Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae

Neta Agmon, Zuojian Tang, Kun Yang, Ben Sutter, Shigehito Ikushima, Yizhi Cai, Katrina Caravelli, James A. Martin, Xiaoji Sun, Woo Jin Choi, Allen Zhang, Giovanni Stracquadanio, Haiping Hao, Benjamin P. Tu, David Fenyo, Joel S. Bader, Jef D. Boeke

Research output: Contribution to journalArticle

Abstract

As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae-based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.

Original languageEnglish (US)
Pages (from-to)E1470-E1479
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number8
DOIs
StatePublished - Feb 21 2017

Fingerprint

Saccharomyces cerevisiae
Yeasts
Genome
Synthetic Biology
Essential Genes
Masks
Growth
Industry
Research Personnel
Ligands
Bacteria
Amino Acids
Costs and Cost Analysis
Food

Keywords

  • Escape mutants
  • Genome safety
  • Histone deacetylase
  • Rpd3L
  • Yeast

ASJC Scopus subject areas

  • General

Cite this

Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae. / Agmon, Neta; Tang, Zuojian; Yang, Kun; Sutter, Ben; Ikushima, Shigehito; Cai, Yizhi; Caravelli, Katrina; Martin, James A.; Sun, Xiaoji; Choi, Woo Jin; Zhang, Allen; Stracquadanio, Giovanni; Hao, Haiping; Tu, Benjamin P.; Fenyo, David; Bader, Joel S.; Boeke, Jef D.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 114, No. 8, 21.02.2017, p. E1470-E1479.

Research output: Contribution to journalArticle

Agmon, N, Tang, Z, Yang, K, Sutter, B, Ikushima, S, Cai, Y, Caravelli, K, Martin, JA, Sun, X, Choi, WJ, Zhang, A, Stracquadanio, G, Hao, H, Tu, BP, Fenyo, D, Bader, JS & Boeke, JD 2017, 'Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae', Proceedings of the National Academy of Sciences of the United States of America, vol. 114, no. 8, pp. E1470-E1479. https://doi.org/10.1073/pnas.1621250114
Agmon, Neta ; Tang, Zuojian ; Yang, Kun ; Sutter, Ben ; Ikushima, Shigehito ; Cai, Yizhi ; Caravelli, Katrina ; Martin, James A. ; Sun, Xiaoji ; Choi, Woo Jin ; Zhang, Allen ; Stracquadanio, Giovanni ; Hao, Haiping ; Tu, Benjamin P. ; Fenyo, David ; Bader, Joel S. ; Boeke, Jef D. / Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae. In: Proceedings of the National Academy of Sciences of the United States of America. 2017 ; Vol. 114, No. 8. pp. E1470-E1479.
@article{c47cf37b89e847b4b62bd736e01b63e7,
title = "Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae",
abstract = "As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae-based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible {"}decoy molecules{"} that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.",
keywords = "Escape mutants, Genome safety, Histone deacetylase, Rpd3L, Yeast",
author = "Neta Agmon and Zuojian Tang and Kun Yang and Ben Sutter and Shigehito Ikushima and Yizhi Cai and Katrina Caravelli and Martin, {James A.} and Xiaoji Sun and Choi, {Woo Jin} and Allen Zhang and Giovanni Stracquadanio and Haiping Hao and Tu, {Benjamin P.} and David Fenyo and Bader, {Joel S.} and Boeke, {Jef D.}",
year = "2017",
month = "2",
day = "21",
doi = "10.1073/pnas.1621250114",
language = "English (US)",
volume = "114",
pages = "E1470--E1479",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "8",

}

TY - JOUR

T1 - Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae

AU - Agmon, Neta

AU - Tang, Zuojian

AU - Yang, Kun

AU - Sutter, Ben

AU - Ikushima, Shigehito

AU - Cai, Yizhi

AU - Caravelli, Katrina

AU - Martin, James A.

AU - Sun, Xiaoji

AU - Choi, Woo Jin

AU - Zhang, Allen

AU - Stracquadanio, Giovanni

AU - Hao, Haiping

AU - Tu, Benjamin P.

AU - Fenyo, David

AU - Bader, Joel S.

AU - Boeke, Jef D.

PY - 2017/2/21

Y1 - 2017/2/21

N2 - As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae-based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.

AB - As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae-based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.

KW - Escape mutants

KW - Genome safety

KW - Histone deacetylase

KW - Rpd3L

KW - Yeast

UR - http://www.scopus.com/inward/record.url?scp=85013304603&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85013304603&partnerID=8YFLogxK

U2 - 10.1073/pnas.1621250114

DO - 10.1073/pnas.1621250114

M3 - Article

C2 - 28174266

AN - SCOPUS:85013304603

VL - 114

SP - E1470-E1479

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 8

ER -