Bioremediation 3.0: Engineering Pollutant-Removing Bacteria in The

1 Bioremediation 3.0: Engineering pollutant-removing bacteria 2 in the times of systemic biology 3 4 by 5 6 Pavel Dvořák1, Pablo I. Nikel2, Jiří Damborský3,4, Víctor de Lorenzo1# 7 8 1 Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), 9 28049 Madrid, Spain 10 2 The Novo Nordisk Foundation Center for Biosustainability, 2800 Lyngby, Denmark 11 3 Loschmidt Laboratories, Centre for Toxic Compounds in the Environment RECETOX 12 and Department of Experimental Biology, Faculty of Science, Masaryk University, 62500 13 Brno, Czech Republic 14 4 International Clinical Research Center, St. Anne's University Hospital, Pekarska 53, 656 15 91 Brno, Czech Republic 16 17 # E-mail: [email protected], Tel.: +34 91-585 4536 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 1 1 2 3 4 Table of Contents 5 6 1. Introduction 7 2. Approaches and tools of systems biology and metabolic engineering for tailoring 8 biodegradation pathways 9 2.1 Step 1: Get to know the contaminant and find a suitable catabolic pathway 10 2.1.1 Databases 11 2.1.2 Pathway prediction systems and toxicity prediction algorithms 12 2.1.3 Detection and quantification of pathway building blocks 13 2.2 Step2: Select and get to know a suitable microbial host 14 2.2.1 Omic techniques in studies of bacterial degraders 15 2.3 Step 3: Build the pathway and optimize its performance in the context of host 16 metabolism 17 2.3.1 Computational tools for pathway and strain optimization 18 2.3.2 Experimental tools for pathway and strain optimization 19 2.3.3 Protein engineering to eliminate bottlenecks of biodegradation pathways 20 3. Synthetic biology approaches and tools for biodegradation pathway engineering 21 3.1 Development of robust microbial chassis for biodegradation of toxic chemicals 22 3.2 Development of synthetic microbial consortia for enhanced biodegradation and 23 bioremediation of pollutants 24 3.3 Development of orthogonal systems in bacteria to enhanced pollutant biodegradation 25 and bioremediation 26 3.4 Engineering microbial biodegradation pathways in vitro 27 4. CO2 capture as a large-scale bioremediation challenge 28 5. Conclusions and perspectives 29 References 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 2 1 2 3 4 Abstract 5 6 Elimination or mitigation of the toxic effects of chemical waste released to the environment 7 by industrial and urban activities relies largely on the catalytic activities of microorganisms— 8 specifically bacteria. Given their capacity to evolve rapidly, they have the biochemical power 9 to tackle a large number of molecules mobilized from their geological repositories through 10 human action (e.g., hydrocarbons, heavy metals) or generated through chemical synthesis 11 (e.g., xenobiotic compounds). Whereas naturally occurring microbes already have 12 considerable ability to remove many environmental pollutants with no external intervention, 13 the onset of genetic engineering in the 1980s allowed the possibility of rational design of 14 bacteria to catabolize specific compounds, which could eventually be released into the 15 environment as bioremediation agents. The complexity of this endeavour and the lack of 16 fundamental knowledge nonetheless led to the virtual abandonment of such a recombinant 17 DNA-based bioremediation only a decade later. In a twist of events, the last few years have 18 witnessed the emergence of new systemic fields (including systems and synthetic biology, and 19 metabolic engineering) that allow revisiting the same environmental pollution challenges 20 through fresh and far more powerful approaches. The focus on contaminated sites and 21 chemicals has been broadened by the phenomenal problems of anthropogenic emissions of 22 greenhouse gases and the accumulation of plastic waste on a global scale. In this article, we 23 analyze how contemporary systemic biology is helping to take the design of bioremediation 24 agents back to the core of environmental biotechnology. We inspect a number of recent 25 strategies for catabolic pathway construction and optimization and we bring them together by 26 proposing an engineering workflow. 27 28 Keywords: bioremediation, biodegradation pathway engineering, emerging pollutants, 29 environmental biotechnology, systemic biology, metabolic engineering, systems biology, 30 synthetic biology. 