Deployment of Engineered Microbes: Contributions to the Bioeconomy and Considerations for Biosecurity

Abstract

Engineering at microscopic scales has an immense effect on the modern bioeconomy. Microbes contribute to such disparate markets as chemical manufacturing, fuel production, crop optimization, and pharmaceutical synthesis, to name a few. Due to new and emerging synthetic biology technologies, and the sophistication and control afforded by them, we are on the brink of deploying engineered microbes to not only enhance traditional applications but also to introduce these microbes to sectors, contexts, and formats not previously attempted. In microbially managed medicine, microbial engineering holds promise for increasing efficacy, improving tissue penetration, and sustaining treatment. In the environment, the most effective areas for deployment are in the management of crops and protection of ecosystems. However, caution is warranted before introducing engineered organisms to new environments where they may proliferate without control and could cause unforeseen effects. We summarize ideas and data that can inform identification and assessment of the risks that these tools present to ensure that realistic hazards are described and unrealistic ones do not hinder advancement. Further, because modes of containment are crucial complements to deployment, we describe the state of the art in microbial biocontainment strategies, current gaps, and how these gaps might be addressed through technological advances in synthetic engineering. Collectively, this work highlights engineered microbes as a foundational and expanding facet of the bioeconomy, projects their utility in upcoming deployments outside the laboratory, and identifies knowns and unknowns that will be necessary considerations and points of focus in this endeavor.

Microbes contribute to such disparate markets as chemical manufacturing, fuel production, crop optimization, and pharmaceutical synthesis. This work highlights engineered microbes as a foundational and expanding facet of the bioeconomy, projects their utility in upcoming deployments outside the laboratory, and identifies knowns and unknowns that will be necessary considerations and points of focus in this endeavor.

Microbial Engineering, Synthetic Biology, and Bioeconomy

The definition of a modern bioeconomy varies,^1-6 but it generally involves the economic activity related to industries associated with biology (eg, healthcare, agriculture, and consumer goods), biochemistry (eg, production of chemicals, materials, and fuels), and sustainability (eg, waste management, remediation, and alternative energy).^1-6 Although estimates of the value that a bioeconomy adds to the national economy are not entirely consistent across studies, such estimates can provide useful reference points for understanding relative contributions. For example, the biobased economy (eg, industrial enzymes, chemicals, textiles, plastics, and agriculture) contributed US$459 billion to the US economy in 2016.⁷ The total revenue from the biotechnology industry (eg, pharmaceuticals, agriculture food and feedstock, biochemicals, biofuels, and materials) contributed more than US$324 billion (at least 2% of gross domestic product [GDP]) and between 5.4% and 8.6% of total US GDP growth in 2012.⁸ Indeed, the “US biotech sector revenue is estimated to have grown on average >10% each year over the past decade—much faster than the rest of the economy.”⁸ Overall, the estimated contribution of the bioeconomy is 5% to 10% of the US GDP.^6,9

Developments in microbial engineering have enabled the biotechnology sector to create new markets, leading to gains in the market share across various sectors of the economy. This is particularly true for the subfield of synthetic biology, in which biological engineering using recombinant DNA approaches and technological innovations in DNA synthesis and sequencing have accelerated advancements in microbial (and higher organism) engineering. Biotechnology companies are heavily investing in synthetic biology startup companies,¹⁰ and private sources and federal agencies are also rapidly increasing their investments. Global venture capital funding for synthetic biology companies increased from US$200 million in 2009 to US$3.8 billion in 2018, a compound annual growth rate (CAGR) of 38.7%, and US federal agencies invested at least $211.2 million in synthetic biology research in 2017.^11-13 The growth of this industry is only projected to increase—the global synthetic biology market is estimated to expand from US$5.3 billion in 2019 to US$18.9 billion by 2024 (CAGR 28.95%).¹⁴ The scale of this growth has generated significant interest from national security experts not only in regard to biosecurity, regulation, and ethics but also in the economic security of the United States as a competitor in the field.¹⁵ Thus, over the last 10 years federal agencies have invested significant resources to better understand the domestic and global bioeconomy and to remain at the leading edge of innovation.^16-21

In this article, we highlight major sectors that have advanced innovation in synthetic biology of engineered microbes, due to the evolving role of microbes in a diverse array of bioeconomic activities. We explore emerging trends in the deployment of microbes in the forms of living therapeutics and environmental enhancements. In addition, we identify and briefly assess the risks associated with these technologies, review US governance relevant to the deployment of engineered microbes, and provide reasoning for continued ingenuity in security measures. This review is intended to inform subsequent discussions and case studies presented in the literature. We conclude by briefly discussing technology for biocontainment and surveillance of engineered microbes to mitigate deployment risks in new contexts.

Applications of Engineered Microbes

Agriculture

Genetically engineered crops are perhaps the most prominent example of the impact of synthetic biology on the economy. In 2012, the estimated revenue for these crops was US$128 billion from 69.5 million hectares grown in the United States, representing 40.8% of global growth. By 2018, global growth of genetically engineered crops had increased 176% to 191.7 million hectares.^22,23 The effects of this technology include marked increases in crop yields (+22%) and farmer profits (+68%), and more environmentally sustainable farming through decreased use of chemical pesticides (-37%).²⁴

The deployment of engineered microbes can further optimize crop benefits. Microbes can allow crops to survive when environmental conditions are not ideal, by providing them with nutrients, pathogen and predator defense, environmental stress hardiness, flavor, development, and normal growth.²⁵ Natural microbes have been used to improve phosphorous availability,²⁶ increase biomass yields,²⁷ enhance plant development,²⁸ and reduce disease incidence,²⁹ among other uses. Expanding the use of these and other applications of engineered microbes is intriguing, and their contributions to the agricultural bioeconomy are likely to extend to domesticated organisms such as honeybees, cattle, and pigs. Engineering, selection, and microbiome modification in these crops and animals have the potential to increase efficiency, health, development, and productivity.³⁰

Healthcare

The production of natural, adapted, and engineered microbes to develop medicine and improve health is a longstanding staple of the bioeconomy. An early example is the fungal antibiotic penicillin for restriction of staphylococcal growth, which revolutionized medical science.³¹ Nearly 90 years later, the global antibiotics market is estimated at US$46.23 billion and is expected to increase to US$63.34 billion by 2026 (CAGR 3.2%).³² In addition to antibiotics, microbes produce other notable products, such as growth factors, hormones, and toxins (eg, Botox).³³ One transformative example is the facilitation of industrial-scale production of insulin for administration to diabetics by recombinant DNA technologies in E coli and S cerevisiae in the 1980s, which replaced previous methods that extracted insulins from the tissues of domesticated animals.³⁴ The impact of microbial production of medicinals extends to disparate applications, a selection of which, along with their market size, is presented in Table 1.

Table 1.

Examples of Health Products Derived from Microbes

Product	Market Size (US$)	Example(s)
Chemotherapeutics	80.0 million	Bacteriocins, phenazines^a
Dietary supplements	115.1 billion	Vitamins, fatty acids, minerals^b
Recombinant proteins	347.2 million	Hormones, growth factors^c
Hyperlipidemia control agents	19.3 billion	Statins^d
Organ transplant immunosuppressants	4.6 billion	Cyclosporine^e

Data were derived from ^a Market Research Future³⁵; ^b Grand View Research³⁶; ^c Coherent Market Insights³⁷; ^d Grand View Research³⁸; and ^e Grand View Research.³⁹

Engineering of microbes is likely to further revolutionize the industrial production of medicinal biologics and to introduce avenues for the discovery of new ones. The implementation of microbes as vehicles for delivery of molecules to sites in vivo is an exciting possible outcome of such work and research in this field is already promising. For cancers, studies in mice have shown antitumor T cell activation in response to vaccination with yeast expressing carcinoma tumor antigens.⁴⁰ In virology, expression of HIV-neutralizing antibody fragments in lactobacilli could provide stable protection against HIV at the vaginal mucosa.⁴¹ And, in the gut, perhaps the most sought-after site of therapeutic intervention, a commensal E coli strain⁴² has shown utility in crowding out pathogens in the contexts of postantibiotic recovery⁴³; biofilm formation,⁴⁴ in local production of enzymes to treat metabolic disorders⁴⁵; and human growth factors to treat inflammatory disease.⁴⁶

Energy, Manufacturing, and Sustainability

Although microbes have been a popular subject of research on engineering new biofuels,^47-51 implementing such applications has remained a challenge. Other renewable and clean energy strategies have well outpaced next-generation biofuels production. Currently, traditional biomass fuels represent 63.6% of global renewable energy use and another 33% is provided through a combination of hydropower, wind, and solar energy.⁵² When considering global investment trends, it appears that these other renewable energy sources will continue to outcompete next-generation biofuels for years to come. In 2018, solar energy commanded 48.9% of new renewables investments, with wind energy close behind at 47.5%, whereas biobased energy represented only 2.7% of investments.⁵³

These data suggest that in the near term, the most effective contributions of engineered microbes toward energy, manufacturing, and sustainability may be through diverse, efficient, and environmentally friendly production of high-value compounds.^33,54 Renewable chemicals appear to be especially promising, with a market value of US$49.22 billion in 2016, which is expected to increase to US$102.76 billion by 2022 (CAGR 13.05%).⁵⁵ Biobased chemicals are projected to make up 11% of the global chemical market in 2020, or about US$375 billion.⁵⁶ But, with this growth comes challenges—the chemicals industry has already maximally reduced fossil fuel consumption and greenhouse gas emissions by intensification and energy efficiency measures.⁵⁷ Thus, chemical innovation via engineered microbes in biofuels, electrification, carbon capture, and waste conversion is poised to contribute to the future growth of the bioeconomy. Examples of industrial products derived from microbes on the market today are presented in Table 2.

Table 2.

