Genes with codon mutations encoding xylanase

Abstract

The present disclosure provides for a polynucleotide sequences encoding a xylanase. More specifically, the present disclosure provides for polynucleotide sequences with codon mutations encoding a xylanase.

Claims

1. An animal feed comprising a polypeptide encoded by a polynucleotide comprising a nucleic acid with the sequence of SEQ ID NO: 1, wherein said polypeptide has xylanase activity.

2. An animal feed comprising a polypeptide encoded by a polynucleotide comprising a nucleic acid sequence having the sequence of SEQ ID NO: 3 with the exception of at least one mutation selected from the group consisting of T6G, A12G, C15T, T33G, A42C, A48C, T57G, A66C, T69C, T72C, and any combination thereof, wherein said polypeptide has xylanase activity.

3. An animal feed comprising a polypeptide encoded by a polynucleotide comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises a sequence exhibiting at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, and wherein said nucleic acid sequence comprises at least one mutation selected from the group consisting of T6G, A12G, C15T, T33G, A42C, A48C, T57G, A66C, T69C, T72C, and any combination thereof, relative to SEQ ID NO:1 or SEQ ID NO: 2, respectively.

4. The animal feed of claim 1, 2 or 3, wherein the polypeptide having xylanase activity is produced in a recombinant bacterial expression system.

5. The animal feed of claim 4, wherein the bacterial expression system is (a) a gram-negative bacteria expression system, (b) the gram-negative bacteria expression system of (a), wherein the gram-negative bacteria expression system is a Pseudomonas, E. coli, Ralstonia, or Caulobacter expression system, or (c) the gram-negative bacteria expression system of (b), wherein the Pseudomonas expression system is a Pseudomonas fluorescens expression system.

6. The animal feed of claim 5, wherein the xylanase that is produced is at least: 65 U/ml, 70 U/ml, 75 U/ml, 80 U/ml, 85 U/ml, 90 U/ml, 95 U/ml, 100 U/ml, 105 U/ml, 110 U/ml, 115 U/ml, 120 U/ml, 125 U/ml, 130 U/ml, 135 U/ml, 140 U/ml, 145 U/ml, 150 U/ml, 155 U/ml, 160 U/ml, 165 U/ml, 170 U/ml, 175 U/ml, or 180 U/ml.

7. The animal feed of claim 4, wherein the polypeptide: (a) does not have a signal sequence, a proprotein sequence, or any combination thereof, or (b) further comprises a heterologous amino acid sequence selected from the group consisting of: a signal sequence, a tag, an epitope, an N-terminal extension, a C-terminal extension, and any combination thereof.

8. The animal feed of claim 4, wherein: (a) the polypeptide further comprises at least a second enzyme, or (b) the polypeptide of (a), wherein the second enzyme is selected from the group consisting of: a lactase, a lipase, a protease, a catalase, a xylanase, a cellulase, a glucanase, a mannanase, an amylase, an amidase, an epoxide hydrolase, an esterase, phospholipase, transaminase, an amine oxidase, cellobiohydrolase, an ammonia lyase, and any combination thereof.

9. The animal feed of claim 2, wherein the polynucleotide has the mutation T6G.

10. The animal feed of claim 2, wherein the polynucleotide has the mutation A12G.

11. The animal feed of claim 2, wherein the polynucleotide has the mutation C15T.

12. The animal feed of claim 2, wherein the polynucleotide has the mutation T33G.

13. The animal feed of claim 2, wherein the polynucleotide has the mutation A42C.

14. The animal feed of claim 2, wherein the polynucleotide has the mutation A48C.

15. The animal feed of claim 2, wherein the polynucleotide has the mutation T57G.

16. The animal feed of claim 2, wherein the polynucleotide has the mutation A66C.

17. The animal feed of claim 2, wherein the polynucleotide has the mutation T69C.

18. The animal feed of claim 2, wherein the polynucleotide has the mutation T72C.

19. A method of making an animal feed, the method comprising: (a) contacting a xylanase with an animal feed, wherein the xylanase is: (i) a xylanase encoded by a polynucleotide comprising a nucleic acid with the sequence of SEQ ID NO: 1; (ii) a xylanase encoded by a polynucleotide comprising a nucleic acid sequence having the sequence of SEQ ID NO: 3 with the exception of at least one mutation selected from the group consisting of T6G, A12G, C15T, T33G, A42C, A48C, T57G, A66C, T69C, T72C, and any combination thereof; or (iii) a xylanase encoded by a polynucleotide comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises a sequence exhibiting at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, and wherein said nucleic acid sequence comprises at least one mutation selected from the group consisting of T6G, A12G, C15T, T33G, A42C, A48C, T57G, A66C, T69C, T72C, and any combination thereof, relative to SEQ ID NO:1 or SEQ ID NO: 2, respectively.

20. The method of claim 19, wherein the animal feed is in the form of a pellet.

21. A method of feeding an animal, the method comprising providing the animal feed made by the method of claim 19 to an animal.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 is an image of SDS PAGE gel electrophoresis displaying various levels of protein expression, as described in Example 2.

