Synthetic Biology-Inspired Biocontainment Strategies of Therapeutic Genetically Engineered Bacteria

Abstract

With the rapid expansion of synthetic gene technologies and engineered bacteria for disease diagnosis or therapy, biosafety concerns have intensified. Substantial efforts have therefore been directed toward developing biocontainment systems that prevent the unintended release of engineered microorganisms and the horizontal transfer of synthetic genetic elements into natural ecosystems. Recent advances in synthetic biology have yielded a diverse suite of biocontainment strategies, including engineered biosafety genetic circuits, genetic isolation approaches, targeted degradation of genetic material, and physical encapsulation of microbial chassis. Furthermore, the incorporation of unnatural nucleic acids and noncanonical amino acid-based orthogonal replication, transcription, and translation systems has markedly improved the robustness and orthogonality of these containment platforms. In this review, we summarize the latest developments in biocontainment strategies for genetically engineered bacteria and discuss how these innovations may address current and emerging biosafety challenges.

Keywords

Biocontainment / Biosafety / Horizontal gene transfer / Auxotrophy / Unnatural nucleic acid / Noncanonical amino acid

Statement of the Use of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this manuscript, the author(s) used ChatGPT (OpenAI GPT-4.1, 2025) in order to polish language. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article.

Acknowledgments

We thank Jingrui Zuo and Youzhi Cao from Soochow University for helpful suggestions.

Author Contributions

Conceptualization, M.G.; Writing—Original Draft Preparation, M.G. and C.C.; Writing—Review & Editing, C.C. and M.G.

Ethics Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No data was used for the research described in the article.

Funding

This research received no external funding.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

[1] Gurbatri CR, Arpaia N, Danino T. Engineering bacteria as interactive cancer therapies. Science 2022, 378, 858-864. doi:10.1126/science.add9667

[2] Kim K, Kang M, Cho BK. Systems and synthetic biology-driven engineering of live bacterial therapeutics. Front. Bioeng. Biotechnol. 2023, 11, 1267378. DOI:10.3389/fbioe.2023.1267378

[3] Raman V, Deshpande CP, Khanduja S, Howell LM, Van Dessel N, Forbes NS. Build-a-bug workshop: Using microbialhost interactions and synthetic biology tools to create cancer therapies. Cell Host Microbe 2023, 31, 1574-1592. doi:10.1016/j.chom.2023.09.006

[4] Ciocan D, Elinav E. Engineering bacteria to modulate host metabolism. Acta Physiol. 2023, 238, e14001. doi:10.1111/apha.14001

[5] Zhai L, Fu L, Wei W, Zheng D. Advances of Bacterial Biomaterials for Disease Therapy. ACS Synth. Biol. 2024, 13, 1400-1411. DOI:10.1021/acssynbio.4c00022

[6] Yan S, Gan Y, Xu H, Piao H. Bacterial carrier-mediated drug delivery systems: A promising strategy in cancer therapy. Front. Bioeng. Biotechnol. 2024, 12, 1526612. DOI:10.3389/fbioe.2024.1526612

[7] Dey S, Sankaran S. Engineered bacterial therapeutics with material solutions. Trends Biotechnol. 2024, 42, 1663-1676. doi:10.1016/j.tibtech.2024.06.011

[8] Lee JW, Chan CTY, Slomovic S, Collins JJ. Next-generation biocontainment systems for engineered organisms. Nat. Chem. Biol. 2018, 14, 530-537. DOI:10.1038/s41589-018-0056-x

[9] Asin-Garcia E, Kallergi A, Landeweerd L, Martins dos Santos VAP. Genetic Safeguards for Safety-by-design: So Close Yet So Far. Trends Biotechnol. 2020, 38, 1308-1312. DOI:10.1016/j.tibtech.2020.04.005

[10] Parker MT, Kunjapur AM. Deployment of Engineered Microbes: Contributions to the Bioeconomy and Considerations for Biosecurity. Health Secur. 2020, 18, 278-296. DOI:10.1089/hs.2020.0010

[11] Arnolds KL, Dahlin LR, Ding L, Wu C, Yu J, Xiong W, et al. Biotechnology for secure biocontainment designs in an emerging bioeconomy. Curr. Opin. Biotechnol. 2021, 71, 25-31. DOI:10.1016/j.copbio.2021.05.004

