ENGINEERED PANTOTHENATE KINASE VARIANT ENZYMES

Abstract

The present invention provides engineered pantothenate kinase (PanK) enzymes, polypeptides having PanK activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing PanK enzymes are also provided. The present invention further provides compositions comprising the PanK enzymes and methods of using the engineered PanK enzymes. The present invention finds particular use in the production of pharmaceutical compounds.

Claims

1. An engineered pantothenate kinase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4, SEQ ID NO: 44, SEQ ID NO: 320, and/or SEQ ID NO: 526, or a functional fragment thereof, wherein said engineered pantothenate kinase comprises at least one substitution or substitution set in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4, SEQ ID NO: 44, SEQ ID NO: 320, and/or SEQ ID NO: 526.

2. The engineered pantothenate kinase of claim 1, comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4, or a functional fragment thereof, wherein said engineered pantothenate kinase comprises at least one substitution or substitution set in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4.

3. The engineered pantothenate kinase of claim 2, wherein said engineered pantothenate kinase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4, or a functional fragment thereof, and wherein said engineered pantothenate kinase comprises at least one substitution or substitution set at one or more positions selected from 103, 103/135/141, 17, 19, 26, 31, 33, 34, 35, 37, 44, 46, 63, 73, 74, 76, 77, 79, 80, 82, 85, 91, 94, 95, 96, 103/135, 103/135/216, 103/135/291, 103/141, 103/141/193/216/238, 103/141/216, 103/141/238, 103/216/238, 120, 124, 126, 128, 129, 130, 135, 135/141/238, 135/141/298/323, 135/193/238/289/291, 135/219, 135/238/291, 135/248/251/291/297, 135/251/323, 135/323, 141, 141/193/251/323, 141/251, 141/251/323, 141/297, 141/323, 145, 154, 164, 193, 193/216, 194, 195, 197, 198, 216, 218, 221, 222, 225, 227, 233, 238, 247, 248, 250, 251, 274, 286, 287, 289, 290, 291, 293, 297, 308, 315, and 323, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4.

4. (canceled)

5. The engineered pantothenate kinase of claim 1, wherein said engineered pantothenate kinase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 44, or a functional fragment thereof, and wherein said engineered pantothenate kinase comprises at least one substitution or substitution set at one or more positions selected from 16/93/120/221/308, 16/141/179/282, 17, 17/34, 17/34/46, 17/34/96/128/129/164, 17/46, 17/46/63, 17/46/63/96, 17/46/63/164, 17/46/126/128/129/164, 17/46/128/129/130/164/193, 17/46/164, 17/63, 17/63/79, 17/63/79/128/129/130/164, 17/63/96, 17/63/96/164, 17/63/129/130, 17/63/164, 17/63/193, 17/79/128/129/130, 17/126, 17/126/129/130/164, 17/126/129/164, 19, 19/35/85/274, 19/35/197/218/275, 19/74/77/141/218, 19/74/145, 19/77/141/218/225, 19/85/145/197/218/308, 19/141, 19/141/225, 20/59/93, 20/59/120/164/263/282, 20/164, 20/164/179/308/320, 20/282, 20/308, 26/31/33/35/76/79/94/221/293, 26/31/33/35/120/141/293, 26/31/33/76/79, 26/31/33/76/79/94, 26/31/120, 26/33/35/126/222/291, 26/33/80/126/291/293, 26/33/94/293, 26/35/79/120/126/194/198, 26/35/120/222/227/293, 26/35/126, 31/33/35/76/79/91/291, 31/33/35/76/141/293, 31/33/35/120/126/227, 31/33/35/126, 31/33/76/79/80/94/291, 31/33/76/79/95, 31/33/120, 31/35/80/94/126/141/293, 31/76/79/120/126/293, 31/76/79/293, 32/46, 33/35, 33/35/76/79/120/141/198/221, 33/35/76/80/126/221/222/227, 33/35/76/80/293, 33/35/94/126/141, 33/35/126/198/293, 33/35/126/222/227, 33/35/126/293, 33/35/221/222, 33/76/79, 33/76/79/126, 33/79/126/221/227/293, 34, 34/46, 34/46/48, 34/46/63, 34/46/63/164, 34/46/79/96/126, 34/46/79/164, 34/46/164, 34/63/126/129/193, 34/79/126/128/130, 34/164, 35, 35/74/77/197/225, 35/74/218, 35/76, 35/76/79/80/126, 35/76/80/126, 35/85, 35/85/141/218, 35/85/218/225, 35/85/218/274, 35/94/120/126/293, 35/145/286, 35/197/274, 35/218/308, 35/274, 46, 46/63, 46/79, 46/79/126/193, 46/96/164, 46/128/129/164, 46/129, 46/164, 46/164/193, 46/193, 59, 59/120, 63, 63/164, 63/193, 74/85/145, 74/141, 76/79/80/120, 77/85/145, 77/218, 79/96/126/129, 79/126/164, 80/126/221/227, 94/95/120/291, 94/126/293, 94/293, 120, 120/164/282/321, 126/128/129/193, 126/129/164, 128/129/130, 141/195/197/218, 164, 164/221/308, 218, 225, 263, 274, and 308, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 44.

6. (canceled)

7. The engineered pantothenate kinase of claim 1, wherein said engineered pantothenate kinase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 320, or a functional fragment thereof, and wherein said engineered pantothenate kinase comprises at least one substitution or substitution set at one or more positions selected from 20/31/35/48/63/74/94/120/164, 20/31/35/74/79/164/218/225/282, 20/35/120/197, 20/48/63/94/120/145/197/218/227, 20/48/218/227, 20/120/145/197/227, 20/197/225/227, 24, 33, 46, 48, 48/63, 48/63/74/79/120/129/227/282, 48/63/94/275, 48/63/145, 63, 63/120/197/218/275, 63/129/145, 63/218/275/282, 74/79/94/275, 94/120, 95, 130, 149, 164, 178, 196, 201, 221, 271, 277, 282, 289, 293, 298, 314, and 321, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 320.

8. (canceled)

9. The engineered pantothenate kinase of claim 1, wherein said engineered pantothenate kinase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 526, or a functional fragment thereof, and wherein said engineered pantothenate kinase comprises at least one substitution or substitution set at one or more positions selected from 24, 24/33, 24/46/221, 24/95/164/178/201/282, 24/149/178/282/293/298, 24/149/201/282, 24/149/282/314, 24/178/201/271/282/293/298/314, 24/201/271/282/293/298, 24/201/282, 24/227, 33/95/104, 46/95/282, 48, 95/201/282/293/314, 164/201/271/314, 178/293/298, 201, 201/282, 221/289, 282/293/298/314/321, and 298, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 526.

10.-18. (canceled)

19. The engineered pantothenate kinase of claim 1, wherein said engineered pantothenate kinase 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 pantothenate kinase variant set forth in the even numbered sequences of SEQ ID NOS: 4-630.

