WO2021219124A1 - Modified threonine transaldolase and application thereof - Google Patents

Abstract

Provided is a modified L-threonine transaldolase (LTTA). Compared with the beginning, the modified LTTA has improved catalytic activity for a reaction between benzaldehyde or a derivative thereof and L-threonine by getting in contact. Also provided are a polynucleotide for encoding the modified LTTA of the present invention, a vector and host cell expressing the modified LTTA of the present invention, and a method for producing 3-phenyl-L-serine and a derivative thereof by using the modified LTTA of the present invention and the host cell.

Description

Modified threonine transaldolase and its application Technical field

The invention relates to the field of enzyme engineering. Specifically, the present invention relates to a modified L-threonine transaldolase (LTTA) and its application in the production of 3-phenyl-L-serine and its derivatives.

Background technique

The general structural formula of 3-phenyl-L-serine and its derivatives is as follows:

3-Phenyl-L-serine and its derivatives are a very important class of organic synthesis intermediates, which are widely used in drug synthesis. For example: (2S,3R)-p-methylsulfonylphenylserine is a key intermediate for the synthesis of veterinary drugs florfenicol and thiamphenicol, (2S,3R)-p-nitrophenylserine can be used as an antibacterial drug chloramphenicol Key intermediates. In addition, 3-phenyl-L-serine and its derivatives can also prepare chiral aziridines through simple transformation (David Tanner, Chiral Aziridines-Their Synthesis and Use in Stereoselective Transformations, Angew. Chem., Int. Ed. Engl., 33, 599 (1994)), which is widely used in the synthesis of chiral drugs. At present, 3-phenyl-L-serine derivatives are mainly prepared by organic synthesis. Such methods often have disadvantages such as many steps, low yield, and poor stereoselectivity. Biologists have also made some attempts to prepare such compounds. For example, Steinreiber et al. (Johannes Steinreiber et al., Threonine aldolases—an emerging tool for organic synthesis, Tetrahedron, 63, 918-926 (2007)) used benzaldehyde derivatives. Synthesize 3-phenyl-L-serine derivatives with glycine under the action of aldolase, the reaction formula is as follows:

At present, this method still has defects such as low conversion rate, poor stereoselectivity, and difficulty in product separation and purification, and does not have the conditions for industrialization. Therefore, exploring a mild, efficient, and economical method to prepare such compounds has attracted the attention of the majority of chemical and biological workers.

The inventor found that LTTA is used to catalyze the reaction between p-methylsulfonyl benzaldehyde and L-threonine, such as (2S,3R)-p-methylsulfonyl phenylserine, see CN 109836362A.

Furthermore, the inventors found that LTTA can catalyze the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives and acetaldehyde. The general reaction formula is as shown in formula (I) ( See, for example, PCT/CN2019/123974):

Through this method, the entire preparation and purification process of 3-phenyl-L-serine or its derivatives has simple operation, mild conditions, and greatly reduces solid waste and production costs. However, there is still a need for LTTA with higher activity to catalyze the reaction of formula (I).

Summary of the invention

In the first aspect, the present invention provides a modified L-threonine transaldolase (LTTA), wherein the modified LTTA comprises amino acid substitutions at one or more positions compared to its starting LTTA, and It has an improved activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine to generate 3-phenyl-L-serine or its derivatives.

In some embodiments, the modified LTTA includes an amino acid substitution at position 70, preferably substitution to H, N or S, more preferably substitution to H, and the position is numbered with reference to SEQ ID NO: 2. In some embodiments, the modified LTTA further comprises amino acids at one or more positions selected from positions 35, 38, 48, 57, 59, 94, 116, 141, 181, 185, 205, 229, and 407 replace. Preferably, position 35 is substituted with A, G or S, more preferably S. Preferably, position 38 is substituted with F. Preferably, position 48 is substituted with A or C. Preferably, position 57 is substituted with S, M or T, more preferably M. Preferably, position 59 is substituted with A or Y. Preferably, position 94 is substituted with E. Preferably, position 116 is substituted with R, S or N. Preferably, position 141 is substituted with C. Preferably, position 181 is substituted with Q. Preferably, position 185 is substituted with G. Preferably, position 205 is selected from S, Q or A. Preferably, position 229 is substituted with C. Preferably, position 407 is substituted with R.

In some embodiments, the modified LTTA comprises the amino acid sequence of one of SEQ ID NO: 13-72 or consists of the amino acid sequence of one of SEQ ID NO: 13-72; or is the same as SEQ ID NO: 13-65 Compared with the amino acid sequence of one of 69-72, the modified LTTA has positions 35, 38, 48, 57, 59, 70, 94, 116, 141, 181, 185, 205, 229 and 407. The position further contains 1-10 amino acid substitutions; or compared with the amino acid sequence of one of SEQ ID NO: 66-68, the modified LTTA also contains 1- 10 amino acid substitutions.

In a second aspect, the present invention provides a polynucleotide encoding the modified LTTA of the present invention, and a vector comprising the polynucleotide of the present invention.

In a third aspect, the present invention provides a host cell containing the modified LTTA of the present invention, its encoding polynucleotide or a vector containing the polynucleotide.

In the fourth aspect, the present invention also provides a method for producing 3-phenyl-L-serine and its derivatives, which comprises combining the modified LTTA of the present invention or the host cell of the present invention with benzaldehyde or its derivatives, and L-threonine contact. Detailed description of the invention

Reference will now be made in detail to certain embodiments of the present invention, examples of which are shown in the attached structures and reaction formulas. Although the invention will be described in conjunction with the enumerated embodiments, it should be understood that they are not intended to limit the invention to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The invention is not limited to the described methods and materials. If one or more of the cited documents, patents and similar materials is different or contradictory to this application, including but not limited to defined terms, term usage, described technology, etc., this application shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although suitable methods and materials are described below, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. Unless otherwise stated, the nomenclature used in this application is based on the IUPAC system nomenclature.

1. Modified L-threonine transaldolase

As used herein, "L-threonine transaldolase" and "LTTA" have the ability to catalyze the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or derivatives and acetaldehyde (ie The reaction of formula (I)) activity. The present invention provides a modified LTTA polypeptide. Compared with the original LTTA polypeptide, the modified LTTA has an improved activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine.