Examples of Industrial Products Derived from Microbes

Chemicals
Product	Market Size (US$)	Application(s)
1,4-butanediol	9.0 billion	Manufacturing solvent^a
Succinic acid	128.8 million	Acidity control, flavoring^b
Oleochemicals	19.1 billion	Soaps, detergents^c
1,3-propanediol	2.6 billion	Solvents, adhesives, laminates^d

Consumer Products
Product	Market Size (US$)	Application(s)
Enzymes	9.3 billion	Food production, detergents, textiles, paper^e
Vitamins	5.2 billion	Dietary supplements^f
Nucleotides	403.1 million	Dietary supplements, pharmaceuticals^g
Amino acids	19.5 billion	Dietary supplements, protein manufacture^h

Data were derived from ^a Biotechnology Innovation Organization⁵⁶; ^b Reports and Data⁵⁸; ^c Grand View Research⁵⁹; ^d Markets and Markets⁶⁰; ^e Grand View Research⁶¹; ^f Research and Markets⁶²; ^g Grand View Research⁶³; and ^h Industry Experts.⁶⁴

Deployment of Engineered Microbes

Emerging Trends and Ramifications for Biosecurity

Although microbes naturally exist in our bodies and our surroundings, environmental release of genetically engineered microbes has typically been restricted to bioreactors. Release of engineered microbes in the United States is governed by a number of regulations (Table 3) but so far has been conducted primarily in agricultural settings. Between 1987 and 2011, 30 field tests of engineered microbes were conducted by agricultural companies on frost management, insecticide development, and other pesticide development.⁶⁵ During the last 20 years, the deliberate release of engineered microbes into contexts outside of bioreactors has been under even greater consideration and scrutiny given their potency for biomedical or environmental impact due to innovations in synthetic biotechnology (Figure 1). In 2018, the US National Academies of Sciences, Engineering, and Medicine identified modifications of existing bacteria to become more dangerous and in situ production of harmful biochemicals as their highest concerns in synthetic biology.⁶⁶ Many microbes are naturally pathogenic, but we will focus on the engineering of innocuous model microbes to perform functions that are intended to benefit humans. Living therapeutics and environmental remediation are 2 emerging and exemplary categories where environmental release of engineered microbes is likely. Both demonstrate a wide range of possible technologies and are trending in academic and commercial research, but they differ in the types of harms they could generate. We will, therefore, expand on the possible risks and the level of safeguarding required for these technologies. In regard to the commercial sector, we focus on the activities of startup companies, as information about their goals, products, and techniques is broadcast more publicly than larger corporations.

Figure 1.

Select Risks and Biocontainment Strategies for Engineered Microbes. Color images are available online.

Table 3.

A Sample of Major US Regulatory Governance Relevant to Engineered Microbe Deployments

Regulation	Year	Citation	Enforcing Agency	Applications and Relevance
Federal Meat Inspection Act	1906	21 USC §601 et seq.	FDA	Regulates safety inspection, packaging, and labeling requirements for nonspecified (eg, bison, rabbit, game animal, zoo animal, and ungulates) meat products. Relevant to environmental release of engineered microbes.
Federal Meat Inspection Act	1906	21 USC §601 et seq.	USDA FSIS	Regulates safety inspection, packaging, and labeling requirements for commercial livestock (eg, cattle, sheep, swine, equine, and goat) meat products. Relevant to animal probiotic use and environmental release of engineered microbes.
Virus, Serum, Toxin Act	1913	21 USC §151 et seq.	USDA APHIS	Ensures veterinary biologics are pure, safe, potent, and effective, and regulates their manufacture and distribution. Relevant to veterinary and research products from and for use in animals.
Federal Food, Drug, and Cosmetic Act	1938	21 USC §301 et seq.	EPA	Requires setting tolerances for pesticide residues that are deemed reasonably certain not to cause harm through aggregate exposure. Relevant to pest control with engineered microbes.
Federal Food, Drug, and Cosmetic Act	1938	21 USC §301 et seq.	FDA	Protects public health and safety through ensuring inspection standards for foods, food additives, drugs, medical devices, and cosmetics. Relevant to food enhancement, animal and human probiotics, animal and human drug manufacture, and chemical manufacture.
Federal Insecticide, Fungicide, and Rodenticide Act	1947	7 USC §136 et seq.	EPA	Regulates production, licensing, labeling, distribution, and inspection of pesticidal substances in plants, herbicide usage on plants, microbial pesticides, and biocontrol of plants and pests. Relevant to pest control with engineered microbes.
Poultry Products Inspection Act	1957	21 USC §451 et seq.	FDA	Regulates safety inspection and labeling requirements for nonspecified (eg, wild turkey, duck, and goose) poultry products. Relevant to environmental release of engineered microbes.
Poultry Products Inspection Act	1957	21 USC §451 et seq.	USDA FSIS	Regulates safety inspection and labeling requirements for commercial (eg, domesticated chicken, turkey, duck, goose, and guinea) poultry products. Relevant to animal probiotic use and environmental release of engineered microbes.
National Environmental Policy Act	1969	42 USC § 4321 et seq.	CEQ	Federal agencies must consider environmental impacts in their decision-making process and must thus prepare an Environmental Impact Statement for all actions significantly affecting the human environment. Relevant to environmental release of engineered microbes.
National Environmental Policy Act	1969	42 USC § 4321 et seq.	EPA	Must prepare and archive their own Environmental Impact Statements. Assesses Environmental Impact Statements of other federal organizations submitted under the Clean Air Act. Internal EPA processes are often viewed as functionally equivalent to NEPA, and are thus excused from it, as are actions under governance by the Clean Air Act and Clean Water Act. Relevant to environmental release of engineered microbes.
Egg Products Inspection Act	1970	21 USC §1031 et seq.	FDA	Regulates safety inspection and labeling requirements for whole (eg, shell) egg products. Relevant to animal probiotic use, environmental release of engineered microbes, and vaccine manufacture.
Egg Products Inspection Act	1970	21 USC §1031 et seq.	USDA FSIS	Regulates safety inspection and labeling requirements for commercial (eg, dried, frozen, and liquid) egg products. Relevant to animal probiotic use and environmental release of engineered microbes.
Endangered Species Act	1973	16 USC § 1531 et seq.	NOAA NMFS	Federal agencies are required to ensure their actions are not likely to jeopardize any imperiled (eg, endangered or threatened) marine or anadromous species or their critical habitats. Relevant to environmental release of engineered microbes, particularly for production of fertilizers, fuels, and pesticides.
Endangered Species Act	1973	16 USC § 1531 et seq.	USFWS	Federal agencies are required to ensure their actions are not likely to jeopardize any imperiled (endangered or threatened) terrestrial or freshwater species or their critical habitats. Relevant to environmental release of engineered microbes, particularly for production of fertilizers, fuels, and pesticides.
Toxic Substances Control Act	1976	15 USC §2601 et seq.	EPA	Requires risk assessment of newly made, intergeneric microorganisms used in applications not assessed by other governance, such as bioremediation, biofertilizers, biosensors, and biofuel production. Relevant to environmental release of engineered microbes, particularly for production of fertilizers, fuels, and pesticides.
Food Quality Protection Act	1996	Public Law 104-170	EPA	Amends Federal Food, Drug, and Cosmetic Act to change residue limit tolerances for pesticide chemicals. Amends Federal Insecticide, Fungicide, and Rodenticide Act to adjust processes for assessment of registrations, pesticide residue tolerances, and data collection. Relevant to pest control with engineered microbes.
Plant Protection Act	2000	7 USC §7701 et seq.	USDA APHIS	Oversees permitting for the importation, interstate movement, and environmental release of biocontrol organisms and biopesticides for plant pests (eg, invertebrate and microbial), diseases, and weeds. Covers both natural (even if native to the United States) and intergeneric microbes. Relevant to pest control and environmental release engineered microbes.

Abbreviations: CEQ, Council on Environmental Quality; EPA, Environmental Protection Agency; FDA, Food and Drug Administration; NOAA NMFS, National Oceanic and Atmospheric Administration National Marine Fisheries Service; USDA APHIS, United States Department of Agriculture Animal and Plant Health Inspection Service; USDA FSIS, United States Department of Agriculture Food Safety and Inspection Service; USFWS, United States Fish and Wildlife Service.

Engineered Microbes as Living Therapeutics

Engineered microbes are emerging as options for living therapeutics, called probiotics—a term more commonly applied to the subset of living therapeutics intended for the gut.⁶⁷ Probiotics are also used for animal health,⁶⁸ thus spanning the agricultural and medical sectors of the bioeconomy. In this article we focus on living therapies designed for humans as they are more likely to have direct human impact.

Microbial therapies can be categorized by their destination (eg, gut, skin, oral, vaginal, or tumor), administration route (eg, oral or intravenous), or target disease (eg, Crohn's disease, colitis, or cancer). Gut probiotics are the most active area of research. In addition to treatment of gastrointestinal disorders, gut probiotics have been shown to play major roles in mediating overall health and neurological health via the gut-blood barrier and the gut-brain axis.^69,70 Probiotics can affect brain activity in rodents⁷¹ and in small cohorts of human patients.^72,73 Several biotechnology companies are working on developing cocktails of natural gut bacteria obtained from fecal samples of healthy individuals (Table 4).

Table 4.

Companies Developing Deployable Engineered Microbial Technologies

Living Therapeutics
Company	Natural or Engineered	Founding Date	Fundraising (US$)^a	Application
Osel	Engineered	1996	Unreported (private)	HIV inhibition
Oragenics	Engineered	1996	76.9 million	Oral health
ActoGeniX (now Precigen)	Engineered	2006	Acquired (60 million)	Colitis
Seres Therapeutics	Natural and engineered	2010	Unreported (public)	Ulcerative colitis, C difficile
Azitra	Engineered	2014	20.8 million	Skin diseases
Synlogic	Engineered	2014	196.4 million	Metabolic disorders
Evelo Therapeutics	Natural	2015	132.5 million	Gut inflammation and oncology
Novome	Engineered	2016	38 million	Metabolic disorders

Environmental Detection
Company	Natural or Engineered	Founding Date	Fundraising (US$)^a	Application
FREDsense	Engineered	2014	0.1 million	Water contaminant detection

Crop Enhancement
Company	Natural or Engineered	Founding Date	Fundraising (US$)^a	Application
Pivot Bio	Natural	2010	86.7 million	Nitrogen fixation
Joyn Bio	Engineered	2017	200 million	Nitrogen fixation

Animal Probiotics
Company	Natural or Engineered	Founding Date	Fundraising (US$)^a	Application
Pando Nutrition	Engineered	2017	0.5 million	Nitrogen fixation

Data were derived from Crunchbase. Accessed January 2020. www.crunchbase.com

In many cases, natural gut bacteria possess insufficient therapeutic efficacy or do not contain the desired active therapeutic molecule(s). For these cases, administration of an engineered living microbe holds promise, but it also constitutes a form of environmental release that could be excreted, if it survives the journey.^74-76 The first clinically administered genetically modified organism was a strain of Lactococcus lactis engineered to secrete murine or human interleukin-10, an anti-inflammatory cytokine, for treatment of colitis and Crohn's disease.^77,78 The strain was engineered with a containment mechanism (a topic addressed in more detail later) based on thymidine auxotrophy.⁷⁹ L lactis has also been engineered to secrete antitumor necrosis factor (antiTNF) single-domain antibody fragments for treatment of chronic colitis in mice.⁸⁰ This technology was later commercialized by ActoGeniX, which was acquired by Intrexon (now Precigen, Inc.) for US$60 million in 2015 (Table 4).

