FIELD OF THE INVENTION

The present invention provides engineered transglutaminase enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, methods of producing these enzymes, and methods of using the engineered transglutaminase enzymes.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently with the specification as an ASCII formatted text file via EFS-Web, with a file name of “CX2-164USP1_ST25.txt”, a creation date of Nov. 7, 2017, and a size of 1,896 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.

BACKGROUND OF THE INVENTION

Transglutaminases (TGase; EP 2.3.2.13)(R-gluaminyl-peptide-aminase-gamma-glutamyltransferase) comprise an enzyme family that catalyze post-translational modifications in proteins, producing covalent amide bonds between a primary amine group in a polyamine or lysine (i.e., an amine donor) and a gamma-carboxyamide group of the glutamyl residue of some proteins and polypeptides (i.e., an amine acceptor). The result of this enzymatic action include modification of the protein's conformation and/or extensive conformation changes resulting from the bonding of the same and different proteins to produce high molecular weight conjugates. These enzymes find use various applications, including in the food, cosmetic, textile, and pharmaceutical industries.

SUMMARY OF THE INVENTION

The present invention provides engineered transglutaminase enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered transglutaminase enzymes.

The present invention provides engineered transglutaminases having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, 6, 34, and/or 256. In some embodiments, the engineered transglutaminases have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6, and at least one substitution or substitution set at one or more positions selected from positions 79, 101, 101/201/212/287, 101/201/285, 101/287, and 327, wherein said positions are numbered with reference to SEQ ID NO:6. In some embodiments, the engineered transglutaminases have at least 85%%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, and at least one substitution or substitution set at one or more positions selected from positions 48, 48/67/70, 48/67/70/181/203/256, 48/67/70/181/256/345, 48/67/70/181/296/345/373, 48/67/70/203/256/296/345, 48/67/70/203/256/345/354/373, 48/67/70/203/345, 48/67/70/256, 48/67/70/256/296/345/373, 48/67/203/256/296/373, 48/67/203/256/345, 48/70/170/203, 48/70/203/254/296/343, 48/70/203/256/345/373, 48/70/203/256/345, 48/70/203/373, 48/170/203, 48/170/203/254/296/346, 48/170/203/254/296/346/373, 48/170/203/254/346/373, 48/170/203/254/346, 48/170/203/296/343/346, 48/170/203/296/346/373, 48/170/203/343/346, 48/170/203/346, 48/170/203/346/373, 48/170/203/373, 48/170/254, 48/170/296, 48/170/296/343/346, 48/170/343/346, 48/181, 48/181/203/256/345, 48/181/203/345, 48/181/256/296/345, 48/181/296, 48/181/296/345, 48/203, 48/203/254/296, 48/203/254/296/343/373, 48/203/254/296/346/373, 48/203/254/346, 48/203/254/346/373, 48/203/256, 48/203/256/296/345, 48/203/296/343/346/373, 48/203/296/343/373, 48/203/296/346, 48/203/296/346/373, 48/203/343/346, 48/203/343/346/373, 48/203/345, 48/203/346, 48/203/346/373, 48/254/296, 48/254/346, 48/256, 48/256/296, 48/256/296/345, 48/296/345, 48/296/373, 48/343/346, 48/345/373, 67/256, 67/296/345, 68/74/190/215/346, 68/136/215/255/282/297/346, 68/136/215/297/346, 68/136/234, 68/158/174/234/282/297/346, 68/158/215/297/346, 68/215/297/346, 68/234, 68/282/297/346, 68/297/346, 74/136/174/282/346, 74/136/174/297/346, 74/136/346, 74/158/255/297, 74/255/346, 74/346, 136/158/190/215/255/297/346, 136/158/215/297/346, 136/174/215/255/282/297/346, 136/190/215/297/346, 136/215/234/282/297, 136/215/234/297/346, 136/215/297, 136/297/346, 158/215/255/346, 158/215/346, 170/203/254/296/343/346, 170/203/254/343/373, 170/203/343/346, 174/190/234/297/346, 174/215/234/297/346, 174/215/255/297/346, 174/282/297/346, 190/255/282/346, 190/297/346, 203/296, 203/343, 203/343/346, 203/346, 215/255/297/346, 215/234/297/346, 215/255/297/346, 215/297, 215/297/346, 215/346, 234/255/346, 255/297/346, 255/346, 297/346, 343/346/373, and 346, wherein said positions are numbered with reference to SEQ ID NO:2. In some additional embodiments, the engineered transglutaminases have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, and at least one substitution or substitution set at one or more positions selected from 33/67/70/181/203/256/296/373, 36/48/203/254/346, 48/67/70/181/203/256/296/373, 48/67/70/203/256/296/373, 48/67/181/203/256/296/373, 48/67/181/203/256/373, 48/67/181/256/296, 48/67/203/256/296/373/378, 48/67/203/256/373, 48/67/203/296/373, 48/67/256/296/373, 48/70/181/203/256/296/373, 48/70/181/203/256/373, 48/70/181/203/296/373, 48/70/203/256/296/373, 48/70/203/256/373, 48/70/203/296, 48/70/203/296/373, 48/70/203/373, 48/70/256/296/373, 48/70/296/373, 48/176/203/254/346/373, 48/181/203/256/296/373, 48/181/203/256/373, 48/181/203/296, 48/181/203/373, 48/181/256/296/373, 48/203/254, 48/203/254/343, 