20. The engineered pantothenate kinase of claim 1, wherein said engineered pantothenate kinase comprises a polypeptide sequence forth in the even numbered sequences of SEQ ID NOS: 4-630.

21. The engineered pantothenate kinase of claim 1, wherein said engineered pantothenate kinase comprises at least one improved property compared to wild-type E. coli pantothenate kinase.

22. The engineered pantothenate kinase of claim 21, wherein said improved property comprises improved activity on a substrate, as compared to a wild-type pantothenate kinase.

23. (canceled)

24. The engineered pantothenate kinase of claim 21, wherein said improved property comprises improved production of phospho-ethynyl glycerol, as compared to a wild-type pantothenate kinase.

25. The engineered pantothenate kinase of claim 1, wherein said engineered pantothenate kinase is purified.

26. A composition comprising at least one engineered pantothenate kinase of claim 1.

27. A polynucleotide sequence encoding at least one engineered pantothenate kinase of claim 1.

28. A polynucleotide sequence encoding at least one engineered pantothenate kinase, wherein said polynucleotide sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 3, SEQ ID NO: 43, SEQ ID NO: 319, and/or SEQ ID NO: 525, wherein the polynucleotide sequence of said engineered pantothenate kinase comprises at least one substitution at one or more positions.

29.-32. (canceled)

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

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

35. The polynucleotide sequence of claim 1, wherein said polynucleotide comprises an odd-numbered sequence of SEQ ID NOS: 3-629.

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

37. A host cell comprising at least one expression vector of claim 36.

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

39. A method of producing an engineered pantothenate kinase in a host cell, comprising culturing the host cell of claim 37, under suitable conditions, such that at least one engineered pantothenate kinase is produced.

40. The method of claim 39, further comprising recovering at least one engineered pantothenate kinase from the culture and/or host cell.

41. The method of claim 39, further comprising the step of purifying said at least one engineered pantothenate kinase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0130] The present invention provides engineered pantothenate kinase (PanK) enzymes, polypeptides having PanK activity, and the polynucleotides encoding these enzymes, as well as the vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing PanK enzymes are also provided. The present invention further provides compositions comprising the PanK enzymes and methods of using the engineered PanK enzymes. The present invention finds particular use in the production of pharmaceutical compounds.

[0131] In some embodiments, the present invention provides enzymes suitable for the production of phosphorylated glycerol derivatives and glyceraldehyde derivatives with bulky substituents on the C2 carbon of glycerol, especially phosphorylated ethynyl-glycerols and ethynyl-glyceraldehydes that are intermediates for the in vitro enzymatic synthesis of the non-natural nucleoside analog shown of compound (1).

##STR00001##

[0132] Production of phosphorylated glyceraldehyde derivatives such as compound (5), can be difficult. However, the corresponding non-phosphorylated glyceraldehyde derivatives (6) can be made by oxidizing the glycerol derivative (7) with an alcohol oxidase. Once the glycerol aldehyde is formed it can be phosphorylated into the desired intermediate (5) by PanK as shown in Scheme I.

##STR00002##

[0133] Alternate methods of producing compound (5) may have advantages in the efficient production of compound (1) at industrial scale. PanK enzymes, including the improved enzymes of the present disclosure, may have improved phosphorylation activity on the triglycerol of compound (7) as compared to the glyceraldehyde of compound (6). Thus, producing compound (5) from compound (7) by first phosphorylating compound (7) using a PanK enzyme to produce compound (8) and then oxidizing to compound (5) using an alcohol oxidase may improve production of compound (1) (see Scheme 2, below). Phosphorylation of compound (7) prior to oxidation by the alcohol oxidase may also allow coupling of the oxidase and aldose reactions may reduce product inhibition and increase enzyme efficiency.

##STR00003##

Engineered PanK Polypeptides

[0134] The present invention provides engineered PanK 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 PanK enzymes with improved properties as compared to wild-type PanK 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 isomerization reaction. In some embodiments, the reaction conditions are modified with regard to concentrations or amounts of engineered PanK, substrate(s), buffer(s), solvent(s), co-factors, pH, conditions including temperature and reaction time, and/or conditions with the engineered PanK polypeptide immobilized on a solid support, as further described below and in the Examples.

[0135] 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.

[0136] 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.

Methods of Using the Engineered Pantothenate Kinase Enzymes

[0137] In some embodiments, the PanK enzymes described herein find use in processes for converting compound (7) to compound (8). Generally, the process for performing the phosphorylation reaction comprises contacting or incubating the substrate compound in presence of a co-substrate, such as adenylate triphosphate (ATP), with an acetate kinase enzyme and acetyl phosphate used to recycle the resulting adenylate triphosphate (ADP) back to ATP.

[0138] 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, reductant, divalent transition metal, 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 PanK polypeptide 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 PanK polypeptide and substrate compound under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the product compound.

[0139] Suitable reaction conditions using the engineered PanK polypeptides typically comprise a co-substrate, which is used stoichiometrically in the phosphorylation reaction. Generally, the co-substrate for PanK enzymes is ATP. Other phosphoryl group donors that are capable of serving as co-substrates for PanK enzymes can be used. Because the co-substrate is used stoichiometrically, the co-substrate is present at an equimolar or higher amount than that of the substrate compound (i.e., the molar concentration of co-substrate is equivalent to or higher than the molar concentration of substrate compound). In some embodiments, the suitable reaction conditions can comprise a co-substrate molar concentration of at least 1 fold, 1.5 fold, 2 fold, 3 fold 4 fold or 5 fold or more than the molar concentration of the substrate compound. In some embodiments, the suitable reaction conditions can comprise a co-substrate concentration, particularly ATP, of about 0.0005 M to about 2 M, 0.01 M to about 2 M, 0.1 M to about 2 M, 0.2 M to about 2 M, about 0.5 M to about 2 M, or about 1 M to about 2 M. In some embodiments, the reaction conditions comprise a co-substrate concentration of about 0.0001 M, 0.001 M, 0.01 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 1 M, 1.5 M, or 2 M. In some embodiments, additional co-substrate can be added during the reaction. In some additional embodiments, the co-substrate is present in lower concentrations due to the presence of a recycling system, such as acetate kinase and acetyl phosphate, that recycles ADP to ATP.

[0140] Substrate compound in the reaction mixtures can be varied, taking into consideration, for example, the desired amount of product compound, the effect of substrate concentration on enzyme activity, stability of enzyme under reaction conditions, and the percent conversion of substrate to product. In some embodiments, the suitable reaction conditions comprise a substrate compound loading of at least about 0.5 to about 200 g/L, 1 to about 200 g/L, 5 to about 150 g/L, about 10 to about 100 g/L, 20 to about 100 g/L or about 50 to about 100 g/L. In some embodiments, the suitable reaction conditions comprise a substrate compound loading 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, at least about 30 g/L, at least about 50 g/L, at least about 75 g/L, at least about 100 g/L, at least about 150 g/L or at least about 200 g/L, or even greater. The values for substrate loadings provided herein are based on the molecular weight of 2-ethynylglycerol; however, it also contemplated that the equivalent molar amounts of various alcohol or aldehyde analogues also can be used in the process.

