invention provides methods of making variant ERED and KRED polypeptides or biologically active fragments thereof. In some embodiments, the method comprises: providing a host cell transformed with a polynucleotide encoding an amino acid sequence that comprises at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, 346, 432, and/or 476, and comprising at least one mutation as provided herein; culturing the transformed host cell in a culture medium under conditions in which the host cell expresses the encoded variant ERED and/or KRED polypeptide; and optionally recovering or isolating the expressed variant ERED and/or KRED polypeptide, and/or recovering or isolating the culture medium containing the expressed variant ERED and/or KRED polypeptide. In some embodiments, the methods further provide optionally lysing the transformed host cells after expressing the encoded ERED and/or KRED polypeptide and optionally recovering and/or isolating the expressed variant ERED or KRED polypeptide from the cell lysate. The present invention further provides methods of making a variant ERED and/or KRED polypeptide comprising cultivating a host cell transformed with a variant ERED and/or KRED polypeptide under conditions suitable for the production of the variant ERED and/or KRED polypeptide and recovering the variant ERED and/or KRED polypeptide. Typically, recovery or isolation of the ERED and/or KRED polypeptide is from the host cell culture medium, the host cell or both, using protein recovery techniques that are well known in the art, including those described herein. In some embodiments, host cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including, but not limited to freeze-thaw cycling, sonication, mechanical disruption, and/or use of cell lysing agents, as well as many other suitable methods well known to those skilled in the art.

    [0218] Engineered ERED and/or KRED enzymes expressed in a host cell can be recovered from the cells and/or the culture medium using any one or more of the techniques known in the art for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography. Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coli, are commercially available under the trade name CelLytic B™ (Sigma-Aldrich). Thus, in some embodiments, the resulting polypeptide is recovered/isolated and optionally purified by any of a number of methods known in the art. For example, in some embodiments, the polypeptide is isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, chromatofocusing, and size exclusion), or precipitation. In some embodiments, protein refolding steps are used, as desired, in completing the configuration of the mature protein. In addition, in some embodiments, high performance liquid chromatography (HPLC) is employed in the final purification steps. For example, in some embodiments, methods known in the art, find use in the present invention (See e.g., Parry et al., Biochem. J., 353:117 [2001]; and Hong et al., Appl. Microbiol. Biotechnol., 73:1331 [2007], both of which are incorporated herein by reference). Indeed, any suitable purification methods known in the art find use in the present invention.

    [0219] Chromatographic techniques for isolation of the ERED and/or KRED polypeptide include, but are not limited to reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., are known to those skilled in the art.

    [0220] In some embodiments, affinity techniques find use in isolating the improved ERED and/or KRED enzymes. For affinity chromatography purification, any antibody which specifically binds the ERED and/or KRED polypeptide may be used. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., may be immunized by injection with the ERED and/or KRED. The ERED and/or KRED polypeptide may be attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette Guerin) and Corynebacterium parvum.

    [0221] In some embodiments, the ERED and/or KRED variants are prepared and used in the form of cells expressing the enzymes, as crude extracts, or as isolated or purified preparations. In some embodiments, the ERED and/or KRED variants are prepared as lyophilisates, in powder form (e.g., acetone powders), or prepared as enzyme solutions. In some embodiments, the ERED and/or KRED variants are in the form of substantially pure preparations.

    [0222] In some embodiments, the ERED and/or KRED polypeptides are attached to any suitable solid substrate. Solid substrates include but are not limited to a solid phase, surface, and/or membrane. Solid supports include, but are not limited to organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof. A solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica or metal, such as gold or platinum. The configuration of the substrate can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface. Surfaces can be planar, substantially planar, or non-planar. Solid supports can be porous or non-porous, and can have swelling or non-swelling characteristics. A solid support can be configured in the form of a well, depression, or other container, vessel, feature, or location. A plurality of supports can be configured on an array at various locations, addressable for robotic delivery of reagents, or by detection methods and/or instruments.

    [0223] In some embodiments, immunological methods are used to purify ERED and/or KRED variants. In one approach, antibody raised against a wild-type or variant ERED and/or KRED polypeptide (e.g., against a polypeptide comprising any of SEQ ID NOs: 10, 20, 162, 262, 282, 294, 322, 346, 432, and/or 476, and/or a variant thereof, and/or an immunogenic fragment thereof) using conventional methods is immobilized on beads, mixed with cell culture media under conditions in which the variant ERED and/or KRED is bound, and precipitated. In a related approach, immunochromatography finds use.

    [0224] In some embodiments, the variant EREDs and/or KREDs are expressed as a fusion protein including a non-enzyme portion. In some embodiments, the variant ERED and/or KRED sequence(s) is fused to a purification facilitating domain. As used herein, the term “purification facilitating domain” refers to a domain that mediates purification of the polypeptide to which it is fused. Suitable purification domains include, but are not limited to metal chelating peptides, histidine-tryptophan modules that allow purification on immobilized metals, a sequence which binds glutathione (e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitope derived from the influenza hemagglutinin protein; See e.g., Wilson et al., Cell 37:767 [1984]), maltose binding protein sequences, the FLAG epitope utilized in the FLAGS extension/affinity purification system (e.g., the system available from Immunex Corp), and the like. One expression vector contemplated for use in the compositions and methods described herein provides for expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography; See e.g., Porath et al., Prot. Exp. Purif., 3:263-281) [1992]) while the enterokinase cleavage site provides a means for separating the variant ERED or KRED polypeptide from the fusion protein. pGEX vectors (Promega) may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.

    [0225] Accordingly, in another aspect, the present invention provides methods of producing the engineered enzyme polypeptides, where the methods comprise culturing a host cell capable of expressing a polynucleotide encoding the engineered enzyme polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the methods further comprise the steps of isolating and/or purifying the enzyme polypeptides, as described herein.

    [0226] Appropriate culture media and growth conditions for host cells are well known in the art. It is contemplated that any suitable method for introducing polynucleotides for expression of the enzyme polypeptides into cells will find use in the present invention. Suitable techniques include, but are not limited to electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.

    Methods of Using the ERED and KRED Enzymes

    [0227] In some embodiments, ERED and KRED enzymes described herein find use in processes for conversion of one or more suitable substrates to a product.

    [0228] In another aspect, the engineered ERED or KRED polypeptides disclosed herein can be used in a process for the conversion of the substrate compound (1), or structural analogs thereof, to the intermediate of compound (2), or structural analogs thereof, to the product of compound (3) or the corresponding structural analog.

    [0229] In the embodiments provided herein and illustrated in the Examples, various ranges of suitable reaction conditions that can be used in the processes, include but are not limited to, substrate loading, co-substrate loading, pH, temperature, buffer, solvent system, polypeptide loading, and reaction time. Further suitable reaction conditions for carrying out the process for biocatalytic conversion of substrate compounds to product compounds using an engineered ERED or KRED described herein can be readily optimized in view of the guidance provided herein by routine experimentation that includes, but is not limited to, contacting the engineered ERED or KRED polypeptide and one or more substrate compounds under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the product compound.

    [0230] The substrate compound(s) in the reaction mixtures can be varied, taking into consideration, for example, the desired amount of product compound, the effect of each substrate concentration on enzyme activity, stability of enzyme under reaction conditions, and the percent conversion of each substrate to product. In some embodiments, the suitable reaction conditions comprise a substrate compound loading for each of one of more substrates of at least about 0.5 to about 25 g/L, 1 to about 25 g/L, 5 to about 25 g/L, about 10 to about 25 g/L, or 20 to about 25 g/L. In some embodiments, the suitable reaction conditions comprise a substrate compound loading for each of one of more substrates of at least about 0.5 g/L, at least about 1 g/L, at least about 5 g/L, at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, or at least about 30 g/L, 40 g/L, 50 g/L, or even greater.

    [0231] In carrying out the ERED or KRED mediated processes described herein, the engineered polypeptide may be added to the reaction mixture in the form of a purified enzyme, partially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, as cell extracts and/or lysates of such cells, and/or as an enzyme immobilized on a solid support. Whole cells transformed with gene(s) encoding the engineered ERED or KRED enzyme or cell extracts, lysates thereof, and isolated enzymes may be employed in a variety of different forms, including solid (e.g., lyophilized, spray-dried, and the like) or semisolid (e.g., a crude paste). The cell extracts or cell lysates may be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment or the like, followed by a desalting procedure prior to lyophilization (e.g., ultrafiltration, dialysis, etc.). Any of the enzyme preparations (including whole cell preparations) may be stabilized by crosslinking using known crosslinking agents, such as, for example, glutaraldehyde or immobilization to a solid phase (e.g., Eupergit C, and the like).

