Synthetic Biology and Its Application in Bioremediation

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1		Valentina Jiménez Díaz	--	1957	2022-04-13 20:44:17	\|
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This entry is adapted from the peer-reviewed paper 10.3390/pr10040712

Synthetic biology is a scientific field based on biology and engineering principles, with the purpose of redesigning and restructuring microorganisms to optimize or create new biological systems with enhanced features. Developing innovative, fast, safe, and cost-effective techniques for elimination of pollutants is of great importance. For this reason, the use of this discipline offers improvement of bioremediation processes.

synthetic biology bioremediation hydrocarbons

1. Synthetic Biology and Bioremediation

Although bioremediation techniques offer great advantages, in many cases they are becoming traditional methods since new tools are emerging. Thanks to multi-omics analysis and advances in genetic engineering, it is possible to obtain the information necessary to choose the best microorganism for the remediation process ^[1].

For example, in order to identify catabolic genes, novel pathways or proteins involved in the biodegradation process, genomic, proteomics and metabolomic tools can be used. Transcriptomics also gives important information, such as how the cell responds after the exposure to toxic compounds and how it affects its metabolic state. On the other hand, in silico tools can give insight into the biochemical reactions that take place in the degradation of contaminants ^[2]

Synthetic biology is a scientific field based on biology and engineering principles, with the purpose of redesigning and restructuring organisms to optimize or create new biological systems with enhanced features ^[3]^[4]^[5]. This field uses molecular tools, systems biology and the reprogramming of the genetic framework, thus constructing synthetic pathways to obtain microorganisms with alternative functions ^[4]^[5]. The use of this discipline brings an improvement of bioremediation processes.

Synthetic biology aims to design and construct an organism with a specific set of characteristics. Using computational models and engineering techniques, genetic circuits and metabolic pathways can be assembled and fine-tuned. To construct these microorganisms, modifications are encoded in vectors that are delivered into suitable hosts, known as chassis. This term in synthetic biology refers to an organism that acts as a carrier for the genetic components and allows them to function ^[6]^[7]^[8]. To build the appropriate chassis, there are two known approaches: (a) top-down, which generates synthetic organisms by manipulating existing genes or metabolic pathways; and (b) bottom-up, where de novo organisms are created from molecular building blocks ^[9]^[10]^[11]^[12].

In any case, a series of general steps can be followed to create an engineered organism for the degradation of pollutants: (1) selection and design of the microorganism, this includes the appropriate choice of the host and preliminary engineering; (2) metabolic or genetic optimization, improvements can be made at different levels to obtain better results in degradation; and (3) tolerance engineering of the chassis, with the aim of regulating or creating a response system to extreme conditions or stress, in order to increase biodegradation (Figure 1) ^[9].

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Figure 1. General workflow of the creation of an engineered microorganism and its possible uses in bioremediation. (a) Steps to create an engineered microorganism; (b) representation of a microorganism designed with synthetic biology for hydrocarbon bioremediation: the first strain functions as a biosensor by producing a bioluminescent signal. At the same time, it carries out the first steps of contaminant degradation, releasing intermediary metabolites. The second strain, having been modified, is now able to assimilate these products to finish the degradation process, reaching the mineralization of the pollutants.

Creation of genetic engineered microorganisms, design of biosensors and the use of consortia, are some of the strategies based on synthetic biology that offer the creation of innovative tools for increasing the efficiency of degradation. Here, this entry will introduce some of the techniques that use synthetic biology as a platform to be used in the area of hydrocarbon bioremediation.

2. Construction of Synthetic Consortia

A microbial consortium is a set of two or more microbial species that work synergistically to create a balanced community where mutual benefit exists ^[13]^[14]. The life cycle of a microbial community depends directly on the relationships existing among them, creating cooperative or competitive dynamics ^[13]^[15], as well as neutral, positive or negative effects ^[16]. These relationships are classified in: (a) symbiotic, where organisms obtain mutual benefit (e.g., mutualisms); and (b) antagonistic, where one species can be harmed and the other benefited (e.g., parasitism) ^[17].

A consortium tends to be more effective than a single microorganism due to the synergistic relationships resulting from complementary activities and metabolic capacity of each species ^[18]. Since metabolites of one microbe can be used by another one, microbial communities can use these mutual interactions to completely degrade contaminants ^[19]. On that account, when designing a consortium it is necessary to establish parameters that guarantee coexistence and stable interactions between all of the species ^[2]^[20].