Lactic acid bacteria such as L lactis are typically the first choice for engineered gut probiotics, but E coli Nissle 1917, a nonpathogenic patient isolate long used as a probiotic,^81,82 was characterized as a safe carrier for targeted delivery of recombinant proteins to the intestinal mucosa in 2005.⁴² This strain was also engineered to secrete antiviral peptides for protection against HIV infection⁸³ and proteins that promote insulin production in human cells.⁸⁴ In recent years, the startup Synlogic has continued to use E coli as a chassis for treating metabolic disorders such as phenylketonuria⁸⁵ and hyperammonemia.⁸⁶ Novome Biotechnologies is also developing therapies for metabolic disorders by engineering Bacteroides,^87,88 the most abundant genus within the gut of US residents, to treat hyperoxaluria⁸⁹ (Table 4). Additionally, while Seres Therapeutics typically harnesses combinations of donor-derived microbes as gut therapeutics, one of the latest additions to their pipeline is a rationally designed, fermented microbiome candidate.

Other sites within the body have been targeted for engineering and pose variable levels of environmental exposure and risks. In the 1980s, the concept of replacement therapy emerged in the treatment and prevention of dental diseases.⁹⁰ Wild-type and laboratory strains of Streptococcus mutans, an oral bacterium, were found to provide lifelong resistance to dental caries following a single application in laboratory rats. This technology formed the basis of the company Oragenics. Microbes from the human oral microbiome have also been applied in other parts of the body. Over 20 years ago, researchers introduced recombinant strains of Streptococcus gordonii, a member of the human oral microbiome that can colonize the vaginal microbiome, into the vaginal microbiota of monkeys to serve as live vaccines by expressing HPV and HIV antigens.^91,92 Later, S gordonii was engineered to function as a therapeutic rather than as a vaccine by secreting a microbiocidal single-chain antibody to treat experimental vaginitis caused by Candida albicans in rats.⁹³ Natural vaginal isolates of Lactobacillus jensenii have also been engineered to secrete various proteins that either bind or inhibit HIV.^94,95 The latter strain generated a 63% reduction in HIV transmission when tested in macaques⁹⁶ and has been commercialized as MucoCept by Osel. The skin microbiome has also been engineered; the company Azitra engineers the natural commensal skin bacterium Staphylococcus epidermidis to address various skin diseases (Table 4).

A final category of microbial therapeutics that merits mention is bacterial cancer therapeutics because of their increased potency and reduced risk of excretion into the environment. Bacteria can be used as cancer therapies because they can penetrate and preferentially reside in tumor microenvironments that are inaccessible to most cancer therapeutics.^97,98 Natural microbes have been used for cancer therapy for several decades, including Bacillus Calmette-Guérin therapy,⁹⁹ which urologists have developed into the most effective therapy currently available in the treatment of nonmuscle invasive bladder cancer. But in general, wild-type bacteria compete weakly against tumors and may seek out more hospitable environments. Thus, engineering can improve their potency and ability to respond¹⁰⁰ while also restricting them to certain tumor environments.¹⁰¹ Common engineering approaches that have been used for bacterial cancer therapies are production of an enzyme that converts a prodrug into a drug (limited by the weak penetration of prodrugs into tumors)^102-104 or production of factors that recruit the immune system to fight against tumor cells (limited by the elevated possibility of a cytokine storm).^105,106 Very rarely have bacteria been engineered to secrete cytotoxic molecules, although bacterial toxins have had independent use in tumor treatment.¹⁰⁷

Application in the Environment

In food webs and elemental life cycles, bacteria and fungi are decomposers and scavengers that accelerate the breakdown of waste and recycle of nutrients. Microbial decomposition can also be used to treat manmade introductions of abiological chemicals as wide-ranging as petroleum to heavy metals.¹⁰⁸ Engineered microbes provide an opportunity to selectively deploy targeted cleanup agents that can detect and destroy environmental toxins,^109,110 such as those for aromatic or chlorinated pollutants, as first reported in the 1980s.¹¹¹ Bacteria also serve wide-ranging roles as symbionts or commensals for plants, and these roles may be more economically attractive to engineer.

The application of engineered microbes in the field has greater value proposition and regulatory acceptance when the function required cannot be found in nature or occurs at an unacceptable timescale—important goals of bioremediation. The first field release of an engineered microbe for bioremediation was Pseudomonas fluorescens strain HK44, which was isolated from a chemical plant contaminated with polyaromatic hydrocarbons.¹¹² This strain was engineered to break down naphthalene and to luminesce upon doing so, enabling detection and tracking.¹¹³ Although the subsequent years presented creative advances in engineered microbial technologies designed to address pesticide,^114,115 heavy metal,¹¹⁶ and radioactive waste contamination,¹¹⁷ none of these technologies were ultimately deployed in the field. An alternative strategy is to use engineered microbes for enhancement of plant-based phytoremediation, as was done for toluene using engineered endophytic bacteria Burkholderia cepacia. This was tested in hydroponic cultivation and greenhouse settings, and plants grown from seeds exposed to the engineered microbe outperformed their counterparts in growth and detoxification.¹¹⁸ As an enabler of the deployment of these technologies, polymer encapsulation techniques such as the use of alginate beads have been tested for strains capable of bioremediation.^119,120 Alginate beads and related encapsulation techniques may help to address the major challenge of survival in the environment through improvement of the resilience of the microbe in toxic environments,¹²¹ as some encapsulated microbes can survive for over 14 years.¹²² Encapsulated engineered microbes can also be embedded within cartridges that interface with electronic devices, as used by the company FREDsense to detect water contaminants.

The emerging problem of microplastic accumulation, particularly in marine environments, presents a new potential opportunity to deploy engineered microbes for bioremediation.^123-125 These plastic particles of less than 5 mm in diameter arise from slow physical disintegration of plastic waste. Microplastics have been found in the guts or stool of numerous organisms, with unknown health consequences and high likelihood of consumption by humans.^126-128 Microbes and associated enzymes have been explored for the degradation of various plastics, with successful breakdown of polyesters achieved by leaf compost cutinases.¹²⁹ Recently, this field was reignited by the discovery of a natural microbe, Ideonella sakaiensis, that breaks down and grows on polyethylene terephthalate (PET) as a sole carbon source.¹³⁰ Given the slow kinetics of PET breakdown in I sakaiensis and its limited growth contexts, the diatom Phaeodactylum tricornutum was engineered to secrete recombinant PET hydrolase originally from I sakaiensis in marine contexts.¹³¹ Such approaches may be promising for tackling distributed sources of amorphous PET from microplastics, drink bottles, and food packaging.

Unlike in medical research, the incentives and infrastructure to perform field-testing of engineered microbes for bioremediation is minimal, concerning to the public, and historically not encouraged by the US Environmental Protection Agency (EPA).^132,133 Thus, very few engineered microbes have been applied outside the laboratory.¹³⁴ Some who propose changes to EPA policy lament the lack of application of engineered microbes designed to break down hydrocarbons¹³⁵ and suggest a more risk-based regulatory approach.¹³⁶ In contrast to the bioremediation sector, the agricultural sector has been an early and prolific proponent of the release of engineered microbes in field tests.^137-139 Initial agricultural objectives for engineered microbes included frost protection,¹³⁷ pest protection,¹⁴⁰ and increased nitrogen uptake for growth enhancement.¹⁴¹ However, even after several successful approvals such as for frost prevention, companies chose to pursue wild-type microbes that could perform similar functions.¹³⁸ As of 1997, only 1 type of living recombinant microbial product requiring environmental release had been commercialized¹³⁸—an engineered strain of Bacillus thuringiensis that combined several insecticidal genes.¹⁴⁰ One detailed technopolicy analysis concluded that the primary reason for the lack of commercialization of engineered microbes has changed over time from regulatory hurdles to a lack of product efficacy and limited potential for profitability.¹³⁸

Enhancement of crops using engineered microbes has recently resurfaced as an area of commercial activity. Nitrogen fixation in agricultural crops is a particularly illustrative example, as microbes can be engineered for improved nitrogen uptake and/or to associate with cereals.^142,143 Because plants rely on soil microbes for nitrogen fixation, nitrogen is often the limiting nutrient for growth.¹⁴⁴ Synthetic fertilizers pose challenges of cost and energy-intensity of production as well as environmental hazards from runoff.^145,146 The company Pivot Bio was originally interested in using engineered microbes to enhance nitrogen uptake^147,148; however, it is now working with native species to produce nitrogenase. In agriculture, there is some precedence for reawakening or modifying genes by using natural processes of mutagenesis (eg, ultraviolet light) and by selecting for traits. Such strategies lie on the continuum between conventional breeding and genetic engineering.^149,150 On the other hand, Joyn Bio, a joint venture between Bayer and Ginkgo Bioworks, has chosen the path of engineering microbes to improve nitrogen fixation, to be introduced at the point of seed treatment¹⁵¹ (Table 4).

In addition to their role in crop enhancement, microbes enhance agricultural processes as animal probiotics. Companies such as Danisco Animal Nutrition, a division of DuPont, have long provided silage probiotic solutions that contain Bacillus species, supporting gut balance and intestinal morphology. Pando Nutrition seeks to provide an antibiotic alternative to poultry producers by genetically engineering a probiotic-secreted enzyme. Advancement of biocontainment strategies may not only responsibly steward the introduction of engineered microbes into new contexts but may also alleviate public perception about such efforts.

Despite the exciting gains that can arise from engineering, the majority of field-deployed microbes so far are wild-type strains, typically natural isolates with unusual traits and a propensity for growth under target field conditions. But, because the introduction of natural or engineered microbes in large quantities to a sensitized ecosystem poses a risk of unanticipated changes, technologies to evaluate environmental health are necessary to ensure the safety of such approaches. Interestingly, microbes could meet this need themselves; the use of natural and engineered microbes as biosensors and bioindicators of environmental perturbance is an area of much interest and has been extensively studied, employed, and reviewed.^152-155 These microbes can be useful in detecting substances of high relevance to sustainability and security (eg, pollutants, chemicals, hormones, pharmaceuticals, and pathogens) and assessing overall quality, indirect effects of perturbances, and to give temporal perspective to these measurements.¹⁵⁶ Biosensors and bioindicators will be crucial to the success and safety of future deployment programs, but the field will also require more tools for assessing and controlling the effects, effectiveness, and risks presented by these approaches.

Deployment Outside the Laboratory

Given the diverse rationale for judiciously introducing engineered microbes into new environments, an organized framework is needed for risk assessment. We encourage scrutinizing the introduction of engineered microbes for each of these 6 risk categories: 1.