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 3 1 2 3 1. Introduction 4 Increasing pollution of air, soils, ground and surface waters constitutes a major threat to 5 public health both in developing countries as well as in industrialized countries including EU 6 states, the USA, India and China. The majority of contaminants that affect soils and waters 7 are heavy metals and organic compounds such as mineral oil hydrocarbons, polyaromatic 8 hydrocarbons, benzene derivatives, and halogenated hydrocarbons. Many of organic polluting 9 compounds for agricultural (the pesticides dichlorodiphenyltrichloroethane, atrazine, and 10 pentachlorophenol), industrial (solvents such as dichloroethane or dielectric fluids such as 11 polychlorinated biphenyls) or military use (explosives such as 2,4,6-trinitrotoluene) are 12 xenobiotics of anthropogenic origin. There is also a spectrum of so-called emerging 13 contaminants (Table 1), i.e., substances long present in the environment whose presence and 14 negative effects have only recently been recognized (Petrie et al., 2015). The list can be 15 further broadened with petroleum-derived plastics and some chemicals originally considered 16 to be green, including certain types of bioplastics or ionic liquids (Amde et al., 2015). Despite 17 the recalcitrant nature of some of these polluting compounds, many are more or less 18 susceptible to biodegradation (Alexander, 1999). In addition to these traditional causes of 19 environmental deterioration, the recent decades have witnessed the onset of ramped-up levels 20 of anthropogenic emissions of CO2 and other greenhouse gases and their ensuing impact on 21 climatic change. Whereas the chemicals themselves are simple (CO2, CH4, N2O), the 22 challenge here is less their biodegradation than their recapture in a non-gaseous form. 23 24 The major entity that causes large-scale transformations in the biosphere are microorganisms 25 and their metabolic pathways. Microbes degrade toxic chemicals via complete mineralization 26 or co-metabolism, in aerobic or anaerobic conditions. Advantageous properties such as small 27 genome size, relative simplicity of the cell, short replication times, rapid evolution and 28 adaptation to the new environmental conditions made microbes, and particularly bacteria, 29 favourable candidates for bioremediation technologies, that is in situ or ex situ removal of 30 polluting chemicals from the environment using biological agents. The removal of 31 environmental pollution caused by the extensive activities of industrial society is a serious 32 topic that draws the attention of biotechnologists. This is because beyond the medical and 33 environmental consequences, the situation signs considerable potential for growth of eco- 34 industry focused on clean-up technologies and removal of environmental contaminants. In 35 fact, valorization of waste chemicals accumulating in industry is one of the pillars of the 36 circular economy and the 4th Industrial Revolution (Schmidt, 2012; Wigginton et al., 2012). 37 38 39 40 41 42 43 44 4 1 2 Table 1 Emerging contaminants. 3 Groups of products Classes of chemicals Examples Amoxicillin, erythromycin, Antibiotics, anti-parasitic metronidazol, tetracycline, agents, ionophores lincomycin, sulfathiazole Stimulants and drugs including Amphetamine, cocaine, caffeine, anti-inflammatory, anti-diabetic, nicotine, propranolol, ibuprofen, anti-epileptic, anti-hypertensive, codeine, carbamazepine, Human and veterinary or anti-cancer drugs, bezafibrate, metformin, pharmaceuticals anticoagulants, hallucinogens, fluoxetine, warfarine, valsartan, analgesics, β-blockers, anti- tramadol, morphine, depressants, lipid regulators, or methandone, diazepam, erectile dysfunction drugs ephedrine, tamoxifen Estrone, estriol, testosterone, Hormones including natural and progesterone, mestranol synthetic estrogens, androgens (ovulation inhibitor), cholesterol Insecticides, plasticizers, Carbaryl, chlorpyrifos, detergents, flame retardants, diethylphtalate, p-nonylphenol, polycyclic aromatic tri(2-chloroethyl)phosphate, Industrial and household hydrocarbons, antioxidants, naphtalene, anthracene, 2,6-di- wastewater products solvents, disinfectants, tert-butylphenol, 1,2,3- fumigants, fragrances, trichloropropane, phenol, 1,4- preservatives dichlorobenzene, acetophenone Insect repellents, polycyclic Bisphenol A, 1-benzophenone, Personal care products musks, sunscreen agents, methylparaben, N,N- fragrances, antiseptics diethyltoluamide, triclosan Nanosilver, alumina Nanomaterials Miscelaneous nanoparticles, titanium dioxide, fullerenes, carbon black 4 Sources: http://toxics.usgs.gov, http://www.eugris.info (Petrie et al., 2015) 5 6 The earliest attempts at directed bioremediation, although not formalized as such at the time, 7 dated back to the late 19th century with the origins of the first wastewater treatment plants 8 (Litchfield, 2005). Bioremediation began in earnest some 45 years ago with the isolation of 9 culturable bacteria from contaminated sites and studying their degradation pathways. The first 10 report on enhanced in situ bioremediation of soil contaminated

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