US 2024/0401096 A1 · BIOMOLECULAR CONDENSATE MANUFACTURING SCAFFOLD

Abstract

Methods and systems for synthesizing biological products within biomimetic condensates that enhance the concentration of enzymes, substrates, co-factors, and other molecules involved in the synthesis. The one or more enzymes involved in catalysis can be engineered to comprise low-complexity amino acid sequences or phase separation domains that can be controlled to drive reversible liquid-liquid phase separation, wherein the resulting biomimetic condensates comprise a traversable phase boundary between their dense internal portion and the less-crowded aqueous composition within which the condensates are maintained. Alternatively, biomimetic condensates can be first formed within the aqueous composition from affinity-tagged scaffold proteins that do not take part in catalysis, but are capable of recruiting into the condensate catalytic enzymes fused to an affinity tag partner. One or more enzymes can comprise or be recruited inside of the biomimetic condensate to generate a wide variety of desired chemical products.

Claims20

Dependent claims

A method for catalyzing the in vitro enzymatic synthesis of a biological product within a biomimetic condensate, the process comprising the following steps:

(a) providing an aqueous composition comprising one or more enzymes, the one or more enzymes having a biological activity, wherein the biological activity consists of producing a biological product upon reacting with one or more substrates;
(b) assembling the one or more enzymes into a biomimetic condensate having an internal portion and an external portion separated by a phase boundary,
wherein the one or more enzymes are contained within the internal portion, and wherein the internal portion is a liquid;
(c) introducing one or more substrates into the aqueous composition,
wherein the one or more substrates can freely traverse the phase boundary between the external portion and internal portion of the biomimetic condensate;
and
(d) initiating a chemical reaction between at least one of the enzymes and at least one of the substrates to synthesize the biological product.

4 more elements

A method for the in vitro enzymatic synthesis of a biological product within a biomimetic condensate, the process comprising the following steps:

(a) providing an aqueous composition comprising one or more scaffold proteins, the one or more scaffold protein comprising at least a fragment of a GKAP protein, at least a fragment of a Homer protein, and at least a fragment of a Shank protein,
wherein at least one of GKAP, Homer, Shank proteins is fused to an affinity tag;
(b) assembling the one or more scaffold proteins into a biomimetic condensate having an internal portion and an external portion separated by a phase boundary,
wherein the one or more scaffold proteins are contained within the internal portion, and
wherein the internal portion is a liquid;
(c) introducing one or more enzymes into the aqueous composition,
wherein at least one of the enzymes is fused to affinity tag partner having an equilibrium dissociation constant (KD) with the affinity tag of less than 1 μM;
(d) binding the affinity tag partner with the affinity tag, thereby recruiting the one or more enzymes into the biomimetic condensate;
(e) introducing one or more substrates into the aqueous composition,
wherein the substrates can freely traverse the phase boundary between the external portion and internal portion of the biomimetic condensate;
and
(f) initiating a chemical reaction between at least one of the enzymes and at least one of the substrates to synthesize the biological product.

9 more elements

Dependencies are inferred from the claim text.

Biomolecular condensate manufacturing scaffold

Life of this patent

Biomolecular condensate manufacturing scaffold

Life of this patent

Abstract

Claims20