WO-2026096748-A2 - GENETICALLY ENGINEERED PEPTIDE SUPERSTRUCTURE AND MICROBE FOR PRODUCTION OF ISOPRENE AND METHOD OF PRODUCTION THEREOF

Abstract

A genetically engineered microbe capable of producing isoprene, wherein the genetically engineered microbe including a heterologous nucleic acid construct encoding at least a portion of a peptide superstructure; the peptide superstructure including a first portion including a first binding site configured to catalyze a first chemical reaction, wherein the first reaction converts a substrate to an intermediate; and a second portion including a second binding site configured to catalyze a second chemical reaction, wherein the second binding site is located in proximity to the first binding site and the second chemical reaction converts the intermediate to isoprene.

Inventors

• GREENHALGH, Jonathan Cloyd
• PRICE, Robert Andrew

Assignees

• CYCLOKINETICS, INC.

Dates

Publication Date

20260507

Application Date

20251030

Priority Date

20241030

Claims (20)

1. 1. A genetically engineered microbe capable of producing isoprene, the genetically engineered microbe comprises: the genetically engineered microbe comprises a heterologous nucleic acid construct encoding at least a portion of a peptide superstructure, wherein the superstructure comprises: a first portion comprising a first binding site configured to catalyze a first chemical reaction, wherein the first reaction converts a substrate to an intermediate; and a second portion comprising a second binding site configured to catalyze a second chemical reaction, wherein: the second binding site is located in proximity to the first binding site; and the second chemical reaction converts the intermediate to isoprene.
2. 2. The genetically engineered microbe of claim 1, wherein: the first portion comprises at least a functional fragment of isopentenyl diphosphate (IPP) isomerase; and the second portion comprises at least a functional fragment of isoprene synthase (IspS).
3. 3. The genetically engineered microbe of claim 1, wherein: the substrate comprises IPP; and the intermediate comprises dimethylallyl pyrophosphate (DMAPP).
4. 4. The genetically engineered microbe of claim 1, wherein the genetically engineered microbe comprises bacteria or fungi, wherein the bacteria or fungi includes one or more members selected from a group consisting of Escherichia Coli, Lactococcus, Vibrio natriegens, Saccharomyces cerevisiae, Aspergillus, Yarrowia lipolytica, Bacillus subtilis, Clostridium ljungdahlii, and Pseudomonas putid.
5. 5. The genetically engineered microbe of claim 1, wherein the genetically engineered microbe comprises a Crabtree-negative microbe.
6. 6. The genetically engineered microbe of claim 1, wherein the genetically engineered microbe comprises a diploid strain.
7. 7. A genetically engineered peptide superstructure for production of isoprene, the superstructure comprising: 47 Attorney Docket No. 1656-002PCT1 a first portion comprising at least a functional fragment of isoprene synthase (TspS) configured to catalyze a first chemical reaction; and a second portion comprising at least a functional fragment of isopentenyl diphosphate (IPP) isomerase configured to catalyze a second chemical reaction, wherein the second chemical reaction converts a substrate to an intermediate, and wherein: the second portion is covalently linked to the first portion through a linker; and the intermediate is converted into isoprene by the first chemical reaction.
8. 8. The superstructure of claim 7, wherein the first portion begins at N-terminal end of the superstructure and the second portion terminates at a C-terminal end of the superstructure.
9. 9. The superstructure of claim 7, wherein: the substrate comprises IPP; and the intermediate comprises dimethylallyl pyrophosphate (DMAPP).
10. 10. The superstructure of claim 7, wherein the linker comprises a rigid linker or a flexible linker.
11. 11. The superstructure of claim 7, wherein a level of isoprene production is correlated to a rigidity of the linker.
12. 12. A genetically engineered peptide superstructure for production of isoprene, the superstructure comprising: a first portion comprising at least a functional fragment of isopentenyl diphosphate (IPP) isomerase configured to catalyze a first chemical reaction, wherein the first chemical reaction converts a substrate to an intermediate; and a second portion comprising at least a functional fragment of isoprene synthase (IspS) configured to catalyze a second chemical reaction, wherein: the second portion is covalently linked to the first portion through a linker; and the intermediate is converted into isoprene by the second chemical reaction.
13. 13. The superstructure of claim 12, wherein the first portion begins at N-terminal end of the superstructure and the second portion terminates at a C-terminal end of the superstructure.
14. 14. The superstructure of claim 12, wherein: the substrate comprises IPP; and 48 Attorney Docket No. 1656-002PCT1 the intermediate comprises dimethylallyl pyrophosphate (DMAPP).
15. 15. The superstructure of claim 12, wherein the linker comprises a rigid linker or a flexible linker.
16. 16. A method of producing isoprene using a genetically engineered peptide superstructure, the method comprising: culturing a genetically engineered microbe under suitable conditions, wherein: the genetically engineered microbe comprises a heterologous nucleic acid construct encoding at least a portion of a peptide superstructure; and the superstructure comprises: a first portion comprising a first binding site; and a second portion comprising a second binding site, the second portion is covalently linked to the first portion through a linker; providing a substrate to the cultured genetically engineered microbe, wherein: the first binding site is configured to bind to the substrate and catalyze a first chemical reaction that converts the substrate to an intermediate; and the second binding site is configured to catalyze a second chemical reaction that converts the intermediate to isoprene.
17. 17. The method of claim 16, wherein the genetically engineered microbe comprises Escherichia coli.
18. 18. The method of claim 16, wherein: the first portion comprises at least a functional fragment of isopentenyl diphosphate (IPP) isomerase; and the second portion comprises at least a functional fragment of isoprene synthase (IspS).
19. 19. The method of claim 16, wherein the first portion begins at C-terminal end of the superstructure and the second portion terminates at a N-terminal end of the superstructure.
20. 20. The method of claim 16, wherein: the substrate comprises IPP; and the intermediate comprises dimethylallyl pyrophosphate (DMAPP). 49 Attorney Docket No. 1656-002PCT1