As used herein, the term "peptide" means a chain of at least two amino acids connected by peptide bonds. The term "polypeptide" is used interchangeably with the term "protein" herein and refers to a chain containing ten or more amino acid residues. All peptide and polypeptide chemical formulas or sequences herein are written from left to right, indicating the direction from the amino terminal to the carboxy terminal.

The term "amino acid" includes naturally occurring amino acids and unnatural amino acids in proteins. The one-letter and three-letter names of amino acids naturally occurring in proteins are commonly used in the field, and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

As used herein, the term "modification" refers to any chemical modification of a polypeptide, such as amino acid substitutions, deletions, insertions, and/or additions.

In some embodiments, the modified LTTA of the present invention contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 compared to its starting LTTA. , 16, 17, 18, 19, 20 or more amino acid substitutions, wherein the modified LTTA of the present invention has an improved catalyzed reaction between benzaldehyde or its derivatives and L-threonine compared with the original LTTA Activity of 3-phenyl-L-serine or derivatives.

In some embodiments, the modified LTTA comprises 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1- 9. 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acid substitutions, wherein the modified LTTA of the present invention has an improvement compared with the original LTTA The activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives.

In some embodiments, the modified LTTA of the present invention contains an amino acid substitution at position 70 compared to its starting LTTA, and the position is numbered with reference to SEQ ID NO: 2, preferably the substitution is H, N or S, more preferably Replaced by H.

In some embodiments, the modified LTTA of the present invention further comprises one or more positions selected from positions 35, 38, 48, 57, 59, 94, 116, 141, 181, 185, 205, 229 and 407. Amino acid substitution. Preferably, position 35 is substituted with A, G or S, more preferably S. Preferably, position 38 is substituted with F. Preferably, position 48 is substituted with A or C. Preferably, position 57 is substituted with S, M or T, more preferably M. Preferably, position 59 is substituted with A or Y. Preferably, position 94 is substituted with E. Preferably, position 116 is substituted with R, S or N. Preferably, position 141 is substituted with C. Preferably, position 181 is substituted with Q. Preferably, position 185 is substituted with G. Preferably, position 205 is selected from S, Q or A. Preferably, position 229 is substituted with C. Preferably, position 407 is substituted with R.

In a preferred embodiment, the modified LTTA of the present invention contains amino acid substitutions at positions 35 and 70, and the positions are numbered with reference to SEQ ID NO: 2, wherein position 35 is substituted with S and position 70 is substituted with H. More preferably, the modified LTTA comprises amino acid substitutions at positions 35, 57 and 70, wherein position 35 is substituted with S, position 57 is substituted with M, and position 70 is substituted with H. Further preferably, the modified LTTA comprises amino acid substitutions at positions 35, 57, 59, 70, 94 and 141, wherein position 35 is substituted with S, position 57 is substituted with M, position 59 is substituted with A, and position 70 is substituted with H, position 94 is substituted with E, and position 141 is substituted with C.

In some embodiments, the modified LTTA of the present invention has an amino acid substitution or a combination of amino acid substitutions selected from the group consisting of the following amino acid substitutions or combinations of amino acid substitutions (positions are numbered with reference to SEQ ID NO: 2):

-35A;

-35S;

-35G;

-57M;

-57T;

-57W;

-59A;

-59Y;

-70H;

-70N;

-70S;

-94E;

-141C;

-205S;

-205Q;

-205A;

-229C;

-59A, 70H;

-181Q, 70H;

-116N, 70H;

-35S, 70H;

-38F, 70H;

-48A, 70H;

-48C, 70H;

-59Y, 70H;

-70H, 94E;

-70H, 116S;

-70H, 116N;

-70H, 141C;

-35S, 70H, 181L;

-35S, 70H, 181Q;

-35S, 38F, 70H;

-35S, 57M, 70H;

-35S, 70H, 94E;

-35S, 70H, 116N;

-35S, 70H, 141C;

-35S, 48A, 70H;

-35S, 38F, 70H, 94E;

-35S, 38F, 70H, 116S;

-35S, 38F, 48A, 70H;

-35S, 48A, 59A, 70H;

-35S, 59A, 70H, 181Q;

-35S, 59Y, 70H, 181Q;

-35S, 48A, 70H, 94E;

-35S, 70H, 116S, 141C;

-35S, 70H, 116N, 141C;

-35S, 48A, 59A, 70H, 94E;

-35S, 59A, 70H, 94E, 141C;

-35S, 38F, 48A, 70H, 94E;

-35S, 38F, 48A, 59A, 70H, 116R;

-35S, 38F, 48A, 94E, 116S, 70H;

-35S, 57T, 59A, 70H, 94E, 141C;

-35S, 57S, 59A, 70H, 94E, 141C;

-35S, 57M, 59A, 70H, 94E, 141C;

-35S, 59A, 70H, 94E, 141C, 185G;

-35S, 59A, 70H, 94E, 141C, 407R; and

-35S, 59A, 70H, 94E, 141C, 205A.

In this article, the LTTA polypeptide on which amino acid modifications are made is referred to as the starting LTTA. The starting LTTA can be wild-type LTTA or a variant of wild-type LTTA. For example, starting from the polypeptide of SEQ ID NO: 2 for modification, compared to the modified LTTA, the polypeptide of SEQ ID NO: 2 is the "starting LTTA"; and if the polypeptide of SEQ ID NO: 2 is modified (for example, SEQ ID NOs: 13-65 and 69-72) start to be modified, compared to the modified LTTA, the variant polypeptide is the "starting LTTA".

As used herein, the term "wild-type LTTA" refers to naturally occurring LTTA. In some embodiments, the wild-type LTTA is LTTA from Pseudomonas. In some embodiments, the wild-type LTTA is shown in SEQ ID NO: 2, 4, or 6. SEQ ID NO: 2 is the amino acid sequence of LTTA from Pseudomonas fluorescens (Genbank accession number AQZ26585.1), SEQ ID NO: 4 is the amino acid sequence of LTTA from Pseudomonas sp.34E 7 (Genbank accession No. CRN02517.1), and SEQ ID NO: 6 is the amino acid sequence of LTTA from Pseudomonas sp. Irchel s3a18 (Genbank accession number WP_095149064). In some embodiments, the wild-type LTTA is an LTTA from Chitiniphilus shinanonensis, and its sequence is shown in SEQ ID NO: 8 (Genbank accession number WP_018749561).

