# US 2016/0251663 A1 · Engineered yeast for production of enzymes

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- Number: US 2016/0251663 A1
- Document ID: US20160251663A1
- Status: Published application
- Kind: A1
- Application number: 15/156,998
- Filed: 2016-05-17
- Published: 2016-09-01
- Earliest claimed: 2011-08-31
- Assignees: Academia Sinica
- Applicants: Academia Sinica
- Inventors: Jui-Jen Chang; Wen-Hsiung Li; Chieh-Chen Huang; Hao-Yeh Lin; Ming-Che Shih
- Web page: https://patentgrep.com/patent/US20160251663A1

## CPC

- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 9/2445: {Beta-glucosidase (3.2.1.21)}
- C12N 15/65: using markers (enzymes used as markers C12N15/52)
- C12N 15/815: {for yeasts other than Saccharomyces}
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C12Y 302/01021: Beta-glucosidase (3.2.1.21)
- C12Y 302/01091: Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Described herein is an engineered Kluyveromyces marxianus cell, the cell comprising in its genome: (i) two different nucleic acid molecules that each contain a promoter operably linked to a gene encoding a functional enzyme, and (ii) a selection nucleic acid molecule that contains a promoter operably linked to a gene encoding a selectable marker, wherein all of the nucleic acids molecules of (i) and (ii) are in tandem and the engineered cell expresses all of the proteins encoded by the genes of (i) and (ii).

## Claims

### Claim 1

An engineered Kluyveromyces marxianus cell, the cell comprising in its genome:
(i) two different nucleic acid molecules that each contain a promoter operably linked to a gene encoding a functional enzyme, and
(ii) a selection nucleic acid molecule that contains a promoter operably linked to a gene encoding a selectable marker,
wherein all of the nucleic acids molecules of (i) and (ii) are in tandem and the engineered cell expresses all of the proteins encoded by the genes of (i) and (ii).

### Claim 2 (depends on 1)

The engineered cell of claim 1, further comprising:
(iii) a reporter nucleic acid molecule that contains a promoter operably linked to a gene encoding a reporter, or
(iv) a scaffold nucleic acid molecule that contains a promoter operably linked to a gene encoding a scaffold polypeptide,
wherein all of the nucleic acids molecules of (i) and (ii), and (iii) or (iv) are in tandem and the engineered cell further expresses all of the proteins encoded by the genes of (iii) or (iv).

### Claim 3 (depends on 1)

The engineered cell of claim 1, wherein each of the promoters of (i), (ii), (iii), and (iv) are selected from the group consisting of an S. cerevisiae GapDHI promoter, an S. cerevisiae AdhI promoter, an S. cerevisiae PGK promoter, an S. cerevisiae Adh4 promoter, an S. cerevisiae TDH3 promoter, an S. cerevisiae TEF1 promoter, an S. cerevisiae TPI1 promoter, an S. cerevisiae GAL1 promoter, an S. cerevisiae GAL7 promoter, an S. cerevisiae GAL10 promoter, an S. cerevisiae CUP1 promoter, an S. cerevisiae ICL1 promoter, a K. lactis GapDHI promoter, a K. lactis PGK promoter, a K. lactis AdhI promoter, a K. lactis Lac4 promoter, a K. lactis Adh4 promoter, and a C. tropicalis ICL promoter.

### Claim 4 (depends on 1)

The engineered cell of claim 1, wherein each gene of (i) is selected from the group consisting of a T. reesei egIII gene, a T. reesei cbhI gene, a T. reesei cbhII gene, a N. patriciarum npabgs gene, an A. niger eglA gene, an A. niger egI gene, a N. crassa cdt-1 gene, a T. aurantiacus lpmo gene, a M. thermophila cdhI gene, a T. fusca lpmo gene, a C. thermocellum celK gene, a C. thermocellum celS gene, a C. thermocellum celA gene, a C. thermocellum celR gene, and a C. thermocellum xynC gene.

