Biological Dual-Use Research and Synthetic Biology of Yeast

In recent years, the publication of the studies on the transmissibility in mammals of the H5N1 influenza virus and synthetic genomes has triggered heated and concerned debate within the community of scientists on biological dual-use research; these papers have raised the awareness that, in some cases, fundamental research could be directed to harmful experiments, with the purpose of developing a weapon that could be used by a bioterrorist. Here is presented an overview regarding the dual-use concept and its related international agreements which underlines the work of the Australia Group (AG) Export Control Regime. It is hoped that the principles and activities of the AG, that focuses on export control of chemical and biological dual-use materials, will spread and become well known to academic researchers in different countries, as they exchange biological materials (i.e. plasmids, strains, antibodies, nucleic acids) and scientific papers. To this extent, and with the aim of drawing the attention of the scientific community that works with yeast to the so called Dual-Use Research of Concern, this article reports case studies on biological dual-use research and discusses a synthetic biology applied to the yeast Saccharomyces cerevisiae, namely the construction of the first eukaryotic synthetic chromosome of yeast and the use of yeast cells as a factory to produce opiates. Since this organism is considered harmless and is not included in any list of biological agents, yeast researchers should take simple actions in the future to avoid the sharing of strains and advanced technology with suspicious individuals.

Abstract In recent years, the publication of the studies on the transmissibility in mammals of the H5N1 influenza virus and synthetic genomes has triggered heated and concerned debate within the community of scientists on biological dual-use research; these papers have raised the awareness that, in some cases, fundamental research could be directed to harmful experiments, with the purpose of developing a weapon that could be used by a bioterrorist. Here is presented an overview regarding the dual-use concept and its related international agreements which underlines the work of the Australia Group (AG) Export Control Regime. It is hoped that the principles and activities of the AG, that focuses on export control of chemical and biological dual-use materials, will spread and become well known to academic researchers in different countries, as they exchange biological materials (i.e. plasmids, strains, antibodies, nucleic acids) and scientific papers. To this extent, and with the aim of drawing the attention of the scientific community that works with yeast to the so called Dual-Use Research of Concern, this article reports case studies on biological dual-use research and discusses a synthetic biology applied to the yeast Saccharomyces cerevisiae, namely the construction of the first eukaryotic synthetic chromosome of yeast and the use of yeast cells as a factory to produce opiates. Since this organism is considered harmless and is not included in any list of biological agents, yeast researchers should take simple actions in the future to avoid the sharing of strains and advanced technology with suspicious individuals.

Definition and Evolution of the Dual-Use Concept
The term dual-use has multiple meanings. It first appeared in discussions over technology transfer between civilian and military applications and, for this reason, it is still associated with a concept related to the application of civilian knowledge to military research. The Research and Development (R&D) advances in commercial technologies such as electronics, informatics, biology and communications have also produced consequences in the military field. In a period in which economies and markets are moving towards integration, the armed forces are increasingly relying on technologies also developed in other countries and sometimes designed for civilian use. In this context, the dual-use concept takes on a dynamic meaning as the development of technologies can fulfill both civilian and military needs. Recently, the term has started to be used in non-proliferation legislation, like export control laws, to address the problem that technologies or knowledge might be used for proliferation purposes; this includes materials, hardware, advanced technologies that have peaceful applications but could also be exploited for the illicit production of biological, nuclear or chemical weapons. Since 9/11, multilateral export regimes have gone beyond the traditional civilian versus military definition, including non-state-actors in state proliferation concerns.

