Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21(DE3) Biosafety Strain.

Synthetic auxotrophs are organisms engineered to require the presence of a particular molecule for viability. They have potential applications in biocontainment and enzyme engineering. We show that these organisms can be generated by engineering ligand-dependence into essential genes. We demonstrate a method for generating a Synthetic auxotroph based on a Ligand-Dependent Essential gene (SLiDE) using 5 essential genes as test cases: pheS, dnaN, tyrS, metG, and adk. We show that a single SLiDE strain can have a 1 × 10(8)-fold increase in viability when chemically complemented with the ligand benzothiazole. The optimized SLiDE strain engineering protocol required less than 1 week and $100 USD. We combined multiple SLiDE strain alleles into the industrial Escherichia coli strain BL21(DE3), yielding an organism that exceeds the biosafety criteria with an escape frequency below the limit of detection of 3 × 10(-11).

[1]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Roberto A Chica,et al.  Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design. , 2005, Current opinion in biotechnology.

[3]  H. Orozco,et al.  Genetic manipulation of longevity-related genes as a tool to regulate yeast life span and metabolite production during winemaking , 2013, Microbial Cell Factories.

[4]  Kate Thodey,et al.  Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways. , 2012, Metabolic engineering.

[5]  Peter F. Stanbury,et al.  Principles of Fermentation Technology , 1984 .

[6]  Gary L. Andersen,et al.  Caulobacter crescentus as a Whole-Cell Uranium Biosensor , 2007, Applied and Environmental Microbiology.

[7]  처치 죠지엠.,et al.  Orthogonal cas9 proteins for rna-guided gene regulation and editing , 2014 .

[8]  Frances H Arnold,et al.  Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450 BM3-catalyzed hydroxylation of small alkanes. , 2012, Protein engineering, design & selection : PEDS.

[9]  P G Schultz,et al.  Designing small-molecule switches for protein-protein interactions. , 2000, Science.

[10]  D. Court,et al.  High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Christopher M. MacDermaid,et al.  Theoretical and computational protein design. , 2011, Annual review of physical chemistry.

[12]  J. L. Stokes,et al.  A Neurospora assay for pyridoxine. , 1943 .

[13]  Tom Ellis,et al.  GeneGuard: A modular plasmid system designed for biosafety. , 2015, ACS synthetic biology.

[14]  P Chambon,et al.  Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. , 1997, Biochemical and biophysical research communications.

[15]  E. Tatum,et al.  X-Ray Induced Growth Factor Requirements in Bacteria. , 1944, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Stanley Fields,et al.  A yeast sensor of ligand binding , 2001, Nature Biotechnology.

[17]  Farren J. Isaacs,et al.  Recoded organisms engineered to depend on synthetic amino acids , 2015, Nature.

[18]  J. Lederberg,et al.  REPLICA PLATING AND INDIRECT SELECTION OF BACTERIAL MUTANTS , 1952, Journal of bacteriology.

[19]  J.D.H. Keatinge,et al.  Why promising technologies fail: the neglected role of user innovation during adoption ☆ , 2001 .

[20]  G. Beadle,et al.  Genetic Control of Biochemical Reactions in Neurospora , 1941 .

[21]  N. Horowitz.  Fifty years ago: the Neurospora revolution. , 1991, Genetics.

[22]  P. Chambon,et al.  Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[23]  John Karanicolas,et al.  Designing allosteric control into enzymes by chemical rescue of structure. , 2012, Journal of the American Chemical Society.

[24]  Srivatsan Raman,et al.  Engineering allostery. , 2014, Trends in genetics : TIG.

[25]  Gerd H. G. Moe-Behrens,et al.  Preparing synthetic biology for the world , 2013, Front. Microbio..

[26]  Kevin D Young,et al.  Indole production by the tryptophanase TnaA in Escherichia coli is determined by the amount of exogenous tryptophan. , 2013, Microbiology.

[27]  Dan S. Tawfik,et al.  Directed enzyme evolution: beyond the low-hanging fruit. , 2012, Current opinion in structural biology.

[28]  Peter D. Karp,et al.  EcoCyc: fusing model organism databases with systems biology , 2012, Nucleic Acids Res..

[29]  Patrick C Cirino,et al.  Design and application of a mevalonate-responsive regulatory protein. , 2011, Angewandte Chemie.

[30]  J. Rossi,et al.  Differential Recovery of Auxotrophs After Penicillin Enrichment in Escherichia coli , 1971, Journal of bacteriology.

[31]  Paul A Dalby,et al.  Strategy and success for the directed evolution of enzymes. , 2011, Current opinion in structural biology.

[32]  L. Eggeling,et al.  Looking for the pick of the bunch: high-throughput screening of producing microorganisms with biosensors. , 2014, Current opinion in biotechnology.

[33]  K. Bayer,et al.  Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production , 2013, Microbial Cell Factories.

[34]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[35]  B. D. Davis,et al.  Isolation of biochemically deficient mutants of bacteria by penicillin. , 1948, Journal of the American Chemical Society.

[36]  N. Costantino,et al.  A set of recombineering plasmids for gram-negative bacteria. , 2006, Gene.

[37]  Ryo Takeuchi,et al.  Biocontainment of genetically modified organisms by synthetic protein design , 2015, Nature.

[38]  H. K. Mitchell,et al.  Neurospora; a temperature-sensitive riboflavinless mutant. , 1946, American journal of botany.