Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae.
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 yeastSaccharomyces 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.
Institute for Systems Genetics, New York University Langone Medical Center, New York, NY 10016.|School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
Neta
N
Agmon
Institute for Systems Genetics, New York University Langone Medical Center, New York, NY 10016.
Zuojian
Z
Tang
School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
Kun
K
Yang
Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX 75390-9038.
Ben
B
Sutter
Institute for Systems Genetics, New York University Langone Medical Center, New York, NY 10016.|Central Laboratories for Key Technologies, Kirin Company Limited, Yokohama, Kanagawa 236-0004, Japan.
Shigehito
S
Ikushima
School of Medicine, Johns Hopkins University, Baltimore, MD 21205.|School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.
Yizhi
Y
Cai
School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
Katrina
K
Caravelli
Institute for Systems Genetics, New York University Langone Medical Center, New York, NY 10016.|School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
James A
JA
Martin
Institute for Systems Genetics, New York University Langone Medical Center, New York, NY 10016.
Xiaoji
X
Sun
School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
Woo Jin
WJ
Choi
School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
Allen
A
Zhang
School of Medicine, Johns Hopkins University, Baltimore, MD 21205.|School of Computer Science and Electronic Engineering, University of Essex, Colchester CO4 3SQ, United Kingdom.
Giovanni
G
Stracquadanio
School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
Haiping
H
Hao
Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX 75390-9038.
Benjamin P
BP
Tu
Institute for Systems Genetics, New York University Langone Medical Center, New York, NY 10016.
David
D
Fenyo
School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
Joel S
JS
Bader
Institute for Systems Genetics, New York University Langone Medical Center, New York, NY 10016; jef.boeke@nyumc.org.|School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
Jef D
0000-0001-5322-4946
JD
Boeke
D020043:Genes, Essential / Q000235:genetics; D016678:Genome / Q000235:genetics*; D060847:Metabolic Engineering / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D058615:Synthetic Biology / Q000379:methods; D013728:Terminator Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
Rpd3L; escape mutants; genome safety; histone deacetylase; yeast