Description

GENETICALLY ENGINEERED PEPTIDE SUPERSTRUCTURE AND MICROBE FOR PRODUCTION OF ISOPRENE AND METHOD OF PRODUCTION THEREOF REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority of U.S. Provisional Application Serial No. 63/713,890, filed on October 30, 2024, and entitled “GENETICALLY ENGINEERED PEPTIDE SUPERSTRUCTURE AND MICROBE FOR PRODUCTION OF ISOPRENE AND METHOD OF PRODUCTION THEREOF,” which is incorporated by reference herein in its entirety. FIELD OF THE INVENTION The present invention generally relates to the field of metabolic engineering. In particular, the present invention is directed to a genetically engineered peptide superstructure and microbe for production of isoprene. REFERENCE TO SEQUENCE LISTING This specification includes a sequence listing submitted herewith, which includes the file entitled 1656-002USU1.xml having the following size: 113,663 bytes which was created October 20, 2025, the contents of which are incorporated by reference herein. BACKGROUND Isoprene is an important commodity chemical used in a wide range of industrial applications, from the production of synthetic rubber for tires and coatings to uses in adhesives and development of specialty elastomers. Current production of isoprene is derived mostly from unsustainable petrochemical sources and often involves resource-intensive methods that require extensive land use, high temperature, and/or use of precious metals. Biosynthesis of isoprene, while showing some progress, is often plagued by competing side reactions that limit the yield of isoprene. SUMMARY OF THE DISCLOSURE In an aspect, a genetically engineered microbe capable of producing isoprene, wherein the genetically engineered microbe including a heterologous nucleic acid construct encoding at least a portion of a peptide superstructure; the peptide superstructure including a first portion including a first binding site configured to catalyze a first chemical reaction, wherein the first reaction converts a substrate to an intermediate; and a second portion including a second binding site configured to catalyze a second chemical reaction, wherein the second binding site is located 1 Attorney Docket No. 1656-002PCT1 in proximity to the first binding site and the second chemical reaction converts the intermediate to isoprene, the first portion may include at least a functional fragment of isopentenyl diphosphate (IPP) isomerase; and the second portion may include at least a functional fragment of isoprene synthase (IspS). The substrate may include IPP; and the intermediate may include dimethylallyl pyrophosphate (DMAPP). The genetically engineered microbe may include bacteria or fungi, wherein the bacteria or fungi may include one or more members selected from a group consisting of Escherichia Coli, Lactococcus, Vibrio natri egens, Saccharomyces cerevisiae, Aspergillus, Yarrowia lipolytica, Bacillus subtilis, Clostridium ljungdahlii, and Pseudomonas putida. The genetically engineered microbe may include a Crabtree-negative microbe. The genetically engineered microbe may include a diploid strain. In another aspect, a genetically engineered peptide superstructure for production of isoprene, the superstructure including a first portion including at least a functional fragment of isoprene synthase (IspS) configured to catalyze a first chemical reaction, and a second portion including at least a functional fragment of isopentenyl diphosphate (IPP) isomerase configured to catalyze a second chemical reaction, wherein the second chemical reaction converts a substrate to an intermediate, wherein the second portion is covalently linked to the first portion through a linker, and wherein the intermediate is converted into isoprene by the first chemical reaction. The first portion may begin at N-terminal end of the superstructure and the second portion may terminate at a C-terminal end of the superstructure. The substrate may include IPP; and the intermediate may include dimethylallyl pyrophosphate (DMAPP). The linker may include a rigid linker or a flexible linker. A level of isoprene production may be correlated to a rigidity of the linker. In another aspect, a genetically engineered peptide superstructure for production of isoprene, the superstructure including a first portion including at least a functional fragment of isopentenyl diphosphate (IPP) isomerase configured to catalyze a first chemical reaction, wherein the first chemical reaction converts a substrate to an intermediate, and a second portion including at least a functional fragment of isoprene synthase (IspS) configured to catalyze a second chemical reaction, wherein the second portion is covalently linked to the first portion through a linker, and wherein the intermediate is converted into isoprene by the second chemical reaction. The first portion may begin at N-terminal end of the superstructure and the second portion may terminate at a C-terminal end of the superstructure. The substrate