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1 Synthetic Auxotrophy Remains Stable After Continuous Evolution and in Co-Culture with 2 Mammalian Cells bioRxiv preprint doi: https://doi.org/10.1101/2020.09.27.315804; this version posted September 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Synthetic auxotrophy remains stable after continuous evolution and in co-culture with 2 mammalian cells 3 Authors: Aditya M. Kunjapur1,2†*, Michael G. Napolitano1,3†, Eriona Hysolli1†, Karen 4 Noguera1, Evan M. Appleton1, Max G. Schubert1, Michaela A. Jones2, Siddharth Iyer4, Daniel J. 5 Mandell1,5 & George M. Church1* 6 Affiliations: 7 1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 238, Boston, 8 MA 02115, USA. 9 2Present Address: Department of Chemical and Biomolecular Engineering, University of 10 Delaware, 150 Academy Street, CLB 215, Newark, DE 19716, USA 11 3Present Address: Ginkgo Bioworks, 27 Drydock Avenue, 8th Floor, Boston, MA 02210, USA. 12 4Present Address: Johns Hopkins University, 3101 Wyman Park Drive, Baltimore, MD 21218, 13 USA 14 5Present Address: GRO Biosciences, 700 Main Street North, Cambridge, MA 02139, USA. 15 *Corresponding authors: AMK ([email protected]) or GMC 16 ([email protected]). 17 †These authors contributed equally to this work. 18 19 Abstract: 20 Understanding the evolutionary stability and possible context-dependence of biological 21 containment techniques is critical as engineered microbes are increasingly under consideration 22 for applications beyond biomanufacturing. While batch cultures of synthetic auxotrophic 23 Escherichia coli previously exhibited undetectable escape throughout 14 days of monitoring, the 24 long-term effectiveness of synthetic auxotrophy is unknown. Here, we report automated 25 continuous evolution of a synthetic auxotroph using custom chemostats that supply a decreasing 26 concentration of essential biphenylalanine (BipA). After 100 days of evolution in three separate 27 trials, populations exhibit no observable escape and are capable of normal growth rates at 10-fold 28 lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction of three 29 proteins implicated in small molecule transport reveals their contribution to increased fitness at 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.27.315804; this version posted September 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 30 low BipA concentrations. Mutations do not appear in orthogonal translation machinery nor in 31 synthetic auxotrophic markers. Based on its evolutionary stability, we introduce the progenitor 32 synthetic auxotroph directly to mammalian cell culture. We observe containment of bacteria 33 without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic 34 auxotrophy is effective on timescales and in contexts that enable diverse applications. 35 36 One Sentence Summary: 37 To ascertain whether life inevitably finds a way, we continuously evolve an Escherichia 38 coli strain that was not able to escape from engineered biocontainment before, and we find that it 39 does not escape even after 100 days of evolution, nor does it escape when added to mammalian 40 cell culture. 41 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.27.315804; this version posted September 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 42 Introduction: 43 New safeguards are needed for the deliberate release of engineered microbes into the 44 environment, which has promise for applications in agriculture, environmental remediation, and 45 medicine1. Genetically encoded biocontainment strategies enable attenuation of engineered live 46 bacteria for diverse biomedical applications2–4, including as potential vaccines5–10, diagnostics11, 47 and therapeutics12–15. Auxotrophy, which is the inability of an organism to synthesize a 48 compound needed for its growth, is an existing strategy for containment. However, foundational 49 studies of auxotrophic pathogens demonstrated proliferation in relevant biological fluids16 and 50 reversion to prototrophy upon serial passaging17,18. Modern genome engineering strategies can 51 prevent auxotrophic reversion, and auxotrophy has been a key component of microbial therapies 52 that have reached advanced clinical trials. However, the ability for auxotrophs to access required 53 metabolites within many host microenvironments, and after leaving the host, remains 54 unaddressed. Auxotrophy may not be effective in scenarios where engineered living bacteria 55 encounter metabolites from dead host cells19 or invade host cells20. Indeed, growth of double 56 auxotrophs is supported in vivo by neoplastic tissue13. Auxotrophy may also be insufficient for 57 tight control of cell proliferation in environments rich with microbial sources of cross-feeding21, 58 such as gut, oral, skin, and vaginal microbiomes. Given that most naturally occurring 59 microorganisms are auxotrophs22, it is also unlikely that auxotrophy will limit the spread of an 60 engineered microbe once it leaves the body and enters the environment. 