Unintended functions due to poor engineering design

Desired functions for a target environment that are undesirable in other environments

Failure to function in the target environment

Engineered DNA sequence acquisition by other organisms

Functional expression of sequences acquired by other organisms

Unintended impacts on the food web due to deployed microbial growth

Speaking very generally, risk 1 is low because microbial engineering techniques are highly targeted and genes subject to heterologous expression are well-annotated in their function. Risks 2 and 3 are much more plausible. An example of risk 2 is a microbe that could perform too much of a desired function, or act on a broader set of environmental molecules that were not provided in the laboratory. Further, within a patient, excess expression of an immunomodulating protein or overcolonization could rapidly jeopardize patient health. These outcomes are more consequential than probable outcomes from risk 3, because a failure to act robustly or proliferate in a desired environment (because laboratory strains are generally believed to be less fit than native environmental strains) have little presumed detriment. Risks 4 and 5 relate to the possibility that engineered DNA sequences become acquired by other microbes, which, in principle, could have the broadest consequences on altering an ecosystem or patient microbiome. For example, bacteria engineered to possess genes encoding human therapeutics could share this genetic material with other gut commensals via horizontal gene transfer, leading to a formerly innocuous microbe gaining biomedical activity. Curiously, while horizontal gene transfer (HGT) is a common mode of DNA exchange in the environment,¹⁵⁷ genetically modified organisms (GMOs) have not been observed to pass on traits through HGT. For example, HGT of herbicide resistance from a GMO plant to bacteria was not detected in a field study.¹⁵⁸ While horizontal gene transfer from GMO plants to humans or the environment is of low probability,¹⁵⁹ the same may not be true of engineered microbes because microbes have elevated rates of gene exchange.¹⁵⁷ Such HGT events can be easily monitored by engineering strains to contain cassettes for expression of reporter proteins only when they are transferred to other organisms.¹⁶⁰ Additionally, technologies have been developed to prevent the potential spread of recombinant traits by HGT.¹⁶¹ Risk 6 is a risk common to the introduction of natural or engineered microbes, as their presence may reshape the local ecology through competition for limited resources or by serving as prey for predators. The latter effects have been observed even in confined but open reactor contexts, such as the predation that occurs when lipid-rich algae are cultivated in open pond reactor systems.^162-164 While this risk is important to acknowledge, we will not discuss it further here.

Perhaps the most salient reason for the need to continue investigation of biocontainment (discussed in more detail in the next section) can be drawn from the lessons of invasive species. In 2012, invasive species cost the US economy an estimated US$150 billion, or about 1% of the GDP.⁸ In response, an “ecological restoration” market in the United States generates revenues of at least US$9.5 billion annually.⁸ While not much is known about invasive microbes, what we know comes from studies of invasive plants. Introduction of nonnative plant species to a target region can have disruptive effects on its microbial community, including introduction and establishment of alien pathogens, perturbations to soil microbiome composition, and alteration of plant–microbe symbioses.¹⁶⁵ These changes could lead to rippling effects on the ecosystem including alteration of food chains, shifts in consumer behavior and range, increased waterway contamination and damage, and modified structural composition and decomposition dynamics of soils, all of which could change landscapes and their resident flora and fauna. It is possible that microbial dynamics could be similarly disruptive in higher organisms as well, with a tangible example being that of white-nose syndrome in hibernating bats in North America.¹⁶⁶

Opportunities for Technology Innovations

Biological Containment of Engineered Microbes

Because biological containment strategies have been reviewed extensively elsewhere,^161,167,168 here we briefly discuss their limitations and opportunities for innovation to guide practitioners and policymakers. Biocontainment systems have classically featured 1 of 3 systems: (1) dependence of the organism on an essential nutrient or metabolic building block that is not easily found in the environment (auxotrophy)¹⁶⁹; (2) dependence of the organism on a nonbuilding block chemical for expression of essential genes (transcriptional control or gene circuit)¹⁷⁰; or (3) restriction of the organism to a limited environment where expression of a toxin or cell lysis mechanism is suppressed (toxin–antitoxin system or kill switch).^171,172 To illustrate challenges and research opportunities associated with published biocontainment approaches, we will use as an example the technique of synthetic auxotrophy, where cells are engineered to rely on synthetic building blocks.^173-177 We chose this biocontainment technique because in ideal settings it has outperformed novel biocontainment strategies in the important metric of escape frequency. Yet, like other recent approaches, it has limitations in its characterization and application that are worth examining.

Measurement of escape frequencies required for large-scale deployment poses a challenge for the advancement of biocontainment research from laboratory settings. The US National Institutes of Health biocontainment standard is defined as less than 1 escape within a population of 10⁸ cells (or an escape rate of 10⁻⁸ escapees per colony forming unit [CFU]).¹⁷⁸ Measurement of escape frequency in batch experiments is limited by the size of liquid culture volumes that can be incubated and the surface area of solid media that can be used for plating. Several synthetic auxotrophs referenced earlier exhibit escape frequencies below the practical detection limits of 10⁻¹² escapees/CFU during a multiday or even multiweek observation period. However, these observations do not imply that escape is impossible, and some escape may be tolerable depending on the application. For personalized therapeutics, escape rates below 10⁻⁸ escapees/CFU may be sufficient given smaller cell counts administered to patients.

Other challenges in the field of biocontainment research include the generalizability of these methods across different organisms, the compatibility of the containment conditions with the intended deployment environment, the compatibility of containment methods with one another, and the evaluation of these strategies over longer timescales. Despite the impressive escape rates obtained, synthetic auxotrophy has only been tested in E coli and requires availability of organism-specific tools for incorporation of synthetic amino acids. Although initially demonstrated in genomically recoded E coli, synthetic auxotrophy functions in nonrecoded hosts.^175-177 Other biocontainment techniques, such as kill switches, generally also require organism-specific genetic elements for expression. In either case, if a biocontainment strategy requires introduction of a nonnative molecule into a new environment, then the properties of the nonnative molecule could have important ramifications and may need to be tested independently. Additionally, the introduction of a synthetic molecule, which is by definition required for synthetic auxotrophy but also a feature of some kill switches, adds cost to a process, creating another barrier to adoption. Another challenge is the compatibility of containment methods with one another. Due to the statistical rarity of escape mechanisms that must simultaneously occur, multilayered approaches may be the easiest and most effective way to implement robust containment.^172,179-186 Finally, how these methods perform over time in the face of evolution is still relatively unknown. To date, most experiments have been in batch, and while applicable to biomanufacturing, the results may not be relevant for environmental deployments. In these situations, an organism may need to survive for weeks or months, timescales subject to significant evolution, and therefore testing of evolutionary stability is required. At least one recent effort to develop kill switches has examined their evolutionary stability.¹⁸⁴ Overall, there is a strong need for research on the development of more generalizable, compatible, and durable biocontainment strategies. An ideal outcome of such research would be the creation of multiple complementary off-the-shelf components that can be easily implemented together for multilayered biocontainment.

Alternative Containment Approaches

If the primary concern is HGT of engineered DNA sequences from engineered microbes to other microbes, then the biocontainment techniques described earlier do not prevent successful DNA exchange. A complementary set of technologies can address HGT through methods that result in destruction of recombinant biological macromolecules if present in the wrong environment (eg, a DNA destruction device¹⁸² or a mutually dependent host-plasmid system¹⁸⁷), methods that overlap a gene of interest with a toxin gene,¹⁸⁸ or methods that use codes other than the universally conserved codon table for decoding of DNA sequence (eg, alternative genetic alphabets¹⁸⁹ or recoded sequences that have codons reassigned¹⁹⁰). Of these approaches, complete destruction of DNA or protein sequences has been difficult to achieve and can be obstructed by mutation. However, the use of alternative genetic alphabets within a protein coding sequence appears relatively foolproof for preventing functional expression if an HGT event were to occur. Challenges include the highly sophisticated level of engineering required to introduce machinery that can recognize synthetic bases. Additionally, while the organism is not necessarily dependent on supplementation of synthetic bases (and, therefore, not a synthetic auxotroph), synthetic bases must be provided to achieve the desired function, thus limiting the affordability of this technique and the context where it can be used.

Surveillance of Engineered Microbes

Given the roles of many natural microbes in infectious diseases, the topic of microbial surveillance techniques has also been reviewed extensively elsewhere.¹⁹¹ In the increasingly likely event of deployment of engineered microbes, surveillance technologies will be needed to monitor release sites and beyond. In this section, we briefly describe recent developments in the use of informatics, next-generation sequencing, and mass spectrometry for surveillance. Although numerous techniques exist for screening at the stage of DNA synthesis,¹⁹² they do not help with the identification of engineered microbes in the environment, where sequencing coupled with DNA amplification are critical. Recent approaches in DNA sequence screening have looked for signatures within large databases such as the Addgene plasmid repository. Engineered microbes can contain natural DNA from other organisms or transgenes produced by DNA synthesis. Because the common paradigm associated with DNA synthesis for heterologous expression is to adjust the codon usage of protein coding sequences, the codon usage of a gene relative to its natural sequence can serve as a signature of engineering from analysis of nucleotide sequences alone.¹⁹³ Deep learning methods have been developed to predict lab-of-origin of a DNA sequence when trained on appropriate datasets.¹⁹⁴ Machine learning has also been used to detect pathogenic sequences from next-generation sequencing reads.^195,196

Identification of a microbe is most straightforward if it can be isolated and cultivated. However, this strategy is not reliable because many microbes defy cultivation under standard laboratory conditions. Approaches for microbial identification have differed across the epidemiological and agricultural communities, which largely search for pathogens or GMOs, respectively. Before the emergence of widely accessible next-generation sequencing techniques, the state of the art in pathogen identification was analysis of chromosomal DNA restriction patterns using pulsed-field gel electrophoresis.¹⁹⁷ Mass spectrometry on organismal proteomes^198,199 or multilocus polymerase chain reaction (PCR) products^200,201 emerged as alternatives for pathogen identification. However, much like sequencing of ribosomal RNA, these approaches are not well-suited to detecting changes in DNA engineered at other loci within a host. In contrast, most GMO screening approaches used in agriculture today are reliant on PCR-based classification methods that are inherently limited to specific transgene sequences.^202-204 Because of the decreasing costs of sequencing, whole-genome DNA sequencing is emerging as a standard in open-view pathogen detection because of its unbiased nature relative to PCR. With this approach, one could search for synthetic DNA sequences within native loci. Moving forward, these approaches can be informed by detection strategies for pathogens,^195,196 invasive species,^205-207 or GMO crops,²⁰⁸ as concepts from each of these categories can apply to engineered microbes (eg, entry into new environments, genetic modification, and smaller size/genomes).

Environmental sampling for engineered microbes is yet another challenge that can be informed by precedents in related fields and strengthened by advances in technologies such as mass spectrometry. Environmental applications and strain choice will likely dictate the best specific locations to search for environmental DNA.²⁰⁹ Samples of environmental DNA have been successfully obtained from sources spanning lake waters²¹⁰ and sewage systems.²¹¹ Sewage is an especially good place to sample for the potential presence of engineered probiotics that may survive in excretions, as sewage has been previously used for successful detection of poliovirus vaccine during a test release.²¹²

The detailed listing of microbial technologies that are likely to be deployed for biomedical or environmental applications can guide researchers in the field of surveillance to better determine where and what to search for. Responsible stewardship of engineered microbes to new contexts will require greater vetting of biocontainment technologies and monitoring frameworks that leverage next-generation sequencing capabilities.