For the present invention, in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (for example, gaps can be introduced in the first amino acid or nucleic acid sequence to match the second amino acid sequence). Or nucleic acid sequence for optimal alignment). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide in the corresponding position in the second sequence, then these molecules are the same at this position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions/total number of positions (i.e. overlapping positions) x 100). Preferably, the two sequences are the same length.

Those skilled in the art know that different computer programs can be used to determine the identity between two sequences.

"Percent amino acid identity" or "percent amino acid sequence identity" refers to comparing the amino acids of two polypeptides, and when optimally aligned, the two polypeptides have approximately the specified percentage of identical amino acids. For example, "95% amino acid identity" refers to comparing the amino acids of two polypeptides. When the two polypeptides are optimally aligned, 95% of the amino acids of the two polypeptides are identical.

In some embodiments, the modified LTTA polypeptide of the present invention has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85% compared to SEQ ID NO: 2, 4, 6 or 8. , 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity.

In some embodiments, the modified LTTA of the present invention includes 1, 2, 3, 4, 5, 6, 7, 8 compared to its starting LTTA (for example, SEQ ID NO: 2, 4, 6, or 8). , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, wherein the modified LTTA of the present invention has improved catalysis compared with the original LTTA The activity of reacting benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives. In some embodiments, the modified LTTA contains 1-20, 1-15, 1-14, 1-13, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acid substitutions. In some embodiments, the modified LTTA of the present invention contains an amino acid substitution at position 70 compared to its starting LTTA, and the position is numbered with reference to SEQ ID NO: 2, preferably the substitution is H, N or S, more preferably Replaced by H. In some embodiments, the modified LTTA has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity.

In some embodiments, the modified LTTA of the present invention further comprises one or more positions selected from positions 35, 38, 48, 57, 59, 94, 116, 141, 181, 185, 205, 229 and 407. Amino acid substitution. Preferably, position 35 is substituted with A, G or S, more preferably S. Preferably, position 38 is substituted with F. Preferably, position 48 is substituted with A or C. Preferably, position 57 is substituted with S, M or T, more preferably M. Preferably, position 59 is substituted with A or Y. Preferably, position 94 is substituted with E. Preferably, position 116 is substituted with R, S or N. Preferably, position 141 is substituted with C. Preferably, position 181 is substituted with Q. Preferably, position 185 is substituted with G. Preferably, position 205 is selected from S, Q or A. Preferably, position 229 is substituted with C. Preferably, position 407 is substituted with R. In some embodiments, the modified LTTA has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity.

In a preferred embodiment, the modified LTTA of the present invention contains amino acid substitutions at positions 35 and 70, and the positions are numbered with reference to SEQ ID NO: 2, wherein position 35 is substituted with S and position 70 is substituted with H. More preferably, the modified LTTA comprises amino acid substitutions at positions 35, 57 and 70, wherein position 35 is substituted with S, position 57 is substituted with M, and position 70 is substituted with H. Further preferably, the modified LTTA comprises amino acid substitutions at positions 35, 57, 59, 70, 94 and 141, wherein position 35 is substituted with S, position 57 is substituted with M, position 59 is substituted with A, and position 70 is substituted with H, position 94 is substituted with E, and position 141 is substituted with C. Preferably, the modified LTTA has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, compared with its starting LTTA. 97%, 98% or 99% sequence identity.

In some embodiments, the modified LTTA of the present invention contains an amino acid substitution at position 70 compared to its starting LTTA, and the position is numbered with reference to SEQ ID NO: 2, preferably the substitution is H, N or S, more preferably Substituted to H, wherein the modified LTTA of the present invention has an improved activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives compared to its starting LTTA . In some embodiments, the initial LTTA and SEQ ID NO: 2, 4, 6, or 8 have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% % Or 99% sequence identity. In some embodiments, the modified LTTA of the present invention further comprises one or more positions selected from positions 35, 38, 48, 57, 59, 94, 116, 141, 181, 185, 205, 229 and 407. Amino acid substitution. Preferably, position 35 is substituted with A, G or S, more preferably S. Preferably, position 38 is substituted with F. Preferably, position 48 is substituted with A or C. Preferably, position 57 is substituted with S, M or T, more preferably M. Preferably, position 59 is substituted with A or Y. Preferably, position 94 is substituted with E. Preferably, position 116 is substituted with R, S or N. Preferably, position 141 is substituted with C. Preferably, position 181 is substituted with Q. Preferably, position 185 is substituted with G. Preferably, position 205 is selected from S, Q or A. Preferably, position 229 is substituted with C. Preferably, position 407 is substituted with R. In a preferred embodiment, the modified LTTA of the present invention contains amino acid substitutions at positions 35 and 70, and the positions are numbered with reference to SEQ ID NO: 2, wherein position 35 is substituted with S and position 70 is substituted with H. More preferably, the modified LTTA comprises amino acid substitutions at positions 35, 57 and 70, wherein position 35 is substituted with S, position 57 is substituted with M, and position 70 is substituted with H. Further preferably, the modified LTTA comprises amino acid substitutions at positions 35, 57, 59, 70, 94 and 141, wherein position 35 is substituted with S, position 57 is substituted with M, position 59 is substituted with A, and position 70 is substituted with H, position 94 is substituted with E, and position 141 is substituted with C.

The term "conservative substitution" is also referred to as "homologous" amino acid residue substitution, and refers to a substitution in which the amino acid residue is replaced by an amino acid residue having a similar side chain, for example, an amino acid with a basic side chain (e.g., lysine) , Arginine and histidine), acidic side chain amino acids (e.g. aspartic acid, glutamic acid), non-charged electroactive side chain amino acids (e.g. glycine, asparagine, glutamine, serine, threonine) Acid, tyrosine, cysteine), non-polar side chain amino acids (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , Tryptophan), β-branched side chain amino acids (e.g. threonine, valine, isoleucine) and aromatic side chain amino acids (e.g. tyrosine, phenylalanine, tryptophan, histidine) acid).