    [0232] The gene(s) encoding the engineered ERED or KRED polypeptides can be transformed into host cell separately or together into the same host cell. For example, in some embodiments one set of host cells can be transformed with gene(s) encoding one engineered ERED or KRED polypeptide and another set can be transformed with gene(s) encoding another ERED or KRED polypeptide. Both sets of transformed cells can be utilized together in the reaction mixture in the form of whole cells, or in the form of lysates or extracts derived therefrom. In other embodiments, a host cell can be transformed with gene(s) encoding multiple engineered ERED or KRED polypeptides. In some embodiments the engineered polypeptides can be expressed in the form of secreted polypeptides and the culture medium containing the secreted polypeptides can be used for the ERED or KRED reaction.

    [0233] In some embodiments, the improved activity and/or regioselectivity and/or stereoselectivity of the engineered ERED or KRED polypeptides disclosed herein provides for processes wherein higher percentage conversion can be achieved with lower concentrations of the engineered polypeptide. In some embodiments of the process, the suitable reaction conditions comprise an engineered polypeptide amount of about 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 20% (w/w), 30% (w/w), 40% (w/w), 50% (w/w), 75% (w/w), 100% (w/w) or more of substrate compound loading.

    [0234] In some embodiments, the engineered polypeptide is present at about 0.01 g/L to about 50 g/L; about 0.05 g/L to about 50 g/L; about 0.1 g/L to about 40 g/L; about 1 g/L to about 40 g/L; about 2 g/L to about 40 g/L; about 5 g/L to about 40 g/L; about 5 g/L to about 30 g/L; about 0.1 g/L to about 10 g/L; about 0.5 g/L to about 10 g/L; about 1 g/L to about 10 g/L; about 0.1 g/L to about 5 g/L; about 0.5 g/L to about 5 g/L; or about 0.1 g/L to about 2 g/L. In some embodiments, the ERED or KRED polypeptide is present at about 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.5 g/L, 1, 2 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L, 40 g/L, or 50 g/L.

    [0235] During the course of the reaction, the pH of the reaction mixture may change. The pH of the reaction mixture may be maintained at a desired pH or within a desired pH range. This may be done by the addition of an acid or a base, before and/or during the course of the reaction. Alternatively, the pH may be controlled by using a buffer. Accordingly, in some embodiments, the reaction condition comprises a buffer. Suitable buffers to maintain desired pH ranges are known in the art and include, by way of example and not limitation, borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine, and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), and the like. In some embodiments, the reaction conditions comprise water as a suitable solvent with no buffer present.

    [0236] In the embodiments of the process, the reaction conditions comprise a suitable pH. The desired pH or desired pH range can be maintained by use of an acid or base, an appropriate buffer, or a combination of buffering and acid or base addition. The pH of the reaction mixture can be controlled before and/or during the course of the reaction. In some embodiments, the suitable reaction conditions comprise a solution pH from about 4 to about 10, pH from about 5 to about 10, pH from about 5 to about 9, pH from about 6 to about 9, pH from about 6 to about 8. In some embodiments, the reaction conditions comprise a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

    [0237] In the embodiments of the processes herein, a suitable temperature is used for the reaction conditions, for example, taking into consideration the increase in reaction rate at higher temperatures, and the activity of the enzyme during the reaction time period. Accordingly, in some embodiments, the suitable reaction conditions comprise a temperature of about 10° C. to about 60° C., about 10° C. to about 55° C., about 15° C. to about 60° C., about 20° C. to about 60° C., about 20° C. to about 55° C., about 25° C. to about 55° C., or about 30° C. to about 50° C. In some embodiments, the suitable reaction conditions comprise a temperature of about 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., or 60° C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature throughout the course of the reaction. In some embodiments, the temperature during the enzymatic reaction can be adjusted over a temperature profile during the course of the reaction.

    [0238] In some embodiments, the processes of the invention are carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymeric solvents, and/or co-solvent systems, which generally comprise aqueous solvents, organic solvents and/or polymeric solvents. The aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. In some embodiments, the processes using the engineered ERED or KRED decarboxylase polypeptides can be carried out in an aqueous co-solvent system comprising an organic solvent (e.g., ethanol, isopropanol (IPA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, and the like), ionic or polar solvents (e.g., 1-ethyl-4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, and the like). In some embodiments, the co-solvent can be a polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or lower alcohol. The non-aqueous co-solvent component of an aqueous co-solvent system may be miscible with the aqueous component, providing a single liquid phase, or may be partly miscible or immiscible with the aqueous component, providing two liquid phases. Exemplary aqueous co-solvent systems can comprise water and one or more co-solvents selected from an organic solvent, polar solvent, and polyol solvent. In general, the co-solvent component of an aqueous co-solvent system is chosen such that it does not adversely inactivate the ERED or KRED decarboxylase enzyme under the reaction conditions. Appropriate co-solvent systems can be readily identified by measuring the enzymatic activity of the specified engineered ERED or KRED decarboxylase enzyme with a defined substrate of interest in the candidate solvent system, utilizing an enzyme activity assay, such as those described herein.

    [0239] In some embodiments of the process, the suitable reaction conditions comprise an aqueous co-solvent, where the co-solvent comprises DMSO at about 1% to about 50% (v/v), about 1 to about 40% (v/v), about 2% to about 40% (v/v), about 5% to about 30% (v/v), about 10% to about 30% (v/v), or about 10% to about 20% (v/v). In some embodiments of the process, the suitable reaction conditions can comprise an aqueous co-solvent comprising ethanol at about 1% (v/v), about 5% (v/v), about 10% (v/v), about 15% (v/v), about 20% (v/v), about 25% (v/v), about 30% (v/v), about 35% (v/v), about 40% (v/v), about 45% (v/v), or about 50% (v/v).

    [0240] In some embodiments, the reaction conditions comprise a surfactant for stabilizing or enhancing the reaction. Surfactants can comprise non-ionic, cationic, anionic and/or amphiphilic surfactants. Exemplary surfactants, include by way of example and not limitation, nonyl phenoxypolyethoxylethanol (NP40), TRITON™ X-100 polyethylene glycol tert-octylphenyl ether, polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodium oleylamidosulfate, polyoxyethylene-sorbitanmonostearate, hexadecyldimethylamine, etc. Any surfactant that may stabilize or enhance the reaction may be employed. The concentration of the surfactant to be employed in the reaction may be generally from 0.1 to 50 mg/ml, particularly from 1 to 20 mg/ml.

    [0241] In some embodiments, the reaction conditions include an antifoam agent, which aids in reducing or preventing formation of foam in the reaction solution, such as when the reaction solutions are mixed or sparged. Anti-foam agents include non-polar oils (e.g., minerals, silicones, etc.), polar oils (e.g., fatty acids, alkyl amines, alkyl amides, alkyl sulfates, etc.), and hydrophobic (e.g., treated silica, polypropylene, etc.), some of which also function as surfactants. Exemplary anti-foam agents include, Y-30® (Dow Corning), poly-glycol copolymers, oxy/ethoxylated alcohols, and polydimethylsiloxanes. In some embodiments, the anti-foam can be present at about 0.001% (v/v) to about 5% (v/v), about 0.01% (v/v) to about 5% (v/v), about 0.1% (v/v) to about 5% (v/v), or about 0.1% (v/v) to about 2% (v/v). In some embodiments, the anti-foam agent can be present at about 0.001% (v/v), about 0.01% (v/v), about 0.1% (v/v), about 0.5% (v/v), about 1% (v/v), about 2% (v/v), about 3% (v/v), about 4% (v/v), or about 5% (v/v) or more as desirable to promote the reaction.

    [0242] The quantities of reactants used in the ERED or KRED reaction will generally vary depending on the quantities of product desired, and concomitantly the amount of ERED or KRED substrate employed. Those having ordinary skill in the art will readily understand how to vary these quantities to tailor them to the desired level of productivity and scale of production.

    [0243] In some embodiments, the order of addition of reactants is not critical. The reactants may be added together at the same time to a solvent (e.g., monophasic solvent, biphasic aqueous co-solvent system, and the like), or alternatively, some of the reactants may be added separately, and some together at different time points. For example, the cofactor, co-substrate and substrate may be added first to the solvent.

