Synthetic bugs on the loose: containment options for deeply engineered (micro)organisms

https://doi.org/10.1016/j.copbio.2016.01.006Get rights and content

Highlights

  • Synthetic Biology raises new questions re environmental fate of engineered microbes.

  • Xenobiology could be the ultimate choice for bringing about certainty-of-containment.

  • Gene drive opens unprecedented possibilities to modify natural ecosystems.

  • Standardized metrics for quantification of containment efficiencies are badly needed.

  • Containment makes risk assessment and IP protection to converge in the same lot.

Synthetic Biology (SynBio) has brought up again questions on the environmental fate of microorganisms carrying genetic modifications. The growing capacity of editing genomes for deployment of man-made programs opens unprecedented biotechnological opportunities. But the same exacerbate concerns regarding fortuitous or deliberate releases to the natural medium. Most approaches to tackle these worries involve endowing SynBio agents with containment devices for halting horizontal gene transfer and survival of the live agents only at given times and places. Genetic circuits and trophic restraint schemes have been proposed to this end in the pursuit of complete containment. The most promising include adoption of alternative genetic codes and/or dependency on xenobiotic amino acids and nucleotides. But the field has to still overcome serious bottlenecks.

Introduction

Since the birth of modern Genetic Engineering in the mid-70s of the past century there has been a recurrent concern that live entities deliberately modified for acquiring non-natural properties could cause unexpected effects when released into the environment. The arch-famous Asilomar conference of 1975 [1, 2] much echoed these concerns, which by that time were limited to the possible accidental escape of the microbes at stake [3]. One decade later, the pioneering work of Ananda Chakrabarty [4] and Kenneth Timmis [5] entered yet one more screw turn to the subject by proposing the deliberate release of genetically engineered (GE) microorganisms (GEMs) for bioremediation of sites polluted extensively with chemical waste. In this case, the challenge was not to contain these agents by making them weaker (as was the main outcome of Asilomar), but to spread them and ensure delivery of the expected activities where and when needed, while avoiding their proliferation beyond the target spatiotemporal scenario [6]. These two main concerns (preventing accidental escape and containing any unplanned dispersion or survival of the GEMs beyond a given time and place) translated along the years into different propositions. These included both barriers to horizontal gene transfer of recombinant DNA and conditional survival of the engineered agents limited only to precise specifications. The many strategies to these ends have been reviewed a number of times [7••, 8••]. In reality none of such earlier containment attempts granted an expected escape frequency that was low enough to be acceptable, but pointing in the way a number of bottlenecks that needed to be tackled for making active or passive containment a reality. In the meantime the lack of any serious health or environmental incidents involving GEMs for the next 20 years made the field to go into some sort of oblivion. It appeared that even in the worst-case scenario, the safety risks associated to modified microorganisms were not worse than naturally occurring counterparts. On the contrary, the rule of thumb was that any man-made genetic modifications resulted in strains that were less fit to compete with the indigenous, native microbial population of wherever site the GEMs could go to [9]. This was good news for the assessment of risks but very bad for the incipient branch of Environmental Biotechnology based on deliberate release of GEMs for bioremediation, biomining or plant-growth promotion. These expectations, which peaked by 1989 [10], came to an end a few years later after realizing how difficult it was to alter the homeostasis of a pre-existing complex biosystem by just adding a new component  whether one/few genes or a new organism [9]. For a number of reasons, the issue of containment of GEMs has been somewhat silent for many years, in part because of the lack of accidents and in part because releasing modified bacteria has not been useful thus far as a sound bioremediation strategy [9]. This is changing with the onset of Synthetic Biology.

Section snippets

What new environmental risks are brought about by Synthetic Biology?

The early 2000s witnessed the birth of Synthetic Biology as conceptual and technical umbrella that adopts electric engineering, mechanical engineering and computation as the interpretative frame for biological objects [11, 12, 13, 14] This has allowed making amazing breakthroughs in our understanding of the relational logic of living objects as well as in the partial or complete redesign of such logic to create new-to-nature-properties. As a result, the concept genetic engineering stops being a

From synthetic auxotrophies to semantic containment

Engineering a GE agent of interest to become dependent on the external addition of a key metabolic intermediate or cell building block for growth and survival has been one of the most widespread stratagems to ensure containment within a desired spatiotemporal frame (called trophic containment). One of the outcomes of the 1975 Asilomar conference was in fact the proposition of using multi-amino acid auxotrophic Escherichia coli strains as the hosts of reference for recombinant constructs [1, 2].

Circuits and more circuits

Since the start of GE, the second grand strategy for containment of engineered microorganisms has involved genetic circuits that limit the survival of the agents at stake beyond specific physicochemical and/or nutritional niches. Typically, they involved a more or less complex network of transcriptional factors, cognate promoters, killer genes and antidotes, the balance of which in response to an environmental signal determined continued existence or programmed death [6]. Many of such earlier

Gene drives and molecular vigilantes

One of the most recent (and fascinating) technologies empowered by Synthetic biology is the so-called gene drive, which enforces inheritance of particular genes or gene variants through entire populations of species that undergo sexual reproduction (i.e. each individual acquires one set of chromosomes from each of the parents). The result is a sort of mutagenic chain reaction [60]. To this end, one individual of the target species is engineered to have the diploid version of one or more genomic

Outlook

While the conceptual rigour of the methodologies just discussed may appear to cover most of the containment needs, in reality there is still a considerable way ahead. As new (meta)genomes are sequenced, novel lethal genes and regulatory elements become available for designing more intricate conditional killing systems that could increase containment efficiencies. Circuits based on familiar biology will however never be absolute, as mutations will inevitably lead to the appearance of surviving

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

MS gratefully acknowledges the EC FP7 projects METACODE (289572) and ST-FLOW (289326). MS is indebted to the other members of the EC SCENIHR working group on synthetic biology for inspiring and challenging discussions. The work in VdL Laboratory is supported by Projects ARISYS (322797) and EMPOWERPUTIDA (635536) of the EU and ERANET Project CONTIBUGS.