International Agreements on Dual-Use Control
In order to counter the production and eventually the use of Weapons of Mass Destruction (WMD), international agreements were signed (Fig. 1). The most important treaties are the Geneva protocol (1925), the Nuclear Non Proliferation Treaty (NPT, 1970), Biological and Toxin Weapons Convention (BTWC, 1972), the Chemical Weapons Convention on nonproliferation of nuclear, chemical and biological weapons (CWC, 1997). The CWC has an Implementation body, the Organisation for the Prohibition of Chemical Weapons (OPCW), while the BWC has none; this is due in part to difficulties associated with the dual use nature of biological research (Enia and Fields 2014). Thus, we are convinced that prevention is the best way to counteract biological threats.
The concept of dual-use was defined in the aforementioned fora and developed in the Nonproliferation Export Control Regimes (Rath et al. 2014;Kelle 2014). Despite the fact that Regimes are less known to the public opinion than treaties, they are very active in preventing the production of WMD by controlling the exports of dual-use products and technologies that might have an unacceptable risk of diversion. While each Regime targets specific threats (the Australia Group is concerned with chemical and biological weapons, the Nuclear Suppliers Group with nuclear weapons, the Missile Technology Control Regime with delivery systems and the Wassenaar Arrangement with conventional arms), together they counter all concerns related to export controls and WMD proliferation in a multilateral and comprehensive way. In particular, Australia Group (AG) controls the exports of chemical and biological materials. This Regime was set up to address the concerns raised by the use of chemical weapons during the Iran-Iraq War (1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988). Moreover, the increased attention to WMD weapons after 9/11 led to the United Nations Security Council Resolution (UNSCR) 1540 (2004), which requires all UN member states to adopt adequate export controls. In this context, the UNSCR 1540 complements and reinforces the nonproliferation regimes. The AG goal is to develop ways to minimize export and transhipping risks so that proliferators are prevented from obtaining the equipment, chemical precursors and biological strains needed to develop chemical and biological weapons. Enforcing a licensing authority over a wide range of chemical weapons precursors is one way to reduce risk. The member States (42 as of February 2016) require licenses to export dual-use chemical manufacturing facilities, equipment, and related technology, plant pathogens, animal pathogens, biological agents and dual-use biological equipment. These items are the basis for the Group's common control lists. The purpose of the AG guidelines is to limit the risks of proliferation and terrorism involving Chemical and Biological Weapons (CBW) by controlling tangible and intangible transfers of technology that could contribute to CBW development activities by states or nonstate actors, in accordance with Article III and X of the Biological and Toxin Weapons Convention (BTWC), Article I and XI of the Chemical Weapons Convention (CWC) and all relevant United Nations Security Council Resolutions (UNSCRs). Since basic science is not controlled by the AG regime, the debate on dual use research is of particular concern in biology because the definitions of basic and applied science sometimes overlap (Evans and Valdivia 2012;Charatsis 2015).

Implication of Dual-Use in Biological Research
While it is rather easy to recognize a dual-use application in the field of chemical or nuclear research, this is particularly complicated in the field of life sciences and biotechnology due to the fact that biotechnology is a rapidly advancing field of endeavor and new techniques and technologies are being developed all the time. Basic research in universities or applied research in pharmaceuticals companies can be considered as dual-use in many cases, such as vaccine research, antibiotic discovery, or in the production of detection systems. These research activities would further increase our knowledge in the defense against pathogens (for humans, animals or crops); however, at the same time they might have malevolent application, as well.
Highly sensitive technological areas include technologies aiming at generating collections of molecules with greater structural or biological diversity, technologies aiming at creating novel molecular or biological diversity and aiming at facilitating the manipulation of complex biological systems, as well as technologies for the production, delivery, and packaging of biological products (Tucker 2012).
In order to avoid the involuntary release of sensitive dual-use information, it would be essential to educate the academic scientific community about the nature of the dual-use dilemma in biotechnology and the responsibilities that researchers have to mitigate its risks. A lot of the debate is based on the assessment of the risk associated with the misuse of research. In 2004, the US National Research Council released the Fink report (National Research Council 2004), (Fig. 2) how to render a vaccine ineffective, how to confer resistance to therapeutically useful antibiotics or antiviral agents, how to enhance the virulence of a pathogen or render a non-pathogen virulent, how to increase the transmissibility of a pathogen, how to alter the host range of a pathogen, how to enable the evasion of diagnostic/detection modalities, how to enable the weaponization of a biological agent or toxin (Miller and Selgelid 2007). Based on the Fink report's recommendations, the US Government established the National Science Advisory Board for Biosecurity (NSABB). This federal advisory committee provides advice and guidance. As the NSABB emphasizes, it can be argued that virtually all life sciences research has a dual-use potential. NSABB also describes a subset of dual-use research that has the highest potential for generating information that could be misused, and calls it 'dual-use research of concern' (DURC). The NSABB definition of DURC implies intent to cause harm and is supportive of a benevolent versus malevolent dual-use concept that includes military applications.