    [0244] The solid reactants (e.g., enzyme, salts, etc.) may be provided to the reaction in a variety of different forms, including powder (e.g., lyophilized, spray dried, and the like), solution, emulsion, suspension, and the like. The reactants can be readily lyophilized or spray dried using methods and equipment that are known to those having ordinary skill in the art. For example, the protein solution can be frozen at −80° C. in small aliquots, then added to a pre-chilled lyophilization chamber, followed by the application of a vacuum.

    [0245] For improved mixing efficiency when an aqueous co-solvent system is used, the ERED or KRED, and co-substrate may be added and mixed into the aqueous phase first. The ERED or KRED substrate may be added and mixed in, followed by the organic phase or the substrate may be dissolved in the organic phase and mixed in. Alternatively, the ERED or KRED may be premixed in the organic phase, prior to addition to the aqueous phase.

    [0246] The processes of the present invention are generally allowed to proceed until further conversion of substrate to product does not change significantly with reaction time (e.g., less than 10% of substrate being converted, or less than 5% of substrate being converted). In some embodiments, the reaction is allowed to proceed until there is complete or near complete conversion of substrate to product. Transformation of substrate to product can be monitored using known methods by detecting substrate and/or product, with or without derivatization. Suitable analytical methods include gas chromatography, HPLC, MS, and the like.

    [0247] In some embodiments of the process, the suitable reaction conditions comprise a substrate loading for each of one or more substrates of at least about 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L or more, and wherein the method results in at least about 50%, 60%, 70%, 80%, 90%, 95% or greater conversion of substrate compound to product compound in about in about 24 h or less, in about 12 h or less, in about 6 h or less, or in about 4 h or less.

    [0248] The engineered ERED or KRED polypeptides of the present invention when used in the process under suitable reaction conditions result in an excess of the desired product in at least 30%, 40%, 50%, 60%, or greater enantiomeric excess over undesired product(s).

    [0249] In some further embodiments of the processes for converting one or more substrate compounds to product compound using the ERED or KRED polypeptides, the suitable reaction conditions can comprise an initial substrate loading for each of one or more substrates to the reaction solution which is then contacted by the polypeptide. This reaction solution is then further supplemented with additional substrate compound as a continuous or batchwise addition over time at a rate of at least about 1 g/L/h, at least about 2 g/L/h, at least about 4 g/L/h, at least about 6 g/L/h, or higher for each of one or more substrate compounds. Thus, according to these suitable reaction conditions, polypeptide is added to a solution having an initial substrate loading of at least about 1 g/L, 5 g/L, or 10 g/L for each of one or more substrate compounds. This addition of polypeptide is then followed by continuous addition of further substrate to the solution at a rate of about 2 g/L/h, 4 g/L/h, or 6 g/L/h for each of one or more substrate compounds until a much higher final substrate loading of at least about 30 g/L or more for each of one or more substrate compounds, is reached. Accordingly, in some embodiments of the process, the suitable reaction conditions comprise addition of the polypeptide to a solution having an initial substrate loading of at least about 1 g/L, 5 g/L, or 10 g/L followed by addition of further substrate to the solution at a rate of about 2 g/L/h, 4 g/L/h, or 6 g/L/h until a final substrate loading of at least about 30 g/L, or more, is reached for each of one or more substrate compounds. This substrate supplementation reaction condition allows for higher substrate loadings to be achieved while maintaining high rates of conversion of substrate to product of at least about 5%, 25%, 50%, 75%, 90% or greater conversion of substrate for either or both of one or more substrate compounds.

    [0250] Any of the processes disclosed herein using the engineered polypeptides for the preparation of compound (3) and/or compound (2) can be carried out under a range of suitable reaction conditions, including but not limited to ranges of ketone substrates, temperature, pH, solvent system, substrate loading, polypeptide loading, cofactor loading, and reaction time. In one example, in some embodiments, the preparation of compound (3) and/or compound (2) can be carried out wherein the suitable reaction conditions comprise: (a) enone substrate compound (1) loading of about 2 g/L to 40 g/L; (b) 1-5 mm MgCl.sub.2 (c) of about 0.1 g/L to 100 g/L of each engineered polypeptide; (d) 0.01M to 1M 70% v/v potassium phosphate buffer with 30% v/v isopropanol; (e) 0.1-2.0 g/L NADPH; (f) pH at 5-8; and (g) temperature of about 20° C. to 60° C. In some embodiments, the suitable reaction conditions comprise: (a) about 5 g/L compound (1) substrate compound); (b) about 2 mM MgCl.sub.2; (c) about 15 g/L of each engineered polypeptide; (d) 70% v/v 140 mm potassium phosphate buffer with 30% v/v isopropanol; (e) 0.5 g/L of NADPH; (f) pH at 6, and (g) about 30° C.

    [0251] In some embodiments, additional reaction components or additional techniques carried out to supplement the reaction conditions. These can include taking measures to stabilize or prevent inactivation of the enzyme, reduce product inhibition, shift reaction equilibrium to formation of the desired product.

    [0252] In further embodiments, any of the above described process for the conversion of one or more substrate compounds to product compound can further comprise one or more steps selected from: extraction; isolation; purification; and crystallization of product compound. Methods, techniques, and protocols for extracting, isolating, purifying, and/or crystallizing the product from biocatalytic reaction mixtures produced by the above disclosed processes are known to the ordinary artisan and/or accessed through routine experimentation. Additionally, illustrative methods are provided in the Examples below.

    [0253] Various features and embodiments of the present invention are illustrated in the following representative examples, which are intended to be illustrative, and not limiting.

    EXPERIMENTAL

    [0254] The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention. Indeed, there are various suitable sources for many of the reagents and equipment described below. It is not intended that the present invention be limited to any particular source for any reagent or equipment item.

    [0255] In the experimental disclosure below, the following abbreviations apply: M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and l (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); um and μ.Math.η (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); psi and PSI (pounds per square inch); ° C. (degrees Centigrade); RT and rt (room temperature); CV (coefficient of variability); CAM and cam (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropyl β-D-1-thiogalactopyranoside); LB (lysogeny broth); TB (terrific broth); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); nt (nucleotide; polynucleotide); aa (amino acid; polypeptide); E. coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, Conn.); HTP (high throughput); HPLC (high pressure liquid chromatography); HPLC-UV (HPLC-Ultraviolet Visible Detector); 1H NMR (proton nuclear magnetic resonance spectroscopy); FIOPC (fold improvements over positive control); Sigma and Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.; Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, Mich.); Microfluidics (Microfluidics, Westwood, Mass.); Life Technologies (Life Technologies, a part of Fisher Scientific, Waltham, Mass.); Amresco (Amresco, LLC, Solon, Ohio); Carbosynth (Carbosynth, Ltd., Berkshire, UK); Varian (Varian Medical Systems, Palo Alto, Calif.); Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); Infors (Infors USA Inc., Annapolis Junction, Md.); and Thermotron (Thermotron, Inc., Holland, Mich.).

    Example 1

    Production of Engineered Polypeptides in pCK110900

    [0256] The polynucleotide (SEQ ID NO: 9) encoding the polypeptide having ene reductase activity (SEQ ID NO:10), was cloned into the pCK110900 vector system (See e.g., U.S. Pat. No. 9,714,437, which is hereby incorporated by reference in its entirety) and subsequently expressed in E. coli W3110fhuA under the control of the lac promoter. This polynucleotide, and associated polypeptide, encodes a chimera derived from OYE1, OYEZ and OYE3 (SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5), as described in U.S. Pat. No. 8,329,438.

    [0257] The polynucleotide (SEQ ID NO: 7) encoding the polypeptide having ketoreductase activity (SEQ ID NO: 8), was cloned into the pCK110900 vector system (See e.g., U.S. Pat. No. 9,714,437, which is hereby incorporated by reference in its entirety) and subsequently expressed in E. coli W3110fhuA under the control of the lac promoter. This polynucleotide, and associated polypeptide, is a variant derived from the wildtype L. kefir.