Conservative amino acid substitutions generally have the least impact on the activity of the resulting protein. This substitution is described below. Conservative substitution is to replace an amino acid with an amino acid that is similar in size, hydrophobicity, charge, polarity, spatial characteristics, and aromaticity. When it is desired to fine-tune the properties of the protein, such substitutions are usually conservative.

As used herein, "homologous" amino acid residues refer to amino acid residues with similar chemical properties related to hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, and the like. Examples of amino acids that are homologous to each other include positively charged lysine, arginine, histidine, negatively charged glutamic acid, aspartic acid, hydrophobic glycine, alanine, valine, and leucine Acid, isoleucine, proline, phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine , Aromatic phenylalanine, tyrosine, tryptophan, serine and threonine with chemically similar side chain groups, or glutamine and asparagine, or leucine and isoleucine.

Examples of conservative amino acid substitutions in proteins include: Ser replaces Ala, Lys replaces Arg, Gln or His replaces Asn, Glu replaces Asp, Ser replaces Cys, Asn replaces Gln, Asp replaces Glu, Pro replaces Gly, Asn or Gln replaces His, Leu Or Val replaces Ile, Ile or Val replaces Leu, Arg or Gln replaces Lys, Leu or Ile replaces Met, Met, Leu or Tyr replaces Phe, Thr replaces Ser, Ser replaces Thr, Tyr replaces Tr, Trp or Phe replaces Tyr, and Ile or Leu replaces Val.

In some embodiments, the modified LTTA comprises the amino acid sequence of one of SEQ ID NO: 13-72 or consists of the amino acid sequence of one of SEQ ID NO: 13-72; or is the same as SEQ ID NO: 13-65 Compared with the amino acid sequence of one of 69-72, the modified LTTA has positions 35, 38, 48, 57, 59, 70, 94, 116, 141, 181, 185, 205, 229 and 407. The position contains 1-10 amino acid substitutions; or compared with the amino acid sequence of one of SEQ ID NO: 66-68, the modified LTTA contains 1-10 positions other than positions 35, 57 and 70 Amino acid substitutions, wherein the modified LTTA of the present invention has an improved activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives compared with the original LTTA. In some embodiments, compared with the amino acid sequence of one of SEQ ID NOs: 13-65 and 69-72, the modified LTTA is at positions 35, 38, 48, 57, 59, 70, 94, 116. The positions other than, 141, 181, 185, 205, 229 and 407 also contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions; or with SEQ ID NO: Compared with the amino acid sequence of one of 66-68, the modified LTTA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 at positions other than positions 35, 57, and 70. Or more amino acid substitutions.

As used herein, the activity of an enzyme refers to a decrease in substrate or an increase in product per unit time in a chemical reaction catalyzed by an enzyme per unit mass under certain conditions. For example, the activity of the modified LTTA of the present invention, under certain conditions (such as the reaction conditions listed in the following examples), under the catalysis of a unit mass of the modified LTTA, 3-phenyl-L generated in a unit time -Serine or its derivatives are expressed in terms of amount.

In this context, the activity of an enzyme can also refer to the relative activity of the enzyme, expressed as the ratio of the activity of the enzyme of interest to the activity of a given enzyme that catalyzes the same reaction, such as percentage relative activity.

In some embodiments, the activity of the modified LTTA of the present invention is expressed as a ratio to the activity of the LTTA of SEQ ID NO: 2. In some embodiments, the modified LTTA has an activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine to produce 3-phenyl-L-serine or its derivatives is at least that of the LTTA of SEQ ID NO: 2 1, 1.05, 1.1, 1.2, 1.3, 1.5, 1.7, 2, 2.5, 3, 3.5, 4 times or more.

As used herein, product selectivity means that when the reaction product contains two or more possible stereoisomers, one of the products is produced more. If the reaction is an enzymatic reaction, the product selectivity is also referred to as the product selectivity of the enzyme. For example, in the reaction of formula (I), the carbons at positions 2 and 3 of the derivatives of 3-phenyl-L-serine are all chiral carbons, which will produce four stereoisomers, namely P(2S,3R ), P(2R,3S), (P(2S,3S) and P(2R,3R).

In this article, the product selectivity of LTTA (wild-type or modified) can be used under certain reaction conditions (such as the reaction conditions listed in the following examples) diastereomeric ratio (DR) and enantiomers The excess percentage (enantiomeric excess percentage, ee%) is characterized, where DR and ee% are calculated using the following formulas (I) and (II).

DR=(P(2S,3R) output+P(2R,3S) output):(P(2S,3S) output+P(2R,3R) output) Formula (I)

ee%=P(2S,3R) output/(P(2S,3R) output+P(2R,3S) output)×100% Formula (II)

In some embodiments, compared to wild-type LTTA, the DR of the modified LTTA of the present invention is increased, for example, to at least 95.5:4.5, at least 96:4, at least 96.5:3.5, at least 97:3, at least 97.5: 2.5, at least 98:2, at least 98.5:1.5, at least 99:1 or higher.

In some embodiments, the ee% of the modified LTTA of the present invention is >99.9%.

In some embodiments, the benzaldehyde derivative is p-methylsulfonyl benzaldehyde. In some embodiments, the derivative of 3-phenyl-L-serine is (2S,3R)-p-methylsulfonylphenylserine.

2. The polynucleotide encoding the modified LTTA.

As used herein, the term "polynucleotide" or "nucleic acid molecule" includes DNA molecules (such as cDNA or genomic DNA) and RNA molecules (such as mRNA) and analogs of DNA or RNA produced using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA. The synthesis of the nucleic acid may use nucleotide analogs or derivatives (for example, inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids with altered base pairing ability or increased nuclease resistance.

The present invention also provides polynucleotides encoding the modified LTTA of the present invention. Therefore, in the present invention, the term modification also includes genetic manipulation of the polynucleotide encoding the LTTA polypeptide of the present invention. The modification may be a substitution, deletion, insertion and/or addition of nucleotides.

As used herein, the term "encoding" refers to the amino acid sequence of a polynucleotide directly specifying its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually starts with the ATG start codon or other start codons such as GTG and TTG, and ends with stop codons such as TAA, TAG, and TGA. The coding sequence can be a DNA, cDNA or recombinant nucleotide sequence.

