Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity

Kun Shi; Ju Mou Li; Mu Qiang Wang; Yi Ke Zhang; Zhi Jun Zhang; Qi Chen; Frank Hollmann; Jian He Xu; Hui Lei Yu

doi:10.1126/sciadv.adp6775

Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity

Kun Shi, Ju Mou Li, Mu Qiang Wang, Yi Ke Zhang, Zhi Jun Zhang, Qi Chen, Frank Hollmann, Jian He Xu, Hui Lei Yu^*

^*Corresponding author for this work

BT/Biocatalysis

Research output: Contribution to journal › Article › Scientific › peer-review

56 Downloads (Pure)

Abstract

Engineering nonribosomal peptide synthetases (NRPSs) has been a “holy grail” in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the “gate-keeper” adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, MabCAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency (kcat/KM) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.

Original language	English
Article number	eadp6775
Number of pages	11
Journal	Science Advances
Volume	10
Issue number	48
DOIs	https://doi.org/10.1126/sciadv.adp6775
Publication status	Published - 2024

Access to Document

10.1126/sciadv.adp6775

sciadv.adp6775Final published version, 3.31 MBLicence: CC BY-NC

Cite this

@article{6c5f8ef533ba4efa8dc80866e537f574,

title = "Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity",

abstract = "Engineering nonribosomal peptide synthetases (NRPSs) has been a “holy grail” in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the “gate-keeper” adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, MabCAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency (kcat/KM) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.",

author = "Kun Shi and Li, {Ju Mou} and Wang, {Mu Qiang} and Zhang, {Yi Ke} and Zhang, {Zhi Jun} and Qi Chen and Frank Hollmann and Xu, {Jian He} and Yu, {Hui Lei}",

year = "2024",

doi = "10.1126/sciadv.adp6775",

language = "English",

volume = "10",

journal = "Science Advances",

issn = "2375-2548",

publisher = "American Association for the Advancement of Science",

number = "48",

}

TY - JOUR

T1 - Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity

AU - Shi, Kun

AU - Li, Ju Mou

AU - Wang, Mu Qiang

AU - Zhang, Yi Ke

AU - Zhang, Zhi Jun

AU - Chen, Qi

AU - Hollmann, Frank

AU - Xu, Jian He

AU - Yu, Hui Lei

PY - 2024

Y1 - 2024

N2 - Engineering nonribosomal peptide synthetases (NRPSs) has been a “holy grail” in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the “gate-keeper” adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, MabCAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency (kcat/KM) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.

AB - Engineering nonribosomal peptide synthetases (NRPSs) has been a “holy grail” in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the “gate-keeper” adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, MabCAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency (kcat/KM) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.

UR - http://www.scopus.com/inward/record.url?scp=85211149281&partnerID=8YFLogxK

U2 - 10.1126/sciadv.adp6775

DO - 10.1126/sciadv.adp6775

M3 - Article

C2 - 39612335

AN - SCOPUS:85211149281

SN - 2375-2548

VL - 10

JO - Science Advances

JF - Science Advances

IS - 48

M1 - eadp6775

ER -

Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity

Abstract

Access to Document

Other files and links

Fingerprint

Cite this