[0141] In carrying out the PanK 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 PanK 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).

[0142] The gene(s) encoding the engineered PanK polypeptides can be transformed into host cells 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 PanK polypeptide and another set can be transformed with gene(s) encoding another engineered PanK 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 PanK 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 PanK reaction.

[0143] In some embodiments, the improved activity and/or selectivity of the engineered PanK 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 0.03% (w/w), 0.05% (w/w), 0.1% (w/w), 0.15% (w/w), 0.2% (w/w), 0.3% (w/w), 0.4% (w/w), 0.5% (w/w), 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 20% (w/w) or more of substrate compound loading.

[0144] In some embodiments, the engineered polypeptide is present at about 0.01 g/L to about 15 g/L; about 0.05 g/L to about 15 g/L; about 0.1 g/L to about 10 g/L; about 1 g/L to about 8 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 PanK 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 g/L, 2 g/L, 5 g/L, 10 g/L, or 15 g/L.

[0145] In some embodiments, the reaction conditions also comprise a divalent metal capable of serving as a cofactor in the reaction. Generally, the divalent metal co-factor is magnesium (i.e., Mg.sup.+2). The magnesium ion may be provided in various forms, such as magnesium chloride (MgCl.sub.2). While magnesium ion is the metal co-factor found in the naturally occurring PanK enzyme and functions efficiently in the engineered enzymes, it is to be understood that other divalent metals capable of acting as a co-factor can be used in the processes. In some embodiments, the reaction conditions can comprises a divalent metal cofactor, particularly Mg, at a concentration of about 1 mM to 1 M, 1 mM to 100 mM, 1 mM to about 50 mM, 25 mM to about 35 mM, about 30 mM to about 60 mM or about 55 mM to about 65 mM. In some embodiments, the reaction conditions comprise a divalent metal co-factor concentration of about 1 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

[0146] 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 buffer is tris. In some embodiments of the process, the suitable reaction conditions comprise a buffer (e.g., tris) concentration of from about 0.01 to about 0.4 M, 0.05 to about 0.4 M, 0.1 to about 0.3 M, or about 0.1 to about 0.2 M. In some embodiments, the reaction condition comprises a buffer (e.g., tris) concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.07, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.3, or 0.4 M.

[0147] In some embodiments, the reaction condition comprises a wet organic solvent. Suitable wet organic solvents are known in the art and include, by way of example and not limitation, wet isopropyl alcohol, wet toluene, and wet methyl tertiary butyl ether.

[0148] In the embodiments of the process, the reaction conditions can 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

[0149] In the embodiments of the processes herein, a suitable temperature can be 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.

[0150] In some embodiments, the reaction conditions can 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, 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.

[0151] In some embodiments, the reaction conditions can 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 (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.

[0152] The quantities of reactants used in the kinase reaction will generally vary depending on the quantities of product desired, and concomitantly the amount of PanK 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.

[0153] 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, PanK enzyme, and substrate may be added first to the solvent.

[0154] 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.

[0155] For improved mixing efficiency when an aqueous co-solvent system is used, the PanK enzyme, and cofactor may be added and mixed into the aqueous phase first. The organic phase may then be added and mixed in, followed by addition of the PanK enzyme substrate and co-substrate. Alternatively, the PanK enzyme substrate may be premixed in the organic phase, prior to addition to the aqueous phase.

[0156] The phosphorylation process is generally allowed to proceed until further conversion of substrate to phosphorylated 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.

[0157] In some embodiments of the process, the suitable reaction conditions comprise a substrate loading of at least about 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 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 48 h or less, in about 36 h or less, in about 24 h or less, or in about 3 h or less.

[0158] In further embodiments of the processes for converting substrate compound to product compound using the engineered PanK polypeptides, the suitable reaction conditions can comprise an initial substrate loading 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. Thus, according to these suitable reaction conditions, polypeptide is added to a solution having an initial substrate loading of at least about 20 g/L, 30 g/L, or 40 g/L. 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 until a much higher final substrate loading of at least about 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L, 150 g/L, 200 g/L or more, 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 20 g/L, 30 g/L, or 40 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, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L or more, is reached. This substrate supplementation reaction condition allows for higher substrate loadings to be achieved while maintaining high rates of conversion of substrate to phosphorylated product of at least about 50%, 60%, 70%, 80%, 90% or greater conversion of substrate.

[0159] In some embodiments, acetate kinase and acetyl phosphate recycle ADP to ATP. In some embodiments, acetate kinase and acetyl phosphate recycle an ADP analogue to an ATP analogue.

[0160] In some embodiments of the processes, the reaction using an engineered PanK polypeptide can comprise the following suitable reaction conditions: (a) substrate loading at about 50 g/L; (b) about 0.5 g/L of the engineered polypeptide; (c) 1.25 eq acetyl phosphate; (d) about 0.1 mol % ATP; (e) about 10 mM MgCl.sub.2; (f) about 0.125 mg/mL acetate kinase; (g) a pH of about 8.0; (h) temperature of about 25 C.; and (i) reaction time of about 18 hrs.

[0161] In some embodiments, additional reaction components or additional techniques are 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 product formation.

[0162] In 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; 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.

[0163] Various features and embodiments of the invention are illustrated in the following representative examples, which are intended to be illustrative, and not limiting.

Engineered PanK Polynucleotides Encoding Engineered Polypeptides, Expression Vectors and Host Cells

[0164] 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) are introduced into appropriate host cells to express the corresponding enzyme polypeptide(s).

[0165] 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., PanK) 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).

[0166] 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.

[0167] 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 comprises SEQ ID NO: 4, while in some other embodiments, the reference polypeptide sequence comprises SEQ ID NO: 44, SEQ ID NO: 320, and/or SEQ ID NO: 526.

[0168] 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.

[0169] 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]).

[0170] 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).

[0171] 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 find 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).

[0172] 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. Biol., 15:5983-5990 [1995]).

[0173] 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 NC1B 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.

[0174] 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.

[0175] 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 GALL 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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 on or the origins of replication of plasmids pBR322, pUC19, pACYC177 (which plasmid has the PISA 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 its functioning temperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433 [1978]).

[0184] 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.

[0185] 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 p3xFLAGTM 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 pBluescriptll 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]).

[0186] Thus, in some embodiments, a vector comprising a sequence encoding at least one variant pantothenate kinase is transformed into a host cell in order to allow propagation of the vector and expression of the variant pantothenate kinase(s). In some embodiments, the variant pantothenate kinases 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 pantothenate kinase(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).

[0187] In another aspect, the present invention provides host cells comprising a polynucleotide encoding an improved pantothenate kinase polypeptide provided herein, the polynucleotide being operatively linked to one or more control sequences for expression of the pantothenate kinase enzyme in the host cell. Host cells for use in expressing the pantothenate kinase 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.