Conclusions

Synthetic biology has afforded power to the field of microbiology for which its innovators have long yearned. The ability to write, rewrite, and unwrite nucleic acid sequences, and, thus, the very identity of organisms, will similarly alter myriad aspects of the bioeconomy. The resultant engineered microbial technologies throughout the bioeconomy have vast potential, but important innovations and considerations are necessary to ensure the proper protections are in place. As deployment of microbes into higher organisms or the larger environment becomes tangible and perhaps even commonplace, the successes and challenges the field faces along the way will be indelibly affected by the work of this new decade. As such, the security of economic markets, biological systems, and the organisms existing within them will be founded on our collective foresight about the topics presented in this work. Players in the bioeconomy must respond to the urgent need to develop innovative engineered microbial solutions for therapeutic, environmental, and energetic scientific problems—and they must design these solutions in ways that protect the world while exhibiting the propriety required of such power.

Footnotes

Acknowledgments

The authors would like to thank Dr. Neil Thompson for his helpful expertise, input, and perspective that contributed to the contents of this manuscript.

References

Organisation for Economic Co-operation and Development (OECD). The Bioeconomy to 2030: Designing a Policy Agenda. Paris: OECD; 2009. Accessed July 11, 2020. https://doi.org/10.1787/9789264056886-en

Bundesministerium für Bildung und Forschung (BMBF). Nationale Forschungsstrategie BioÖkonomie 2030. Berlin: BMBF; 2010. Accessed July 11, 2020. https://www.bmbf.de/upload_filestore/pub/Nationale_Forschungsstrategie_Biooekonomie_2030.pdf

European Commission. Innovating for Sustainable Growth: A Bioeconomy for Europe. Brussels: European Commission; 2012. Accessed July 11, 2020. https://op.europa.eu/en/publication-detail/-/publication/1f0d8515-8dc0-4435-ba53-9570e47dbd51

Staffas

, Gustavsson

, McCormick

. Strategies and policies for the bioeconomy and bio-based economy: an analysis of official national approaches. Sustainability. 2013; 5(6):2751-2769.

Dubois

, San Juan

. How Sustainability is Addressed in Official Bioeconomy Strategies at International, National, and Regional Levels. Rome: Food and Agriculture Organization of the United Nations; 2016. Accessed July 11, 2020. http://www.fao.org/3/a-i5998e.pdf

National Academies of

Sciences

, Engineering, and Medicine . Safeguarding the Bioeconomy. Washington, DC: The National Academies Press; 2020. Accessed July 11, 2020. https://www.nap.edu/catalog/25525/safeguarding-the-bioeconomy

Daystar

, Handfield

, Golden

, McConnell

, Morrison

. An Economic Impact Analysis of the U.S. Biobased Products Industry (2018). Washington, DC: US Department of Agriculture; 2018. Accessed July 11, 2020. https://www.biopreferred.gov/BPResources/files/BiobasedProductsEconomicAnalysis2018.pdf

Carlson

Estimating the biotech sector's contribution to the US economy. Nat Biotechnol. 2016; 34(3):247-255.

Carlson

Biology Is Technology: The Promise, Peril, and New Business of Engineering Life. Cambridge, MA: Harvard University Press; 2011.

10.

CB Insights. Hacking DNA: all the corporates investing in synthetic biology startups in one timeline. CB Insights website. Published March 10, 2017. Accessed July 11, 2020. https://www.cbinsights.com/research/synthetic-biology-corporate-investments/

11.

Shaw

Synthetic biology poised for a big impact. Biocompare website. Published September 25, 2018. Accessed July 11, 2020. https://www.biocompare.com/Editorial-Articles/353545-Synthetic-Biology-Poised-for-a-Big-Impact/

12.

Schmidt

These 98 synthetic biology companies raised $3.8 billion in 2018. SnBioBeta website. Published December 19, 2018. Accessed July 11, 2020. https://synbiobeta.com/these-98-synthetic-biology-companies-raised-3-8-billion-in-2018/

13.

Engineering Our Way to a Sustainable Bioeconomy, Hearing Before the Subcommittee on Research and Technology; Committee on Science, Space, and Technology, 114th Cong, 1st Sess (2019). Accessed July 11, 2020. https://www.govinfo.gov/content/pkg/CHRG-116hhrg35583/pdf/CHRG-116hhrg35583.pdf

14.

Bergin

Synthetic Biology: Global Markets. Boston: BCC Research LLC; 2020. Accessed July 11, 2020. https://www.bccresearch.com/market-research/biotechnology/synthetic-biology-global-markets.html

15.

Gronvall

GK.

US competitiveness in synthetic biology. Health Secur. 2015; 13(6):378-389.

16.

Executive Office of the President. National Bioeconomy Blueprint. Washington, DC: The White House; 2012. Accessed July 11, 2020. https://obamawhitehouse.archives.gov/sites/default/files/microsites/ostp/national_bioeconomy_blueprint_april_2012.pdf

17.

Executive Office of the President. Putting the Bioeconomy Blueprint to Work. The White House: Washington, DC; 2012. Accessed July 11, 2020. https://obamawhitehouse.archives.gov/sites/default/files/microsites/ostp/bioeconomy_fact_sheet_april_26_2012_0.pdf

18.

US Department of Energy. Synthetic Biology: Report to Congress. Washington, DC: US Department of Energy; 2013. Accessed July 11, 2020. https://haseloff.plantsci.cam.ac.uk/resources/SynBio_reports/DOE-Synthetic-Biology-Report-to-Congress_Fnl.pdf

19.

Office of Technical Intelligence. Technical Assessment: Synthetic Biology. Washington, DC: US Department of Defense; 2015. Accessed July 11, 2020. https://defenseinnovationmarketplace.dtic.mil/wp-content/uploads/2018/02/OTI-SyntheticBiologyTechnicalAssessment.pdf

20.

, Zhao

. A brief overview of synthetic biology research programs and roadmap studies in the United States. Synth Syst Biotechnol. 2016; 1(4):258-264.

21.

Engineering Biology Research and Development Act, HR 4373, 116th Cong (2019). Accessed July 11, 2020. https://www.congress.gov/bill/116th-congress/house-bill/4373

22.

James

. Global Status of Commercialized Biotech/GM Crops: 2012. Brief 44. Manila: International Service for the Acquisition of Agri-biotech Applications; 2012. Accessed July 11, 2020. https://www.isaaa.org/resources/publications/briefs/44/download/isaaa-brief-44-2012.pdf

23.

International Service for the Acquisition of Agri-biotech Applications (ISAAA). Global Status of Commercialized Biotech/GM Crops in 2018: Biotech Crops Continue to Help Meet the Challenges of Increased Population and Climate Change. Manila: ISAAA; 2018. Accessed July 11, 2020. http://www.isaaa.org/resources/publications/briefs/54/

24.

Klümper

, Qaim

. A meta-analysis of the impacts of genetically modified crops. PLoS One. 2014; 9(11):e111629.

25.

Reid

, Greene

. How Microbes Can Help Feed the World. Sterling, VA: American Academy of Microbiology; 2012. Accessed July 11, 2020. https://www.asmscience.org/content/report/colloquia/colloquia.18

26.

Baas

, Bell

, Mancini

, Lee

, Conant

, Wallenstein

. Phosphorus mobilizing consortium Mammoth P(^TM) enhances plant growth. PeerJ. 2016; 4:e2121.

27.

Panke-Buisse

, Poole

, Goodrich

, Ley

, Kao-Kniffin

. Selection on soil microbiomes reveals reproducible impacts on plant function. ISME J. 2015; 9(4):980-989.

28.

Marulanda

, Barea

J-M

, Azcón

. Stimulation of plant growth and drought tolerance by native microorganisms (AM fungi and bacteria) from dry environments: mechanisms related to bacterial effectiveness. J Plant Growth Regul. 2009; 28(2):115-124.

29.

Mendes

, Kruijt

, de Bruijn

, et al. Deciphering the rhizosphere microbiome for disease-suppressive bacteria. Science. 2011; 332(6033):1097-1100.

30.

Mueller

, Sachs

. Engineering microbiomes to improve plant and animal health. Trends Microbiol. 2015; 23(10):606-617.

31.

Fleming

On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzæ. Br J Exp Pathol. 1929; 10(3):226-236.

32.

Reports and Data. Antibiotics Market By Type (Protein, DNA, RNA, Cell Wall Synthesis Inhibitors, Mycolic Acid Inhibitors), By End-User (Hospital pharmacies, Retail pharmacies, Online pharmacies) By Product Type, By Administration Mode and Segment Forecasts, 2016-2026. New York: Reports and Data; 2019. Accessed July 11, 2020. https://www.reportsanddata.com/report-detail/antibiotics-market

33.

Demain

, Adrio

. Contributions of microorganisms to industrial biology. Mol Biotechnol. 2007; 38(1):41.

34.

Walsh

Therapeutic insulins and their large-scale manufacture. Appl Microbiol Biotechnol. 2005; 67(2):151-159.

35.

Market Research Future. Chemotherapy Market Research Report - Global Forecast till 2024. Maharashtra, India: Market Research Future; 2019. Accessed July 11, 2020. https://www.marketresearchfuture.com/reports/chemotherapy-market-5791

36.

Grand View Research. Dietary Supplements Market Size Analysis Report by Ingredient (Botanicals, Vitamins), By Form, By Application (Immunity, Cardiac Health), By End User, By Distribution Channel, And Segment Forecasts, 2019–2025. San Francisco: Grand View Research; 2019.

37.

Coherent Market Insights. Recombinant Protein Market: Global Industry Insights and Forecast, 2025. Seattle: Coherent Market Insights; 2016.

38.

Grand View Research. Hyperlipidemia Drugs Market Analysis Report by Drug Class (Statins, PCSK9 Inhibitors, Bile Acid Sequestrants, Cholesterol Absorption Inhibitors, Fibric Acid Derivatives, Combination), and Segment Forecasts, 2016–2022. San Francisco: Grand View Research; 2018. Accessed July 11, 2020. https://www.grandviewresearch.com/industry-analysis/hyperlipidemia-drugs-market

39.

Grand View Research. Organ Transplant Immunosuppressant Drugs Market Size, Share and Trends Analysis Report by Drug Class (Calcineurin Inhibitors, Antibodies), by Transplant, by Distribution Channel, and Segment Forecasts, 2019–2026. San Francisco: Grand View Research; 2019. Accessed July 2020 . https://www.grandviewresearch.com/industry-analysis/organ-transplant-immunosuppressant-drugs-market

40.

Wansley

, Chakraborty

, Hance

, et al. Vaccination with a recombinant Saccharomyces cerevisiae expressing a tumor antigen breaks immune tolerance and elicits therapeutic antitumor responses. Clin Cancer Res. 2008; 14(13):4316-4325.

41.

Marcobal

, Liu

, Zhang

, et al. Expression of human immunodeficiency virus type 1 neutralizing antibody fragments using human vaginal Lactobacillus. AIDS Res Hum Retroviruses. 2016; 32(10-11):964-971.

42.

Westendorf

, Gunzer

, Deppenmeier

, et al. Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules. FEMS Immunol Med Microbiol. 2005; 43(3):373-384.