[12] Ou Y, Guo S. Safety risks and ethical governance of biomedical applications of synthetic biology. Front. Bioeng. Biotechnol. 2023, 11, 1292029. DOI:10.3389/fbioe.2023.1292029

[13] Halawa EM, Fadel M, Al-Rabia MW, Behairy A, Nouh NA, Abdo M, et al. Antibiotic action and resistance: Updated review of mechanisms, spread, influencing factors, and alternative approaches for combating resistance. Front. Pharmacol. 2024, 14, 1305294. DOI:10.3389/fphar.2023.1305294

[14] Angles AP, Valle-Pérez AU, Hauser C, Mahfouz MM.Microbial Biocontainment Systems for Clinical, Agricultural, and Industrial Applications. Front. Bioeng. Biotechnol. 2022, 10, 830200. DOI:10.3389/fbioe.2022.830200

[15] Huang Y, Lin X, Yu S, Chen R, Chen W. Intestinal Engineered Probiotics as Living Therapeutics: Chassis Selection, Colonization Enhancement, Gene Circuit Design, and Biocontainment. ACS Synth. Biol. 2022, 11, 3134-3153. doi:10.1021/acssynbio.2c00314

[16] Arboleda-García A, Alarcon-Ruiz I, Boada-Acosta L, Boada Y, Vignoni A, Jantus-Lewintre E. Advancements in synthetic biology-based bacterial cancer therapy: A modular design approach. Crit. Rev. Oncol./Hematol. 2023, 190, 104088. doi:10.1016/j.critrevonc.2023.104088

[17] Moe-Behrens GHG, Davis R, Haynes KA. Preparing synthetic biology for the world. Front. Microbiol. 2013, 4, 5. doi:10.3389/fmicb.2013.00005

[18] Wilson DJ. NIH guidelines for research involving recombinant DNA molecules. Acc. Res. 1993, 3, 177-185. DOI:10.1080/08989629308573848

[19] Feng G, Huang H, Chen Y. Effects of emerging pollutants on the occurrence and transfer of antibiotic resistance genes: A review. J. Hazard. Mater. 2021, 420, 126602. DOI:10.1016/j.jhazmat.2021.126602

[20] Wang S, Li W, Xi B, Cao L, Huang C. Mechanisms and influencing factors of horizontal gene transfer in composting system: A review. Sci. Total Environ. 2024, 955, 177017. DOI:10.1016/j.scitotenv.2024.177017

[21] Hirota R, Abe K, Katsuura Z-I, Noguchi R, Moribe S, Motomura K, et al. A Novel Biocontainment Strategy Makes Bacterial Growth and Survival Dependent on Phosphite. Sci. Rep. 2017, 7, 44748. DOI:10.1038/srep44748

[22] Agmon N, Tang Z, Yang K, Sutter B, Ikushima S, Cai Y, et al. Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 2017, 114, E1470-E1479. doi:10.1073/pnas.1621250114

[23] Asin-Garcia E, Batianis C, Li Y, Fawcett JD, de Jong I, dos Santos VAPM. Phosphite synthetic auxotrophy as an effective biocontainment strategy for the industrial chassis Pseudomonas putida. Microb. Cell Factories 2022, 21, 156. doi:10.1186/s12934-022-01883-5

[24] Sebesta J, Xiong W, Guarnieri MT, Yu J.Biocontainment of Genetically Engineered Algae. Front. Plant Sci. 2022, 13, 839446. DOI:10.3389/fpls.2022.839446

[25] Bongaerts N, Edoo Z, Abukar AA, Song X, Sosa-Carrillo S, Haggenmueller S, et al. Low-cost anti-mycobacterial drug discovery using engineered E. coli. Nat. Commun. 2022, 13, 3905. DOI:10.1038/s41467-022-31570-3

[26] Hedin KA, Kruse V, Vazquez-Uribe R, Sommer MOA. Biocontainment strategies for in vivo applications of Saccharomyces boulardii. Front. Bioeng. Biotechnol. 2023, 11, 1136095. DOI:10.3389/fbioe.2023.1136095

