US-12618050-B2 - Engineered polypeptides that exhibit increased catalytic efficiency for unnatural cofactors and uses thereof

US12618050B2US 12618050 B2US12618050 B2US 12618050B2US-12618050-B2

Abstract

Provided are engineered polypeptides having dehydrogenase activity that exhibit increased catalytic efficiency for nicotinamide-based unnatural cofactors relative to wild-type or parent polypeptides that encode a dehydrogenase that utilizes nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. Also provided are cell-free and whole cell biotransformation systems for converting a substrate into a product using a redox reaction with a re-cycled unnatural cofactor that utilizes an engineered polypeptide that has dehydrogenase activity and which exhibits increased catalytic efficiency for nicotinamide-based unnatural cofactor.

Inventors

Han Li
Justin B. Siegel
Youtian CUI
Wai Shun Mak

Assignees

THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Dates

Publication Date: 20260505
Application Date: 20191210

Claims (7)

1 . An engineered polypeptide having dehydrogenase activity that exhibits catalytic efficiency for a nicotinamide-based unnatural cofactor, wherein the engineered polypeptide comprises 1, 2, 3, 4, 5, 6, 7, or 8 introduced amino acid substitutions in comparison to the sequence of a wild-type or parent polypeptide having the polypeptide sequence of SEQ ID NO:1, wherein the introduced amino acid substitutions comprise at least the substitution of I195R in comparison to SEQ ID NO:1, wherein the cofactor normally utilized by the wild-type or parent polypeptide is nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+), wherein the nicotinamide-based unnatural cofactor is a cofactor that is not normally utilized by the dehydrogenase encoded by the wild-type or parent polypeptide to catalyze a reaction and which is selected from the group consisting of nicotinamide mononucleotide (NMN + ), 1-phenyl-1,4,-dihydronicotinamide (PNA + ), 1-benzyl-1,4-dihydronicotinamide (BNA + ), 1-(4-hydroxyphenyl) 1,4-dihydronicotinamide (HPNA + ), 1-methyl-1,4-dihydronicotinamide (MNA + ), nicotinamide flucytosine dinucleotide (NFCD + ), and nicotinamide mononucleoside (NR + ).
2 . The engineered polypeptide of claim 1 , wherein the nicotinamide-based unnatural cofactor is NMN+.
3 . The engineered polypeptide of claim 1 , wherein the engineered polypeptide further comprises the introduced amino acid substitutions of A93K and Y39Q in comparison to SEQ ID NO:1.
4 . The engineered polypeptide of claim 1 , wherein the engineered polypeptide comprises a sequence selected from SEQ ID NO:2 or SEQ ID NO:6.
5 . The engineered polypeptide of claim 1 , wherein the engineered polypeptide further comprises the introduced amino acid substitution of S17E in comparison to SEQ ID NO:1.
6 . The engineered polypeptide of claim 3 , wherein the engineered polypeptide further comprises the introduced amino acid substitution of S17E in comparison to SEQ ID NO:1.
7 . The engineered polypeptide of claim 6 , wherein the engineered polypeptide comprises the sequence of SEQ ID NO:7.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Ser. No. 62/777,725, filed Dec. 10, 2018 and to U.S. Provisional Application Ser. No. 62/852,936, filed May 24, 2019, the disclosures of which are incorporated herein by reference in their entirety. STATEMENT OF GOVERNMENT SUPPORT This application is a U.S. National Stage Application filed under 35 U.S.C. § 371 and claims priority to International Application No. PCT/US2019/065553, filed Dec. 10, 2019, which application claims priority to U.S. Provisional Application Ser. No. 62/777,725, filed Dec. 10, 2018 and to U.S. Provisional Application Ser. No. 62/852,936, filed May 24, 2019, the disclosures of which are incorporated herein by reference in their entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 10, 2019, is named Sequence_ST25.txt and is 74,131 bytes in size. TECHNICAL FIELD Provided herein are engineered polypeptides that exhibit increased catalytic efficiency for unnatural cofactors and uses of said polypeptides in engineered unnatural redox cofactor systems for whole-cell biomanufacturing and in cell-free applications. BACKGROUND Oxidoreductases are some of the best-established enzymatic tools for chemical synthesis due to their versatile functionality, broad substrate ranges, and high regio- and enantio-selectivity. Large-scale industrial processes using oxidoreductases to install the desired chiral centers have been developed, which feature lower cost, high product yield, mild reaction conditions, and environmental friendliness compared to their chemical catalyst-mediated counterparts. Oxidoreductases utilize cofactors which must be regenerated with each product molecule. An efficient redox cofactor recycling system is an indispensable component of an economically viable oxidoreductase process, owing to the formidable cost of using electron donors NAD(P)H at stoichiometric amounts. To this end, various methods have been employed to regenerate the reduced cofactors. Among them, formate dehydrogenases (FDHs) and glucose dehydrogenases (GDHs) are most widely used because they consume inexpensive substrates to reduce NAD(P)+ in a virtually irreversible way. SUMMARY Enzymatic biotransformation is a convenient way to manufacture chiral chemicals. Typically, an enzymatic biotransformation requires an enzyme, such as oxidoreductase, and a redox cofactor system to mediate the regeneration of the enzyme. These cofactors are expensive to use in vitro and difficult to control in vivo. Although simple and efficient redox cofactor analogs, also known as biomimetics, have been synthesized, native enzymes seem to display low activities towards these simple unnatural cofactor analogs. A potentially elegant solution is to utilize unnatural cofactors which operate in an orthogonal manner to naturally occurring cofactors. Provided herein is the development of an unnatural redox cofactor system that utilizes a nicotinamide-based unnatural cofactor (e.g., NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase (GDH) with a 107-fold cofactor specificity switch towards the nicotinamide-based unnatural cofactor over NAD(P)+. It is demonstrated herein that this system can be used to support diverse redox chemistries in vitro with high total turnover number (˜39,000); to specifically channel reducing power in Escherichia coli whole cells from glucose to a pharmaceutical intermediate; levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Accordingly, the disclosure provides for computationally designed engineered polypeptides and proteins that efficiently regenerate unnatural cofactors to support diverse redox chemistries, including for applications, like the synthesis of compounds, chiral compounds in particular, and in vitro biotransformations. Additionally, the disclosure provides for unnatural redox cofactor systems, comprising said engineered polypeptides for efficient whole-cell biomanufacturing and other applications. In a particular embodiment, the disclosure provides for an engineered polypeptide that exhibits increased catalytic efficiency for an unnatural cofactor than a wild-type or parent polypeptide sequence, wherein the engineered polypeptide comprises one or more introduced amino acid substitutions in comparison to the wild-type or parent polypeptide sequence, and wherein the one or more amino acid substitutions increases electrostatic complementarity between the engineered polypeptide and the unnatural cofactor. In a further embodiment of the foregoing embodiment, the engineered polypeptide encodes an oxidoreductase. In yet a further embodiment of any of the foregoing embodiments, the engineered polypeptide encodes a dehydrogenase. In yet a fu