### Claim 5 (depends on 1)

The engineered cell of claim 1, wherein the promoter and the gene in each nucleic acid molecule of (i) are selected from the group consisting of:
(a) an S. cerevisiae GapDHI promoter and a T. reesei egIII gene,
(b) an S. cerevisiae AdhI promoter and a N. patriciarum npabgs gene,
(c) an S. cerevisiae PGK promoter and a T. reesei cbhI gene,
(d) an S. cerevisiae PGK promoter and an A. niger eglA gene,
(e) an S. cerevisiae Adh4 promoter and a T. reesei cbhI gene,
(f) a K. lactis GapDHI promoter and an A. niger eglA gene,
(g) a K. lactis PGK promoter and a T. reesei cbhII gene,
(h) a K. lactis AdhI promoter and a N. crassa cdt-1 gene,
(i) a K. lactis Lac4 promoter and an A. niger egI gene,
(j) a K. lactis AdhI promoter and a T. reesei cbhI gene,
(k) a K. lactis Adh4 promoter and a N. patriciarum npabgs gene,
(l) a K. lactis Lac4 promoter and a T. aurantiacus lpmo gene,
(m) a K. lactis Lac4 promoter and a M. thermophila cdhI gene,
(n) a C. tropicalis ICL promoter and a T. reesei cbhII gene,
(o) a K. lactis AdhI promoter and a T. aurantiacus lpmo gene, and
(p) an S. cerevisiae Adh1 promoter and a M. thermophila cdhI gene.

### Claim 6 (depends on 1)

The engineered cell of claim 1, wherein the promoter and the gene in (ii) are selected from the group consisting of:
(a) a K. lactis Lac4 promoter and a kanamycin resistance gene,
(b) a K. lactis GapDHI promoter and a kanamycin resistance gene,
(c) an C. tropicalis ICL promoter and an aureobasidin A resistance gene,
(d) an S. cerevisiae ADH1 promoter and a kanamycin resistance gene,
(e) an S. cerevisiae ADH1 promoter and a Zeocin resistance gene,
(f) an S. cerevisiae ADH1 promoter and a hygromycin resistance gene,
(g) an S. cerevisiae ADH1 promoter and an aureobasidin A resistance gene,
(h) an S. cerevisiae ADH4 promoter and an aureobasidin A resistance gene, and
(i) an S. cerevisiae ADH1 promoter and a blasticidin resistance gene.

### Claim 7 (depends on 1)

The engineered cell of claim 1, wherein the cell contains:
(1) a first nucleic acid molecule that contains the K. lactis Lac4 promoter operably linked to the KanMx gene,
(2) a second nucleic acid molecule that contains the S. cerevisiae GapDHI promoter operably linked to the T. reesei egIII gene,
(3) a third nucleic acid molecule that contains the K. lactis GapDHI promoter operably linked to the T. reesei cbhI gene,
(4) a fourth nucleic acid molecule that contains the K. lactis AdhI promoter operably linked to the green fluorescent protein gene, and
(5) a fifth nucleic acid molecule that contains the S. cerevisiae AdhI promoter operably linked to the N. patriciarum npabgs gene,
(1)-(5) being adjacent to each other in the order of (1)-(2)-(3)-(4)-(5) in the 5′ to 3′ direction.

### Claim 8 (depends on 7)

The engineered cell of claim 7, wherein (1)-(5) are inserted within the Lac4 promoter region of the parent Kluyveromyces marxianus cell of the engineered cell.

### Claim 9 (depends on 1)

The engineered cell of claim 1, wherein the cell contains:
(1) a first nucleic acid molecule that contains the K. lactis Lac4 promoter operably linked to the KanMx gene,
(2) a second nucleic acid molecule that contains the S. cerevisiae GapDHI promoter operably linked to the T. reesei egIII gene,
(3) a third nucleic acid molecule that contains the S. cerevisiae PGK promoter operably linked to the T. reesei cbhI gene,
(4) a fourth nucleic acid molecule that contains the K. lactis GapDHI promoter operably linked to the A. niger eglA gene,
(5) a fifth nucleic acid molecule that contains the K. lactis PGK promoter operably linked to the T. reesei cbhII gene,
(6) a sixth nucleic acid molecule that contains the K. lactis AdhI promoter operably linked to the N. crassa cdt-1 gene, and
(7) a seventh nucleic acid molecule that contains the S. cerevisiae AdhI promoter is operably linked to the N. patriciarum npabgs gene,
(1)-(7) being adjacent to each other in the order of (1)-(2)-(3)-(4)-(5)-(6)-(7) in the 5′ to 3′ direction.

### Claim 10 (depends on 9)

The engineered cell of claim 9, wherein (1)-(7) are inserted within the Lac4 promoter region of the parent Kluyveromyces marxianus cell of the engineered cell.