[27] Rueda-Mejia MP, Bühlmann A, Ortiz-Merino RA, Lutz S, Ahrens CH, Künzler M, et al. Pantothenate Auxotrophy in a Naturally Occurring Biocontrol Yeast. Appl. Environ. Microbiol. 2023, 89, e00884. DOI:10.1128/aem.00884-23

[28] Lindner SN, Ramirez LC, Krüsemann JL, Yishai O, Belkhelfa S, He H, et al. NADPH-Auxotrophic E. coli: A Sensor Strain for Testing In Vivo Regeneration of NADPH. ACS Synth. Biol. 2018, 7, 2742-2749. DOI:10.1021/acssynbio.8b00313

[29] Isabella VM, Ha BN, Castillo MJ, Lubkowicz DJ, Rowe SE, Millet YA, et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat. Biotechnol. 2018, 36, 857-864. DOI:10.1038/nbt.4222

[30] Nelson MT, Charbonneau MR, Coia HG, Castillo MJ, Holt C, Greenwood ES, et al. Characterization of an engineered live bacterial therapeutic for the treatment of phenylketonuria in a human gut-on-a-chip. Nat. Commun. 2021, 12, 2805. doi:10.1038/s41467-021-23072-5

[31] Kurtz CB, Millet YA, Puurunen MK, Perreault M, Charbonneau MR, Isabella VM, et al. An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. Sci. Transl. Med. 2019, 11, eaau7975. DOI:10.1126/scitranslmed.aau7975

[32] Leventhal DS, Sokolovska A, Li N, Plescia C, Kolodziej SA, Gallant CW, et al. Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor immunity. Nat. Commun. 2020, 11, 2739. DOI:10.1038/s41467-020-16602-0

[33] Luke JJ, Piha-Paul SA, Medina T, Verschraegen CF, Varterasian M, Brennan AM, et al. Phase I Study of SYNB1891, an Engineered E. coli Nissle Strain Expressing STING Agonist, with and without Atezolizumab in Advanced Malignancies. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2023, 29, 2435. DOI:10.1158/1078-0432.CCR-23-0118

[34] Ata Ö, Mattanovich D. Into the metabolic wild: Unveiling hidden pathways of microbial metabolism. Microb. Biotechnol. 2024, 17, e14548. DOI:10.1111/1751-7915.14548

[35] Chan CTY, Lee JW, Cameron DE, Bashor CJ, Collins JJ. “Deadman” and “Passcode” microbial kill switches for bacterial containment. Nat. Chem. Biol. 2015, 12, 82. DOI:10.1038/nchembio.1979

[36] Stirling F, Naydich A, Bramante J, Barocio R, Certo M, Wellington H, et al.Synthetic Cassettes for pH-Mediated Sensing, Counting, and Containment. Cell Rep. 2020, 30, 3139-3148.e4. DOI:10.1016/j.celrep.2020.02.033

[37] Jones BS, Lamb LS, Goldman F, Stasi AD. Improving the safety of cell therapy products by suicide gene transfer. Front. Pharmacol. 2014, 5, 254. DOI:10.3389/fphar.2014.00254

[38] Čelešnik H, Tanšek A, Tahirović A, Vižintin A, Mustar J, Vidmar V, et al. Biosafety of biotechnologically important microalgae: Intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803. Biol. Open 2016, 5, 519. DOI:10.1242/bio.017129

[39] Zhou Y, Sun T, Chen Z, Song X, Chen L, Zhang W. Development of a New Biocontainment Strategy in Model Cyanobacterium Synechococcus Strains. ACS Synth. Biol. 2019, 8, 2576-2584. DOI:10.1021/acssynbio.9b00282

[40] Hoffmann SA, Cai Y. Engineering stringent genetic biocontainment of yeast with a protein stability switch. Nat. Commun. 2024, 15, 1060. DOI:10.1038/s41467-024-44988-8

[41] Varma S, Gulati KA, Sriramakrishnan J, Ganla RK, Raval R. Environment signal dependent biocontainment systems for engineered organisms: Leveraging triggered responses and combinatorial systems. Synth. Syst. Biotechnol. 2024, 10, 356. doi:10.1016/j.synbio.2024.12.005