[0188] Polynucleotides for expression of the pantothenate kinase 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.

[0189] 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.

[0190] 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, 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.

[0191] 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 pyperi, Pichia Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia hpolytica.

[0192] 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).

[0193] 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. anthracia, 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. herbi cola, 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).

[0194] 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).

[0195] 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 pantothenate kinase variant(s) within the host cell and/or in the culture medium. For example, knockout of Alp 1 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).

[0196] 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 herein) finds use.

[0197] 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 pantothenate kinase 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.

[0198] In some embodiments, cells expressing the variant pantothenate kinase 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 are 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.

[0199] In some embodiments of the present invention, cell-free transcription/translation systems find use in producing variant pantothenate kinase(s). Several systems are commercially available and the methods are well-known to those skilled in the art.

[0200] The present invention provides methods of making variant pantothenate kinase 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 NO: 4, SEQ ID NO: 44, SEQ ID NO: 320, and/or SEQ ID NO: 526, 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 pantothenate kinase polypeptide; and optionally recovering or isolating the expressed variant pantothenate kinase polypeptide, and/or recovering or isolating the culture medium containing the expressed variant pantothenate kinase polypeptide. In some embodiments, the methods further provide optionally lysing the transformed host cells after expressing the encoded pantothenate kinase polypeptide and optionally recovering and/or isolating the expressed variant pantothenate kinase polypeptide from the cell lysate. The present invention further provides methods of making a variant pantothenate kinase polypeptide comprising cultivating a host cell transformed with a variant pantothenate kinase polypeptide under conditions suitable for the production of the variant pantothenate kinase polypeptide and recovering the variant pantothenate kinase polypeptide. Typically, recovery or isolation of the pantothenate kinase 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.

[0201] Engineered pantothenate kinase 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 BTM (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.

[0202] Chromatographic techniques for isolation of the pantothenate kinase 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.

[0203] In some embodiments, affinity techniques find use in isolating the improved pantothenate kinase enzymes. For affinity chromatography purification, any antibody which specifically binds the pantothenate kinase 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 pantothenate kinase. The pantothenate kinase 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.

[0204] In some embodiments, the pantothenate kinase 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 pantothenate kinase variants are prepared as lyophilisates, in powder form (e.g., acetone powders), or prepared as enzyme solutions. In some embodiments, the pantothenate kinase variants are in the form of substantially pure preparations.

[0205] In some embodiments, the pantothenate kinase 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.

[0206] In some embodiments, immunological methods are used to purify pantothenate kinase variants. In one approach, antibody raised against a variant pantothenate kinase polypeptide (e.g., against a polypeptide comprising SEQ ID NO: 4, SEQ ID NO: 44, SEQ ID NO: 320, and/or SEQ ID NO: 526, and/or an immunogenic fragment thereof) using conventional methods is immobilized on beads, mixed with cell culture media under conditions in which the variant pantothenate kinase is bound, and precipitated. In a related approach, immunochromatography finds use.

[0207] In some embodiments, the variant pantothenate kinases are expressed as a fusion protein including a non-enzyme portion. In some embodiments, the variant pantothenate kinase sequence 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 pantothenate kinase polypeptide from the fusion protein. pGEX vectors (Promega) may also be used to express foreign polypeptides as fusion proteins with glutathione S-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.

[0208] 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.

[0209] 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.

[0210] 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

[0211] 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.

[0212] In the experimental disclosure below, the following abbreviations apply: M (molar); mM (millimolar), uM and M (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and g (micrograms); L and 1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); um and tri (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); RH (relative humidity); CV (coefficient of variability); CAM and cam (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropyl -D-1-thiogalactopyranoside); LB (Luria 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, CT); 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, MI); Microfluidics (Microfluidics, Westwood, MA); Life Technologies (Life Technologies, a part of Fisher Scientific, Waltham, MA); Amresco (Amresco, LLC, Solon, OH); Carbosynth (Carbosynth, Ltd., Berkshire, UK); Varian (Varian Medical Systems, Palo Alto, CA); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Infors (Infors USA Inc., Annapolis Junction, MD); and Thermotron (Thermotron, Inc., Holland, MI).

Example 1

E. coli Expression Hosts Containing Recombinant PanK Genes

[0213] The initial engineered PanK enzyme used to produce the variants of the present invention, SEQ ID NO: 4, was cloned into the expression vector pCK110900 (See, FIG. 3 of US Pat. Appln. Publn. No. 2006/0195947) operatively linked to the lac promoter under control of the lacl repressor. The expression vector also contains the P15a origin of replication and the chloramphenicol resistance gene. The resulting plasmids were transformed into E. coli W3110, using standard methods known in the art. The transformants were isolated by subjecting the cells to chloramphenicol selection, as known in the art (See e.g., U.S. Pat. No. 8,383,346 and WO2010/144103).

Example 2

Preparation of HTP PanK-Containing Wet Cell Pellets

[0214] E. coli cells containing recombinant PanK-encoding genes from monoclonal colonies were inoculated into 180 l LB containing 1% glucose and 30 g/mL chloramphenicol (CAM) in the wells of 96-well, shallow-well microtiter plates. The plates were sealed with O.sub.2-permeable seals, and cultures were grown overnight at 30 C., 200 rpm, and 85% humidity. Then, 10 l of each of the cell cultures were transferred into the wells of 96-well, deep-well plates containing 390 mL TB and 30 g/mL CAM. The deep-well plates were sealed with O.sub.2-permeable seals and incubated at 30 C., 250 rpm, and 85% humidity until OD.sub.600 0.6-0.8 was reached. The cell cultures were then induced by IPTG to a final concentration of 1 mM and incubated overnight under the same conditions as originally used. The cells were then pelleted using centrifugation at 4,000 rpm for 10 min. The supernatants were discarded, and the pellets were frozen at 80 C. prior to lysis.

Example 3

Preparation of HTP PanK-Containing Cell Lysates

[0215] First, 200 IA lysis buffer containing 20 mM potassium phosphate buffer, pH 7.5, 1 mg/mL lysozyme, and 0.5 mg/mL PMBS were added to the cell paste in each well, produced as described in Example 2. The cells were lysed at room temperature for 2 hours with shaking on a bench top shaker. The plate was then centrifuged for 15 min at 4,000 rpm and 4 C. The clear supernatants were used in biocatalytic reactions to determine their activity levels.