In addition, nucleic acid molecules covering all or part of the nucleic acid sequence of the present invention can be separated by polymerase chain reaction (PCR), which uses the design of synthetic oligonucleotide primers based on the sequence information contained in the sequence.

The polynucleotide of the present invention can be amplified using cDNA, mRNA or genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.

The polynucleotide of the present invention can be prepared by standard synthesis techniques, for example, by using an automated DNA synthesizer.

The invention also relates to the complementary strands of the nucleic acid molecules described herein. A nucleic acid molecule that is complementary to other nucleotide sequences is a molecule that is sufficiently complementary to the nucleotide sequence so that it can hybridize with other nucleotide sequences to form a stable duplex.

As used herein, the term "hybridization" refers to nucleotides that are at least about 90%, preferably at least about 95%, more preferably at least about 96%, and more preferably at least 98% homologous to each other under given stringent hybridization and washing conditions. The sequences generally remain hybridized to each other.

Those skilled in the art know various conditions for hybridization, such as stringent hybridization conditions and highly stringent hybridization conditions. See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.

Of course, the polynucleotide of the present invention does not include a polynucleotide that only hybridizes to a poly A sequence (such as the 3'end poly (A) of mRNA) or a complementary stretch of poly T (or U) residues.

3. Expression and production of modified LTTA

In order to express the modified LTTA of the present invention, nucleic acid constructs and vectors containing the polynucleotide of the present invention, such as expression vectors, are also provided.

As used herein, the term "expression" includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule, which is isolated from a naturally occurring gene or modified to contain a nucleic acid segment that does not occur in nature. When the nucleic acid construct contains the control sequences required to express the coding sequence of the present invention, the term nucleic acid construct is synonymous with the term "expression cassette".

The term "expression vector" refers herein to a linear or circular DNA molecule, which comprises a polynucleotide encoding a polypeptide of the present invention, the polynucleotide and additional nucleotides provided for the expression of the polynucleotide, for example, Control sequence, operably connected. The expression vector includes a viral vector or a plasmid vector.

The term "control sequence" refers herein to include all elements necessary or advantageous for the expression of the polynucleotide encoding the polypeptide of the present invention. Each control sequence may be natural or foreign to the nucleotide sequence encoding the polypeptide, or natural or foreign to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminator. At a minimum, control sequences include promoters and transcription and translation termination signals.

For example, the control sequence may be a suitable promoter sequence, a nucleotide sequence recognized by the host cell to express the polynucleotide encoding the polypeptide of the present invention. The promoter sequence contains transcription control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, for example, the Escherichia coli lac operon. The promoters also include mutated, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell.

The term "operably linked" herein refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence, whereby the control sequence directs the expression of the polypeptide coding sequence.

The polynucleotide encoding the polypeptide of the present invention can be subjected to various operations to allow expression of the polypeptide. Before inserting it into the vector, it is desirable or necessary to manipulate the polynucleotide according to the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

In order to identify and select host cells containing the expression vector of the present invention, the vector of the present invention preferably contains one or more selectable markers, which allow simple selection of transformed, transfected, transduced, etc. cells. A selectable marker is a gene whose product provides biocide or virus resistance, heavy metal resistance, supplementation of auxotrophs, etc. For example, the bacterial selectable marker is the dal gene from Bacillus subtilis or Bacillus licheniformis, or a marker that confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.

The vector of the present invention can be integrated into the genome of the host cell or can replicate autonomously in the cell without relying on the genome. Elements required for integration into the host cell genome or autonomous replication are known in the art (see, for example, the aforementioned Sambrook et al., 1989).

Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing foreign nucleic acids (such as DNA) into host cells, which can be found in, for example, the aforementioned Sambrook et al., 1989; Davis et al. .,Basic Methods in Molecular Biology (1986) and other laboratory manuals.

The present invention also relates to a recombinant host cell, which contains the polynucleotide of the present invention, which is advantageously used for the recombinant production of LTTA polypeptides. The vector containing the polynucleotide of the present invention is introduced into a host cell, whereby the vector is retained as a chromosomal integrant or as a self-replicating extrachromosomal vector. Those skilled in the art know conventional vectors and host cells for expressing proteins.

In some embodiments, the host cell of the present invention is an E. coli cell, such as E. coli BL21 (DE3). In some embodiments, the expression vector is pET-30a(+).

4. Production of 3-phenyl-L-serine and its derivatives

In addition, the present invention provides a method for preparing 3-phenyl-L-serine or a derivative thereof, which comprises contacting the modified LTTA or host cell of the present invention with benzaldehyde or a derivative thereof and L-threonine.

In some embodiments, the method for preparing 3-phenyl-L-serine or a derivative thereof of the present invention includes the following steps:

(a) Providing the activity of the modified LTTA of the present invention, benzaldehyde or its derivatives, and L-threonine to the reaction medium;

(b) incubating the reaction medium so that benzaldehyde or its derivative reacts with L-threonine to generate 3-phenyl-L-serine or its derivative and acetaldehyde;

(c) Optionally, recover 3-phenyl-L-serine or a derivative thereof.

In some embodiments, acetaldehyde reductase and reduced nicotinamide adenine dinucleotide (NADH) are added to the reaction medium to reduce acetaldehyde to ethanol. In some embodiments, the acetaldehyde reductase is an acetaldehyde reductase from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO: 10.

In some embodiments, glucose dehydrogenase and glucose are added to the reaction medium to achieve coenzyme (NADH) regeneration. In some embodiments, the glucose dehydrogenase is derived from Bacillus subtilis (Bacillus subtilis), and its amino acid sequence is shown in SEQ ID NO: 12.

The process of acetaldehyde reduction and coenzyme regeneration is shown in formula (II):

In some embodiments, formate or formate and formate dehydrogenase are added to the reaction medium to achieve coenzyme (NADH) regeneration. In some embodiments, the formate dehydrogenase is derived from Candida boidinii, and its amino acid sequence is shown in SEQ ID NO: 73.