References (77)

  • M. Schmidt et al.

    Synthetic constructs in/for the environment: managing the interplay between natural and engineered biology

    FEBS Lett

    (2012)
  • S. Lindow et al.

    Genetic engineering of bacteria from managed and natural habitats

    Science

    (1989)
  • D. Endy

    Foundations for engineering biology

    Nature

    (2005)
  • E. Andrianantoandro et al.

    Synthetic biology: new engineering rules for an emerging discipline

    Mol Sys Biol

    (2006)
  • V. de Lorenzo et al.

    Synthetic biology: discovering new worlds and new words

    EMBO Rep

    (2008)
  • C.G. Acevedo-Rocha

    The synthetic nature of biology

    Ambivalences of Creating Life

    (2016)
  • M. Porcar et al.

    Confidence, tolerance, and allowance in biological engineering: the nuts and bolts of living things

    Bioessays

    (2015)
  • M. Schmidt

    Xenobiology: a new form of life as the ultimate biosafety tool

    Bioessays

    (2010)
  • M. Schmidt

    Safeguarding the genetic firewall with xenobiology

    21st Century Borders/Synthetic Biology: Focus on Responsibility and Governance

    (2012)
  • C.G. Acevedo-Rocha et al.

    How many biochemistries are available to build a cell?

    Chembiochem

    (2015)
  • P. Marliere

    The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world

    Syst Synth Biol

    (2009)
  • G.H. Moe-Behrens et al.

    Preparing synthetic biology for the world

    Front Microbiol

    (2013)
  • Risks SCoEaNIH

    Opinion on Synthetic Biology I. Definition

    (2014)
  • Risks SCoEaNIH

    Opinion on Synthetic Biology II. Risk Assessment Methodologies and Safety Aspects

    (2015)
  • Risks SCoEaNIH

    Final Opinion on Synthetic Biology III: Risks to the Environment and Biodiversity Related to Synthetic Biology and Research Priorities in the Field of Synthetic Biology

    (2015)
  • K.H. Redford et al.

    Synthetic biology and conservation of nature: wicked problems and wicked solutions

    PLoS Biol

    (2013)
  • L. Alphey et al.

    Five things to know about genetically modified (GM) insects for vector control

    PLoS Pathog

    (2014)
  • R.E. Carmichael et al.

    An introduction to synthetic biology in plant systems

    New Phytol

    (2015)
  • N.J. Patron et al.

    Standards for plant synthetic biology: a common syntax for exchange of DNA parts

    New Phytol

    (2015)
  • Y. Zhang et al.

    A controllable on-off strategy for the reproductive containment of fish

    Sci Rep

    (2015)
  • L. Alphey

    Genetic control of mosquitoes

    Annu Rev Entomol

    (2014)
  • V.M. Gantz et al.

    Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi

    Proc Natl Acad Sci U S A

    (2015)
  • A. Hammond et al.

    A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae

    Nat Biotechnol

    (2016)
  • A.F. Harris et al.

    Field performance of engineered male mosquitoes

    Nat Biotechnol

    (2011)
  • R. Lacroix et al.

    Open field release of genetically engineered sterile male Aedes aegypti in Malaysia

    PLoS One

    (2012)
  • M.J. Lajoie et al.

    Genomically recoded organisms expand biological functions

    Science

    (2013)
  • D.J. Mandell et al.

    Biocontainment of genetically modified organisms by synthetic protein design

    Nature

    (2015)
  • A.J. Rovner et al.

    Recoded organisms engineered to depend on synthetic amino acids

    Nature

    (2015)
  • Cited by (60)

    • Ecological firewalls for synthetic biology

      2022, iScience
      Citation Excerpt :

      Is this an uncertain domain where little can be predicted or controlled? A major issue of deployed engineered microorganisms concerns the potential implications for local ecosystems and unknown evolutionary outcomes [Schmidt and de Lorenzo, 2012, 2016]. As a consequence of an almost complete lack of experimental evidence (because of moratoria and the absence of a proper theoretical framework) this issue has been very often discussed in non-scientific terms.

    • Biotechnology for secure biocontainment designs in an emerging bioeconomy

      2021, Current Opinion in Biotechnology
      Citation Excerpt :

      Synthetic auxotrophy has recently been demonstrated through the use of non-canonical amino acids (ncAA). In this approach, a rare codon is first reassigned to a synonymous codon to create an unused codon in the genome; this codon can then be used for the incorporation of a non-standard amino acid into essential genes [15•]. This makes cell survival dependent on an exogenous supply of the ncAA, and if applied to transgenes, would greatly decrease the implications of HGT [7,16•,17,18••].

    View all citing articles on Scopus
    View full text