(2) FIG. 2 is a bar graph showing the level of activity of protein preparations, as described in Example 3.

(3) FIG. 3 is SEQ ID NO: 1, the polynucleotide with 10 silent mutations: T6G, A12G, C15T, T33G, A42C, A48C, T57G, A66C, T69C, T72C, as compared to SEQ ID NO: 3.

(4) FIG. 4 is SEQ ID NO: 2, the polynucleotide with 10 silent mutations: as compared to SEQ ID NO: 3.

(5) FIG. 5 is SEQ ID NO: 3, the unmodified parent polynucleotide sequence of SEQ ID NO: 1 and 2.

(6) FIG. 6 is SEQ ID NO: 4, the polypeptide encoded by SEQ ID NO: 1, 2, and 3.

(7) FIG. 7 is a sequence alignment of SEQ ID NO: 1 and SEQ ID NO: 3.

(8) FIG. 8 is a sequence alignment of SEQ ID NO: 2 and SEQ ID NO: 3.

TERMS TO DEFINE

(9) A codon is a three polynucleotide sequence that specifies the identity of an amino acid to be added to a protein.

(10) A silent mutation is a mutation in a codon that does not result in the specification of a different amino acid.

(11) An Open Reading Frame is a series of codons that specifies the sequence of amino acids in a protein.

(12) A base position is the numerical location of a base in a polynucleotide sequence, counted consecutively from the start of the open reading frame or from some other reference marker.

(13) To encode a protein means to specify the amino acid sequence of that protein.

(14) Codon optimization as used herein refers to modifying the codons of a gene or ORF to those that are more frequently or most frequently used in the target and host organisms, but does not alter the amino acid sequence of the original translated protein. An examples of codon optimization software is Aptagen's Gene Forge codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171). Other publicly available databases for optimized codons are available and can work equally in some embodiments as well.

(15) A mutation is a change in a nucleotide sequence or an amino acid sequence compared to a reference.

(16) A nucleotide refers to one of the four bases which comprise DNA sequenceAdenine (A), Thymidine (T), Guanidine (G), and Cytosine (C).

(17) Xylanase is an enzyme with xylanase, mannanase, and/or a glucanase activity.

(18) A Unit (abbreviated as U) is defined as 1 mole of reducing sugars per minute at pH 6.5 and 40 C.

Example 1

(19) Method of making enhanced expression variants.

(20) Two variants (SEQ ID NO: 1 and NO: 2) were designed based on SEQ ID NO: 3 to mutate at the DNA level to improve the gene expression. The design of the two mutant variants takes into account the two dimensional, three dimensional structures of the DNA sequences, as well as the preferred codons of the host, all of which that may influence gene expression. The mutations were introduced on the PCR primers. Both genes were PCR-amplified and clones into the Pseudomonas vector pDOW1169 (DOW AgroSciences, IN) using standard molecular cloning techniques. The resulting expression constructs were transformed into Pseudomonas fluorescens DC454 (DOW AgroSciences, IN) and expression levels of the proteins were determined. A transformant with the SEQ ID NO:1 was designated as the lead showing significantly enhanced expression levels over the parent SEQ ID NO: 3.

Example 2

(21) Method of preparing SDS-PAGE to visually determine expression levels of variants. Criterion precast Tris-HCl polyacrylamide gel (Bio-rad Laboratories, Inc) was used to separate proteins. The gel was run at 150V using Tris-glycine buffer (FIG. 1). Protein loading was normalized to load proteins from 0.33 OD600 cells for each lane. SeeBlue pre-stained protein standard was used (Life Technologies). As shown in FIG. 1, the expression level of SEQ ID NO: 2 was lower than the expression level of parent SEQ ID NO: 3, and SEQ ID NO: 1.

Example 3

(22) Method of determining relative expression levels for variants.

(23) SEQ ID NO: 1, 2, and 3 genes were expressed in shake flask. The cultures were grown at 30 C. and 220 rpm to an OD600 of 19 in a designed complex medium, and induced with 0.3 mM IPTG (Isopropyl-D-1-thiogalactopyranoside) for 24 hours. Cells were harvested and lysed either by sonication or heat-treatment at 65 C. for 1 hour. Xylanase activity was measured by Nelson-Somogyi reducing sugar assay using wheat arabinoxylan as substrate. (Roger A. O'Neill, Alan Darvill, Peter Albersheim, A fluorescence assay for enzymes that cleave glycosidic linkages to produce reducing sugars, Analytical Biochemistry, Volume 177, Issue 1, (1989)11-15). Activity levels were measured in U/ml as shown in FIG. 2 to determine relative expression levels from each culture. A Unit (abbreviated as U) is defined as 1 mole of reducing sugars per minute at pH 6.5 and 40 C. The specific activity for the assay in this assay was determined to be 533 units/mg protein. As shown in FIG. 2, the expression level of SEQ ID NO: 2 was lower than the expression level of parent SEQ ID NO: 3, and SEQ ID NO: 1.