In some embodiments, the reaction medium is a buffer, such as PBS or Tris-HCl buffer. In one embodiment, the reaction medium is a PBS buffer, such as 0.1M, pH 7.0 PBS buffer.

In some embodiments, the incubation is performed at 25-40°C, preferably 28-35°C, such as 30°C.

Example

Through the following examples, those skilled in the art will understand the present invention more clearly. It should be understood that the embodiments are only for illustration, but not for limiting the scope of the present invention.

Example 1: Materials and methods

Unless otherwise specified, the experimental methods used in the present invention are all conventional methods. For specific gene cloning operations, please refer to the aforementioned Sambrook et al., 1989.

i) Reagents and instruments:

DNA polymerase (PrimeSTAR Max DNA Polymerase) and DpnI endonuclease were purchased from TaKaRa;

The plasmid extraction kit was purchased from Axygen;

P-Methylsulfonyl benzaldehyde was purchased from Aladdin, item number M185093, purity 98%;

L-threonine was purchased from Macleans, catalog number C10393311, analytically pure;

Pyridoxal phosphate was purchased from Aladdin, item number P136795, purity ≥98%;

Magnesium chloride was purchased from Aladdin, catalog number A2006034, analytically pure;

Oxidized nicotinamide adenine dinucleotide (NAD) was purchased from Aladdin, catalog number N196974, with a purity of 95%.

ii) Vector and strain: The expression vector used was pET-30a(+), the plasmid was purchased from Novagen, and the host cell used was E. coli BL21 (DE3), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

iii) Sequencing, primer synthesis and gene synthesis were completed by Suzhou Hongxun Biotechnology Co., Ltd. The genes (SEQ ID NO: 1, 3, 5, 7, 9 and 11) were synthesized and constructed into the vector pET-30a.

iv) Site-directed mutation:

Design specific primer pairs, and introduce the required substitutions at the corresponding bases at the amino acid positions of the required mutations. Using the extracted pre-mutation plasmid (containing the wild-type LTTA coding sequence, pET-30a(+) skeleton) as a template, the mutant was prepared using the method described by Packer and Liu (Methods for the directed evolution of proteins. Nat Rev Genet, 2015, 16(7):379-394).

v) Protein expression and preparation of enzyme solution:

1. Shake flask culture for expression

The E. coli cells transformed with the plasmid containing the target gene were inoculated into LB liquid medium containing 50mg/L of kanamycin (50mL medium in a 250mL bottle, peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH7.0), incubate overnight at 37°C with shaking. Transfer 1mL culture to TB liquid medium (50mL medium in a 250mL bottle, peptone 12g/L, yeast extract 24g/L, glycerol 4mL/L, potassium dihydrogen phosphate 2.31g/L, dipotassium hydrogen phosphate 12.54g/L), incubate with shaking at 37°C until OD600 reaches 0.6-0.8, add IPTG (final concentration of 0.4mM) and incubate at 30°C overnight to induce protein expression.

After incubation, the culture was centrifuged at 4,000 g at 4°C for 10 min, the supernatant was discarded, and E. coli cells were collected. The collected E. coli cells were resuspended in 20 mL of pre-cooled phosphate buffered saline (PBS) at pH 7.0, and the E. coli cells were sonicated at 4°C. The cell disruption solution was centrifuged at 6,000 g at 4°C for 15 min to remove the precipitate, and the supernatant obtained was a solution containing the recombinant enzyme, which was used to catalyze the reaction. The enzyme solution can also be freeze-dried into enzyme powder and stored at 4°C for later use.

2. Cultivate on 96-well plates for expression

The E. coli cells transformed with the plasmid containing the target gene were inoculated into the wells of a 96-well shallow-well plate, and each well was filled with 100 μL of LB medium containing 50 μg/mL kanamycin. The culture was incubated in a shaker for 18 hours (250 rpm, 30°C and 85% relative humidity). Transfer 20 μL of the incubated culture to the wells of a 96-well deep well plate filled with 180 μL of TB medium containing 50 μg/mL kanamycin. The plate was incubated at 30°C and 250 rpm for 2 hours, and then IPTG (final concentration of 0.4 mM) was added for induction, and incubated in a shaker at 30°C and 250 rpm for 20 hours. The E. coli cells were collected by centrifugation, and 300 μL of lysate (20mM phosphate buffer pH 7.0, 1mM MgSO ₄ , 1mg/mL lysozyme and 0.5mg/mL polymyxin B sulfate) was added. The mixture was shaken at room temperature for 2h, centrifuged and the supernatant was taken for enzyme activity determination.

vi) Enzyme activity determination

method 1:

The following system was prepared to determine the enzyme activity of the LTTA mutant (200μl):

P-methylsulfonyl benzaldehyde 0.7g/L, L-threonine 0.6g/L, pyridoxal phosphate 0.025g/L, magnesium chloride 0.1g/L, NADH 0.2g/L, add acetaldehyde reductase (final concentration 0.05g/L) and L-threonine transaldolase or its mutant (final concentration 0.2g/L), 100mM phosphate buffer (pH6.5).

The reactants were added to the wells of a 96-well plate, mixed briefly, and the SpectraMax190 (Molecular Devices) light absorption microplate reader was used to detect the activity of LTTA and its mutants by changing the absorbance at 340 nm.

Method 2:

The following system was prepared to determine the enzyme activity of the LTTA mutant (20 mL):

P-methylsulfonyl benzaldehyde 50g/L, L-threonine 42g/L, magnesium chloride 1g/L, NAD ⁺ 0.1g/L, pyridoxal phosphate 0.15g/L, D-glucose 65g/L, 100mM phosphate buffer Liquid (pH6.5).

Heat to 30℃ and stir evenly with magnetic force. Add 20mg of L-threonine transaldolase enzyme powder, 20mg of acetaldehyde reductase enzyme powder and 8mg of glucose dehydrogenase enzyme powder. Sulfone phenylserine.

Method 3:

The following system was prepared to determine the enzyme activity of the LTTA mutant (1mL):

P-methylsulfonyl benzaldehyde 28.4g/L, L-threonine 23.8g/L, magnesium chloride 1g/L, pyridoxal phosphate 0.066g/L, sodium formate 20.4g/L, NADH 0.325g/L, phosphate buffer 100mM, pH7.0.