    [0258] In a 96-well format, single colonies were picked and grown in 190 μL LB media containing 1% glucose and 30 μg/mL CAM at 30° C., 200 rpm, and 85% humidity. Following overnight growth, 20 μL of the grown cultures were transferred into a deep-well plate containing 380 μL of TB media with 30 μg/mL CAM, with 1 mM MgSO.sub.4 for KRED cultures. The cultures were grown at 30° C. at 250 rpm with 85% humidity for approximately 2.5 hours. When the optical density (OD.sub.600) of the cultures reached 0.4-0.6, expression of the ene reductase or ketoreductase gene was induced by the addition of IPTG to a final concentration of 1 mM. Following induction, growth continued for 18-20 hours at 30° C., 250 rpm, and 85% humidity. Cells were harvested by centrifugation at 4,000 rpm at 4° C. for 10 minutes; the supernatant was then discarded. The cell pellets were stored at −80° C. until ready for use.

    [0259] Prior to performing the assay, the cell pellets were thawed and resuspended in 200 μL of lysis buffer containing 1 g/L lysozyme, 0.5 g/L PMBS and 0.025 μL/mL of commercial DNAse (New England BioLabs, M0303L) in 0.1 M potassium phosphate buffer at pH 6.0 or 0.2 M sodium phosphate buffer pH 7.0. The plates were agitated with medium-speed shaking for 2 hours on a microtiter plate shaker at room temperature. The plates were then centrifuged at 4,000 rpm for 10 minutes at 4° C., and the clarified supernatants were used in the HTP assay reaction described in the following examples.

    [0260] Shake-flask procedures can be used to generate engineered ene reductase or ketoreductase shake-flask powders (SFP), which are useful for secondary screening assays and/or use in the biocatalytic processes described herein. Shake flask powder preparation of enzymes provides a more purified preparation (e.g., up to 30% of total protein) of the engineered enzyme, as compared to the cell lysate used in HTP assays and also allows for the use of more concentrated enzyme solutions. To start the culture, 10-uL aliquot of a glycerol stock of E. coli containing a plasmid encoding an engineered polypeptide of interest was inoculated into 8 mL of LB cell culture media with 30 μg/mL CAM and 1% glucose. The culture was grown overnight (at least 16 hours) in an incubator at 30° C. with shaking at 250 rpm. The grown culture was then added to 250 mL of TB media with 30 μg/mL CAM in a 1 L shake-flask. The 250-mL culture was grown at 30° C. and 250 rpm for 3.5 hours until OD.sub.600 reached 0.6-0.8. Expression of the ene reductase or keto reductase gene was induced by the addition of IPTG to a final concentration of 1 mM, and growth was continued for an additional 18-20 hours. Cells were harvested by transferring the culture into a pre-weighed centrifuge bottle which was then centrifuged at 4,000 rpm for 10 minutes at 4° C. The supernatant was discarded, and the remaining cell pellet was weighed. In some embodiments, the cell pellet was stored at −80° C. until ready to use. For lysis, the cell pellet was resuspended in 6 mL/g wet cell weight of 10 mM sodium phosphate or potassium buffer at pH 6.0, or pH 7.0 with 2 mM MgSO.sub.4 added for the KRED lysis, and lysed using a 110L MICROFLUIDIZER® processor system (Microfluidics). Cell debris was removed by centrifugation at 10,000 rpm for 60 minutes at 4° C. The clarified lysate was collected, frozen at −80° C., and then lyophilized, using standard methods known in the art. Lyophilization of frozen clarified lysate provides a dry shake-flask powder comprising crude engineered polypeptide.

    Example 2

    Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 10 for Improved Production of Compound (3)

    [0261] The engineered polynucleotide (SEQ ID NO: 9) encoding the polypeptide with ene reductase activity of SEQ ID NO: 10 was used to generate the engineered polypeptides of Table 2.2. These polypeptides displayed improved ene reductase activity under the desired conditions e.g., the improvement in the formation of the alcohol of compound (3) from the substrate enone of compound (1), in a coupled reaction with keto reductase (SEQ ID NO: 8), as compared to the starting polypeptide. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 10, as described below.

    [0262] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 9. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods that measured the polypeptides' ability to produce compound (3).

    [0263] The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 50 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 5 g/L (1), 0.5 g/L NADPH, 15 g/L KRED (KRED P2-G03, Codexis, Inc.), and dissolved in a mixture of 70% (v/v) 140 mM potassium phosphate buffer at pH 6 with 2 mM magnesium chloride and 30% (v/v) isopropyl alcohol. The reaction plates were heat-sealed and shaken at 600 rpm at 30° C. for 18 hours.

    [0264] After overnight incubation (˜18 hours), 300 μL/well of acetonitrile were added to the reaction plates and mixed well. The plates were sealed and centrifuged at 4,000 rpm for 10 min. An aliquot of the supernatant was removed and further diluted 800-fold into water. After dilution, the reactions were analyzed on the Agilent RapidFire 365 high throughput mass spectrometer according the details in Table 2.1, below.

    TABLE-US-00001 TABLE 2.1 RapidFire SPE-MS/MS Conditions for 3-Hydroxyhexanoid Acid Detection Agilent RapidFire Conditions Pump1 Buffer Water LCMS grade; 0.6 mL/min flow rate Pump2 Buffer ACC Juice (50% water:25% ACN:25% Acetone) LCMS grade + 100 uM ammonium acetate; 1.25 mL/min flow rate Pump3 Buffer ACC Juice (50% water:25% ACN:25% Acetone) LCMS grade + l00 uM ammonium acetate; 1.25 mL/min flow rate Aqueous wash Water Organic wash Acetonitrile SPE cartridge Graphilic carbon type D RF state 1 Aspirate 600 ms RF state 2 Load/Wash 1750 ms RF state 3 Extra Wash 0 RF state 4 Elute 6000 ms RF state 5 Reequilibrate 1000 ms Agilent Jet Stream source parameters Drying gas temperature 300° C. Drying gas flow 9 L/min Nebulizer pressure 45 psi Sheath gas temperature 300° C. Sheath gas flow 12 L/min Capillary voltage −2500 V Nozzle voltage −2000 V Agilent 6470 Triple Quadrupole MRM parameters Compound Q1 Q3 Dwell Fragmentor CE CAV 3-HydroxyCycloHexenoid- 143 97.1 40 100 14 4 Acid (Quant ion) 3-HydroxyCycloHexenoid- 143 95.1 40 100 26 4 Acid 3-HydroxyCycloHexenoid- 143 69 40 100 45 4 Acid 3-HydroxyCycloHexenoid- 143 79.2 40 100 31 4 Acid 3-HydroxyCycloHexenoid- 143 58.9 40 100 27 4 Acid

    TABLE-US-00002 TABLE 2.2 ERED Activity for the Production of Compound (3) Relative to SEQ ID NO: 10 Percent Conversion Fold SEQ ID NO: Amino Acid Differences Improvement Relative (nt/aa) (Relative to SEQ ID NO: 10) to SEQ ID NO: 10 11/12 E19K/T260P/E363K/D394K +++ 13/14 V250T/T309S/T384I +++ 15/16 V250T/E290K/T384I +++ 17/18 V250T/T384I +++ 19/20 A32V/N127T/V250T/T384I +++ 21/22 K168A ++ 23/24 A183G + 25/26 R13G + 27/28 L103S +++ 29/30 A183E ++ 31/32 K399E ++ 33/34 N209R +++ 35/36 K399V + 37/38 K5S + 39/40 N398G + 41/42 Q109G ++ 43/44 L103R/K154G + 45/46 K399G + 47/48 L103G ++ 49/50 K99R + 51/52 K5Q + 53/54 K369E + 55/56 H30Y + 57/58 E307G ++ 59/60 N100R + 61/62 D107E +++ 63/64 S306V + 65/66 P341G ++ 67/68 Y359Q + 69/70 K168Q ++ 71/72 A183W + 73/74 N156G ++ 75/76 D92E + 77/78 Y359R ++ 79/80 D107A ++ 81/82 F18L/L103G ++ 83/84 K154R + 85/86 V56Q + 87/88 D394A + 89/90 H44Y ++ 91/92 K172R + 93/94 A279L + 95/96 A279Q + 97/98 A183F ++  99/100 F124S +++ 101/102 Q10L + 103/104 K369A + 105/106 K168G + 107/108 N398E + 109/110 E149F + 111/112 L103Q ++ 113/114 Q169V + 115/116 H44F + 117/118 L103A ++ 119/120 Y359V + 121/122 D107R ++ 123/124 L103R +++ 125/126 L103T +++ 127/128 D107N ++ 129/130 V56M + 131/132 D107Q ++ 133/134 V250R +++ Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 10 and defined as follows: “+” .88 to 1.21, “++” > 1.21, “+++” > 1.58

    [0265] Selected enzymes were regrown and retested under similar conditions with 2 equivalents of NADPH and without a cofactor recycling system to look for the presence of the undesired enantiomer of the intermediate. These samples were derivatized with TMS-diazomethane and analyzed by GC as their methyl esters according to the method in Table 2.3. >99% e.e. was observed in all samples tested.