[42] Mu Z, Zou Z, Yang Y, Wang W, Xu Y, Huang J, et al. A genetically engineered Escherichia coli that senses and degrades tetracycline antibiotic residue. Synth. Syst. Biotechnol. 2018, 3, 196. DOI:10.1016/j.synbio.2018.05.001

[43] Halvorsen TM, Ricci DP, Park DM, Jiao Y, Yung MC. Comparison of Kill Switch Toxins in Plant-Beneficial Pseudomonas fluorescens Reveals Drivers of Lethality, Stability, and Escape. ACS Synth. Biol. 2022, 11, 3785-3796. doi:10.1021/acssynbio.2c00386

[44] Jurėnas D, Fraikin N, Goormaghtigh F, Van Melderen L. Biology and evolution of bacterial toxin-antitoxin systems. Nat. Rev. Microbiol. 2022, 20, 335-350. DOI:10.1038/s41579-021-00661-1

[45] Ferry QRV, Lyutova R, Fulga TA. Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs. Nat. Commun. 2017, 8, 14633. DOI:10.1038/ncomms14633

[46] Soucy SM, Huang J, Gogarten JP. Horizontal gene transfer: Building the web of life. Nat. Rev. Genet. 2015, 16, 472-482. doi:10.1038/nrg3962

[47] Wolff JH, Mikkelsen JG. Delivering genes with human immunodeficiency virus-derived vehicles: Still state-of-the-art after 25 years. J. Biomed. Sci. 2022, 29, 79. DOI:10.1186/s12929-022-00865-4

[48] Wright O, Delmans M, Stan G-B, Ellis T. GeneGuard: A Modular Plasmid System Designed for Biosafety. ACS Synth. Biol. 2015, 4, 307-316. DOI:10.1021/sb500234s

[49] Eng A, Borenstein E.Microbial Community Design:Methods, Applications, and Opportunities. Curr. Opin. Biotechnol. 2019, 58, 117. DOI:10.1016/j.copbio.2019.03.002

[50] Ibrahim M, Raajaraam L, Raman K. Modelling microbial communities: Harnessing consortia for biotechnological applications. Comput. Struct. Biotechnol. J. 2021, 19, 3892-3907. DOI:10.1016/j.csbj.2021.06.048

[51] Forti AM, Jones MA, Elbeyli DN, Butler ND, Kunjapur AM. Engineered orthogonal and obligate bacterial commensalism mediated by a non-standard amino acid. Nat. Microbiol. 2025, 10, 1404-1416. DOI:10.1038/s41564-025-01999-5

[52] Mimee M, Citorik RJ, Lu TK.Microbiome Therapeutics—Advances and Challenges. Adv. Drug Deliv. Rev. 2016, 105, 44. doi:10.1016/j.addr.2016.04.032

[53] Ke J, Wang B, Yoshikuni Y. Microbiome Engineering: Synthetic Biology of Plant-Associated Microbiomes in Sustainable Agriculture. Trends Biotechnol. 2021, 39, 244-261. DOI:10.1016/j.tibtech.2020.07.008

[54] Rapp KM, Jenkins JP, Betenbaugh MJ. Partners for life: Building microbial consortia for the future. Curr. Opin. Biotechnol. 2020, 66, 292-300. DOI:10.1016/j.copbio.2020.10.001

[55] Ozdemir T, Fedorec AJH, Danino T, Barnes CP. Synthetic Biology and Engineered Live Biotherapeutics: Toward Increasing System Complexity. Cell Syst. 2018, 7, 5-16. DOI:10.1016/j.cels.2018.06.008

[56] Dou J, Bennett MR. Synthetic biology and the gut microbiome. Biotechnol. J. 2017, 13, e1700159. doi:10.1002/biot.201700159

[57] Nevot G, Santos-Moreno J, Campamà-Sanz N, Toloza L, Parra-Cid C, Jansen PAM, et al. Synthetically programmed antioxidant delivery by a domesticated skin commensal. Cell Syst. 2025, 14, 101169. DOI:10.1016/j.cels.2025.101169