43.

Leatham

, Banerjee

, Autieri

, Mercado-Lubo

, Conway

, Cohen

. Precolonized human commensal Escherichia coli strains serve as a barrier to E. coli O157:H7 growth in the streptomycin-treated mouse intestine. Infect Immun. 2009; 77(7):2876-2886.

44.

Hancock

, Dahl

, Klemm

. Probiotic Escherichia coli strain Nissle 1917 outcompetes intestinal pathogens during biofilm formation. J Med Microbiol. 2010; 59(4):392-399.

45.

Isabella

, Ha

, Castillo

, et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat Biotechnol. 2018; 36(9):857-864.

46.

Hamady

ZZR

, Scott

, Farrar

, et al. Xylan-regulated delivery of human keratinocyte growth factor-2 to the inflamed colon by the human anaerobic commensal bacterium Bacteroides ovatus. Gut. 2010; 59(4):461-469.

47.

Agler

, Wrenn

, Zinder

, Angenent

. Waste to bioproduct conversion with undefined mixed cultures: the carboxylate platform. Trends Biotechnol. 2011; 29(2):70-78.

48.

Kosa

, Ragauskas

. Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol. 2011; 29(2):53-61.

49.

Qian

, Morse

. Miniaturizing microbial fuel cells. Trends Biotechnol. 2011; 29(2):62-69.

50.

Wood

, Hong

, Ma

. Engineering biofilm formation and dispersal. Trends Biotechnol. 2011; 29(2):87-94.

51.

Gleizer

, Ben-Nissan

, Bar-On

, et al. Conversion of Escherichia coli to generate all biomass carbon from CO₂. Cell. 2019; 179(6):1255-1263.e12.

52.

Ritchie

, Roser

Renewable energy. Our World in Data website. Published 2019. Accessed July 11, 2020. https://ourworldindata.org/renewable-energy

53.

Frankfurt School-UNEP Centre/BNEF. Global Trends in Renewable Energy Investment. Frankfurt: Frankfurt School of Finance and Management; 2019. Accessed July 11, 2020. https://wedocs.unep.org/bitstream/handle/20.500.11822/29752/GTR2019.pdf

54.

National Research Council. Industrialization of Biology: A Roadmap to Accelerate the Advanced Manufacturing of Chemicals. Washington, DC: The National Academies Press; 2015.

55.

Zion Market Research. Renewable Chemicals Market (Ketones, Biopolymers, Alcohols, Organic Acids, Platform Chemicals and Others) for Agriculture, Textiles, Housing, Environment, Transportation, Food and Beverage Packaging, Communication, Bio-Medical and Other Applications: Global. Maharashtra, India: Zion Market Research; 2018. Accessed July 11, 2020. https://www.zionmarketresearch.com/report/renewable-chemicals-market

56.

Biotechnology Innovation Organization (BIO). Advancing the Biobased Economy: Renewable Chemical Biorefinery Commercialization, Progress, and Market Opportunities, 2016 and Beyond. Washington, DC: BIO; 2016. Accessed July 11, 2020. https://archive.bio.org/advancing-biobased-economy-renewable-chemical-biorefinery-commercialization-progress-and-market

57.

Artz

, Müller

, Thenert

, et al. Sustainable conversion of carbon dioxide: an integrated review of catalysis and life cycle assessment. Chem Rev. 2018; 118(2):434-504.

58.

Reports and Data. Succinic Acid Market by Type (Petro-Based, and Bio-Based), by Applications (Food and Beverages, Pharmaceutical, Personal Care, Coatings, Industrial, and Others), by Regions, Forecasts to 2026. New York: Reports and Data; 2019.

59.

Grand View Research. Lactic Acid Market Size, Share & Trend Analysis Report by Raw Material (Corn, Sugarcane), by Application (Industrial, Food & Beverages, Polylactic Acid), by Region, and Segment Forecasts, 2019–2025. San Francisco: Grand View Research; 2019.

60.

Markets and Markets. 1,3-Propanediol (PDO) Market by Application (Polytrimethylene terephthalate (PTT), Cosmetics, Personal Care & Cleaning Products, Polyurethane (PU)) and Region (Americas, APAC, Europe, Middle East & Africa (EMEA)) - Global Forecast to 2024. Markets and Markets; 2019.

61.

Grand View Research. Enzymes Market Size, Share & Trends Analysis Report by Source (Plants, Animals, Microorganisms), by Application (Industrial, Specialty), by Product (Carbohydrase, Proteases), And Segment Forecasts, 2019–2025. San Francisco: Grand View Research; 2019.

62.

Research and Markets. Vitamins Market by Type (Vitamin B, Vitamin E, Vitamin D, Vitamin C, Vitamin A, and Vitamin K), Application (Healthcare Products, Food & Beverages, Feed, and Personal Care Products), Source (Synthetic and Natural), etc. - Global Forecast to 2023. Research and Markets; 2018.

63.

Grand View Research. Nucleotide Market Analysis by Technology (Taqman Allelic Discrimination, Gene Chips and Microarrays, SNP by Pyrosequencing), by Application (Pharmaceuticals, Diagnostics Research, Food and Beverage Additive, Animal Feed Additive) and Segment Forecasts to 2022. San Francisco: Grand View Research; 2016.

64.

Industry Experts. Global Amino Acids Market – Products and Applications. Industry Experts; 2017.

65.

Wozniak

, McClung

, Gagliardi

, Segal

, Matthews

. Regulation of genetically engineered microorganisms under FIFRA, FFDCA and TSCA. In: Wozniak

, McHugen

, eds. Regulation of Agricultural Biotechnology: The United States and Canada. Dordrecht: Springer Netherlands; 2013:57-94.

66.

National Academies of

Sciences

, Engineering, and Medicine . Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press; 2018.

67.

Fuller

. Probiotics in human medicine. Gut. 1991(4):439-442.

68.

Fuller

Probiotics in man and animals. J Appl Bacteriol. 1989; 66(5):365-378.

69.

Cryan

, Dinan

. Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nat Rev Neurosci. 2012; 13(10):701-712.

70.

Bienenstock

, Kunze

, Forsythe

. Microbiota and the gut–brain axis. Nutr Rev. 2015; 73(suppl 1):28-31.

71.

Ait-Belgnaoui

, Colom

, Braniste

, et al. Probiotic gut effect prevents the chronic psychological stress-induced brain activity abnormality in mice. Neurogastroenterol Motil. 2014; 26(4):510-520.

72.

Tillisch

, Labus

, Kilpatrick

, et al. Consumption of fermented milk product with probiotic modulates brain activity. Gastroenterology. 2013; 144(7):1394-401.

73.

Pinto-Sanchez

, Hall

, Ghajar

, et al. Probiotic Bifidobacterium longum NCC3001 reduces depression scores and alters brain activity: a pilot study in patients with irritable bowel syndrome. Gastroenterology. 2017; 153(2):448-459.e8.

74.

Piñero-Lambea

, Ruano-Gallego

, Fernández

LÁ

. Engineered bacteria as therapeutic agents. Curr Opin Biotechnol. 2015; 35:94-102.

75.

Ozdemir

, Fedorec

AJH

, Danino

, Barnes

. Synthetic biology and engineered live biotherapeutics: toward increasing system complexity. Cell Syst. 2018; 7(1):5-16.

76.

Bober

, Beisel

, Nair

. Synthetic biology approaches to engineer probiotics and members of the human microbiota for biomedical applications. Annu Rev Biomed Eng. 2018; 20(1):277-300.

77.

Steidler

, Hans

, Schotte

, et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science. 2000; 289(5483):1352-1355.

78.

Braat

, Rottiers

, Hommes

, et al. A Phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin Gastroenterol Hepatol. 2006; 4(6):754-759.

79.

Steidler

, Neirynck

, Huyghebaert

, et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol. 2003; 21(7):785-789.

80.

Vandenbroucke

, De Haard

, Beirnaert

, et al. Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis. Mucosal Immunol. 2010; 3(1):49-56.

81.

, Yang

, Tham

, Chen

, Guo

, Zhu

. Genetic engineering of probiotic Escherichia coli Nissle 1917 for clinical application. Appl Microbiol Biotechnol. 2016; 100(20):8693-8699.

82.

Wassenaar

TM.

Insights from 100 years of research with probiotic E. coli. Eur J Microbiol Immunol. 2016; 6(3):147-161.

83.

Rao

, Hu

, McHugh

, Lueders

, Henry

, Zhao

, et al. Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide. Proc Natl Acad Sci U S A. 2005; 102(34):11993-11998.

84.

Duan

, Curtis

, March

. Secretion of insulinotropic proteins by commensal bacteria: rewiring the gut to treat diabetes. Appl Environ Microbiol. 2008; 74(23):7437-7438.

85.

Isabella

, Ha

, Castillo

, et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat Biotechnol. 2018; 36(9):857-867.

86.

Kurtz

, Millet

, Puurunen

, 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(475).

87.

Whitaker

, Shepherd

, Sonnenburg

. Tunable expression tools enable single-cell strain distinction in the gut microbiome. Cell. 2017; 169(3):538-546.e12.

88.

Shepherd

, Deloache

, Pruss

, Whitaker

, Sonnenburg

. An exclusive metabolic niche enables strain engraftment in the gut microbiota. Nature. 2018; 557(7705):434-438.

89.

Abratt

, Reid

. Oxalate-degrading bacteria of the human gut as probiotics in the management of kidney stone disease. In: Laskin

, Gadd

, Sariaslani

, eds. Advances in Applied Microbiology. Volume 10. Cambridge, MA: Academic Press Inc.; 2010:63-87.

90.

Hillman

, Socransky

. Replacement therapy of the prevention of dental disease. Adv Dent Res. 1987; 1(1):119-125.

91.

Medaglini

, Rush

, Sestini

, Pozzi

. Commensal bacteria as vectors for mucosal vaccines against sexually transmitted diseases: vaginal colonization with recombinant streptococci induces local and systemic antibodies in mice. Vaccine. 1997; 15(12-13):1330-1337.

92.

Di Fabio

, Medaglini

, Rush

, et al. Vaginal immunization of Cynomolgus monkeys with Streptococcus gordonii expressing HIV-1 and HPV 16 antigens. Vaccine. 1998; 16(5):485-492.

93.

Beninati

, Oggioni

, Boccanera

, et al. Therapy of mucosal candidiasis by expression of an anti-idiotype in human commensal bacteria. Nat Biotechnol. 2000; 18(10 suppl):1060-1064.

94.

Chang

TLY

, Chang

, Simpson

, et al. Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4. Proc Natl Acad Sci U S A. 2003; 100(20):11672-11677.

95.

Liu

, Lagenaur

, Simpson

, et al. Engineered vaginal Lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-N. Antimicrob Agents Chemother. 2006; 50(10):3250-3259.

96.