    TABLE-US-00003 TABLE 2.3 GC Parameters Method for Determining Conversion and Stereoselectivity of ERED and KRED variants Instrument Agilent 6890 GC Column BGB-174, 30 m × 250 μm × 0.25 μm film thickness (p/n 27430-025) Carrier gas and flow rate Helium, 2.0 mL/min (constant flow control) Column temperature 125° C. hold for 0.5 minutes, ramp to 150° C. at 20° C./min, hold at 150° C. for 3.9 min, ramp to 200° C. at 55.sup.o C./min, hold at 200° C. for 0.5 minutes Detector and temperature FID, 250° C. Inlet temperature and 250° C., 50:1 split ratio Injection volume 2 μL Compound retention times Trans alcohol (both enantiomers): 5.67 min (all retention times are for the (R)-intermediate: 5.81 min methyl esters following Cis alcohol (both enantiomers): 5.81 min derivatization with TMS- (S)-intermediate: 5.89 min diazomethane) Substrate: 6.18 Allylic Alcohols (unknown stereochemistry): 6.67, 6.72 min

    Example 3

    Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 20 for Improved Production of Compound (3)

    [0266] The engineered polynucleotide (SEQ ID NO: 19) encoding the polypeptide with ene reductase activity of SEQ ID NO: 20 was used to generate the engineered polypeptides of Table 3.1. These polypeptides displayed improved ene reductase activity under the desired conditions e.g., the improvement in the formation of the alcohol of compound (3) from the substrate enone of compound (1), in a coupled reaction with keto reductase KRED-P2-G03, as compared to the starting polypeptide. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 20, as described below.

    [0267] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 19, Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods that measured the polypeptides' ability to produce compound (3).

    [0268] The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 25 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 5 g/L (1), 0.5 g/L NADPH, 10 g/L KRED-P2-G03, and dissolved in a mixture of 40% (v/v) 250 mM potassium phosphate buffer at pH 6 with 2 mM magnesium chloride and 60% (v/v) isopropyl alcohol. The reaction plates were heat-sealed and shaken at 600 rpm at 30° C. for 18 hours.

    [0269] After overnight incubation (˜18 hours), 300 μL/well of acetonitrile were added to the reaction plates and mixed well. The plates were sealed and centrifuged at 4,000 rpm for 10 min. An aliquot of the supernatant was removed and further diluted 400-fold into water. After dilution, the reactions were analyzed on the Agilent RapidFire 365 high throughput mass spectrometer measuring the amount of alcohol compound (3) using the conditions in Table 2.1.

    TABLE-US-00004 TABLE 3.1 ERED Activity for the Production of Compound (3) Relative to SEQ ID NO: 20 SEQ Percent Conversion Fold ID NO: Improvement Relative to (nt/aa) Amino Acid Differences (Relative to SEQ ID NO: 20) SEQ ID NO: 20 135/136 V32A/L103G/Q109G/K154R/K168Q/A183G/P341R/N398E +++ 137/138 V32A/A183G/P341R/K399E + 139/140 V32A/A183G + 141/142 H44Y/L103R/D107E/F124S/T127N/A183E +++ 143/144 L103R/D107E/F124S/N209R/D394E +++ 145/146 Q148M + 147/148 T38S ++ 149/150 Q148R + 151/152 V114M + 153/154 Y83V +++ 155/156 Y83M + 157/158 M40G + 159/160 G261S + 161/162 G261C + 163/164 Y83F + 165/166 V118A + 167/168 Q148A + 169/170 L103R/D107Q/F124S +++ 171/172 F124S/E150M +++ 173/174 V32A/H44Y/D107Q/F124S/T127N/E150M/A183E + 175/176 V32A/H44Y/L103T/D107Q/F124S/T127N/E150M/T250R ++ 177/178 H44Y/L103Q/F124S/T127N/E150M/A183E/T260P ++ 179/180 V32A/L103R/D107R/F124S/N209R/T250R +++ 181/182 V32A/L103A/D107Q/F124S + 183/184 V32A/F124S/T127N/T250R ++ 185/186 V32A/D107R/F124S/N209R/D394E ++ 187/188 V32A/L103R/D107Q/F124S/T127N/E150M/T250R + 189/190 H44Y/L103Q/D107Q/F124S/T127N ++ 191/192 V32A/H44Y/L103Q/D107R/F124S/T127N/P341G/D394E ++ 193/194 H44Y/L103T/F124S/T250R/D394E ++ 195/196 V32A/L103R/D107E/F124S/T250R/D394E ++ 197/198 V32A/L103T/D107R/F124S/A183E/T250R ++ 199/200 L103R/D107Q/F124S/T250R + 201/202 L103R/D107R/F124S/T127N/E150M + Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 20 and defined as follows: “+” 1.06 to 2.37, “++” > 2.37, “+++” > 2.89

    [0270] Selected enzymes were regrown and retested under similar conditions with 2 equivalents of NADPH and without a cofactor recycling system to look for the presence of the undesired enantiomer of the intermediate. These samples were derivatized with TMS-diazomethane and analyzed by GC as their methyl esters according to the method in Table 2.3. >99% e.e. was observed in all samples tested.

    Example 4

    Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 162 for Improved Production of Compound (3)

    [0271] The engineered polynucleotide (SEQ ID NO: 161) encoding the polypeptide with ene reductase activity of SEQ ID NO: 162 was used to generate the engineered polypeptides of Table 4.1. These polypeptides displayed improved ene reductase activity under the desired conditions e.g., the improvement in the formation of the alcohol of compound (3) from the substrate enone or compound (1), in a coupled reaction with keto reductase KRED-P2-G03, as compared to the starting polypeptide. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 162, as described below.

    [0272] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 161. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods that measured the polypeptides' ability to produce compound (3).

    [0273] The enzyme assays were carried out in 96-well deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 25 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 20 g/L (1), 0.5 g/L NADPH, 2 g/L KRED-P2-G03, 2.5 g/L GDH-105, 100 g/L glucose dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0274] After overnight incubation (˜18 hours), 700 μL/well of acetonitrile were added to the reaction plates and mixed well. The plates were sealed and centrifuged at 4,000 rpm for 20 min. An aliquot of the supernatant was removed and further diluted 400-fold into water. After dilution, the reactions were analyzed on the Agilent RapidFire 365 high throughput mass spectrometer, measuring the amount of alcohol compound (3) using the conditions in Table 2.1.

    TABLE-US-00005 TABLE 4.1 ERED Activity for the Production of Compound (3) Relative to SEQ ID NO: 162 Percent Conversion Fold SEQ ID NO: Amino Acid Differences Improvement Relative (nt/aa) (Relative to SEQ ID NO: 162) to SEQ ID NO: 162 203/204 L298K + 205/206 F7G + 207/208 M378T ++ 209/210 T127V + 211/212 S306P ++ 213/214 S161A + 215/216 E307G ++ 217/218 Q109G + 219/220 Y359T + 221/222 N100E + 223/224 E336G + 225/226 V333T ++ 227/228 N209R/M378E ++ 229/230 V95I ++ 231/232 F7G/E307G ++ 233/234 E307Q + 235/236 N209R + 237/238 S297Y +++ 239/240 V56Y/M378Q +++ 241/242 M378G +++ 243/244 A258K + 245/246 E302A + 247/248 V4S + 249/250 V299G + 251/252 P341G +++ 253/254 Y359E + 255/256 A146P/V333A + 257/258 M378R ++ 259/260 S297F ++ 261/262 S297W +++ 263/264 M378P +++ Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 162 and defined as follows: “+” 1.20 to 1.36, “++” > 1.36, “+++” > 1.63

    [0275] Select variants were retested without KRED-P2-G03 to check the selectivity of the ERED variants. The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 25 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 5 g/L (1), 0.5 g/L NADPH, 2.5 g/L GDH-105, 12.9 g/L glucose dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0276] After overnight incubation (˜18 hours), a 100-uL aliquot of a mixture of 20% 6 M HCl and 80% isopropyl alcohol, ˜5 mg NaCl and 0.5 mL EtOAc was added to each well. The plates were sealed, shaken for 15 minutes, and centrifuged at 4,000 rpm for 5 min. A 200-uL aliquot of the EtOAc supernatant was removed. 10 uL of 2 M TMS-diazomethane were added to each well to derivatize the samples as their methyl-esters. After 1 hr at RT, the excess TMS-diazomethane was quenched with 4 uL acetic acid. These samples were then analyzed by chiral GC to determine the activity and selectivity of the enzyme variants using the analytical method described in Table 2.3. >99% enantiomeric excess was observed in all variants tested.