[58] VanArsdale E, Navid A, Chu MJ, Halvorsen TM, Payne GF, Jiao Y, et al. Electrogenetic signaling and information propagation for controlling microbial consortia via programmed lysis. Biotechnol. Bioeng. 2023, 120, 1366-1381. doi:10.1002/bit.28337

[59] Huang S, Lee AJ, Tsoi R, Wu F, Zhang Y, Leong KW, et al. Coupling spatial segregation with synthetic circuits to control bacterial survival. Mol. Syst. Biol. 2016, 12, 859. DOI:10.15252/msb.20156567

[60] Hayashi N, Lai Y, Fuerte-Stone J, Mimee M, Lu TK. Cas9-assisted biological containment of a genetically engineered human commensal bacterium and genetic elements. Nat. Commun. 2024, 15, 2096. DOI:10.1038/s41467-024-45893-w

[61] Che H, Wang Z, Li Y, Nie Y, Tian X. A Stable and Sensitive Engineering Bacterial Sensor via Physical Biocontainment and Two-Stage Signal Amplification. Anal. Chem. 2024, 96, 8807-8813. DOI:10.1021/acs.analchem.4c01341

[62] Caliando BJ, Voigt CA. Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nat. Commun. 2015, 6, 6989. DOI:10.1038/ncomms7989

[63] Rottinghaus AG, Ferreiro A, Fishbein SRS, Dantas G, Moon TS. Genetically stable CRISPR-based kill switches for engineered microbes. Nat. Commun. 2022, 13, 672. DOI:10.1038/s41467-022-28163-5

[64] Asin-Garcia E, Martin-Pascual M, Garcia-Morales L, van Kranenburg R, Martins dos Santos VAP. ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida. ACS Synth. Biol. 2021, 10, 2672. DOI:10.1021/acssynbio.1c00297

[65] Hartig AM, Dai W, Zhang K, Kapoor K, Rottinghaus AG, Moon TS, et al. Influence of Environmental Conditions on the Escape Rates of Biocontained Genetically Engineered Microbes. Environ. Sci. Technol. 2024, 58, 22657-22667. DOI:10.1021/acs.est.4c10893

[66] Asin-Garcia E, Martin-Pascual M, de Buck C, Allewijn M, Müller A,Martins dos Santos VAP. GenoMine: A CRISPRCas9-based kill switch for biocontainment of Pseudomonas putida. Front. Bioeng. Biotechnol. 2024, 12, 1426107. doi:10.3389/fbioe.2024.1426107

[67] Pantoja Angles A, Ali Z, Mahfouz M. CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing. ACS Synth. Biol. 2022, 11, 430-440. DOI:10.1021/acssynbio.1c00516

[68] Yang B, Wu C, Teng Y, Chou KJ, Guarnieri MT, Xiong W. Tailoring microbial fitness through computational steering and CRISPRi-driven robustness regulation. Cell Syst. 2024, 15, 1133-1147.e4. DOI:10.1016/j.cels.2024.11.012

[69] Foo GW, Leichthammer CD, Saita IM, Lukas ND, Batko IZ, Heinrichs DE, et al. Intein-based thermoregulated meganucleases for containment of genetic material. Nucleic Acids Res. 2024, 52, 2066. DOI:10.1093/nar/gkad1247

[70] Tang T-C, Tham E, Liu X, Yehl K, Rovner AJ, Yuk H, et al. Hydrogel-Based Biocontainment of Bacteria for Continuous Sensing and Computation. Nat. Chem. Biol. 2021, 17, 724. DOI:10.1038/s41589-021-00779-6

[71] Datta D, Weiss EL, Wangpraseurt D, Hild E, Chen S, Golden JW, et al. Phenotypically complex living materials containing engineered cyanobacteria. Nat. Commun. 2023, 14, 4742. DOI:10.1038/s41467-023-40265-2

[72] Wu F, Lin S, Luo H, Wang C, Liu J, Zhu X, et al. Noncontact microbiota transplantation by core-shell microgel-enabled nonleakage envelopment. Sci. Adv. 2025, 11, eadr7373. DOI:10.1126/sciadv.adr7373

[73] Zou Z-P, Cai Z, Zhang X-P, Zhang D, Xu C-Y, Zhou Y, et al. Delivery of Encapsulated Intelligent Engineered Probiotic for Inflammatory Bowel Disease Therapy. Adv. Healthc. Mater. 2025, 14, 2403704. DOI:10.1002/adhm.202403704