Example 4

Preparation of Lyophilized Lysates from Shake Flask (SF) Cultures

[0216] Selected HTP cultures grown as described above were plated onto LB agar plates with 1% glucose and 30 g/ml CAM and were grown overnight at 37 C. A single colony from each culture was transferred to 6 ml of LB with 1% glucose and 30 g/ml CAM. The cultures were grown for 18 h at 30 C., 250 rpm, and subcultured approximately 1:50 into 250 ml of TB containing 30 g/ml CAM, to a final OD.sub.600 of 0.05. The cultures were grown for approximately 195 minutes at 30 C. and 250 rpm, to an OD.sub.600 between 0.6-0.8 and induced with 1 mM IPTG. The cultures were then grown for 20 h at 30 C. and 250 rpm. The cultures were centrifuged at 4,000 rpm for 20 min. The supernatant was discarded, and the pellets were resuspended in 30 ml of 25 mM potassium phosphate buffer, pH 7.5. The cells were pelleted (4,000 rpm for 20 min) and frozen at 80 C. for 120 minutes. Frozen pellets were resuspended in 30 ml of 25 mM potassium phosphate buffer, pH 7.5, and lysed using a Microfluidizer system (Microfluidics) at 18,000 psi. The lysates were pelleted (10,000 rpm for 60 min), and the supernatants were frozen and lyophilized to generate shake flake (SF) enzymes.

Example 5

Improvements Over SEQ ID NO: 4 in the Phosphorylation Activity of 2-Ethynyl Glycerol

[0217] SEQ ID NO: 4 was selected as the parent enzyme after screening variants disclosed in U.S. patent application Ser. No. 16/460,147 for the phosphorylation activity of ethynyl glycerol substrate. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3. Each variant was screened in a 50 L reaction that comprised of 50 mg/mL 2-ethynylglycerol (EGO) substrate, 0.1 mol % ATP, 0.25 mg/mL acetate kinase (SEQ ID NO: 632), 1.25 eq (NH.sub.4).sub.2 SEQ ID NO: 4 was selected as the parent enzyme after screening variants disclosed in U.S. patent application Ser. No. 16/460,147 for the phosphorylation activity of ethynyl glycerol substrate. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3. Each variant was screened in a 50 L reaction that comprised of 50 mg/mL 2-ethynylglycerol (EGO) substrate, 0.1 mol % ATP, 0.25 mg/mL acetate kinase (SEQ ID NO: 632), 1.25 eq (NH.sub.4).sub.2acetyl phosphate (AcP), 10 mM MgCl.sub.2 in 50 mM Bis-Tris buffer, pH 7.5 and 32 diluted soluble lysate for 15 hours at 30 C. The 96-well plates were heat-sealed and incubated in a Thermotron shaker at 600 rpm to produce a chromophore containing species and enable simple reaction monitoring. The reaction samples were derivatized using tripotassium 5,5,5 42,2,2-nitrilotris(methylene-tris(1H-benzimidazole-2,1-diyOltripentanoate hydrate ((BimC4A)3) at the following conditions 5 equivalents (eq) benzyl azide, 5 mol % Copper sulfate, 7.5 mol % (BimC4A)3, 20 mol % sodium ascorbate, 9:1 water:DMSO to achieve click chemistry. Post reaction, 5 uL of reaction were combined with 220 uL of (BimC4A)3 derivatization solution in new 96-well plates. The click chemistry reaction was incubated for 1 hour at 45 C. The samples were then filtered by centrifugation using 0.22 micron 96-well filter plates in preparation for analysis by UPLC-UV (Example 9) or RapidFire MS (Example 10).

[0218] Activity relative to SEQ ID NO: 4 (Activity FIOP) was calculated as the percent conversion of the product formed by the variant over the percent conversion produced by SEQ ID NO: 4 and shown in Table 5.1. The percent conversion was calculated by dividing the area of the product peak by the sum of the areas of the substrate, product and impurities/side product peaks as observed by the UPLC-UV analysis (Example 9).

TABLE-US-00001 TABLE 5.1 Activity of Variants Relative to SEQ ID NO: 4 Activity FIOP 1 SEQ ID NO: Amino Acid Differences (Relative to (nt/aa) (Relative to SEQ ID NO: 4) SEQ ID NO: 4) 5/6 K154H +++ 7/8 H141L + 9/10 I287L + 11/12 Y250F + 13/14 I251L + 15/16 K289A + 17/18 K154R + 19/20 I103V + 21/22 K297V + 23/24 E290G + 25/26 T135S + 27/28 I193V + 29/30 Q247T + 31/32 S216G + 33/34 Y238F + 35/36 K289S + 37/38 T248S + 39/40 T248A + 41/42 K297R + 43/44 I103V/T135S/H141L +++ 45/46 T135S/H141L/Y238F ++ 47/48 A315L + 49/50 I103V/H141L/S216G + 51/52 V33I + 53/54 N79S + 55/56 T135S/I193V/Y238F/K289A/ + M291K 57/58 I103V/H141L/Y238F + 59/60 S293L + 61/62 R129S + 63/64 T135S + 65/66 R129G + 67/68 A120T + 69/70 R96P + 71/72 V227T + 73/74 D195A + 75/76 I103V/T135S/S216G + 77/78 P194H + 79/80 R34A + 81/82 M19L + 83/84 H222G + 85/86 I103V/H141L/I193V/S216G/ + Y238F 87/88 I103V/T135S + 89/90 I103V/H141L + 91/92 T135S/R323C + 93/94 M218C + 95/96 I193V/S216G + 97/98 E63L + 99/100 K274A + 101/102 T135S/I251L/R323C + 103/104 A31R + 105/106 D197G + 107/108 T135S/H141L/Q298A/R323C + 109/110 R82T + 111/112 T286D + 113/114 N37F + 115/116 S77L + 117/118 N73G + 119/120 T135S/D219G + 121/122 H141L/K297V + 123/124 D195G + 125/126 D44W + 127/128 M291G + 129/130 P221E + 131/132 R130Y + 133/134 T17V + 135/136 D35R + 137/138 Q85S + 139/140 R124G + 141/142 P126S + 143/144 N79A + 145/146 P126V + 147/148 I46K + 149/150 H141L/I193V/I251L/R323C + 151/152 T135S/T248A/I251L/M291K/ + K297R 153/154 F74L + 155/156 H128S + 157/158 D197V + 159/160 S308A + 161/162 P221S + 163/164 K198R + 165/166 H141L/I251L/R323C + 167/168 P126Q + 169/170 V145I + 171/172 G91C + 173/174 H141L/R323C + 175/176 T135S/Y238F/M291K + 177/178 H141L/I251L + 179/180 N79Y + 181/182 I251L + 183/184 G94S + 185/186 H225Y + 187/188 I103V/T135S/M291K + 189/190 V233C + 191/192 L80E + 193/194 Q95G + 195/196 H164P + 197/198 R323C + 199/200 I103V/S216G/Y238F + 201/202 D35S + 203/204 I103V + 205/206 A120L + 207/208 I76V + 209/210 D26A + 211/212 D26T + 1 Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 4 and defined as follows: + > than 1-fold but less than 2.0-fold increased activity; ++ > than 2.0-fold but less than 4-fold increased activity; +++ > than 4-fold increased activity

Example 6

Improvements Over SEQ ID NO: 44 in the Phosphorylation Activity of 2-Ethynyl Glycerol