Heat to 30℃ and mix well, add LTTA to a final concentration of 0.6mg/mL, formate dehydrogenase to a final concentration of 0.6mg/mL and acetaldehyde reductase to a final concentration of 0.3mg/mL, start the stirring reaction, and pass after 1 hour of reaction The product was detected by HPLC.

vii) HPLC analysis

For content analysis, the HPLC parameters are as follows:

Column Aliglent TC-C18 column (250mm*4.6mm*5μm);

Mobile phase PBS (50mM, pH8.0): Acetonitrile=85:15 (0-2min), 50:50 (7-8min), 85:15 (9-13min);

Flow rate 1mL/min;

Column temperature 30°C;

Detection wavelength 235nm;

Retention time p-methylsulfonyl benzaldehyde: 9.91min,

P-Methylsulfonylphenylserine: 3.25min.

For stereoisomer analysis, the HPLC parameters are as follows:

Column Aliglent TC-C18 column (250mm*4.6mm*5μm);

Mobile phase PBS (50mM, pH8.0): Acetonitrile=80:20;

Flow rate 1mL/min;

Column temperature 30°C;

Detection wavelength 340nm;

Retention time (2R,3S)-p-methylsulfonyl phenylserine: 5.4min,

(2S,3R)-p-methylsulfonylphenylserine: 5.9min,

(2R, 3R)-p-methylsulfonyl phenylserine: 6.3min,

(2S, 3S)-p-methylsulfonylphenylserine: 6.8min.

Example 2: Preparation and detection of mutants of LTTA (LTTA01) of Pseudomonas fluorescens

Using the nucleic acid encoding LTTA01 (SEQ ID NO:1) as a template, a mutant was prepared according to the method of Example 1. The resulting mutants are shown in Table 1. According to the method of Example 1, the expression LTTA01 and its mutants were cultured by shaking flasks, and enzyme powder was prepared. The activities of LTTA01 and its mutants were measured according to Method 2 in section vi) of Example 1, and the results are shown in Table 1, where the relative activity refers to the activity of the mutant/the activity of LTTA01.

Table 1

Substitutions in mutants	SEQ ID NO:	Relative activity
N35A	13	1.3
N35S	14	1.15
C57M	15	1.61
C57T	16	1.36
F59A	17	1.01
F59Y	18	1.08
F70H	19	1.38
Q94E	20	1.18
M141C	twenty one	1.12
M205S	twenty two	1.27
M205Q	twenty three	1.22
M205A	twenty four	1.3
H229C	25	1.26
F59A-F70H	26	1.71
F181Q-F70H	27	1.68
K116N-F70H	28	1.73
N35S-F70H	29	3.19
Y38F-F70H	30	1.60
G48A-F70H	31	1.59
G48C-F70H	32	1.42
F59Y-F70H	33	1.70
F70H-Q94E	34	1.61
F70H-K116S	35	1.53
F70H-K116N	36	1.63
F70H-M141C	37	1.45
N35S-F70H-F181L	38	2.36
N35S-F70H-F181Q	39	2.49
N35S-Y38F-F70H	40	3.51
N35S-C57M-F70H	41	4.23
N35S-F70H-Q94E	42	3.45
N35S-F70H-K116N	43	3.06
N35S-F70H-M141C	44	3.09
N35S-G48A-F70H	45	3.80
N35S-Y38F-F70H-Q94E	46	3.51
N35S-Y38F-F70H-K116S	47	2.87
N35S-Y38F-G48A-F70H	48	3.32
N35S-G48A-F59A-F70H	49	3.60
N35S-F59A-F70H-F181Q	50	2.36
N35S-F59Y-F70H-F181Q	51	2.62
N35S-G48A-F70H-Q94E	52	3.83
N35S-F70H-K116S-M141C	53	2.46
N35S-F70H-K116N-M141C	54	2.62
N35S-G48A-F59A-F70H-Q94E	55	3.76
N35S-F59A-F70H-Q94E-M141C	56	3.32
N35S-Y38F-G48A-F70H-Q94E	57	3.70
N35S-Y38F-G48A-F59A-F70H-K116R	58	3.73

N35S-Y38F-G48A-Q94E-K116S-F70H	59	2.65
N35S-C57T-F59A-F70H-Q94E-M141C	60	4.02
N35S-C57S-F59A-F70H-Q94E-M141C	61	3.95
N35S-C57M-F59A-F70H-Q94E-M141C	62	4.32
N35S-F59A-F70H-Q94E-M141C-W185G	63	2.19
N35S-F59A-F70H-Q94E-M141C-K407R	64	2.69
N35S-F59A-F70H-Q94E-M141C-M205A	65	4.02

Example 3: Preparation and detection of mutants of L-threonine transaldolase from different sources

According to the method of Example 1, a mutant containing the substitution combination N35S-C57M-F70H was prepared based on the following LTTA encoding nucleic acid:

-Pseudomonas sp.34 E 7's LTTA: LTTA02, encoding nucleic acid SEQ ID NO: 3;

-LTTA of Pseudomonas sp. Irchel s3a18: LTTA03, encoding nucleic acid SEQ ID NO: 5; and

-LTTA of Chitiniphilus shinanonensis: LTTA04, encoding nucleic acid SEQ ID NO: 7.

According to the method of Example 1, the expression LTTA01 and its mutants were cultured by shaking flasks, and enzyme powder was prepared. The activity of each wild-type LTTA and its mutants was measured according to method 2 in section vi) of Example 1, and the results are shown in Table 2, where the relative activity refers to the activity of the mutant/the activity of LTTA01.