61 Synthetic auxotrophy may overcome these hurdles by requiring provision of a synthetic 62 molecule for survival of the engineered bacteria. This strategy was first implemented 63 successfully in E. coli by engineering an essential protein to depend on incorporation of a non- 64 standard amino acid (nsAA)23,24. We previously engineered E. coli strains for dependence on the 65 nsAA biphenylalanine (BipA) by computer-aided redesign of essential enzymes in conjunction 66 with expression of orthogonal translation machinery for BipA incorporation23. Among several 67 synthetic auxotrophs originally constructed, one strain harbored three redesigned, nsAA- 68 dependent genes: Adenylate kinase (adk.d6), tyrosyl-tRNA synthetase (tyrS.d8), and BipA- 69 dependent aminoacyl-tRNA synthetase, for aminoacylation of BipA (BipARS.d6). This BipA- 70 dependent strain, dubbed “DEP”, exhibited undetectable escape throughout 14 days of 71 monitoring at an assay detection limit of 2.2x10-12 escapees per colony forming unit (CFU)23. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.27.315804; this version posted September 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 72 Though this strain demonstrates effective biocontainment in 1 L batch experiments, its precise 73 escape frequency and long-term stability remained unexplored. 74 Here, we perform the first study of evolutionary stability of a synthetic auxotroph, with 75 the aid of automated continuous evolution. Continuous evolution better emulates scenarios where 76 biocontainment may be needed by fostering greater genetic variability within a population. We 77 posited that decreasing BipA concentrations would add selective pressure for adaptation or for 78 escape, either of which would be enlightening. Adaptive laboratory evolution of DEP may 79 improve its fitness in relevant growth contexts, as previously demonstrated for its non- 80 auxotrophic but recoded ancestor, C321.ΔA25. We report that DEP maintains its inability to grow 81 in the absence of synthetic nutrient, even after three parallel 100-day chemostat trials. 82 Additionally, we find evidence of adaptation, with evolved DEP isolates requiring 10-fold lower 83 BipA concentration to achieve optimal growth than ancestral DEP (0.5 μM rather than 5 μM). 84 We resequence evolved populations and perform allelic reconstruction in ancestral DEP using 85 multiplex automatable genome engineering (MAGE), identifying alleles that partially restore the 86 adaptive phenotype. Finally, we advance this technology towards host-microbe co-culture 87 applications, demonstrating direct mixed culture of DEP and mammalian cells without need for 88 physical barriers nor complex fluidics. 89 Results: 90 Continuous evolution 91 To perform continuous evolution of E. coli, we constructed custom chemostats for 92 parallelized and automated culturing (Fig. 1A). Our design and construction was based on the 93 eVOLVER system26, an open-source, do-it-yourself automated culturing platform (Figs. S1-S4). 94 By decreasing BipA concentration over time in our chemostats, we provide an initial mild 95 selection for escape and steadily increase its stringency. This design is analogous to a 96 “morbidostat”, where a lethal drug is introduced dynamically at sub-lethal concentrations to 97 study microbial drug resistance27, but with synthetic auxotrophy providing selective pressure. 98 Our working algorithm for automated adjustment of BipA concentration as a function of 99 turbidity is shown in Fig. 1B, and a representative image of our hardware is shown in Fig. 1C. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.27.315804; this version posted September 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who
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