[0219] SEQ ID NO: 44 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3. Each variant was screened in a 50 L reaction that comprised of 50 mg/mL 2-ethynylglycerol (EGO) substrate, 0.1 mol % ATP, 0.25 mg/mL acetate kinase (SEQ ID NO: 632), 1.25 eq (NH.sub.4).sub.2AcP, 10 mM MgCl.sub.2 in 50 mM Bis-Tris buffer, pH 7.0 (final master mix pH 6.8) and 64 diluted soluble lysate for 15 hours at 30 C. The 96-well plates were heat-sealed and incubated in a Thermotron shaker at 600 rpm. To produce a chromophore containing species and enable simple reaction monitoring, the reaction samples were derivatized using tripotassium 5,5,5-[2,2,2-nitrilotris(methylene-tris(1H-benzimidazole-2,1-diyl)]tripentanoate hydrate ((BimC4A)3) at the following conditions 5 eq benzyl azide, 5 mol % Copper sulfate, 7.5 mol % (BimC4A)3, 20 mol % sodium ascorbate, 9:1 water:DMSO to achieve click chemistry. Post reaction, 5 uL of reaction were combined with 220 uL of (BimC4A)3 derivatization solution in new 96-well plates. The click chemistry reaction was incubated for 1 hour at 45 C. The samples were then filtered by centrifugation using 0.22 micron 96-well filter plates in preparation for analysis by UPLC-UV (Example 9) or RapidFire MS (Example 10).

[0220] Activity relative to SEQ ID NO: 44 (Activity FIOP) was calculated as the click product peak intensity, of each variant, per click product peak intensity of SEQ ID NO: 44 using Analytical Method 10.1, and the results are shown in Table 6.1.

TABLE-US-00002 TABLE 6.1 Activity of Variants Relative to SEQ ID NO: 44 SEQ ID Amino Acid Differences Activity FIOP .sup.1 (Relative NO: (nt/aa) (Relative to SEQ ID NO: 44) to SEQ ID NO: 44) 213/214 D35R/Q85S/M218C/H225Y +++ 215/216 D35R/M218C/S308A +++ 217/218 D35R/D197V/K274A +++ 219/220 D35R/F74L/M218C ++ 221/222 M19L/D35R/D197G/M218C/L275M ++ 223/224 T20G/H164S/N179V/S308M/E320K ++ 225/226 T20G/E59R/A120V/H164S/T263M/K282A ++ 227/228 T20G/S308M ++ 229/230 F74L/L141H ++ 231/232 S77L/M218C ++ 233/234 E59R/A120V ++ 235/236 T17V/E63L/R96P ++ 237/238 T20G/H164S + 239/240 M19L/Q85S/V145I/D197G/M218C/S308A + 241/242 T20G/K282A + 243/244 T17V/P126V/R129S/R130Y/H164P + 245/246 T17V/I46K/H128S/R129G/R130Y/H164P/I193V + 247/248 T17V/I46K/E63L/R96P + 249/250 T263M + 251/252 H164S/P221C/S308M + 253/254 M19L/L141H + 255/256 T17V/P126Q/R129G/H164P + 257/258 T20G/E59R/N93G + 259/260 I46K/H164P + 261/262 D35R/L141H/D195G/M218C/K274A + 263/264 E59R + 265/266 T17V/E63L/H164P + 267/268 M19L/F74L/V145I + 269/270 T17V/I46K/E63L + 271/272 V33I/D35S/I76V/L80E/P126S/P221E/H222G/V227T + 273/274 T17V/R34A/R96P/H128S/R129G/H164P + 275/276 L141H/D195G/D197G/M218C + 277/278 A31R/I76V/N79A/A120T/P126S/S293L + 279/280 A120V/H164S/K282A/E321L + 281/282 A31R/V33I/D35S/A120T/P126S/V227T + 283/284 T20G/E59A/N93G/L141H/E321L + 285/286 D35R/V145I/T286D + 287/288 S308A + 289/290 R34A/I46K/E63L/H164P + 291/292 T17V/I46K/E63L/R96P + 293/294 D35R + 295/296 T17V/E63L/R96P/H164P + 297/298 T17V/I46K/H164P + 299/300 T17V/E63L/N79S/H128S/R129G/R130Y/H164P + 301/302 M218C + 303/304 H225Y + 305/306 Q16R/N93G/A120V/P221C/S308M + 307/308 T17V/N79S/H128S/R129S/R130Y + 309/310 H128S/R129G/R130Y + 311/312 D35S/I76V/L80E/P126S + 313/314 R34A/N79S/P126Q/H128S/R130Y + 315/316 I46K/N79S + 317/318 T17V/P126V + 319/320 V33I/D35S/P126S/H222G/V227T + 321/322 D35S/G94S/A120T/P126S/S293L + 323/324 T17V/E63L + 325/326 E63L/H164P + 327/328 N79S/P126V/H164P + 329/330 D26T/V33I/L80E/P126S/M291G/S293L + 331/332 R34A/H164P + 333/334 M19L/S77L/L141H/M218C/H225Y + 335/336 G94S/P126S/S293L + 337/338 I46K/I193V + 339/340 V33I/N79A/P126S/P221E/V227T/S293L + 341/342 I46K/H128S/R129G/H164P + 343/344 V33F/D35S/P126S/S293P + 345/346 P126V/R129G/H164P + 347/348 T17V/E63L/N79S + 349/350 A32S/I46K + 351/352 L80E/P126S/P221E/V227T + 353/354 T17V/E63L/I193V + 355/356 D35R/S77L/M218C + 357/358 D26T/A31R/V33I/D35S/I76V/N79A/ + G94S/P221E/S293L 359/360 V33I/I76V/N79A + 361/362 I46K/R96P/H164P + 363/364 R34A/I46K/N79S/H164P + 365/366 R34A/E63L/P126V/R129S/I193V + 367/368 I46K/E63L + 369/370 K274A + 371/372 A31R/V33I/D35S/I76V/L141H/S293L + 373/374 E63L + 375/376 M19L/L141H/H225Y + 377/378 A31R/V33I/A120T + 379/380 D26T/A31R/V33I/I76V/N79A + 381/382 V33I/D35S/P126S/K198R/S293L + 383/384 A31R/V33I/I76V/N79A/L80E/G94S/M291G + 385/386 Q16R/L141H/N179V/K282A + 387/388 S308M + 389/390 D26T/A31R/A120T + 391/392 I46K/H164P/I193V + 393/394 I76V/N79A/L80E/A120T + 395/396 T17V/I46K/E63L/H164P + 397/398 F74L/Q85S/V145I + 399/400 A31R/I76V/N79A/S293L + 401/402 I46K/R129G + 403/404 D26T/V33I/D35S/P126S/H222G/M291G + 405/406 D35R/F74L/S77L/D197V/H225Y + 407/408 R34A/I46K/E63L + 409/410 P126V/H128S/R129G/I193V + 411/412 T17V/R34A/I46K + 413/414 A31R/V33I/D35S/I76V/N79A/G91C/M291G + 415/416 E63L/I193V + 417/418 V33I/D35S/P221E/H222G + 419/420 D35R/Q85S/M218C/K274A + 421/422 D26T/D35S/A120T/H222G/V227T/S293L + 423/424 D35S/I76V + 425/426 T17V + 427/428 T17V/E63L/R129S/R130Y + 429/430 V33I/I76V/N79A/P126S + 431/432 D26T/A31R/V33I/I76V/N79A/G94S + 433/434 R34A/I46K/N79S/R96P/P126V + 435/436 A31R/D35S/L80E/G94S/P126S/L141H/S293L + 437/438 T17V/I46K/P126V/H128S/R129S/H164P + 439/440 I46K/N79S/P126V/I193V + 441/442 V33I/D35S/I76V/N79A/A120T/L141H/K198R/P221E + 443/444 G94S/Q95G/A120T/M291G + 445/446 N79S/R96P/P126Q/R129S + 447/448 V33I/D35S + 449/450 D35R/Q85S/L141H/M218C + 451/452 T17V/I46K + 453/454 G94S/S293L + 455/456 R34A/I46K/H164P + 457/458 R34A/I46K + 459/460 A31R/V33I/D35S/P126S + 461/462 H164S + 463/464 R34A + 465/466 M19L/D35R/Q85S/K274A + 467/468 D26T/D35S/N79A/A120T/P126S/P194H/K198R + 469/470 T17V/R34A + 471/472 A31R/V331/I76V/N79A/Q95G + 473/474 D26T/V33I/G94S/S293L + 475/476 D35R/K274A + 477/478 D35R/Q85S + 479/480 A120V + 481/482 D26T/A31R/V33I/D35S/A120T/L141H/S293L + 483/484 D35S/I76V/N79A/L80E/P126S + 485/486 M19L/F74L/S77L/L141H/M218C + 487/488 S77L/Q85S/V145I + 489/490 D26T/D35S/P126S + 491/492 V33I/D35S/G94S/P126S/L141H + 493/494 M19L + 495/496 V33I/D35S/I76V/L80E/S293L + 497/498 R34A/I46K/R48C + 499/500 I46K + .sup.1 Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 44 and defined as follows: + > than 1-fold but less than 2.0-fold increased activity; ++ > than 2.0-fold but less than 4-fold increased activity; +++ > than 4-fold increased activity

