期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：6
页码：1803-1808
DOI：10.1073/pnas.1424704112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe advance of biotechnology opens up greater possibilities of bioterror and bioerror. Here, we propose multiplexed safeguard switches rooted in the development of foundational genomic, regulatory, and metabolic technologies. Safeguard switches can be regulated by submicromolar small molecule(s) and combined in a modular fashion. The resulting safeguard strains show high fitness and low reversion rates. Moreover, two distinct classes of safeguard switches are orthogonal, providing a potential fail-safe mechanism. The safeguard technologies provide a practical and generic approach to containing engineered microbes within defined laboratory and/or industrial environments, and can in principle be used in the field as well. Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer's yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational "safeguard" control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10-10), consistent with their orthogonal nature and the individual escape frequencies of <10-6. Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance.
