# US 12,110,489 B2 · Gene site saturation mutagenesis (GSSM) method

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- Number: US 12,110,489 B2
- Document ID: US12110489B2
- Status: Granted
- Kind: B2
- Application number: 16/954,092
- Filed: 2018-12-18
- Published: 2022-08-11
- Granted: 2024-10-08
- Earliest claimed: 2017-12-20
- Assignees: BASF SE
- Applicants: BASF SE
- Inventors: Jared Dennis; Xuqiu Tan; Robin Cai
- Web page: https://patentgrep.com/patent/US12110489B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/1031: {mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR}
- C12N 1/20: Bacteria; Culture media therefor
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 2800/101: for bacteria
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed herein is an improved Gene Site Saturation Mutagenesis (GSSM) method for producing a plurality of modified polynucleotides and/or polypeptides, creating specific changes to a gene, and reassembling mutations or changes at one or more sites.

## Claims

### Claim 1

A method of producing a plurality of modified polynucleotides from a parent polynucleotide comprising the coding region of a subject protein, wherein each of the plurality of modified polynucleotides encode a modified polypeptide that comprises an amino acid substitution at a first amino acid position of interest in the protein of interest, the method comprising:
(a) providing a plurality of primers capable of binding to a desired region of the parent polynucleotide, wherein the desired region comprises the codon for the amino acid at the first position of interest of the subject protein, wherein at least N of the plurality of primers each comprise a first codon for a different alternative amino acid at the first amino acid position of interest, wherein N is the number of desired amino acid substitutions at the first position of interest;
(b) contacting the plurality of primers with the parent polynucleotide to form a reaction mixture; and
(c) subjecting the reaction mixture to a polymerase extension reaction to generate the plurality of modified polynucleotides;
wherein the plurality of primers does not comprise degenerate primers.

### Claim 2 (depends on 1)

The method of claim 1, wherein at least one of the plurality of primers comprises a second codon for one of the alternative amino acids.

### Claim 3 (depends on 1)

The method of claim 1, wherein the plurality of primers comprises at least two or more primers each comprising a second codon for a different alternative amino acid.

### Claim 4 (depends on 1)

The method of claim 1, wherein N is an integer between 1 and 19.

### Claim 5 (depends on 1)

The method of claim 1, wherein N is 19.

### Claim 6 (depends on 1)

The method of claim 1, wherein the plurality of polynucleotides comprises polynucleotides that encode for polypeptides having all possible standard amino acid substitutions at the first amino acid position of interest as compared to the subject protein.

### Claim 7 (depends on 1)

The method of claim 1, further comprising designing the plurality of primers based on the sequence of the desired region of the parent polynucleotide.

### Claim 8 (depends on 7)

The method of claim 7, wherein at least one of the plurality of primers is capable of binding to a region between 20 nucleotides upstream and 20 nucleotides downstream of the codon for the amino acid at the first amino acid position of interest.

### Claim 9 (depends on 1)

The method of claim 1, further comprising treating the plurality of modified polynucleotides generated in step (c) with a restriction enzyme to remove the parent polynucleotide.

### Claim 10 (depends on 1)

The method of claim 1, further comprising recovering the plurality of modified polynucleotides.

### Claim 11 (depends on 1)

The method of claim 1, further comprising introducing one or more of the modified polynucleotides to a host cell to express one or more mutants of the subject protein.

### Claim 12 (depends on 11)

The method of claim 11, wherein at least one of the codons in the primers for the different alternative amino acids at the first amino acid position of interest is a preferred codon of the host cell.

### Claim 13 (depends on 11)

The method of claim 11, further comprising analyzing the one or more mutants of the subject protein for stability, pH, product inhibition, salt tolerance, thermostability, substrate specificity, activity, stereoselectivity, expression, or a combination thereof.

### Claim 14 (depends on 13)

The method of claim 13, further comprising selecting one or more mutants of the subject protein based on their stability, thermostability, substrate specificity, activity, stereoselectivity, expression, pH profile change (higher or lower pH), co-factor specificity, product inhibition, salt tolerance, sensitivity to salt concentration, sensitivity to salt, binding affinity, or a combination thereof.

### Claim 15 (depends on 11)

The method of claim 11, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

### Claim 16 (depends on 15)

The method of claim 15, wherein the host cell is a cell from an organism selected from the group consisting of Pichia pastoris, Bacillus licheniformis, Bacillus subtilis, Pseudomonas fluorescens, Myceliopthora thermophile fungus, Aspergillus niger, Trichoderma reesei, and Escherichia coli.

### Claim 17 (depends on 1)

The method of claim 1, wherein the parent polynucleotide is a circular double-stranded DNA.

### Claim 18 (depends on 1)

The method of claim 1, wherein the plurality of primers comprises at least M additional primers each comprising codon for a different alternative amino acid at a second amino acid position of interest of the subject protein, wherein M is the number of desired amino acid substitutions at the second position of interest.