Example 7

Improvements Over SEQ ID NO: 320 in the Phosphorylation Activity of 2-Ethynyl Glycerol and Stability of the PanK Enzyme

[0221] SEQ ID NO: 320 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3. Each variant was screened in a 50 L reaction that comprised of 50 mg/mL 2-ethynylglycerol (EGO) substrate, 0.1 mol % ATP, 0.125 mg/mL acetate kinase (SEQ ID NO: 634), 1.25 eq (NH.sub.4).sub.2AcP, 10 mM MgCl.sub.2 in 50 mM Bis-Tris buffer, pH 7.5 (final master mix pH 8.0) and 64 diluted soluble lysate for 15 hours at 30 C. For stability testing, the soluble lysates, generated as described in Example 3, were pre-incubated at 40 C. for 1.5 hours and then diluted 32 for screening. The 96-well plates were heat-sealed and incubated in a Thermotron shaker at 600 rpm. To produce a chromophore containing species and enable simple reaction monitoring, the reaction samples were derivatized using tripotassium 5,5,5-[2,2,2-nitrilotris(methylene-tris(1H-benzimidazole-2,1-diyl)]tripentanoate hydrate ((BimC4A)3) at the following conditions 5 eq benzyl azide, 5 mol % Copper sulfate, 7.5 mol % (BimC4A)3, 20 mol % sodium ascorbate, 9:1 water:DMSO to achieve click chemistry. Post reaction, 5 uL of reaction were combined with 220 uL of (BimC4A)3 derivatization solution in new 96-well plates. The click chemistry reaction was incubated for 1 hour at 45 C. The samples were then filtered by centrifugation using 0.22 micron 96-well filter plates in preparation for analysis by UPLC-UV (Example 9) or RapidFire MS (Example 10).

[0222] Activity and stability relative to SEQ ID NO: 320 (Activity FIOP) were calculated as the click product peak intensity, of each variant, per click product peak intensity of SEQ ID NO: 320 using Analytical method 10.1, and the results are shown in Table 7.1.

TABLE-US-00003 TABLE 7.1 Activity and Stability of Variants Relative to SEQ ID NO: 320 Activity FIOP .sup.1 Stability FIOP .sup.2 SEQ ID Amino Acid Differences (Relative to SEQ (Relative to SEQ NO: (nt/aa) (Relative to SEQ ID NO: 320) ID NO: 320) ID NO: 320) 501/502 T20G/G94S/A120T + + 503/504 R48C/E63L + + 505/506 T20G/R48C/M218C/T227V + + 507/508 G94S/A120T + + 509/510 T20G/A31R/S35D/R48C/E63L/F74L/ + + G94S/A120T/H164P 511/512 E63L + + 513/514 E63L/A120T/D197G/M218C/L275M + + 515/516 E63L/R129S/V145I + + 517/518 R48C/E63L/V145I + + 519/520 T20G/A120T/V145I/D197G/T227V ++ + 521/522 R48C/E63L/F74L/N79S/A120T/R129G/ + + T227V/K282A 523/524 F74L/N79A/G94S/L275M + + 525/526 R48C/E63L/G94S/L275M ++ + 527/528 T20G/S35D/A120T/D197G + + 529/530 E63L/M218C/L275M/K282A + + 531/532 T20G/R48C/E63L/G94S/A120T/V145I/ + + D197G/M218C/T227V 533/534 T20G/A31R/S35D/F74L/N79A/H164S/ + + M218C/H225Y/K282A 535/536 T20G/D197G/H225Y/T227V + + 537/538 Q95D + + 539/540 Q95E + 541/542 K289C + 543/544 I33V + 545/546 G271M + + 547/548 Q24H + 549/550 P178H + 551/552 G196I + + 553/554 K277R + + 555/556 P178D + + 557/558 Q24V + ++ 559/560 V201G + 561/562 P221G ++ 563/564 R130L + 565/566 I46A + 567/568 S293Y + + 569/570 E321L + + 571/572 R149A ++ + 573/574 H164G ++ + 575/576 Q298E ++ + 577/578 K282D + ++ 579/580 S314G ++ + 581/582 Q298D + ++ 583/584 R48V + 585/586 G271C + + .sup.1 Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 320 and defined as follows: + > than 1-fold but less than 1.75-fold increased activity; ++ > than 1.75-fold .sup.2 Levels of increased stability were determined relative to the reference polypeptide of SEQ ID NO: 320 and defined as follows: + > than 1-fold but less than 1.75-fold increased activity; ++ > than 1.75-fold

Example 8

Improvements Over SEQ ID NO: 526 in the Phosphorylation Activity of 2-Ethynyl Glycerol and Stability of the PanK Enzyme