48/203/254/343/346/373, 48/203/254/343/355/373, 48/203/254/343/373, 48/203/254/346/373, 48/203/254/373, 48/203/256/296, 48/203/256/296/373, 48/203/256/373, 48/203/296/373, 48/203/296/373/374, 48/203/343/373, 48/203/373, 48/254, 48/254/343/346/373, 48/254/343/373, 48/254/346/373, 48/254/373, 48/256/296/373, 48/256/373, 48/373, 67/70/181/203/256/296/373, 67/70/181/256/296/373, 67/70/181/373, 67/181/203/256/296, 67/181/203/256/296/373, 67/181/203/256/373, 67/203/256/296/373, 67/256/296/373, 70/181/203/256/296/373, 70/181/203/296/373, 70/203, 70/203/256/296/373, 70/203/256/373, 70/203/296/373, 74/136/215/234/282/297/346, 74/136/215/234/282/346, 74/136/215/234/297, 74/136/215/234/297/343/346, 74/136/215/234/297/346, 74/136/215/234/346, 74/136/215/282/297/346, 74/136/215/282/346, 74/136/215/297/346, 74/136/215/346, 74/136/234/282/297/346, 74/136/234/346, 74/136/282/297/346, 74/215, 74/215/234/282/297/346, 74/215/282/297/346, 74/215/346, 136/215/234/282/297/346, 136/215/282/297, 136/215/282/297/346, 136/215/282/346, 136/215/297/346, 136/215/346, 136/234/297, 136/234/297/346, 136/234/346, 136/282/297, 181/203/256, 181/203/256/296, 181/203/256/296/373, 181/203/256/373, 181/203/296/373, 181/203/373, 181/256/296/373, 181/296, 203/224/254/373, 203/254, 203/254/343/346/373, 203/254/343/373, 203/254/346, 203/254/346/373, 203/254/373, 203/346/373, 203/373, 203/209/256/373, 203/256, 203/256/296, 203/256/296/320/373, 203/256/296/373, 203/256/296/373/386, 203/256/373, 203/296/373, 203/373, 215/234/282/297/346, 215/234/282/346, 215/234/346, 234/282/346, 254, 254/346, 254/346/373, 254/373, 256/296, 256/296/373, 256/373, 282/297/346, 343/373, and 373, wherein said positions are numbered with reference to SEQ ID NO: 2. In some further embodiments, the engineered transglutaminases have at least 850/%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%6 or more sequence identity to SEQ ID NO: 34, and at least one substitution or substitution set at one or more positions selected from 48/49, 49, 50, 50, 331, 291, 292, 330, and 331, wherein said positions are numbered with reference to SEQ ID NO: 34. In yet some additional embodiments, the engineered transglutaminases have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 256, and at least one substitution or substitution set at one or more positions selected from 27/48/67/70/74/234/256/282/346/373, 27/48/67/70/136/203/215/256/282/346/373, 27/48/67/70/346/373, 27/48/67/74/203/256/346/373, 27/67/234/296/373, 45/287/328/333, 45/292/328, 48, 48/284/292/333, 48/287/292/297, 48/287/297/328/333, 48/292, 48/292/297, 48/49/50/292/331, 48/49/50/292, 48/49/50/331, 48/49/330/331, 48/49/50/349, 48/49/50/291/292/331, 48/49/50/292/331, 48/67/70/203/215/234/256/346, 48/67/70/234/256/282/297/346, 48/67/70/346, 48/67/74/203/234/256/282/346/373, 48/67/74/234/297/346/373, 48/67/74/346, 48/67/203/346/373, 48/67/234/256/297/346/373, 48/67/234/256/346/373, 48/67/215/282/297/346/373, 48/67/346/373, 48/70/74/297/346/373, 48/70/203/215/256/282/346/373, 48/70/215/234/256/346/373, 48/74/203/234/256/346/373, 48/74/234/256/297/346/373, 48/136/256/346/373, 48/203/234/256/297/346/373, 48/203/234/256/346/373, 48/203/234/346/373, 48/203/296/373, 48/215/234/346/373, 48/215/346/373, 48/234/256/296/346/373, 48/234/256/346/373, 48/256/373, 49/50/292/331, 49/50/292/331/349, 49/50/331, 49/50/331/349, 50, 67/70/74/136/203/215/256/346/373, 67/70/74/203/215/234/346/373, 67/70/74/215/234/297/346/373, 67/70/74/215/256/373, 67/70/136/203/297/346/373, 67/70/203/215/256/346/373, 67/70/203/373, 67/70/215, 67/74/136, 67/74/203/234/256, 67/74/215/256/297/346/373, 67/74/215/346/373, 67/74/256/346/373, 67/136/203/215/256/346/373, 67/136/203/256/346/373, 67/203/234/256/346/373, 67/203/297/346/373, 67/215/234/297/346/373, 67/297/346, 70/74/203/215/346/373, 136, 136/346/373, 203/234/346, 203/234/346/373, 203/373, 234/282, 287, 234/346/373, 287/292, 287/292/295/297, 287/292/297, 287/295/297, 287/330/333, 292, 292/297, 292/330/331, 292/330/331, 292/331, 292/331/349, 292/349, 295, 295/297/333, 297/328, 297/373, 328/333, 330, 330/331, 331, 331/349, 333, 346/373, and 373, wherein said positions are numbered with reference to SEQ ID NO: 256. In some embodiments, the engineered transglutaminases comprise a polypeptide sequence comprising a sequence having at least 90% sequence identity to SEQ ID NO:2, 6, 34, and/or 256. In some alternative embodiments, the engineered transglutaminases comprise a polypeptide sequence comprising a sequence having at least 95% sequence identity to SEQ ID NO:2, 6, 34, and/or 256. In yet some additional embodiments, the engineered transglutaminases comprise a polypeptide sequence set forth in SEQ ID NO:2, 6, 34, or 256. In yet some additional embodiments, the engineered transglutaminases comprise a polypeptide sequence encoding a variant provided in Table 8.1, 9.1, 9.2, 10.1, and/or 11.1. In some further embodiments, the engineered transglutaminase comprises a polypeptide sequence selected from the even-numbered sequences set from in SEQ ID NOS: 4 to 756.

