# US 2021/0277428 A1 · Novel malonyl-coa biosensor based on type iii polyketide synthase and use thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2021/0277428 A1
- Document ID: US20210277428A1
- Status: Published application
- Kind: A1
- Application number: 16/477,897
- Filed: 2018-08-30
- Published: 2021-09-09
- Earliest claimed: 2018-06-08
- Applicants: Korea Advanced Institute of Science and Technology
- Inventors: Sang Yup Lee; Dongsoo YANG
- Web page: https://patentgrep.com/patent/US20210277428A1

## CPC

- C12P 7/22: aromatic
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12P 7/42: Hydroxy-carboxylic acids
- C12P 17/06: containing a six-membered hetero ring, e.g. fluorescein
- C12Q 1/008: {for determining co-enzymes or co-factors, e.g. NAD, ATP}
- C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)}
- C12Q 1/48: involving transferase
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention relates to a recombinant microorganism for malonyl-CoA detection in which a type III polyketide synthase-encoding gene is inserted in the genome or in which a recombinant vector containing the gene is introduced; a method of screening a malonyl-CoA production-inducing substance using the recombinant microorganism; a method of screening a gene which is involved in increased malonyl-CoA production; and a method comprising knocking down the gene, screened by the method, in a microorganism, thus increasing the production of malonyl-CoA in the microorganism, and producing a useful substance in the microorganism using malonyl-CoA as a precursor. The use of the biosensor according to the present invention provides single-step signal generation, utilization in various microorganisms, utilization in self-fluorescent microorganisms, a simple construction method, and a simple screening method. In addition, when the present invention is combined with high-throughput screening, it has advantages in that strains having increased malonyl-CoA producing ability can be screened very easily and rapidly (˜3 days) and can be applied directly to the malonyl-CoA-based production of useful compounds.

## Claims

### Claim 1

A recombinant microorganism for indicating intracellular malonyl-CoA level in which a type III polyketide synthase-encoding gene is inserted in the genome of the microorganism or in which a recombinant vector containing the type III polyketide synthase-encoding gene is introduced.

### Claim 2 (depends on 1)

The recombinant microorganism of claim 1, wherein the type III polyketide synthase is:
RppA derived from a microorganism selected from the group consisting of Streptomyces griseus, Streptomyces coelicolor, Streptomyces avermitilis, Saccharopolyspora erythraea, Streptomyces peucetius, and Streptomyces aculeolatus;
PhlD (polyketide synthase) derived from Pseudomonas fluorescens;
DpgA (polyketide synthase) derived from Amycolatopsis mediterranei;
ALS (aloesone synthase) derived from Rheum palmatum; or
PCS (5,7-dihydroxy-2-methylchromone synthase), OKS (octaketide synthase), PKS3 (aloesone synthase), PKS4 (octaketide synthase 2) or PKS5 (octaketide synthase 3), which is derived from Aloe arborescens.

### Claim 3 (depends on 1)

The recombinant microorganism of claim 1, wherein a gene encoding the type III polyketide synthase is operably linked to a promoter selected from the group consisting of tac, trc, T7, BAD, λPR an Anderson synthetic promoter.

### Claim 4 (depends on 1)

The recombinant microorganism of claim 1, wherein the recombinant microorganism is selected from the group consisting of E. coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Cyanobacterium, and Cyclobacterium.

### Claim 5 (depends on 1)

A method for screening a malonyl-CoA production-inducing substance, comprising the steps of:
(a) culturing the recombinant microorganism of claim 1;
(b) adding a candidate substance to the recombinant microorganism;
(c) comparing the color of a culture supernatant of the recombinant microorganism after addition of the candidate substance with the color of a culture supernatant of the recombinant microorganism without addition of the candidate substance; and
(d) selecting the candidate substance as the malonyl-CoA production-inducing substance, when the culture supernatant of the recombinant microorganism after addition of the candidate substance shows a deeper red color than the culture supernatant of the recombinant microorganism without addition of the candidate substance.

### Claim 6 (depends on 5)

The method of claim 5, wherein the comparison between the colors in step (c) is made by the naked eyes.

### Claim 7 (depends on 5)

The method of claim 5, wherein the comparison between the colors in step (c) is made by measuring absorbance, and step (d) comprises selecting the candidate substance as the malonyl-CoA production-inducing substance, when the absorbance measured after addition of the candidate substance is higher than that measured without addition of the candidate substance.

### Claim 8 (depends on 1)

A method for screening a gene involved in increase of malonyl-CoA production, the method comprising the steps of:
(a) introducing a gene regulation library, which changes gene expression in a recombinant microorganism, into the recombinant microorganism of claim 1, thereby constructing a recombinant microorganism library in which gene expression in the recombinant microorganism is changed;
(b) culturing the constructed recombinant microorganism library and the recombinant microorganism before introduction of the gene regulation library, and comparing the color of the culture supernatant of the recombinant microorganism library with the color of the culture supernatant of the recombinant microorganism before introduction of the gene regulation library; and
(c) selecting the gene introduced in the recombinant microorganism library as a gene involved in increase of malonyl-CoA production, when the culture supernatant of the recombinant microorganism library shows a deeper red color than the culture supernatant of the recombinant microorganism before introduction of the gene regulation library.

### Claim 9 (depends on 8)

The method of claim 8, wherein the comparison between the colors in step (b) is made by the naked eyes.

### Claim 10 (depends on 8)

The method of claim 8, wherein the comparison between the colors in step (b) is made by measuring absorbance, and step (c) comprises selecting the gene introduced in the recombinant microorganism library, when the absorbance of the culture supernatant of the recombinant microorganism library is higher than that of the culture supernatant of the recombinant microorganism before introduction of the gene regulation library.

### Claim 11 (depends on 8)

The method of claim 8, wherein the gene regulation library is a library selected from an sRNA library, a genomic library, a cDNA library, a gRNA library, and an oligonucleotide library for construction of knockout or mutant strains.

### Claim at position 12

-33. (canceled)