[0223] SEQ ID NO: 526 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 2, and the soluble lysate was generated as described in Example 3. Each variant was screened in a 50 L reaction that comprised of 50 mg/mL 2-ethynylglycerol (EGO) substrate, 0.1 mol % ATP, 0.125 mg/mL acetate kinase (SEQ ID NO: 634), 1.25 eq (NH.sub.4).sub.2AcP, 10 mM MgCl.sub.2 in 50 mM Bis-Tris buffer, pH 7.5 (final master mix pH 8.0) and 128 diluted soluble lysate for 15 hours at 30 C. For stability testing, the soluble lysates, generated as described in Example 3, were pre-incubated at 45 C. for 1.5 hours and then diluted 64 for screening. The 96-well plates were heat-sealed and incubated in a Thermotron shaker at 600 rpm. To produce a chromophore containing species and enable simple reaction monitoring, the reaction samples were derivatized using tripotassium 5,5,5-[2,2,2-nitrilotris(methylene-tris(1H-benzimidazole-2,1-diyl)]tripentanoate hydrate ((BimC4A)3) at the following conditions 5 eq benzyl azide, 5 mol % Copper sulfate, 7.5 mol % (BimC4A)3, 20 mol % sodium ascorbate, 9:1 water:DMSO to achieve click chemistry. Post reaction, 5 uL of reaction were combined with 220 uL of (BimC4A)3 derivatization solution in new 96-well plates. The click chemistry reaction was incubated for 1 hour at 45 C. The samples were then filtered by centrifugation using 0.22 micron 96-well filter plates in preparation for analysis by UPLC-UV (Example 9) or RapidFire MS (Example 10).

[0224] Activity and stability relative to SEQ ID NO: 526 (Activity FIOP) were calculated as the click product peak intensity, of each variant, per click product peak intensity of SEQ ID NO: 526 using Analytical method 10.1, and the results are shown in Table 8.1.

TABLE-US-00004 TABLE 8.1 Activity and Stability of Variants Relative to SEQ ID NO: 526 Activity FIOP .sup.1 Stability FIOP .sup.2 SEQ ID Amino Acid Differences (Relative to SEQ (Relative to SEQ NO: (nt/aa) (Relative to SEQ ID NO: 526) ID NO: 526) ID NO: 526) 587/588 I33V/Q95N/A104T ++ + 589/590 P221G/K289C ++ + 591/592 Q24H/I46V/P221G + 593/594 Q24H/T227V + 595/596 I46V/Q95E/K282A + + 597/598 Q24H/I33V + + 599/600 C48V + + 601/602 V201G + + 603/604 P178D/S293Y/Q298D + + 605/606 Q95D/V201G/K282D/S293Y/S314G + + 607/608 Q24V/V201G/K282D + + 609/610 H164G/V201G/G271C/S314G + + 611/612 Q298D + + 613/614 Q24V/R149A/V201G/K282D + + 615/616 Q24V/Q95D/H164G/P178D/V201G/K282D + ++ 617/618 Q24V/P178D/V201G/G271C/K282D/ + + S293Y/Q298E/S314G 619/620 Q24V + + 621/622 Q24V/R149A/P178D/K282D/S293Y/ + + Q298E 623/624 K282D/S293Y/Q298E/S314G/E321L + + 625/626 V201G/K282D + + 627/628 Q24V/V201G/G271C/K282D/S293Y/ + + Q298E 629/630 Q24V/R149A/K282D/S314G + + .sup.1 Levels of increased activity were determined relative to the reference polypeptide of SEQ ID NO: 526 and defined as follows: + > than 1.2-fold but less than 1.70-fold increased activity; ++ > than 1.70-fold .sup.2 Levels of increased stability were determined relative to the reference polypeptide of SEQ ID NO: 526 and defined as follows: + > than 1.2-fold but less than 1.70-fold increased activity; ++ > than 1.70-fold

Example 9

Analytical Detection of (BimC4A)3-Derived 2-Ethynylglycerol Phosphate by UPLC-UV

[0225] Data described in Example 5 was collected using the analytical method provided in Table 9.1. The method provided herein finds use in analyzing the variants produced using the present invention. However, it is not intended that present invention be limited to the methods described herein, as there are other suitable methods known in the art that are applicable to the analysis of the variants provided herein and/or produced using the methods provided herein.

TABLE-US-00005 TABLE 9.1 Analytical Method Instrument Agilent 1290 - UPLC Column Waters Acquity HSS T3, 2.1 50 mm, 1.8 um Mobile Phase A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile Gradient Time (min) % A % B 0 98 2 0.1 98 2 0.7 80 20 1.1 0 100 1.5 98 2 2.0 98 2 Flow Rate 1.25 mL/min Run Time 2.0 min Product Elution order (BimC4A)3 -Derived 2-Ethynylglycerol phosphate: ~0.98 min 2- ethynylglycerol: ~1.35 min Column Temperature 40 C. Injection Volume 5 L Detection UV 210 nm

Example 10

Analytical Detection of (BimC4A)3-Derived 2-Ethynylglycerol Phosphate by Rapid Fire-MS

[0226] Data described in Examples 6, 7, and 8 were collected using the analytical method provided in Table 10.1. The method provided herein finds use in analyzing the variants produced using the present invention. However, it is not intended that present invention be limited to the methods described herein, as there are other suitable methods known in the art that are applicable to the analysis of the variants provided herein and/or produced using the methods provided herein.

TABLE-US-00006 TABLE 10.1 Analytical Method Instrument Agilent RapidFire Pump1 Buffer 0.1% Formic water LCMS grade water; 1.5 mL/min flow rate Pump2 Buffer ACC Juice (50% water LCMS grade, 25% acetonitrile LCMS grade, 25% acetone GCMS grade) + 100 um ammonium acetate ; 1.25 mL/min flow rate Pump3 Buffer ACC Juice (50% water LCMS grade, 25% acetonitrile LCMS grade, 25% acetone GCMS grade) + 100 um ammonium acetate; 1.25 mL/min flow rate Aqueous wash Water Organic wash Acetonitrile SPE cartridge C18 RF state 1 Aspirate 600 ms RF state 2 Load/Wash 3000 ms RF state 3 Extra Wash 0 RF state 4 Elute 6500 ms RF state 5 Reequilibrate 1000 ms Agilent Jet Stream source parameters Drying gas temperature .sup.350 C. Drying gas flow 13 L/min Nebulizer pressure 45 psi Sheath gas temperature .sup.330 C. Sheath gas flow 12 L/min Capillary voltage 2500 V Nozzle voltage 2500 V Agilent 6470 Triple Quadrupole MRM parameters Compound Q1 Q3 Dwell Fragmentor CE CAV (BimC4A)3 -Derived 2- 330 231.8 109 80 10 3 Ethynylglycerol phosphate (BimC4A)3 -Derived 2- 330 214 109 80 10 3 Ethynylglycerol phosphate (BimC4A)3 -Derived 2- 330 90.8 109 80 30 3 Ethynylglycerol phosphate