The present disclosure relates to methods of using transaminase polypeptides in the synthesis of chiral amines from prochiral ketones.

The present invention provides engineered transaminase polypeptides useful for the synthesis of chiral amine compounds under industrially relevant conditions. The invention also provides polynucleotides encoding the engineered transaminase polypeptides, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases for the production of chiral amine compounds.

The present invention discloses a ω-transaminase mutant obtained through DNA synthetic shuffling combined mutation. The ω-transaminase mutant is obtained through point mutation of a wild type ω-transaminase from Aspergillus terrus. The amino acid sequence of the wild type ω-transaminase is shown in SEQ ID NO: 1. The mutation site of the ω-transaminase mutant is any one of: (1) F115L-H210N-M150C-M280C; (2) F115L-H210N; (3) F115L-H210N-E253A-I295V; (4) I77L-F115L-E133A-H210N-N245D; (5) I77L-Q97E-F115L-L118T-E253A-G292D; (6) I77L-E133A-N245D-G292D; and (7) H210N-N245D-E253A-G292D. According to the present invention, forward mutations obtained in the previous stage are randomly combined through a DNA synthetic shuffling combined mutation method. It is verified through experiments that this method can effectively improve the probability of forward mutation and increase experimental efficiency and feasibility, and is capable of obtaining mutant enzymes with thermodynamic stability remarkably superior to that of wild enzymes via screening.

A microorganism is transformed to be capable of producing guanidinoacetic acid (GAA). A method can be used for the fermentative production of GAA using such a microorganism. A corresponding method can be used for the fermentative production of creatine.

The present disclosure provides engineered transaminase polypeptides for the production of amines, polynucleotides encoding the engineered transaminases, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases to prepare compounds useful in the production of active pharmaceutical agents.

A non-naturally occurring microbial organism includes a microbial organism having a 1,3-butanediol (1,3-BDO) pathway having at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme expressed in a sufficient amount to produce 1,3-BDO. The pathway includes an enzyme selected from a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP dehydrogenase, a 2-amino-4-hydroxypentanoate aminotransferase, a 2-amino-4-hydroxypentanoate oxidoreductase (deaminating), a 2-oxo-4-hydroxypentanoate decarboxylase, a 3-hydroxybutyraldehyde reductase, an AKP aminotransferase, an AKP oxidoreductase (deaminating), a 2,4-dioxopentanoate decarboxylase, a 3-oxobutyraldehyde reductase (ketone reducing), a 3-oxobutyraldehyde reductase (aldehyde reducing), a 4-hydroxy-2-butanone reductase, an AKP decarboxylase, a 4-aminobutan-2-one aminotransferase, a 4-aminobutan-2-one oxidoreductase (deaminating), a 4-aminobutan-2-one ammonia-lyase, a butenone hydratase, an AKP ammonia-lyase, an acetylacrylate decarboxylase, an acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), an acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), an acetoacetyl-CoA reductase (ketone reducing), a 3-hydroxybutyryl-CoA reductase (aldehyde forming), a 3-hydroxybutyryl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA dehydratase, and a crotonase. A method for producing 1,3-BDO, includes culturing such microbial organisms under conditions and for a sufficient period of time to produce 1,3-BDO.

The present disclosure provides engineered transaminase polypeptides for the production of amines, polynucleotides encoding the engineered transaminases, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases to prepare compounds useful in the production of active pharmaceutical agents.

The present disclosure provides engineered transaminase polypeptides useful for the synthesis of chiral amine compounds under industrially relevant conditions. The disclosure also provides polynucleotides encoding the engineered transaminase polypeptides, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases for the production of chiral amine compounds.

The present disclosure provides engineered transaminase polypeptides for the production of amines, polynucleotides encoding the engineered transaminases, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases to prepare compounds useful in the production of active pharmaceutical agents.

The present disclosure relates to transaminase polypeptides capable of aminating a dicarbonyl substrate, and polynucleotides, vectors, host cells, and methods of making and using the transaminase polypeptides.