Lagenaur

, Sanders-Beer

, Brichacek

, et al. Prevention of vaginal SHIV transmission in macaques by a live recombinant Lactobacillus. Mucosal Immunol. 2011; 4(6):648-657.

97.

Forbes

NS.

Engineering the perfect (bacterial) cancer therapy. Nat Rev Cancer. 2010; 10(11):785–794.

98.

Zhou

, Gravekamp

, Bermudes

, Liu

. Tumour-targeting bacteria engineered to fight cancer. Nat Rev Cancer. 2018; 18(12):727-743.

99.

Gandhi

, Morales

, Lamm

. Bacillus Calmette-Guérin immunotherapy for genitourinary cancer. BJU Int. 2013; 112(3):288-297.

100.

Anderson

, Clarke

, Arkin

, Voigt

. Environmentally controlled invasion of cancer cells by engineered bacteria. J Mol Biol. 2006; 355(4):619-627.

101.

Din

, Danino

, Prindle

, et al. Synchronized cycles of bacterial lysis for in vivo delivery. Nature. 2016; 536(7614):81-85.

102.

Theys

, Landuyt

, Nuyts

, et al. Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum. Cancer Gene Ther. 2001; 8(4):294-297.

103.

Mei

, Theys

, Landuyt

, Anne

, Lambin

. Optimization of tumor-targeted gene delivery by engineered attenuated Salmonella typhimurium. Anticancer Res. 2002; 22(6A):3261-3266.

104.

Theys

, Pennington

, Dubois

, et al. Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo. Br J Cancer. 2006; 95(9):1212-1219.

105.

Barbé

, Mellaert

, Theys

, et al. Secretory production of biologically active rat interleukin-2 by Clostridium acetobutylicum DSM792 as a tool for anti-tumor treatment. FEMS Microbiol Lett. 2005; 246(1):67-73.

106.

Chowdhury

, Castro

, Coker

, Hinchliffe

, Arpaia

, Danino

. Programmable bacteria induce durable tumor regression and systemic antitumor immunity. Nat Med. 2019; 25(7):1057-1063.

107.

Nauts

, Swift

, Coley

. The treatment of malignant tumors by bacterial toxins as developed by the late William B. Coley, M.D., reviewed in the light of modern research. Cancer Res. 1946; 6(4):205-216.

108.

Alexander

Biodegradation and Bioremediation. San Diego: Academic Press; 1994.

109.

Singh

, Kang

, Mulchandani

, Chen

. Bioremediation: environmental clean-up through pathway engineering. Curr Opin Biotechnol. 2008; 19(5):437-444.

110.

Dvořák

, Nikel

, Damborský

, de Lorenzo

. Bioremediation 3.0: engineering pollutant-removing bacteria in the times of systemic biology. Biotechnol Adv. 2017; 35(7):845-866.

111.

Menn

, Easter

, Sayler

. Genetically engineered microorganisms and bioremediation. In: Rehm

H-J

, Reed

, eds. Biotechnology Set. Hoboken, NJ: Wiley; 2001:441-463.

112.

Ripp

, Nivens

, Ahn

, Werner

, Jarrell IV

, Easter

, et al. Controlled field release of a bioluminescent genetically engineered microorganism for bioremediation process monitoring and control. Environ Sci Technol. 2000; 34(5):846-853.

113.

King

JMH

, Digrazia

, Applegate

, et al. Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegradation. Science. 1990; 249(4970):778-781.

114.

Richins

, Kaneva

, Mulchandani

, Chen

. Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase. Nat Biotechnol. 1997; 15(10):984-987.

115.

Shimazu

, Mulchandani

, Chen

. Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase. Biotechnol Bioeng. 2001; 76(4):318-324.

116.

Valls

, Atrian

, De Lorenzo

, Fernández

. Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil. Nat Biotechnol. 2000; 18(6):661-665.

117.

Brim

, Venkateswaran

, Kostandarithes

, Fredrickson

, Daly

. Engineering Deinococcus geothermalis for bioremediation of high-temperature radioactive waste environments. Appl Environ Microbiol. 2003; 69(8):4575-4582.

118.

Barac

, Taghavi

, Borremans

, et al. Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants. Nat Biotechnol. 2004; 22(5):583-588.

119.

Bettmann

, Rehm

. Degradation of phenol by polymer entrapped microorganisms. Appl Microbiol Biotechnol. 1984; 20(5):285-290.

120.

Sahasrabudhe

, Modi

. Dehalogenation of 3-chlorobenzoate by immobilized Pseudomonas sp. B13 cells. Biotechnol Bioeng. 1988; 31(8):889-893.

121.

Cassidy

, Lee

, Trevors

. Environmental applications of immobilized microbial cells: A review. J Ind Microbiol. 1996; 16:79-101.

122.

Bashan

, Gonzalez

. Long-term survival of the plant-growth-promoting bacteria Azospirillum brasilense and Pseudomonas fluorescens in dry alginate inoculant. Appl Microbiol Biotechnol. 1999; 51(2):262-266.

123.

Browne

, Crump

, Niven

, et al. Accumulation of microplastic on shorelines woldwide: sources and sinks. Environ Sci Technol. 2011; 45(21):9175-9179.

124.

Cheang

, Ma

, Fok

. Occurrence and composition of microplastics in the seabed sediments of the coral communities in proximity of a metropolitan area. Int J Environ Res Public Health. 2018; 15(10):2270.

125.

Cohen

, Internicola

, Mason

, Kukulka

. Observations and simulations of microplastic debris in a tide, wind, and freshwater-driven estuarine environment – the Delaware Bay. Environ Sci Technol. 2019; 53(24):14204–14211.

126.

Setälä

, Fleming-Lehtinen

, Lehtiniemi

. Ingestion and transfer of microplastics in the planktonic food web. Environ Pollut. 2014; 185:77-83.

127.

Gall

, Thompson

. The impact of debris on marine life. Mar Pollut Bull. 2015; 92(1-2):170-179.

128.

Cox

, Covernton

, Davies

, Dower

, Juanes

, Dudas

. Human consumption of microplastics. Environ Sci Technol. 2019; 53(12):7068-7074.

129.

Silva

, Carneiro

, O'Neill

, et al. Cutinase - A new tool for biomodification of synthetic fibers. J Polym Sci Part A Polym Chem. 2005; 43(11):2448-2450.

130.

Yoshida

, Hiraga

, Takehana

, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science. 2016; 351(6278):1196-1199.

131.

Moog

, Schmitt

, Senger

, et al. Using a marine microalga as a chassis for polyethylene terephthalate (PET) degradation. Microb Cell Fact. 2019; 18(1):171.

132.

Miller

The EPA's war on bioremediation. Nat Biotechnol. 1997; 15(6):486.

133.

Sivasubramaniam

, Franks

. Bioengineering microbial communities: their potential to help, hinder and disgust. Bioengineered. 2016; 7(3):137-144.

134.

Sayler

, Ripp

. Field applications of genetically engineered microorganisms for bioremediation processes. Curr Opin Biotechnol. 2000; 11(3):286-289.

135.

Chakrabarty

AM.

Genetically-manipulated microorganisms and their products in the oil service industries. Trends Biotechnol. 1985; 3(2):32-39.

136.

Ezezika

, Singer

. Genetically engineered oil-eating microbes for bioremediation: prospects and regulatory challenges. Technol Soc. 2010; 32(4):331-335.

137.

Lindow

, Panopoulos

. Field tests of recombinant ice–Pseudomonas syringae for biological frost control in potato. In: Sussman

, Collins

, Skinner

, Stewart-Tull

, eds. Release of Genetically-Engineered Micro-Organisms. Cambridge, MA: Academic Press; 1988:121-138.

138.

Wrubel

, Krimsky

, Anderson

. Regulatory oversight of genetically engineered microorganisms: Has regulation inhibited innovation?. Environ Manage. 1997; 21(4):571-586.

139.

Wozniak

, McClung

, Gagliardi

, Segal

, Matthews

. Regulation of genetically engineered microorganisms under FIFRA, FFDCA and TSCA. In: Wozniak

, McHughen

, eds. Regulation of Agricultural Biotechnology: The United States and Canada. Dordrecht: Springer Netherlands; 2012:57-94.

140.

Baum

, Kakefuda

, Gawron-Burke

. Engineering Bacillus thuringiensis bioinsecticides with an indigenous site-specific recombination system. Appl Environ Microbiol. 1996; 62(12):4367-4373.

141.

Ronson

, Bosworth

, Genova

, et al. Field release of genetically-engineered Rhizobium meliloti and Bradyrhizobium japonicum strains. In: Gresshoff

, Roth

, Stacey

, Newton

, eds. Nitrogen Fixation. New York: Springer US; 1990:397-403.

142.

Geddes

, Ryu

, Mus

, et al. Use of plant colonizing bacteria as chassis for transfer of N2-fixation to cereals. Curr Opin Biotechnol. 2015; 32:216-222.

143.

Mus

, Crook

, Garcia

, et al. Symbiotic nitrogen fixation and the challenges to its extension to nonlegumes. Appl Environ Microbiol. 2016; 82(13):3698-3710.

144.

, Fan

, Miller

. Plant nitrogen assimilation and use efficiency. Annu Rev Plant Biol. 2012; 63(1):153-182.

145.

Lundberg

, Weitzberg

, Cole

, Benjamin

. Nitrate, bacteria and human health. Nat Rev Microbiol. 2004; 2(7):593-602.

146.

Erisman

, Bleeker

, Galloway

, Sutton

. Reduced nitrogen in ecology and the environment. Environ Pollut. 2007; 150(1):140-149.

147.

Temme

, Zhao

, Voigt

. Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. Proc Natl Acad Sci U S A. 2012; 109(18):7085-7090.

148.

Bloch

, Temme

, Tamsir

, inventors; Pivot

Bio

, Inc., assignee. Methods and compositions for improving engineered microbes. World patent WO 2019/032926 A1, February 14, 2019.

149.

National Research Council; Institute of Medicine; Board on Life Sciences; Food and Nutrition Board; Board on Agriculture and Natural Resources; Committee on Identifying and Assessing Unintended Effects of Genetically Engineered Foods on Human Health. Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects. Washington, DC: The National Academies Press; 2004.

150.

Podevin

, Devos

, Davies

, Nielsen

. Transgenic or not? No simple answer! New biotechnology-based plant breeding techniques and the regulatory landscape. EMBO Rep. 2012; 13(12):1057-1061.

151.

Splitter

These super-microbes could fix agriculture's nitrogen problem. Forbes. September 20, 2018. Accessed July 12, 2020. https://www.forbes.com/sites/jennysplitter/2018/09/20/these-super-microbes-could-fix-agricultures-nitrogen-problem/#4b8c16d443db

152.

, Jia

, Hou

, Lei

. Microbial biosensors: a review. Biosens Bioelectron. 2011; 26(5):1788-1799.

153.

Vogrinc

, Vodovnik

, Marinsek-Logar

. Microbial biosensors for environmental monitoring. Acta Agric Slov. 2015; 106(2):67-75.