### Claim 19 (depends on 18)

The method of claim 18, wherein M is an integer between 1 and 19.

### Claim 20 (depends on 18)

The method of claim 18, wherein M is 19.

### Claim 21 (depends on 18)

The method of claim 18, wherein at least one of the plurality of modified polynucleotides encodes a polypeptide comprising an amino acid substitution at the second amino acid position of interest.

### Claim 22 (depends on 18)

The method of claim 18, wherein the plurality of polynucleotides comprises polynucleotides that encode for polypeptides having all possible standard amino acid substitutions at the second amino acid position of interest as compared to the subject protein.

### Claim 23

A method of producing a plurality of modified polypeptides of a parent polypeptide comprising the coding region of a subject protein, wherein each of the plurality of modified polypeptides comprises an amino acid substitution at a first amino acid position of interest, the method comprising:
providing a plurality of primers capable of binding to a desired region of a template polynucleotide encoding the parent polypeptide, wherein the desired region comprises the codon for the amino acid at the first amino acid position of interest in the parent polypeptide, wherein at least N of the plurality of primers each comprise a first codon for a different alternative amino acid for the amino acid at the first amino acid position of interest, wherein N is the number of desired amino acid substitutions at the first position of interest;
contacting the plurality of primers with the template polynucleotide to form a reaction mixture;
subjecting the reaction mixture to a polymerase extension reaction to generate the plurality of modified polynucleotides; and
introducing the modified polynucleotides to a recombinant expression system to produce the plurality of modified polypeptides;
wherein the plurality of primers does not comprise degenerate primers.

### Claim 24 (depends on 23)

The method of claim 23, wherein at least one of the plurality of primers comprises a second codon for one of the alternative amino acids.

### Claim 25 (depends on 23)

The method of claim 23, wherein the plurality of primers comprises at least two or more primers each comprising a second codon for a different alternative amino acid.

### Claim 26 (depends on 23)

The method of claim 23, wherein N is an integer between 1 and 19.

### Claim 27 (depends on 23)

The method of claim 23, wherein N is 19.

### Claim 28 (depends on 23)

The method of claim 23, wherein the plurality of polynucleotides comprises polynucleotides that encode for polypeptides having all possible standard amino acid substitutions at the first amino acid position of interest as compared to the parent polypeptide.

### Claim 29 (depends on 23)

The method of claim 23, further comprising designing the plurality of primers based on the sequence of the desired region.

### Claim 30 (depends on 23)

The method of claim 23, wherein at least one of the plurality of primers is capable of binding to a region between 20 nucleotides upstream and 20 nucleotides downstream of the codon for the amino acid at the first amino acid position of interest.

### Claim 31 (depends on 23)

The method of claim 23, wherein the recombinant expression system is a host cell.

### Claim 32 (depends on 31)

The method of claim 31, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

### Claim 33 (depends on 31)

The method of claim 31, wherein at least one of the codons in the primers for the different alternative amino acids at the first amino acid position of interest is a preferred codon of the host cell.

### Claim 34 (depends on 31)

The method of claim 31, wherein at least one of the codons in the primers for the different alternative amino acids at the first amino acid position of interest is the most preferred codon for an amino acid in the host cell.

### Claim 35 (depends on 23)

The method of claim 23, further comprising analyzing one or more of the modified polypeptides for stability, thermostability, substrate specificity, activity, stereoselectivity, expression, pH profile change, co-factor specificity, product inhibition, salt tolerance, sensitivity to salt concentration, sensitivity to salt, binding affinity, or a combination thereof.

### Claim 36 (depends on 34)

The method of claim 34, further comprising selecting one or more mutants of the subject protein based on their stability, thermostability, substrate specificity, activity, stereoselectivity, expression, pH profile change, co-factor specificity, product inhibition, salt tolerance, sensitivity to salt concentration, sensitivity to salt, binding affinity, or a combination thereof.

### Claim 37 (depends on 23)

The method of claim 23, wherein the template polynucleotide is a circular double-stranded DNA.

### Claim 38 (depends on 23)

The method of claim 23, wherein the plurality of primers comprises at least M additional primers each comprising codon for a different alternative amino acid at a second amino acid position of interest of the subject protein, wherein M is the number of desired amino acid substitutions at the second position of interest.

### Claim 39 (depends on 38)

The method of claim 38, wherein M is an integer between 1 and 19.

### Claim 40 (depends on 38)

The method of claim 38, wherein M is 19.

### Claim 41 (depends on 38)

The method of claim 38, wherein at least one of the plurality of modified polypeptides comprises an amino acid substitution at the second amino acid position of interest.

### Claim 42 (depends on 38)

The method of claim 38, wherein the plurality of modified polynucleotides have all possible standard amino acid substitutions at the second amino acid position of interest as compared to the parent polypeptide.