### Claim 11 (depends on 1)

The engineered cell of claim 1, wherein the cell contains:
(1) a first nucleic acid molecule that contains the K. lactis Lac4 promoter operably linked to the A. niger egI gene,
(2) a second nucleic acid molecule that contains the S. cerevisiae GapDHI promoter operably linked to the T. reesei egIII gene,
(3) a third nucleic acid molecule that contains the S. cerevisiae PGK promoter operably linked to the A. niger eglA gene,
(4) a fourth nucleic acid molecule that contains the K. lactis GapDHI promoter operably linked to the KanMx gene,
(5) a fifth nucleic acid molecule that contains the K. lactis PGK promoter operably linked to the T. reesei cbhII gene,
(6) a sixth nucleic acid molecule that contains the K. lactis AdhI promoter operably linked to the T. reesei cbhI gene, and
(7) a seventh nucleic acid molecule that contains the S. cerevisiae AdhI promoter operably linked to the N. patriciarum npabgs gene,
(1)-(7) being adjacent to each other in the order of (1)-(2)-(3)-(4)-(5)-(6)-(7) in the 5′ to 3′ direction.

### Claim 12 (depends on 11)

The engineered cell of claim 11, wherein (1)-(7) are inserted within the Lac4 promoter region of the parent Kluyveromyces marxianus cell of the engineered cell.

### Claim 13 (depends on 1)

The engineered cell of claim 1, wherein the cell contains:
(1) a first nucleic acid molecule that contains the K. lactis Lac4 promoter operably linked to the A. niger egI gene,
(2) a second nucleic acid molecule that contains the S. cerevisiae GapDH promoter operably linked to the T. reesei egIII gene,
(3) a third nucleic acid molecule that contains the S. cerevisiae PGK promoter operably linked to the A. niger eglA gene,
(4) a fourth nucleic acid molecule that contains the S. cerevisiae Adh4 promoter operably linked to the T. reesei cbhI gene,
(5) a fifth nucleic acid molecule that contains the C. tropicalis ICL promoter operably linked to the S. cerevisiae AUR1 gene,
(6) a sixth nucleic acid molecule that contains the K. lactis Adh4 promoter operably linked to the N. patriciarum npabgs gene,
(7) a seventh nucleic acid molecule that contains the K. lactis PGK promoter operably linked to the T. reesei cbhII gene,
(8) an eighth nucleic acid molecule that contains the K. lactis AdhI promoter operably linked to the T. reesei cbhI gene, and
(9) a ninth nucleic acid molecule that contains the S. cerevisiae AdhI promoter operably linked to the N. patriciarum npabgs gene,
(1)-(9) being adjacent to each other in the order of (1)-(2)-(3)-(4)-(5)-(6)-(7)-(8)-(9) in the 5′ to 3′ direction.

### Claim 14 (depends on 13)

The engineered cell of claim 13, wherein (1)-(9) are inserted within the Lac4 promoter region of the parent Kluyveromyces marxianus cell of the engineered cell.

### Claim 15 (depends on 1)

The engineered cell of claim 1, wherein the cell contains:
(1) a first nucleic acid molecule that contains the K. lactis Lac4 promoter operably linked to the A. niger egI gene,
(2) a second nucleic acid molecule that contains the S. cerevisiae GapDHI promoter operably linked to the T. reesei egIII gene,
(3) a third nucleic acid molecule that contains the S. cerevisiae PGK promoter operably linked to the A. niger eglA gene,
(4) a fourth nucleic acid molecule that contains the K. lactis GapDHI promoter operably linked to the KanMx gene,
(5) a fifth nucleic acid molecule that contains the K. lactis PGK promoter operably linked to the T. reesei cbhII gene,
(6) a sixth nucleic acid molecule that contains the K. lactis PGK promoter operably linked to the T. reesei cbhI gene,
(7) a seventh nucleic acid molecule that contains the S. cerevisiae AdhI promoter is operably linked to the N. patriciarum npabgs gene,
(8) an eighth nucleic acid molecule that contains the K. lactis Lac4 promoter operably linked to the T. aurantiacus lpmo gene, and
(9) a ninth nucleic acid molecule that contains the K. lactis Lac4 promoter operably linked to the M. thermophila cdhI gene,
(1)-(7) being adjacent to each other in the order of (1)-(2)-(3)-(4)-(5)-(6)-(7) in the 5′ to 3′ direction.

### Claim 16 (depends on 15)

The engineered cell of claim 15, wherein (1)-(9) are inserted within the Lac4 promoter region of the parent Kluyveromyces marxianus cell of the engineered cell.

### Claim 17 (depends on 2)

The engineered cell of claim 2, wherein the scaffold polypeptide of (iv) is a C. thermocellum cellulosomal scaffoldin