Case Studies/Examples for Biological Dual-Use Research
In 2012, two studies on transmissibility of the H5N1 virus in mammals appeared on Nature and Science journals. The two study groups approached the subject in a slightly different way but, by using ferrets as a model, they obtained the same results, identifying the mutations that are sufficient to make H5N1 virus airborne and transmissible to mammals (Herfst et al. 2012;Imai et al. 2012). This type of research is an example of dual-use research of concern (DURC) because the publication of these results could help bioterrorists create a human pandemic flu strain. Creating in a laboratory a new and not naturally engineered virus transmissible to humans is considered potentially dangerous, even though the original research had peaceful purposes. Since both papers described how and which mutations were produced, a proliferation risk became evident and this type of research generated a long debate (H5N1 special issue on Science, 22 June 2012; Berns et al. 2012;Yong 2012;Webster 2012;Gronvall 2013), making scientists well aware of dual-use research (Hunter 2012;Edwards et al. 2014). One of the two studies was conducted at the Rotterdam University in the Netherlands.The Dutch government considered the manuscript as a dual-use item and required the authors to apply for an export license, in order to resubmit the manuscript to Science (Enserink 2012). The Dutch government officials applied the 2009 EU regulation on export control to prevent proliferation of nuclear, chemical and biological weapons (Enserink 2013;Charatsis 2015) and finally the license was released. The two papers were published in 2012 with the positive position of NSABB and the World Health Organization (WHO) expert panel. Another example of dual-use research of concern (DURC) was the publication of the discovery of a new Clostridium botulinum toxin (BoNT), the H toxin. In this case, the bacterium producing this new toxin was a natural strain isolated from a patient and was not a genetically modified organism (Hooper and Hirsch 2014).
Nevertheless, two manuscripts were published without the sequence of the toxin H gene, in order to withhold it at least until the development of an antidote Dover et al. 2014). While the discussion on this topic was still ongoing (Enserink 2015), a new research suggested that the newly reported H toxin might be a hybrid of BoNT serotype A and serotype F, for which antidotes are available (Kalb et al. 2015). Despite this specific discovery, the release of sensitive biological results, considered as DURC, remains a big issue and should be addressed by national rules and guidelines (Suk et al. 2011).

