Enzyme scaffolds and methods of use

Patent metadata

Field	Value
Publication number	US-10577633-B2
Application number	US-201715467340-A
Country	US
Kind code	B2
Filing date	Mar 23, 2017
Priority date	Mar 23, 2016
Publication date	Mar 3, 2020
Grant date	Mar 3, 2020

Abstract

Polypeptide scaffolds comprising enzymatic proteins are provided. The enzymatic polypeptide scaffolds comprise heterologous enzymes to form a heterologous metabolic pathway, and can be targeted to a substrate through a surface anchoring domain. The enzymatic polypeptide scaffolds leverage the high specificity and affinity protein/protein interaction between the cohesins and dockerins of microorganismal cellulosomes to form custom enzymatic arrays.

First claim

What is claimed is:

1. An enzymatic polypeptide scaffold comprising:
a first linker domain, a first cohesin domain, and a second cohesin domain, wherein the first linker domain interconnects the first and second cohesin domains;
a first recombinant polypeptide comprising a first dockerin domain and an acetolactate synthase catalytic domain, wherein the first dockerin domain selectively binds to the first cohesin domain and the acetolactate synthase catalytic domain has a sequence identity of at least 90% to the polypeptide of SEQ ID NO: 21; and
a second recombinant polypeptide comprising a second dockerin domain and an acetolactate decarboxylase domain, wherein the second dockerin domain selectively binds to the second cohesin domain, and the acetolactate decarboxylase catalytic domain has a sequence identity of at least 90% to the polypeptide of SEQ ID NO: 22.

2. The enzymatic polypeptide scaffold of claim 1 , further comprising:
a second linker domain and a third cohesin domain, wherein the second linker domain interconnects the second and third cohesin domains; and
a third recombinant polypeptide comprising a third dockerin domain and a butanediol dehydrogenase catalytic domain, wherein the third dockerin domain selectively binds to the third cohesin domain and the butanediol dehydrogenase catalytic domain has a sequence identity of at least 90% to the polypeptide of SEQ ID NO: 23.

3. The enzymatic polypeptide scaffold of claim 1 , further comprising a surface anchoring domain and an anchoring linker domain, wherein the anchoring linker domain interconnects the surface anchoring domain and the first cohesin domain.

4. The enzymatic polypeptide scaffold of claim 2 , further comprising:
a first polypeptide linker between the first dockerin domain and the acetolactate synthase catalytic domain, wherein the acetolactate synthase catalytic domain has a sequence identity of at least 90% to the polypeptide of SEQ ID NO: 21,
a second polypeptide linker between the second dockerin domain and the acetolactate decarboxylase catalytic domain, wherein the acetolactate decarboxylase catalytic domain has a sequence identity of at least 90% to the polypeptide of SEQ ID NO: 22, and
a third polypeptide linker between the third dockerin domain and the butanediol dehydrogenase catalytic domain, wherein the butanediol dehydrogenase catalytic domain has a sequence identity of at least 90% to the polypeptide of SEQ ID NO: 23.

5. The enzymatic polypeptide scaffold of claim 3 , wherein the surface anchoring domain is a cellulose binding domain.

6. The enzymatic polypeptide scaffold of claim 2 , wherein the first linker and the second linker are each independently a synthetic linker, the first linker has an amino acid sequence that is 95% identical to SEQ ID NO: 4, and the second linker has an amino acid sequence that is 95% identical to SEQ ID NO: 6.

7. An enzymatic polypeptide scaffold array comprising:
a first enzymatic polypeptide scaffold according to claim 1 , further comprising a first adapter linker and a first adapter dockerin, wherein the first adapter linker interconnects the first adapter dockerin and the first cohesin domain of the first scaffold;
a second enzymatic polypeptide scaffold according to claim 1 , further comprising a second adapter linker and a second adapter dockerin, wherein the second adapter linker interconnects the second adapter dockerin and the first cohesin domain of the second scaffold; and
an adapter scaffold comprising two adapter cohesin domains and an adapter linker domain that interconnects the adapter cohesins,
wherein the first and second adapter dockerins selectively bind to the adapter cohesin domains; and
wherein the adapter scaffold interconnects the first and second enzymatic polypeptide scaffolds.

8. A method for producing 2,3 butanediol from pyruvate, comprising:
(i) contacting pyruvate with the enzymatic polypeptide scaffold of claim 1 , and
(ii) recovering 2,3 butanediol.

Assignees

• Alliance Sustainable Energy

Inventors

• Bomble Yannick J
• Himmel Michael E
• Linger Jeffrey
• Brunecky Roman
• Aikens John

Classifications

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12P7/18 Primary

  polyhydric · CPC title

• C12N9/0036

  acting on NADH or NADPH (1.6) · CPC title

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12Y102/01003

  Aldehyde dehydrogenase (NAD+) (1.2.1.3) · CPC title