[74] Inda-Webb ME, Jimenez M, Liu Q, Phan NV, Ahn J, Steiger C, et al.Sub-1.4 cm 3 capsule for detecting labile inflammatory biomarkers in situ. Nature 2023, 620, 386-392. DOI:10.1038/s41586-023-06369-x

[75] Marlière P, Patrouix J, Döring V, Herdewijn P, Tricot S, Cruveiller S, et al. Chemical Evolution of a Bacterium’s Genome. Angew. Chem. Int. Ed. 2011, 50, 7109-7114. DOI:10.1002/anie.201100535

[76] Malyshev DA, Dhami K, Lavergne T, Chen T, Dai N, Foster JM, et al. A semi-synthetic organism with an expanded genetic alphabet. Nature 2014, 509, 385-388. DOI:10.1038/nature13314

[77] Zhang Y, Ptacin JL, Fischer EC, Aerni HR, Caffaro CE, San Jose K, et al. A semi-synthetic organism that stores and retrieves increased genetic information. Nature 2017, 551, 644-647. DOI:10.1038/nature24659

[78] Mukai T, Lajoie MJ, Englert M, Söll D.Rewriting the Genetic Code. Annu. Rev. Microbiol. 2017, 71, 557-577. doi:10.1146/annurev-micro-090816-093247

[79] Lajoie MJ, Rovner AJ, Goodman DB, Aerni H-R, Haimovich AD, Kuznetsov G, et al. Genomically recoded organisms expand biological functions. Science 2013, 342, 357-360. DOI:10.1126/science.1241459

[80] Wang L, Brock A, Herberich B, Schultz PG. Expanding the Genetic Code of Escherichia coli. Science 2001, 292, 498-500. doi:10.1126/science.1060077

[81] Kato Y. Translational Control using an Expanded Genetic Code. Int. J. Mol. Sci. 2019, 20, 887. DOI:10.3390/ijms20040887

[82] Chang T, Ding W, Yan S, Wang Y, Zhang H, Zhang Y, et al. A robust yeast biocontainment system with two-layered regulation switch dependent on unnatural amino acid. Nat. Commun. 2023, 14, 6487. DOI:10.1038/s41467-023-42358-4

[83] Kato Y. An engineered bacterium auxotrophic for an unnatural amino acid: A novel biological containment system. PeerJ 2015, 3, e1247. DOI:10.7717/peerj.1247

[84] Kato Y. Extremely Low Leakage Expression Systems Using Dual Transcriptional-Translational Control for Toxic Protein Production. Int. J. Mol. Sci. 2020, 21, 705. DOI:10.3390/ijms21030705

[85] Kuru E, Määttälä R-M, Noguera K, Stork DA, Narasimhan K, Rittichier J, et al. Release Factor Inhibiting Antimicrobial Peptides Improve Nonstandard Amino Acid Incorporation in Wild-type Bacterial Cells. ACS Chem. Biol. 2020, 15, 1852-1861. DOI:10.1021/acschembio.0c00055

[86] Gao X, Sun Y, Yang Y, Yang X, Liu Q, Guo X, et al. Directed evolution of hydroxylase XcP4H for enhanced 5-HTP production in engineered probiotics to treat depression. Int. J. Biol. Macromol. 2025, 307, 142250. doi:10.1016/j.ijbiomac.2025.142250

[87] Rovner AJ, Haimovich AD, Katz SR, Li Z, Grome MW, Gassaway BM, et al. Recoded organisms engineered to depend on synthetic amino acids. Nature 2015, 518, 89-93. DOI:10.1038/nature14095

[88] Xuan W, Schultz PG. A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids. Angew. Chem. Int. Ed. 2017, 56, 9170-9173. DOI:10.1002/anie.201703553

[89] Tack DS, Ellefson JW, Thyer R, Wang B, Gollihar J, Forster MT, et al. Addicting diverse bacteria to a noncanonical amino acid. Nat. Chem. Biol. 2016, 12, 138-140. DOI:10.1038/nchembio.2002