    Example 5

    Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 262 for Improved Production of Compound (3)

    [0277] The engineered polynucleotide (SEQ ID NO: 261) encoding the polypeptide with ene reductase activity of SEQ ID NO: 262 was used to generate the engineered polypeptides of Table 5.1. These polypeptides displayed improved ene reductase activity under the desired conditions e.g., the improvement in the formation of the alcohol of compound (3) from the substrate enone of compound (1), in a coupled reaction with keto reductase KRED-P2-G03, as compared to the starting polypeptide. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 262, as described below.

    [0278] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 261. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods that measured the polypeptides' ability to produce compound (3).

    [0279] The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 20 v/v % of undiluted ene reductase lysate, prepared as described in EXAMPLE 1: 25 g/L (1), 0.5 g/L NADPH, 2 g/L KRED-P2-G03, 2.5 g/L GDH-105, 200 g/L glucose dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0280] After overnight incubation (˜18 hours), 700 μL/well of acetonitrile were added to the reaction plates and mixed well. The plates were sealed and centrifuged at 4,000 rpm for 20 min. An aliquot of the supernatant was removed and further diluted 500-fold into water. After dilution, the reactions were analyzed on the Agilent RapidFire 365 high throughput mass spectrometer, according to the details in Table 2.1.

    TABLE-US-00006 TABLE 5.1 ERED Activity for the Production of Compound (3) Relative to SEQ ID NO: 262 SEQ ID NO: Amino Acid Differences Percent Conversion Fold Improvement (nt/aa) (Relative to SEQ ID NO: 262) Relative to SEQ ID NO: 262 265/266 V4T/N209R/Y359E/M378P + 267/268 N209R/A258K + 269/270 V4T/K151R/V333A ++ 271/272 V4T/K151R/E307G/M378P +++ 273/274 N209R/V333A/Y359T + 275/276 N209R + 277/278 N100E/N209R/A258K/Y359T + 279/280 V4T/K151R/Y359E/M378Q + 281/282 V4T/N100E/N209R/A258K/Y359T/M378Q + 283/284 A146P/K151R/Y359T/M378P ++ 285/286 V4T/N209R/Y359T + 287/288 M378P + 289/290 N209R/L298K/Y359E/M378P ++ 291/292 N100E/A146P/K151R/A258K/Y359E/M378Q ++ 293/294 N100K/M378G + 295/296 M378E ++ 297/298 F7G/V95I/N100K +++ 299/300 A258R/M378G + 301/302 V95I/A258R/M378G ++ 303/304 V95I/N100K/N326G/M378T + 305/306 P341G +++ 307/308 V95I/N100K/N326G/V333T/M378T ++ 309/310 V95I/V333T +++ Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 262 and defined as follows: “+” 1.65 to 2.11, “++” > 2.11 , “+++” > 2.56

    [0281] Select variants were retested without KRED-P2-G03 to check the selectivity of the ERED variants. The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 20 v/v % of undiluted ene reductase lysate, prepared as described in EXAMPLE 1: 5 g/L (1), 0.5 g/L NADPH, 2.5 g/L GDH-105, 25 g/L glucose dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0282] After overnight incubation (˜18 hours), a 100-uL aliquot of a mixture of 20% 6 M HCl and 80% isopropyl alcohol, ˜5 mg NaCl and 0.5 mL EtOAc was added to each well. The plates were sealed, shaken for 15 minutes, and centrifuged at 4,000 rpm for 5 min. A 200-uL aliquot of the EtOAc supernatant was removed. 10 uL of 2 M TMS-diazomethane were added to each well to derivatize the samples as their methyl-esters. After 1 hr at RT, the excess TMS-diazomethane was quenched with 4 uL acetic acid. These samples were then analyzed by chiral GC to determine the activity and selectivity of the enzyme variants using the analytical method described in Table 1-3. >99% enantiomeric excess was observed in all variants tested.

    Example 6

    Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 282 for Improved Production of Compound (3)

    [0283] The engineered polynucleotide (SEQ ID NO: 281) encoding the polypeptide with ene reductase activity of SEQ ID NO: 282 was used to generate the engineered polypeptides of Table 6.1. These polypeptides displayed improved ene reductase activity under the desired conditions e.g., the improvement in the formation of the alcohol of compound (3) from the substrate enone of compound (1), in a coupled reaction with keto reductase KRED-P2-G03, as compared to the starting polypeptide. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 282, as described below.

    [0284] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 281. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods that measured the polypeptides' ability to produce compound (3).

    [0285] The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL. total reaction volume per well. The reactions contained 25 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 35 g/L (1), 0.5 g/L NADPH, 2 g/L KRED-P2-G03, 2.5 g/L GDH-105, 200 g/L glucose dissolved in 200 mM sodium phosphate buffer at pH 7.3 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0286] After overnight incubation (˜18 hours), 700 μL/well of acetonitrile were added to the reaction plates and mixed well. The plates were sealed and centrifuged at 4,000 rpm for 10 min. An aliquot of the supernatant was removed and further diluted 500-fold into water. After dilution, the reactions were analyzed on the Agilent RapidFire 365 high throughput mass spectrometer, measuring the amount of alcohol compound (3) using the conditions in Table 2.1.

    TABLE-US-00007 TABLE 6.1 ERED Activity for the Production of Compound (3) Relative to SEQ ID NO: 282 Percent Conversion SEQ ID NO: Amino Acid Differences Fold Improvement (nt/aa) (Relative to SEQ ID NO: 282) Relative to SEQ ID NO: 282 311/312 V89I + 313/314 L283C + 315/316 L243I + Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 282 and defined as follows: “+” from 1.02 to 1.80

    [0287] Select variants were retested to check the selectivity of the ERED variants. This test was done in the presence of KRED-P2-G03, looking for the presence of the cis-isomer of the product. The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 5 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 5 g/L (1), 0.5 g/L NADPH, 2 g/L KRED-P2-G03, 2.5 g/L GDH-105, 25 g/L glucose, 200 g/L sodium gluconate, dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0288] After overnight incubation (˜18 hours), a 100-uL aliquot of a mixture of 20% 6 M HCl and 80% isopropyl alcohol, ˜5 mg NaCl and 0.5 mL EtOAc was added to each well. The plates were sealed, shaken for 15 minutes, and centrifuged at 4,000 rpm for 5 min. A 200-uL aliquot of the EtOAc supernatant was removed. 10 uL of 2 M TMS-diazomethane were added to each well to derivatize the samples as their methyl-esters. After 1 hr at RT, the excess TMS-diazomethane was quenched with 4 uL acetic acid. These samples were then analyzed by chiral GC to determine the activity and selectivity of the enzyme variants using the analytical method described in Table 1-3. >99% enantiomeric excess was observed in all variants tested.

    Example 7

    Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 294 for Improved Production of Compound (3)

    [0289] The engineered polynucleotide (SEQ ID NO: 293) encoding the polypeptide with ene reductase activity of SEQ ID NO: 294 was used to generate the engineered polypeptides of Table 7.1. These polypeptides displayed improved ene reductase activity under the desired conditions e.g., the improvement in the formation of the alcohol of compound (3) from the substrate enone of compound (1), in a coupled reaction with keto reductase KRED-P2-G03, as compared to the starting polypeptide. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 294, as described below.

    [0290] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 293. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods that measured the polypeptides' ability to produce compound (3).

    [0291] The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 5 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 5 g/L (1), 0.5 g/L NADPH, 2 g/L KRED-P2-G03, 2 g/L GDH-105, 100 g/L glucose dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0292] After overnight incubation (˜18 hours), 700 μL/well of acetonitrile were added to the reaction plates and mixed well. The plates were sealed and centrifuged at 4,000 rpm for 10 min. An aliquot of the supernatant was removed and further diluted 500-fold into water. After dilution, the reactions were analyzed on the Agilent RapidFire 365 high throughput mass spectrometer, measuring the amount of alcohol compound (3) using the conditions in Table 2.1.