The present invention also provides engineered polynucleotide sequences encoding the engineered transglutaminases provided herein. In some embodiments, the engineered polynucleotide sequences comprise polynucleotide sequences that are at least 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or more identical to a sequence selected from the odd-numbered sequences set forth in SEQ ID NOS: 3 to 755. The present invention also provides vectors comprising the engineered polynucleotide sequences provided herein. In some embodiments, the vectors further comprise at least one control sequence. The present invention also provides host cells comprising the vectors provided herein.

The present invention also provides methods for producing the engineered transglutaminases provided herein, comprising culturing a host cell under conditions that at least one engineered transglutaminase is produced by said host cell. In some embodiments, the host cell produces an engineered transglutaminase. In some embodiments, the methods further comprise the step of recovering said engineered transglutaminase produced by said host cell.

The present invention also provides engineered transglutaminases capable of modifying a free amine in insulin in the presence of a glutamine donor. In some embodiments, the engineered transglutaminases provided herein are capable of modifying a glutamine in insulin in the presence of a lysine donor.

The present invention also provides methods of modifying insulin comprising: providing insulin and at least one engineered transglutaminase provided herein, combining said insulin, glutamine, and at least one engineered transglutaminase under conditions such that said insulin is modified. The present invention also provides methods of modifying insulin comprising: providing insulin and at least one engineered transglutaminase provided herein, combining said insulin, lysine, and at least one engineered transglutaminase under conditions such that said insulin is modified.

