A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

Lakshmeesha K. Nagappa; Wakana Sato; Farzana Alam; Kameshwari Chengan; Christopher M. Smales; Tobias Von Der Haar; Karen M. Polizzi; Katarzyna P. Adamala; Simon J. Moore

doi:10.3389/fbioe.2022.992708

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

Lakshmeesha K. Nagappa, Wakana Sato, Farzana Alam, Kameshwari Chengan, Christopher M. Smales, Tobias Von Der Haar, Karen M. Polizzi, Katarzyna P. Adamala, Simon J. Moore

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

5 Scopus citations

Abstract

Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta^™ 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.

Original language	English (US)
Article number	992708
Journal	Frontiers in Bioengineering and Biotechnology
Volume	10
DOIs	https://doi.org/10.3389/fbioe.2022.992708
State	Published - Sep 16 2022

Bibliographical note

Funding Information:
LN was supported by Leverhulme Trust award RPG-2021-018. WS was supported by the Funai Overseas Scholarship of The Funai Foundation for Information Technology and by the National Science Foundation award 2123465. KC was supported by a Global Challenges Research Fund studentship and University of Kent. We gratefully acknowledge funding from the EPSRC Future Targeted Healthcare Manufacturing Hub (Grant Reference: EP/P006485/1) for financial assistance with publication costs.

Publisher Copyright:
Copyright © 2022 Nagappa, Sato, Alam, Chengan, Smales, Von Der Haar, Polizzi, Adamala and Moore.

Keywords

TX-TL
cell-free gene expression
cell-free protein synthesis
industrial biotechnology
pichia pastoris cell-free
protein production

Publisher link

10.3389/fbioe.2022.992708

Find It

Find It at U of M Libraries

Cite this

@article{245f0d9b38194127bb82670311a1df04,

title = "A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems",

abstract = "Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta{\texttrademark} 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.",

keywords = "TX-TL, cell-free gene expression, cell-free protein synthesis, industrial biotechnology, pichia pastoris cell-free, protein production",

author = "Nagappa, \{Lakshmeesha K.\} and Wakana Sato and Farzana Alam and Kameshwari Chengan and Smales, \{Christopher M.\} and \{Von Der Haar\}, Tobias and Polizzi, \{Karen M.\} and Adamala, \{Katarzyna P.\} and Moore, \{Simon J.\}",

note = "Funding Information: LN was supported by Leverhulme Trust award RPG-2021-018. WS was supported by the Funai Overseas Scholarship of The Funai Foundation for Information Technology and by the National Science Foundation award 2123465. KC was supported by a Global Challenges Research Fund studentship and University of Kent. We gratefully acknowledge funding from the EPSRC Future Targeted Healthcare Manufacturing Hub (Grant Reference: EP/P006485/1) for financial assistance with publication costs. Publisher Copyright: Copyright {\textcopyright} 2022 Nagappa, Sato, Alam, Chengan, Smales, Von Der Haar, Polizzi, Adamala and Moore.",

year = "2022",

month = sep,

day = "16",

doi = "10.3389/fbioe.2022.992708",

language = "English (US)",

volume = "10",

journal = "Frontiers in Bioengineering and Biotechnology",

issn = "2296-4185",

publisher = "Frontiers Media SA",

}

TY - JOUR

T1 - A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

AU - Nagappa, Lakshmeesha K.

AU - Sato, Wakana

AU - Alam, Farzana

AU - Chengan, Kameshwari

AU - Smales, Christopher M.

AU - Von Der Haar, Tobias

AU - Polizzi, Karen M.

AU - Adamala, Katarzyna P.

AU - Moore, Simon J.

N1 - Funding Information: LN was supported by Leverhulme Trust award RPG-2021-018. WS was supported by the Funai Overseas Scholarship of The Funai Foundation for Information Technology and by the National Science Foundation award 2123465. KC was supported by a Global Challenges Research Fund studentship and University of Kent. We gratefully acknowledge funding from the EPSRC Future Targeted Healthcare Manufacturing Hub (Grant Reference: EP/P006485/1) for financial assistance with publication costs. Publisher Copyright: Copyright © 2022 Nagappa, Sato, Alam, Chengan, Smales, Von Der Haar, Polizzi, Adamala and Moore.

PY - 2022/9/16

Y1 - 2022/9/16

N2 - Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta™ 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.

AB - Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta™ 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.

KW - TX-TL

KW - cell-free gene expression

KW - cell-free protein synthesis

KW - industrial biotechnology

KW - pichia pastoris cell-free

KW - protein production

UR - http://www.scopus.com/inward/record.url?scp=85139133164&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85139133164&partnerID=8YFLogxK

U2 - 10.3389/fbioe.2022.992708

DO - 10.3389/fbioe.2022.992708

M3 - Article

C2 - 36185432

AN - SCOPUS:85139133164

SN - 2296-4185

VL - 10

JO - Frontiers in Bioengineering and Biotechnology

JF - Frontiers in Bioengineering and Biotechnology

M1 - 992708

ER -

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

Abstract

Bibliographical note

Keywords

Publisher link

Find It

Other files and links

Fingerprint

Cite this