Engineered phosphopentomutase variant enzymes
The present invention provides engineered phosphopentomutase (PPM) enzymes, polypeptides having PPM activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing PPM enzymes are also provided. The present invention further provides compositions comprising the PPM enzymes and methods of using the engineered PPM enzymes. The present invention finds particular use in the production of pharmaceutical compounds.
1 . An engineered phosphopentomutase comprising a polypeptide sequence having at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4, and/or 12 or a functional fragment thereof, wherein the polypeptide sequence of said engineered phosphopentomutase comprises the substitution set 22/188/257/266/341 and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4.
2 . The engineered phosphopentomutase of claim 1 , wherein said polypeptide sequence has at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:4, and wherein the polypeptide sequence of said engineered phosphopentomutase comprises the substitution set 22K/188E/257V/266G/341L wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 4.
3 . The engineered phosphopentomutase of claim 1 , wherein said polypeptide sequence of said engineered phosphopentomutase has at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 12, and wherein said polypeptide sequence of said engineered phosphopentomutase further comprises at least one substitution or substitution set at one or more positions selected from 118, 150, 155, 191, 235, 238, 239, 244, 256, 263, 265, 267, 284, and 355, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 12.
4 . The engineered phosphopentomutase of claim 1 , wherein said engineered phosphopentomutase comprises a polypeptide sequence that is at least 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered phosphopentomutase variant set forth in the even numbered sequences of SEQ ID NOS: 12 and 36-88.
5 . The engineered phosphopentomutase of claim 1 , wherein said engineered phosphopentomutase comprises a polypeptide sequence forth in at least one of the even numbered sequences of SEQ ID NOS: 12 and 36-88.
6 . The engineered phosphopentomutase of claim 1 , wherein said engineered phosphopentomutase comprises at least one improved property compared to the phosphopentomutase set forth in SEQ ID NO:4.
7 . The engineered phosphopentomutase of claim 6 , wherein said improved property comprises improved activity on a substrate.
8 . The engineered phosphopentomutase of claim 5 , wherein said substrate comprises compound (4).
9 . The engineered phosphopentomutase of claim 1 , wherein said improved property comprises improved production of compound (1) and/or compound (3).
10 . The engineered phosphopentomutase of claim 1 , wherein said engineered phosphopentomutase is purified.
11 . A composition comprising at least one engineered phosphopentomutase of claim 1 .