Table 2

LTTA or mutant	SEQ ID NO:	Relative activity
LTTA01	2	1
LTTA01-N35S-C57M-F70H	41	4.23
LTTA02	4	1.02
LTTA02-N35S-C57M-F70H	66	4.30
LTTA03	6	0.49
LTTA03-N35S-C57M-F70H	67	2.79
LTTA04	8	0.13
LTTA04-N35S-C57M-F70H	68	0.17

Example 4: Introduce different substitutions at positions 35, 57 and 70 to prepare single mutants and compare their enzyme activities

Using the nucleic acid encoding LTTA01 (SEQ ID NO:1) as a template, a mutant was prepared according to the method of Example 1, and different substitutions were introduced at positions 35, 57, and 70. The resulting mutants are shown in Table 1. According to the method of Example 1, the expression LTTA01 and its mutants were cultured in a 96-well plate. The activity of LTTA01 and the mutant was measured according to Method 1 in Section vi) of Example 1, and the results are shown in Table 3, where the relative activity refers to the activity of the mutant/the activity of LTTA01.

table 3

Substitutions in mutants	SEQ ID NO:	Relative activity
N35A	13	2.1
N35E	To	0.06
N35G	69	1.77
N35H	To	0.69
N35M	To	0.41

N35P	To	0.22
N35Q	To	0.42
N35S	14	1.65
N35V	To	0.08
C57A	To	0.07
C57E	To	0.32
C57F	To	0.6
C57G	To	0.81
C57M	15	1.3
C57P	To	0.2
C57T	16	1.16
C57W	70	1.54
C57Y	To	0.36
F70E	To	0.33
F70H	19	1.88
F70L	To	0.5
F70N	71	1.29
F70Q	To	0.71
F70S	72	1.53
F70T	To	0.8
F70V	To	0.6

Example 5. The effect of introducing different mutations in LTTA01 on enzyme activity and product selectivity

Using the nucleic acid encoding LTTA01 (SEQ ID NO:1) as a template, a mutant was prepared according to the method of Example 1. According to the method of Example 1, the expression LTTA01 and its mutants were cultured by shaking flasks, and enzyme powder was prepared. The activity and product selectivity of LTTA01 and the mutant were determined according to Method 3 in Article vi) of Example 1, and the results are shown in Table 4. The relative activity refers to the activity of the mutant/the activity of LTTA01, and the product selectivity is represented by DR , And for each enzyme, ee%>99.9%.

Table 4

LTTA or mutant (indicated by its substitution)	Relative activity	DR
WT(LTTA01)	1.00	95.2:4.8
N35S	0.41	96.8:3.2
C57N	1.17	94.0:6.0
N35S-C57N	0.49	97.2:2.8
F70H	1.67	96.7:3.3
N35S-F70H	1.71	98.1:1.9
C57N-F70H	1.66	95.5:4.5
N35S-C57N-F70H	2.13	98.1:1.9
N35S-C57M-F59A-F70H-Q94E-M141C	3.25	99.4:0.6

It can be seen that the product selectivity of mutants containing F70H or N35S is higher than that of wild type, and the product selectivity of mutants containing N35S and F70H mutations is further improved. The mutant containing the single mutation of N35S was not as active as WT under the above reaction conditions, but it did not significantly affect the enzyme activity after being combined with F70H.

Example 6. The effect of introducing different mutations at position 70 of LTTA01 on enzyme activity and product selectivity

Using the nucleic acid encoding LTTA01 (SEQ ID NO:1) as a template, a mutant was prepared according to the method of Example 1, and a substitution was introduced at position 70 of LTTA01. According to the method of Example 1, the expression LTTA01 and its mutants were cultured by shaking flasks, and enzyme powder was prepared. The activity and product selectivity of LTTA01 and the mutant were determined according to Method 3 in Article vi) of Example 1, and the results are shown in Table 5. The relative activity refers to the activity of the mutant/the activity of LTTA01, and the product selectivity is represented by DR , And for each enzyme, ee%>99.9%.

table 5

LTTA and mutants	Relative activity	DR
WT	1.00	95.3:4.7
F70G	0.00	-
F70H	2.69	97.1:2.9
F70N	3.62	97.7:2.3
F70P	0.11	91.4:8.6
F70R	0.14	88.2:11.8
F70S	3.17	97.8:2.2
F70W	0.08	91.5:8.5
F70Y	0.22	92.4:7.6

Claims (10)

1. A modified L-threonine transaldolase (LTTA),

   Compared with its starting LTTA, the amino acid substitution at position 70 is included, and the amino acid substitution at position 70 is H, N, or S, and optionally, the modified LTTA also includes positions 35, 38, 48, 57, 59, 94, 116, 141, 181, 185, 205, 229 and 407 substitutions at one or more positions, wherein the amino acid positions are numbered with reference to SEQ ID NO: 2,

   Wherein, compared with the starting LTTA, the modified LTTA has an improved activity of catalyzing the reaction of benzaldehyde or its derivatives with L-threonine to generate 3-phenyl-L-serine and its derivatives.
2. The modified LTTA of claim 1, comprising one or more amino acid substitutions selected from the group consisting of: A, G, or S at position 35, F at position 38, A or C at position 48, and A or C at position 57, S, M, T or W, position 59 is substituted with A or Y, position 94 is substituted with E, position 116 is substituted with R, S or N, position 141 is substituted with C, position 181 is substituted with Q or L, position 185 is substituted with G, position 205 is selected from S, Q or A, position 229 is substituted with C, and position 407 is substituted with R.
3. The modified LTTA of claim 1 or 2, wherein the modified LTTA comprises amino acid substitutions at positions 35 and 70, wherein position 35 is substituted to S and position 70 is substituted to H.
4. The modified LTTA of any one of claims 1-3, wherein the modified LTTA comprises amino acid substitutions at positions 35, 57, and 70, wherein position 35 is substituted with S, position 57 is substituted with M, and position 70 is substituted with H.
5. The modified LTTA of any one of claims 1 to 4, wherein the modified LTTA comprises amino acid substitutions at positions 35, 57, 59, 70, 94 and 141, wherein the substitution at position 35 is S and the substitution at position 57 is M, position 59 is substituted with A, position 70 is substituted with H, position 94 is substituted with E, and position 141 is substituted with C.
6. A modified LTTA comprising the amino acid sequence of one of SEQ ID NO: 13-72.
7. A polynucleotide encoding the modified LTTA of any one of claims 1-6.
8. An expression vector comprising the polynucleotide of claim 7.
9. A host cell comprising the modified LTTA of any one of claims 1-6, the polynucleotide of claim 7 or the vector of claim 8.
10. A method for producing 3-phenyl-L-serine and its derivatives, comprising combining the modified LTTA of any one of 1-6 or the host cell of claim 9 with benzaldehyde or its derivatives and L-threonine Acid contact.