154.

Meet

FRED

, the field-ready electrochemical detector. FREDsense Technologies website. Accessed July 17, 2020. https://media-openideo-rwd.oiengine.com/attachments/211eb0f3-d4d5-4631-aaf3-f83e98082f32.pdf

155.

Astudillo-García

, Hermans

, Stevenson

, Buckley

, Lear

. Microbial assemblages and bioindicators as proxies for ecosystem health status: potential and limitations. Appl Microbiol Biotechnol. 2019; 103(16):6407-6421.

156.

Holt

, Miller

. Bioindicators: using organisms to measure environmental impacts. Nat Educ Knowl. 2010;3(10).

157.

Aminov

RI.

Horizontal gene exchange in environmental microbiota. Front Microbiol. 2011; 2:158.

158.

Mohr

, Tebbe

. Field study results on the probability and risk of a horizontal gene transfer from transgenic herbicide-resistant oilseed rape pollen to gut bacteria of bees. Appl Microbiol Biotechnol. 2007; 75(3):573-582.

159.

Keese

Risks from GMOs due to horizontal gene transfer. Environ Biosafety Res. 2008; 7(3):123-149.

160.

Jaenecke

, De Lorenzo

, Timmis

, Díaz

. A stringently controlled expression system for analysing lateral gene transfer between bacteria. Mol Microbiol. 1996; 21(2):293-300.

161.

Schmidt

, de Lorenzo

. Synthetic bugs on the loose: containment options for deeply engineered (micro)organisms. Curr Opin Biotechnol. 2016; 38:90-96.

162.

Kunjapur

, Eldridge

. Photobioreactor design for commercial biofuel production from microalgae. Ind Eng Chem Res. 2010; 49(8):3516-3526.

163.

Day

, Thomas

, Achilles-Day

UEM

, Leakey

RJG

. Early detection of protozoan grazers in algal biofuel cultures. Bioresour Technol. 2012; 114:715-719.

164.

Flynn

, Kenny

, Mitra

. Minimising losses to predation during microalgae cultivation. J Appl Phycol. 2017; 29(4):1829-1840.

165.

van der Putten

, Klironomos

, Wardle

. Microbial ecology of biological invasions. ISME J. 2007; 1(1):28-37.

166.

Blehert

, Hicks

, Behr

, et al. Bat white-nose syndrome: an emerging fungal pathogen?. Science. 2009; 323(5911):227.

167.

Wright

, Stan

, Ellis

. Building-in biosafety for synthetic biology. Microbiology. 2013; 159(Pt 7):1221-1235.

168.

Lee

, Chan

CTY

, Slomovic

, Collins

. Next-generation biocontainment systems for engineered organisms. Nat Chem Biol. 2018; 14(6):530-537.

169.

Boyer

, Roulland-Dussoix

. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969; 41(3):459-472.

170.

Cai

, Agmon

, Choi

, et al. Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes. Proc Natl Acad Sci U S A. 2015; 112(6):1803-808.

171.

Molin

, Boe

, Jensen

, et al. Suicidal genetic elements and their use in biological containment of bacteria. Annu Rev Microbial. 1993; 47:139-166.

172.

Chan

CTY

, Lee

, Cameron

, Bashor

, Collins

. “Deadman” and “Passcode” microbial kill switches for bacterial containment. Nat Chem Biol. 2016; 12(2):82-86.

173.

Mandell

, Lajoie

, Mee

, et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature. 2015; 518(7537):55-60.

174.

Rovner

, Haimovich

, Katz

, et al. Recoded organisms engineered to depend on synthetic amino acids. Nature. 2015; 518(7537):89-93.

175.

Xuan

, Schultz

. A strategy for creating organisms dependent on noncanonical amino acids. Angew Chemie - Int Ed. 2017; 56(31):9170-9173.

176.

Gan

, Liu

, Wang

, Schultz

. Functional replacement of histidine in proteins to generate noncanonical amino acid dependent organisms. J Am Chem Soc. 2018; 140(11):3829-3832.

177.

Koh

, Yao

, Gleason

, Mills

, Schultz

. A general strategy for engineering noncanonical amino acid dependent bacterial growth. J Am Chem Soc. 2019; 141(41):16213-16216.

178.

National Institutes of Health (NIH). NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines). Bethesda, MD: NIH; 2016.

179.

Contreras

, Molin

, Ramos

. Conditional-suicide containment system for bacteria which mineralize aromatics. Appl Environ Microbiol. 1991; 57(5):1504-1508.

180.

Ronchel

, Ramos

. Dual system to reinforce biological containment of recombinant bacteria designed for rhizoremediation. Appl Environ Microbiol. 2001; 67(6):2649-2656.

181.

Cai

, Agmon

, Choi

, et al. Intrinsic biocontainment: multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes. Proc Natl Acad Sci U S A. 2015; 112(6):1803-1808.

182.

Caliando

, Voigt

. Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nat Commun. 2015; 6(1):6989.

183.

Gallagher

, Patel

, Interiano

, Rovner

, Isaacs

. Multilayered genetic safeguards limit growth of microorganisms to defined environments. Nucleic Acids Res. 2015; 43(3):1945-1954.

184.

Stirling

, Bitzan

, O'Keefe

, et al. Rational design of evolutionarily stable microbial kill switches. Mol Cell. 2017; 68(4):686-697.e3.

185.

Dimas

, Jiang

X-L

, Alberto de la Paz

, Morcos

, Chan

CTY

. Engineering repressors with coevolutionary cues facilitates toggle switches with a master reset. Nucleic Acids Res. 2019; 47(10):5449-5463.

186.

Stirling

, Naydich

, Bramante

, et al. Synthetic cassettes for pH-mediated sensing, counting and containment. Cell Rep. 2020; 30(9):3139-3148.e4.

187.

Wright

, Delmans

, Stan

, Ellis

. GeneGuard: a modular plasmid system designed for biosafety. ACS Synth Biol. 2015; 4(3):307-316.

188.

Blazejewski

, Ho

, Wang

. Synthetic sequence entanglement augments stability and containment of genetic information in cells. Science. 2019; 365(6453):595-598.

189.

Malyshev

, Dhami

, Lavergne

, et al. A semi-synthetic organism with an expanded genetic alphabet. Nature. 2014; 509(7500):385-388.

190.

, Isaacs

. Genomic recoding broadly obstructs the propagation of horizontally transferred genetic elements. Cell Syst. 2016; 3(2):199-207.

191.

O'Brien

, Stelling

. Integrated multilevel surveillance of the world's infecting microbes and their resistance to antimicrobial agents. Clin Microbiol Rev. 2011; 24(2):281-295.

192.

Bügl

, Danner

, Molinari

, et al. DNA synthesis and biological security. Nat Biotechnol. 2007; 25(6):627-629.

193.

Kunjapur

, Pfingstag

, Thompson

. Gene synthesis allows biologists to source genes from farther away in the tree of life. Nat Commun. 2018; 9(1):4425.

194.

Nielsen

AAK

, Voigt

. Deep learning to predict the lab-of-origin of engineered DNA. Nat Commun. 2018; 9(1):3135.

195.

Deneke

, Rentzsch

, Renard

. PaPrBaG: a machine learning approach for the detection of novel pathogens from NGS data. Sci Rep. 2017; 7:39194.

196.

Bartoszewicz

, Seidel

, Rentzsch

, Renard

. DeePaC: predicting pathogenic potential of novel DNA with reverse-complement neural networks. Bioinformatics. 2020; 26(1):81-89.

197.

Tenover

, Arbeit

, Goering

, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995; 33(9):2233-2239.

198.

Spinali

, Van Belkum

, Goering

, et al. Microbial typing by matrix-assisted laser desorption ionization-time of flight mass spectrometry: Do we need guidance for data interpretation?. J Clin Microbiol. 2015; 53(3):760-765.

199.

Singhal

, Kumar

, Kanaujia

, Virdi

. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Front Microbiol. 2015; 6:791.

200.

Ecker

, Sampath

, Blyn

, et al. Rapid identification and strain-typing of respiratory pathogens for epidemic surveillance. Proc Natl Acad Sci U S A. 2005; 102(22):8012-8017.

201.

Sampath

, Hall

, Massire

, et al. Rapid identification of emerging infectious agents using PCR and electrospray ionization mass spectrometry. Ann N Y Acad Sci. 2007; 1102(1):109-120.

202.

James

, Schmidt

A-M

, Wall

, Green

, Masri

. Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis. J Agric Food Chem. 2003; 51(20):5829-5834.

203.

Petrillo

, Angers-Loustau

, Henriksson

, Bonfini

, Patak

, Kreysa

. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms. Database (Oxford). 2015; 2015:bav101.

204.

Bonfini

, van den Bulcke

, Mazzara

, Ben

, Patak

. GMOMETHODS: The European Union Database of Reference Methods for GMO Analysis. J AOAC Int. 2012; 95(6):1713-1719.

205.

US Fish and Wildlife Service (FWS). Invasive Species Strike Teams: National Wildlife Refuge System (Fiscal Year 2010 Update). Washington, DC: FWS; 2011. https://www.fws.gov/invasives/pdfs/Strike%20Team%20Fact%20Sheet%202010.pdf

206.

Examining Innovative Solutions to Control Invasive Species and Promote Wildlife Conservation, Hearing before the Senate Committee on Environment and Public Works (2017) (Testimony of Jim Kurth, Acting Director, US Fish and Wildlife Service, Department of the Interior). Accessed July 12, 2020. https://www.epw.senate.gov/public/_cache/files/7/e/7ea3e6ee-d457-4fdb-9c76-04af5c1f50fe/184B7724A239EA0750B2F11755E8D2C1.kurth-witness-testimony-03.15.2017.pdf

207.

Fisheries Program: eDNA. US Fish and Wildlife Services website. Last updated October 28, 2019. Accessed July 12, 2020. https://www.fws.gov/midwest/fisheries/eDNA.html

208.

Holst-Jensen

Testing for genetically modified organisms (GMOs): past, present and future perspectives. Biotechnol Adv. 2009; 27(6):1071-1082.

209.

Mahon

, Jerde

. Using environmental DNA for invasive species surveillance and monitoring. In: Walker

, ed. Methods in Molecular Biology. Totowa, NJ: Humana Press Inc.; 2016:131-142.

210.

Bain

, Cangelosi

, Eder

. Monitoring microbes in the Great Lakes. Environ Monit Assess. 2011; 182(1-4):431-442.

211.

Sinclair

, Choi

, Riley

, Gerba

. Chapter 9: Pathogen surveillance through monitoring of sewer systems. In: Laskin

, Gadd

, Sariaslani

, eds. Advances in Applied Microbiology. Cambridge, MA: Academic Press Inc.; 2008:249-269.

212.

Nelson

, Circo

, Evans

. Strategic viral surveillance of sewage during and following an oral poliovirus vaccine campaign. Am J Epidemiol. 1967; 86(3):641-652.