[90] Mandell DJ, Lajoie MJ, Mee MT, Takeuchi R, Kuznetsov G, Norville JE, et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 2015, 518, 55-60. DOI:10.1038/nature14121

[91] Koh M, Nasertorabi F, Han GW, Stevens RC, Schultz PG. Generation of an Orthogonal Protein-Protein Interface with a Noncanonical Amino Acid. J. Am. Chem. Soc. 2017, 139, 5728-5731. DOI:10.1021/jacs.7b02273

[92] Gan F, Liu R, Wang F, Schultz PG. Functional Replacement of Histidine in Proteins To Generate Noncanonical Amino Acid Dependent Organisms. J. Am. Chem. Soc. 2018, 140, 3829-3832. DOI:10.1021/jacs.7b13452

[93] Fredens J, Wang K, de la Torre D, Funke LFH, Robertson WE, Christova Y, et al. Total synthesis of Escherichia coli with a recoded genome. Nature 2019, 569, 514. DOI:10.1038/s41586-019-1192-5

[94] Nyerges A, Vinke S, Flynn R, Owen SV, Rand EA, Budnik B, et al. A swapped genetic code prevents viral infections and gene transfer. Nature 2023, 615, 720-727. DOI:10.1038/s41586-023-05824-z

[95] Choi Y-N, Kim D, Lee S, Shin YR, Lee JW. Quadruplet codon decoding-based versatile genetic biocontainment system. Nucleic Acids Res. 2025, 53, gkae1292. DOI:10.1093/nar/gkae1292

[96] Cai Y, Agmon N, Choi WJ, Ubide A, Stracquadanio G, Caravelli K, et al. Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes. Proc. Natl. Acad. Sci. USA 2015, 112, 1803-1808. DOI:10.1073/pnas.1424704112

[97] Callaway E. ‘Minimal’ cell raises stakes in race to harness synthetic life. Nature 2016, 531, 557-558. doi:10.1038/531557a

[98] Yang Z-K, Luo H, Zhang Y, Wang B, Gao F. Pan-genomic analysis provides novel insights into the association of E. coli with human host and its minimal genome. Bioinformatics 2019, 35, 1987-1991. DOI:10.1093/bioinformatics/bty938

[99] Lawson CE, Martí JM, Radivojevic T, Jonnalagadda SVR, Gentz R, Hillson NJ, et al. Machine learning for metabolic engineering: A review. Metab. Eng. 2021, 63, 34-60. DOI:10.1016/j.ymben.2020.10.005

[100] Dianawati D, Mishra V, Shah NP. Survival of Microencapsulated Probiotic Bacteria after Processing and during Storage: A Review. Crit. Rev. Food Sci. Nutr. 2016, 56, 1685-1716. DOI:10.1080/10408398.2013.798779

[101] Nguyen T-T, Nguyen P-T, Pham M-N, Razafindralambo H, Hoang Q-K, Nguyen H-T. Synbiotics: A New Route of Selfproduction and Applications to Human and Animal Health. Probiotics Antimicrob. Proteins 2022, 14, 980-993. doi:10.1007/s12602-022-09960-2

[102] Hassanisaadi M, Vatankhah M, Kennedy JF, Rabiei A, Saberi Riseh R. Advancements in xanthan gum: A macromolecule for encapsulating plant probiotic bacteria with enhanced properties. Carbohydr. Polym. 2025, 348, 122801. doi:10.1016/j.carbpol.2024.122801

[103] de Lorenzo V, Krasnogor N, Schmidt M. For the sake of the Bioeconomy: Define what a Synthetic Biology Chassis is! New Biotechnol. 2021, 60, 44-51. DOI:10.1016/j.nbt.2020.08.004

[104] Wang F, Zhang W. Synthetic biology: Recent progress, biosafety and biosecurity concerns, and possible solutions. J. Biosaf. Biosecur. 2019, 1, 22-30. DOI:10.1016/j.jobb.2018.12.003

[105] Kalvapalle PB, Staubus A, Dysart MJ, Gambill L, Reyes Gamas K, Lu LC, et al. Information storage across a microbial community using universal RNA barcoding. Nat. Biotechnol. 2025, 1-8. DOI:10.1038/s41587-025-02593-0