    TABLE-US-00008 TABLE 7.1 ERED Activity for the Production of Compound (3) Relative to SEQ ID NO: 294 Percent Conversion Fold SEQ ID NO: Amino Acid Differences Improvement Relative (nt/aa) (Relative to SEQ ID NO: 294) to SEQ ID NO: 294 317/318 V118C ++ 319/320 T377K/G378Q ++ 321/322 T374S/G378Q + 323/324 V118A ++ 325/326 Y314L + 327/328 N47H +++ 329/330 Q148K ++ 331/332 Q148L + 333/334 G378Q + 335/336 A258K/C261V + 337/338 A258K/C261L +++ 339/340 A258K/C261M + Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 294 and defined as follows: “+” .91 to 1.07, “++” > 1.07, “+++” > 1.13

    [0293] Select variants were retested to check the selectivity of the ERED variants. This test was done in the presence of KRED-P2-G03, looking for the presence of the cis-isomer of the product. The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 5 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 5 g/L (1), 0.5 g/L NADPH, 2 g/L KRED-P2-G03, 2.5 g/L GDH-105, 25 g/L glucose, 200 g/L sodium gluconate, dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0294] After overnight incubation (˜18 hours), a 100-uL aliquot of a mixture of 20% 6 M HCl and 80% isopropyl alcohol, ˜5 mg NaCl and 0.5 mL EtOAc was added to each well. The plates were sealed, shaken for 15 minutes, and centrifuged at 4,000 rpm for 5 min. A 200-uL aliquot of the EtOAc supernatant was removed. 10 uL of 2 M TMS-diazomethane were added to each well to derivatize the samples as their methyl-esters. After 1 hr at RT, the excess TMS-diazomethane was quenched with 4 uL acetic acid. These samples were then analyzed by chiral GC to determine the activity and selectivity of the enzyme variants using the analytical method described in Table 1.3. >99% enantiomeric excess was observed in all variants tested.

    Example 8

    Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 322 for Improved Production of Compound (3)

    [0295] The engineered polynucleotide (SEQ ID NO: 321) encoding the polypeptide with ene reductase activity of SEQ ID NO: 322 was used to generate the engineered polypeptides of Table 8.1. These polypeptides displayed improved ene reductase activity under the desired conditions e.g., the improvement in the formation of the alcohol of compound (3) from the substrate enone of compound (1), in a coupled reaction with keto reductase KRED-P2-G03, as compared to the starting polypeptide. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 322, as described below.

    [0296] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 321. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods that measured the polypeptides' ability to produce compound (3).

    [0297] The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL. total reaction volume per well. The reactions contained 2 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 10 g/L (1), 0.5 g/L NADPH, 2 g/L KRED-P2-G03, 2 g/L GDH-105, 200 g/L glucose dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0298] After overnight incubation (˜18 hours), 700 μL/well of acetonitrile were added to the reaction plates and mixed well. The plates were sealed and centrifuged at 4,000 rpm for 10 min. An aliquot of the supernatant was removed and further diluted 500-fold into water. After dilution, the reactions were analyzed on the Agilent RapidFire 365 high throughput mass spectrometer, measuring the amount of alcohol compound (3) using the conditions in Table 2.1.

    TABLE-US-00009 TABLE 8.1 ERED Activity for the Production of Compound (3) Relative to SEQ ID NO: 322 Percent Conversion Fold Improvement SEQ ID NO: Amino Acid Differences Relative to SEQ ID (nt/aa) (Relative to SEQ ID NO: 322) NO: 322 341/342 K100N/L243I ++ 343/344 K100N/L243I/S374T ++ 345/346 N47H/V89I/V95I/Q148L/A258K/C261V +++ 347/348 V95I/Q148L/A258K/C261V ++ 349/350 V95I/Q148L +++ 351/352 V95I/L243I + 353/354 N47H/V89I/A258K + 355/356 N47H/V95I + 357/358 V95I/Q148L/L243I/A258K/C261V + 359/360 V89I/V95I/L243I + 361/362 V95I/Q148L/L243I/C261V + 363/364 Q148L/L243I ++ 365/366 N47H/V89I/V95I/L243I/A258K/ + C261L/Q378P Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 322 and defined as follows: “+” .82 to 1.33, “++” > 1.33, “+++” > 2.71

    [0299] Select variants were retested to check the selectivity of the ERED variants. This test was done in the presence of KRED-P2-G03, looking for the presence of the cis-isomer of the product. The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 5 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 5 g/L (1), 0.5 g/L NADPH, 2 g/L KRED-P2-G03, 2.5 g/L GDH-105, 25 g/L glucose, 200 g/L sodium gluconate, dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0300] After overnight incubation (˜18 hours), a 100-uL aliquot of a mixture of 20% 6 M HCl and 80% isopropyl alcohol, ˜5 mg NaCl and 0.5 mL EtOAc was added to each well. The plates were sealed, shaken for 15 minutes, and centrifuged at 4,000 rpm for 5 min. A 200-uL aliquot of the EtOAc supernatant was removed. 10 uL of 2 M TMS-diazomethane were added to each well to derivatize the samples as their methyl-esters. After 1 hr at RT, the excess TMS-diazomethane was quenched with 4 uL acetic acid. These samples were then analyzed by chiral GC to determine the activity and selectivity of the enzyme variants using the analytical method described in Table 2.3. >99% enantiomeric excess was observed in all variants tested.

    Example 9

    Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 346 for Improved Production of Compound (3)

    [0301] The engineered polynucleotide (SEQ ID NO: 345) encoding the polypeptide with ene reductase activity of SEQ ID NO: 346 was used to generate the engineered polypeptides of Table 9.1. These polypeptides displayed improved ene reductase activity under the desired conditions e.g., the improvement in the formation of the alcohol of compound (3) from the substrate enone of compound (1), in a coupled reaction with keto reductase KRED-P2-G03, as compared to the starting polypeptide. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 346 as described below.

    [0302] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 345. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods that measured the polypeptides' ability to produce compound (3).

    [0303] The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL. total reaction volume per well. The reactions contained 40 v/v % of fold-fold diluted ene reducase lysate, prepared as described in EXAMPLE 1 and then heated to 45° C. for 2 hours, 10 g/L (1), 0.5 g/L NADPH, 0.5 g/L KRED (SEQ ID NO: 476), 0.25 g/L GDH-105, 200 g/L glucose dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0304] After overnight incubation (˜18 hours), 700 μL/well of acetonitrile were added to the reaction plates and mixed well. The plates were sealed and centrifuged at 4,000 rpm for 10 min. An aliquot of the supernatant was removed and further diluted 500-fold into water. After dilution, the reactions were analyzed on the Agilent RapidFire 365 high throughput mass spectrometer, measuring the amount of alcohol compound (3) with conditions in Table 2.1.

    TABLE-US-00010 TABLE 9.1 ERED Activity for the Production of Compound (3) Relative to SEQ ID NO: 346 Percent Conversion Fold SEQ ID NO: Amino Acid Differences Improvement Relative (nt/aa) (Relative to SEQ ID NO: 346) to SEQ ID NO: 346 367/368 T388G +++ 369/370 V393A + 371/372 W397A ++ 373/374 C108S ++ 375/376 A11V ++ 377/378 R13V +++ 379/380 A235G +++ 381/382 A392G ++ 383/384 R64V ++ 385/386 P20E ++ 387/388 R64N ++ 389/390 W397F ++ 391/392 V175R + 393/394 P320R + 395/396 C108L + 397/398 K99V + 399/400 A29V + 401/402 W397C +++ 403/404 K99M + 405/406 V393E + 407/408 R64A ++ 409/410 A11G + 411/412 T388V + 413/414 K99V/N398K + 415/416 V333Q + 417/418 W397D ++ 419/420 R13Q +++ 421/422 V393T + 423/424 I21L + 425/426 Q10R +++ 427/428 R64E + 429/430 L243M + Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 346 and defined as follows: “+” 2.30 to 2.94, “++” > 2.94, “+++” > 3.39

    [0305] Select variants were retested to check the selectivity of the ERED variants. This test was done in the presence of KRED (SEQ ID NO: 476), looking for the presence of the cis-isomer of the product. The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 5 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1: 5 g/L (1), 0.5 g/L NADPH, 0.5 g/L KRED (SEQ ID NO: 476), 0.25 g/L GDH-105, 25 g/L glucose, 200 g/L sodium gluconate, dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0306] After overnight incubation (˜18 hours), a 50-uL aliquot of a mixture of 20% 6 M HCl and 80% isopropyl alcohol, ˜5 mg NaCl and 0.5 mL EtOAc was added to each well. The plates were sealed, shaken for 15 minutes, and centrifuged at 4,000 rpm for 5 min. A 200-uL aliquot of the EtOAc supernatant was removed. 10 uL of 2 M TMS-diazomethane were added to each well to derivatize the samples as their methyl-esters. After 1 hr at RT, the excess TMS-diazomethane was quenched with 4 uL acetic acid. These samples were then analyzed by chiral GC to determine the activity and selectivity of the enzyme variants using the analytical method described in Table 6-2. >99% enantiomeric excess was observed in all variants tested.