Synthetic Biology and Synthetic Genome in Yeast
New evolving biological technologies, such as synthetic biology, receive great attention; the synthetic biology top-down approach is widely used to create new metabolic pathways in bacteria (Escherichia coli) or unicellular eukaryotes, such as the yeast Saccharomyces cerevisiae, in order to produce new compounds such as biofuels, molecules (often of pharmaceutical value) and chemicals (for a recent review see Cameron et al. 2014). With this technique, the synthesis and the insertion of functional biological components into natural genomes is achieved, often combining genes from different organisms. Just to mention a few examples, yeast has been recently used as a biomanufacturing platform for the synthesis of the antimalarial artemisinin precursor (Paddon et al. 2013) and the production of codeine and morphine (Thodey et al. 2014); in 2015, different groups successfully inserted genes from bacteria, opium poppy and sugar beet into yeast, reconstructing the different steps of the pathway for morphine or codeine production (DeLoache et al. 2015;Fossati et al. 2015;Beaudoin 2015). Recently the entire biosynthetic pathway of thebaine (precursor of morphine) and hydrocodone has been achieved by inserting genes from plants, bacteria and mammals (Galanie et al. 2015). The expression of the entire opiate pathway in a single yeast cell has now become a reality but could be subject to misuse so the authors, being aware of the possible dual-use of these strains, of their own accord contacted experts in biotechnology policy to explain that anyone with access to morphine-producing yeast strains and with basic fermentation skills, could easily produce morphine starting from glucose (Oye et al. 2015). The interest in synthetic biology research as a proliferation risk (van der Bruggen 2012; Kelle 2013), made a leap forward when the first synthetic bacterial chromosome and the first synthetic living organism were created (Lartigue et al. 2009;Gibson et al. 2010). With a synthetic biology bottom-up approach, whole genomes can be synthesised from scratch, allowing researchers to reshuffle and/or eliminate non-essential genes from the genome. Synthetic viruses were created previously (Fig. 2). The possibility to create a synthetic genome from scratch could inspire the production of a human pathogenic virus (Atlas et al. 2003;Bügl et al. 2007;Collett 2006) or bacterium for which no vaccine is available. Following these experiments, President Barack Obama asked the Presidential Commission for the Study of Bioethical Issues to review the developing field of synthetic biology and identify appropriate ethical boundaries to minimize risks. The study was released in 2010, Fig. 2. On June, 2015, The European Commission's Scientific Committees published the second of their three Opinions on: ''Synthetic Biology II: Risk assessment methodologies and safety aspects'' (http://ec.europa.eu/health/scientific_ committees/emerging/docs/scenihr_o_048.pdf). In 2014, the production of the first synthetic yeast chromosome gave a boost to the field of synthetic biology (Annaluru et al. 2014;Gibson and Venter 2014); the experiment is part of a collaborative project called Sc 2.0, that aims at producing the first synthetic eukaryotic genome, by employing scientists from academic and commercial institutions across the globe (http://syntheticyeast.org). In September 2015, at the 27th International Conference on Yeast Genetics and Molecular Biology in Levico Terme, Trento, Italy, J. Boeke, who leads the Sc 2.0 project, announced that 6 synthetic chromosomes were already completed. The yeast synthetic chromosomes have unique features, that allow researchers to reshuffle and/or eliminate non-essential genes from the genome by using the SCRaMbLE technique (Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution) (Dymond et al. 2011). The genome reduction and the construction of a minimal cell factory for industrial applications are made possible by the insertion of LoxP sites near the genes during the construction of synthetic DNA. The value of this technique is to create engineering cells for the efficient production of a desired compound; nevertheless this aim often fails due to inhibitory interactions caused by the introduction of the heterologous pathway into an existing natural metabolic network. In the short to medium term, synthetic biology is unlikely to pose new risks or threats, but the long-term possibility to insert foreign synthetic genes into a natural organism, with the aim of producing biological weapons or drugs could turn even a harmless organism, such as the Saccharomyces cerevisiae into a danger. For example, bacterial toxins from Escherichia coli, Vibrio cholerae, Bacillus thuringiensis, Clostridium botulinum and Clostridium tetani were successfully expressed in the yeast Pichia pastoris (Gurkan and Ellar 2005). In particular, the non-enzymatic heavy chain of the botulinum toxin B was expressed in yeast with the aim of expressing a secretory toxin antigen (Liu et al. 2015), while the enzymatic domain of the botulinum toxin A was successfully expressed in E. coli (Kumar Singh et al. 2011), suggesting that the heterologous expression of the catalytic domain of this potent toxin could be achieved by using yeast strains for bioterrorist purposes.
Many scientists working on yeast simply do not consider the possibility that by sharing plasmids and strains, they could inadvertently assist in a biological weapons programme, since yeast strains are Generally Regarded as Safe (GRAS) and researchers are not familiar with dual-use research. Advanced biotechnological capabilities are becoming more accessible to non-experts and indeed the production of synthetic chromosome 3 was a project accomplished by an international team made up mostly of undergraduate students. The Sc 2.0 Project also includes a group of scientists from the United States, in partnership with both LA Biohackers and high school students. The Sc 2.0 project Statement of Ethics and Governance is available on the web page http://syntheticyeast.org/. In a near future, it will be possible to determine the minimal genome necessary for life, using it as a versatile platform to easily express synthetic pathways and bypassing the cell's physiological limit to produce new compounds, such as not only antibiotics, pharmaceutical agents or biofuels, but also toxic agents or drugs.
The examples reported indicate that we are facing a real genomic and biotechnological revolution in life sciences and that researchers who manipulates GRAS organisms should also be aware of bio-threats in the future. Simple actions would be sufficient to prevent the unconscious spread of intangible technologies or strains and plasmids among potential bioterrorists. First, periodic outreaches to universities are essential: life scientists should attend obligatory seminars in their universities, not only on biosafety but also on biosecurity issues. In Italy, this is of particular concern because we do not even use two different words to translate ''biosafety'' and ''biosecurity'', generating confusion for non-experts. Second, it is important that scientists could recognise and understand the potential harm that can be caused by the evolving technologies and by the research they are conducting. Sometimes a harmless experiment could lead to a dangerous result, as happened to australian scientists who, while studying a contraceptive for mice, unintentionally produced a modified lethal mousepox (Jackson et al. 2001). Third, scientists are usually prone to sharing their biological products with colleagues; once they have described their results in scientific papers, they only take care that the asker does not develop the same research they are developing. A researcher, contacted by an individual who is asking for scientific material, should take time at least to verify that the end user is a real scientist and that his address corresponds to an existing laboratory. If the individual is unknown as a scientist (e.g. he has no scientific publications, he is not affiliated with universities or official research centres), his attempt to acquire biological material or intangible technology (knowledge and information) shall be notified to the law enforcement authorities.

Conclusions
The community of synthetic biology should be more active in evaluating the costs and benefits of dual-use technologies (Herrlich 2013;Uhlenhaut et al. 2013). In the pursuit of knowledge, scientists need to recognize ethical and moral responsibilities towards society. In particular, Kuhlau and colleagues described a set of moral obligations for scientists that are particularly relevant to dual-use research of concern (Kuhlau et al. 2008;Kelle 2009). The authors suggested that the obligation to prevent unintended harm imposes a requirement on scientists for awareness of risks, and for reasonable efforts to minimize risks of unintended misuse. The assessments of threat, such as the likelihood of deliberate misuse, should be considered by scientists, and an extensive engagement and dialogue across all sectors of civil society and academia would be useful (Relman 2013). In the light of the fast development of biotechnology, the outreach to academia should be as broad as possible, in order to capture the attention also of researchers involved in basic science.