Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design

Jake W. Saunders; Adam M. Damry; Vanessa Vongsouthi; Matthew A. Spence; Rebecca L. Frkic; Chloe Gomez; Patrick A. Yates; Dana S. Matthews; Nobuhiko Tokuriki; Malcolm D. McLeod; Colin J. Jackson

doi:10.1021/acs.biochem.4c00165

Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design

Jake W. Saunders, Adam M. Damry, Vanessa Vongsouthi, Matthew A. Spence, Rebecca L. Frkic, Chloe Gomez, Patrick A. Yates, Dana S. Matthews, Nobuhiko Tokuriki, Malcolm D. McLeod, Colin J. Jackson^*

^*Corresponding author for this work

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

6 Citations (Scopus)

Abstract

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

Original language	English
Pages (from-to)	1663-1673
Number of pages	11
Journal	Biochemistry
Volume	63
Issue number	13
Early online date	17 Jun 2024
DOIs	https://doi.org/10.1021/acs.biochem.4c00165
Publication status	Published - 2 Jul 2024

Access to Document

10.1021/acs.biochem.4c00165

Cite this

Saunders, J. W., Damry, A. M., Vongsouthi, V., Spence, M. A., Frkic, R. L., Gomez, C., Yates, P. A., Matthews, D. S., Tokuriki, N., McLeod, M. D., & Jackson, C. J. (2024). Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design. Biochemistry, 63(13), 1663-1673. https://doi.org/10.1021/acs.biochem.4c00165

@article{55e98e11e66e48ffa0d71d79948cc0ba,

title = "Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design",

abstract = "The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.",

keywords = "Proteins, Molecules, Dye, Model, Insight, Sequence, Petase",

author = "Saunders, \{Jake W.\} and Damry, \{Adam M.\} and Vanessa Vongsouthi and Spence, \{Matthew A.\} and Frkic, \{Rebecca L.\} and Chloe Gomez and Yates, \{Patrick A.\} and Matthews, \{Dana S.\} and Nobuhiko Tokuriki and McLeod, \{Malcolm D.\} and Jackson, \{Colin J.\}",

note = "{\textcopyright} 2024 American Chemical Society.",

year = "2024",

month = jul,

day = "2",

doi = "10.1021/acs.biochem.4c00165",

language = "English",

volume = "63",

pages = "1663--1673",

journal = "Biochemistry",

issn = "0006-2960",

publisher = "American Chemical Society",

number = "13",

}

TY - JOUR

T1 - Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design

AU - Saunders, Jake W.

AU - Damry, Adam M.

AU - Vongsouthi, Vanessa

AU - Spence, Matthew A.

AU - Frkic, Rebecca L.

AU - Gomez, Chloe

AU - Yates, Patrick A.

AU - Matthews, Dana S.

AU - Tokuriki, Nobuhiko

AU - McLeod, Malcolm D.

AU - Jackson, Colin J.

PY - 2024/7/2

Y1 - 2024/7/2

N2 - The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

AB - The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

KW - Proteins

KW - Molecules

KW - Dye

KW - Model

KW - Insight

KW - Sequence

KW - Petase

UR - https://www.scopus.com/pages/publications/85196492733

U2 - 10.1021/acs.biochem.4c00165

DO - 10.1021/acs.biochem.4c00165

M3 - Article

AN - SCOPUS:85196492733

SN - 0006-2960

VL - 63

SP - 1663

EP - 1673

JO - Biochemistry

JF - Biochemistry

IS - 13

ER -

Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design

Abstract

Access to Document

Other files and links

Fingerprint

Cite this