    Example 10

    Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 432 for Improved Production of Compound (3)

    [0307] The engineered polynucleotide (SEQ ID NO: 431) encoding the polypeptide with ketoreductase activity of SEQ ID NO: 432 was used to generate the engineered polypeptides of Table 10.1. These polypeptides displayed improved ketoreductase activity under the desired conditions e.g., the improvement in the formation of the alcohol of compound (3), from the intermediate ketone of compound (2), as compared to the starting polypeptide. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 432, as described below.

    [0308] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 431. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods that measured the polypeptides' ability to produce compound (3).

    [0309] The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 2 v/v % of undiluted ketoreductase lysate, prepared as described in EXAMPLE 1: 2 g/L (1), 8 g/L (2), 0.5 g/L NADPH, 1 g/L GDH-105, 100 g/L glucose, 100 g/L sodium gluconate, dissolved in 200 mM sodium phosphate buffer at pH 7.2 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 40° C. for 18 hours.

    [0310] After overnight incubation (˜18 hours), a 50-uL aliquot of a mixture of 20% HCl and 80% isopropyl alcohol, ˜5 mg NaCl and 1 mL EtOAc was added to each well. The plates were sealed, shaken for 15 minutes, and centrifuged at 4,000 rpm for 5 min. A 200-uL aliquot of the EtOAc supernatant was removed for analysis. 10 uL of 2 M TMS-diazomethane were added to each well to derivatize the samples as their methyl-esters. After 1 hr at RT, the excess TMS-diazomethane was quenched with 4 uL acetic acid. These samples were then analyzed by chiral GC to determine the activity and selectivity of the enzyme variants using the analytical method described in Table 1-3.

    [0311] The allylic alcohol of compound (4) is a byproduct that is formed if a KRED reacts directly with the substrate compound (1). The first round of the KRED screen (and the second round retest) were done using GC to check for selectivity and to look for this undesired side product.

    TABLE-US-00011 TABLE 10.1 KRED Activity for the Production of Compound (3) Relative to SEQ ID NO: 432 Percent Conversion Fold Improvement SEQ ID NO: Amino Acid Differences Relative to (nt/aa) (Relative to SEQ ID NO: 432) SEQ ID NO: 432 433/434 L21K/K72T/T103D/L147I + 435/436 L21K/T103D/L147I + 437/438 L21K/S65R/T103D/T152K + 439/440 L21K/S65R/L147I/T152K + 441/442 L21K/K72T/N131Y/V181I/D197R + 443/444 L21K + 445/446 L21K/K72T/L73V/T103D + 447/448 L21K/K72T/T152K/V181I + 449/450 L21K/L73V/V181I ++ 451/452 L21K/S65R/K72T/L73V/T103D + 453/454 L21K/L73V/T103D ++ 455/456 L21K/T103D/L147I/T152K + 457/458 L21K/K72T/T103D ++ 459/460 L21K/L73V/N131Y/L147I + 461/462 L21K/L73V/L147I + 463/464 L21K/S65R/N131Y/T152K +++ 465/466 L21K/S65R/T152K ++ 467/468 L21K/S65R/K72T/L147I +++ 469/470 L21K/T103D +++ 471/472 L21K/S65R/K72T/N131Y/L147/V181I + 473/474 L21K/L147I +++ 475/476 L21K/K72T/T103D/L147I/T152K/V181I ++ 477/478 L21K/K72T/T103D/N131Y/T152K/I226L ++ 479/480 T2S/K72D ++ 481/482 T2S/K72D/L172V +++ 483/484 K26R/D173Y/N221D ++ Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 432 and defined as follows: “+” 1.07 to 1.20, “++” > 1.20, “+++” > 1.25

    Example 11

    Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 476 for Improved Production of Compound (3)

    [0312] The engineered polynucleotide (SEQ ID NO: 475) encoding the polypeptide with ketoreductase activity of SED ID NO: 476 was used to generate the engineered polypeptides of Table 11.1. These polypeptides displayed improved ketoreductase activity under the desired conditions e.g., the improvement in the formation of the alcohol of compound (3), from the substrate enone of compound (1), in a coupled reaction with ene reductase (SEQ ID NO: 346), as compared to the starting polypeptide. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 476, as described below.

    [0313] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 475. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods that measured the polypeptides' ability to produce compound (3).

    [0314] The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL. total reaction volume per well. The reactions contained 0.25 v/v % of undiluted ketoreductase lysate, prepared as described in EXAMPLE 1: 5 g/L (1), 0.5 g/L NADPH, 0.25 g/L GDH-105, 100 g/L glucose, 100 g/L sodium gluconate, dissolved in 200 mM sodium phosphate buffer at pH 7.5 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0315] After overnight incubation (˜18 hours), 700 μL/well of acetonitrile were added to the reaction plates and mixed well. The plates were sealed and centrifuged at 4,000 rpm for 10 min. An aliquot of the supernatant was removed and further diluted 500-fold into water. After dilution, the reactions were analyzed on the Agilent RapidFire 365 high throughput mass spectrometer, measuring the amount of alcohol compound (3) using the conditions in Table 2.1.

    TABLE-US-00012 TABLE 11.1 KRED Activity for the Production of Compound (3) Relative to SEQ ID NO: 476 Percent Conversion Fold Improvement SEQ ID NO: Amino Acid Differences Relative to (nt/aa) (Relative to SEQ ID NO: 476) SEQ ID NO: 476 485/486 E190R +++ 487/488 L17R ++ 489/490 V43R + 491/492 T250L + 493/494 L17M ++ 495/496 L17K ++ 497/498 E45H + 499/500 T54P + 501/502 P194R + 503/504 M205E ++ 505/506 D198A +++ 507/508 E190L +++ 509/510 E190V ++ 511/512 E190Q ++ 513/514 T71G + 515/516 L195M + 517/518 D198R +++ 519/520 E190A + 521/522 T71E + 523/524 S96R + Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 476 and defined as follows: “+” 1.38 to 1.99, “++” > 1.99, “+++” > 3.32

    [0316] Select variants were retested to check the selectivity of the KRED variants. This test was done in the presence of ERED (SEQ ID NO: 346), looking for the presence of the cis-isomer of the product and the allylic alcohol. The enzyme assays were carried out in 96-well, deep-well (2.1 mL total volume) plates, in 100 μL total reaction volume per well. The reactions contained 1 v/v % of undiluted ene reducase lysate, prepared as described in EXAMPLE 1, 5 g/L (1), 0.5 g/L NADPH, 0.25 g/L ERED (SEQ ID NO: 346), 0.25 g/L GDH-105, 100 g/L glucose, 100 g/L sodium gluconate, dissolved in 200 mM sodium phosphate buffer at pH 7.6 with 2 mM magnesium chloride. The reaction plates were heat-sealed and shaken at 600 rpm at 45° C. for 18 hours.

    [0317] After overnight incubation (˜18 hours), a 50-uL aliquot of a mixture of 20% 6 M HCl and 80% isopropyl alcohol, ˜5 mg NaCl and 0.5 mL EtOAc was added to each well. The plates were sealed, shaken for 15 minutes, and centrifuged at 4,000 rpm for 5 min. A 200-uL aliquot of the EtOAc supernatant was removed. 10 uL of 2 M TMS-diazomethane were added to each well to derivatization the samples as their methyl-esters. After 1 hr at RT, the excess TMS-diazomethane was quenched with 4 uL acetic acid. These samples were then analyzed by chiral GC to determine the activity and selectivity of the enzyme variants using the analytical method described in Table 1.3. >99% enantiomeric excess was observed in all variants tested. All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

    [0318] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).