# US 10,480,003 B2 · Constructs and systems and methods for engineering a CO2 fixing photorespiratory by-pass pathway

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- Number: US 10,480,003 B2
- Document ID: US10480003B2
- Status: Granted
- Kind: B2
- Application number: 15/230,332
- Filed: 2016-08-05
- Published: 2017-03-30
- Granted: 2019-11-19
- Earliest claimed: 2014-02-06
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Cheryl A. Kerfeld; Patrick Shih; Jan Zarzycki; Krishna K. Niyogi
- Web page: https://patentgrep.com/patent/US10480003B2

## CPC

- C12N 15/8269: {Photosynthesis}
- C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine}
- Y02P 60/20: Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

Methods and compositions to introduce a synthetic pathway based on the 3-hydroxypropionate bicycle into an organism such as the model cyanobacterium, Synechococcus elongatus sp. PCC 7942. The heterologously expressed pathway acts as a photorespiratory bypass as well as an additional carbon fixation cycle orthogonal to the endogenous Calvin-Benson cycle. We demonstrate the function of all six introduced enzymes, which not only has implications on increasing net-photosynthetic productivity, but also key enzymes in the pathway are involved in high-value products that are of biotechnological interest, such as 3-hydroxypropionate.

## Claims

### Claim 1

A recombinant host cell comprising heterologously expressed genes mcr, mcl, mch, mct, meh, and pcs, and a second heterologously expressed mct gene, and wherein the recombinant host cell does not naturally express the heterologously expressed genes and wherein the host cell produces a synthetic photorespiratory bypass pathway.

### Claim 2 (depends on 1)

The recombinant host cell of claim 1, wherein at least one of the heterologously expressed genes is from Chloroflexus aurantiacus J-10-fl.

### Claim 3 (depends on 1)

The recombinant host cell of claim 1, wherein at least one of the heterologously expressed genes is from Erythrobacter sp. NAP1.

### Claim 4 (depends on 1)

The recombinant host cell of claim 1, wherein the heterologously expressed genes comprise genes having a sequence of SEQ ID NOs: 19-24 or functional homologs thereof.

### Claim 5 (depends on 4)

The recombinant host cell of claim 4, wherein the functional homologs comprise genes having a sequence of SEQ ID NOs: 25-30.

### Claim 6 (depends on 1)

The recombinant host cell of claim 1, wherein the second heterologous mct gene is from the same organism as the mct gene.

### Claim 7 (depends on 1)

The recombinant host cell of claim 1, wherein the second heterologous mct gene is from a different organism than the mct gene.

### Claim 8 (depends on 1)

The recombinant host cell of claim 1, wherein one or more of the heterologously expressed genes is encoded from a DNA expression cassette.

### Claim 9 (depends on 8)

The recombinant host cell of claim 8, wherein the DNA expression cassette comprises a promoter operably linked to the one or more heterologously expressed genes.

### Claim 10 (depends on 8)

The recombinant host cell of claim 8, wherein the expression cassette has a sequence in accordance with any of SEQ ID NOs: 31-34.

### Claim 11 (depends on 8)

The recombinant host cell of claim 8, wherein the expression cassette comprises a selectable marker.

### Claim 12 (depends on 8)

The recombinant host cell of claim 8 wherein the expression cassette comprises an inducible promoter.

### Claim 13 (depends on 12)

The recombinant host cell of claim 12 wherein the inducible promoter is an IPTG-inducible promoter.

### Claim 14 (depends on 1)

The recombinant host cell of claim 1, wherein the heterologously expressed genes are stably incorporated into the recombinant host cell.

### Claim 15 (depends on 1)

The recombinant host cell of claim 1, wherein the heterologously expressed genes are stably incorporated into the genome of the recombinant host cell.

### Claim 16 (depends on 1)

The recombinant host cell of claim 1, wherein the heterologously expressed genes are transiently expressed from a plasmid.

### Claim 17 (depends on 1)

The recombinant host cell of claim 1, wherein the recombinant host cell is a bacterial cell, a cyanobacterial cell, an oxygenic photoautotroph or a plant cell.

### Claim 18

A recombinant host cell comprising stably transformed heterologous genes having sequences of SEQ ID Nos: 19-24 or functional homologs thereof wherein SEQ ID No: 23 encodes a first heterologously expressed mct gene, and wherein the recombinant host cell comprises a second heterologously expressed mct gene, wherein the host cell produces a synthetic photorespiratory bypass pathway.

### Claim 19 (depends on 18)

The recombinant host cell of claim 18, wherein the recombinant host cell is a bacterial cell, a cyanobacterial cell, an oxygenic photoautotroph or a plant cell.

### Claim 20 (depends on 18)

The recombinant host cell of claim 18, wherein the functional homologs comprise genes having a sequence of SEQ ID NOs: 25-30.