Each environment has autochthonous microorganisms with different degradation capacities. However, the degradation of hydrocarbons by these individual microorganisms can be low. Consortia are capable of achieving complete mineralization of contaminants thanks to a sequential degradation due to the synergistic and metabolic activities they possess as a group ^[21]. Therefore, an alternative in hydrocarbon bioremediation is the addition and combination of allochthonous microorganisms for the creation of consortia, which is more effective and sustainable than traditional methods ^[22]^[23].

Molecular tools, along with systems biology, enable the analysis of genetic information and cell to cell interactions ^[24]. Omics tools can provide information on genes, proteins and metabolic pathways that will help to understand microbial behavior to enhance the biodegradation processes. For example, they can be used to evaluate the structure and dynamics of microbial consortia in different environmental scenarios ^[2]. Thanks to this, synthetic biology offers the possibility of creating engineered microbial communities to make strong cellular functions and improve its microbial capabilities and cooperation ^[24]. Assembling synthetic consortia will improve efficacy in bioremediation. These modifications can be made by manipulating environmental conditions, communication networks, syntrophic interactions or the genetic framework and new genetic modules ^[2]^[8]^[24].

Synthetic biology tools can be used in microbial consortia to facilitate the interaction among microorganisms. Some of these tools are: (a) syntrophic interactions, to create a metabolic network where metabolites produced by one organism can be used by another one; (b) exogenous molecules, adding external inputs to control cell communities and gene expression; and (c) intercellular signaling, to control communication between cells and gene expression (e.g., quorum sensing) ^[8]^[25].

One example of a synthetic community is the study of a consortium consisting of two bacterial strains that were modified for phenanthrene degradation, by Jia et al. ^[26]. The used strains were: (a) Escherichia coli HY, with two terminal dioxygenase modules and an electron transfer chain; and (b) Pseudomonas aeruginosa PH2, with a catechol 1,2-dioxygenase module. A gas chromatography-mass spectrometry (GC-MS) was performed to identify metabolites. The initial oxidation steps were made by E. coli HY1 (phenanthrene into 9,10-dihydroxy phenanthrene or 1,2-dihydroxy phenanthrene), and then ring cleavage was performed by P. aeruginosa PH2 to produce catechol. Further conversion between intermediates was through the tricarboxylic acid cycle (TCA). The modified consortium was able to degrade 71% of phenanthrene in nine days, while the wild type (P. aeruginosa PH1) only degraded 45% ^[26]. This confirmed the improved removal capacity of a constructed consortium compared to unmodified strains, confirming once again a new alternative for bioremediation of polycyclic aromatic hydrocarbons (PAHs).

Engineered consortia offer great advantages due to their efficient and improved levels of degradation in comparison to individual strains. This means that synthetic communities are a useful and a valid platform for bioremediation of hydrocarbons, even with efficient results. Analysis of each microbial community, separately and as a whole, is needed when designing and constructing one. It is important to remember synergistic relationships are the key to the consortium’s success since metabolic capabilities and characteristics of each species can be integrated, enhancing biodegradation. Developing a microbial consortium with specific parameters to bioremediate hydrocarbon-contaminated areas is one of the most promising benefits that synthetic biology offers.

3. Risk Assessment of Synthetic Biology

Considering the bioremediation process will take place at the site of contamination, genetically engineered microorganisms (GEMs) will be leaving the laboratory and entering a natural environment, which may entail risks or difficulties since it stops being a controlled environment ^[24]^[27]. The environmental risks relate to gene contamination, toxicity and competition with native species. The problems of gene contamination are related to horizontal gene transfer which leads to the delivery of the recombinant genes ^[28], modifying autochthonous microorganisms and altering their natural genetics ^[27]. One of the major risks is posed by plasmids containing antibiotics as resistance genes, as they can lead to the formation of antibiotic-resistant superbugs in nature ^[29]. It is also important to consider the release of compounds toxic to the environment or related to human health due to the change in microbial metabolism ^[27]^[29]. In regards to the competition with native species, as noted by de Lorenzo ^[30], the risk of altering the microbial composition by introducing GEMs into natural ecosystems is not as high as commonly thought. Due to the homeostasis of biological ecosystems and resistance to colonization, engineered microorganism have difficulties at establishing in a new environment, meaning that it is unlikely that the modified microorganisms could displace the indigenous community ^[29]^[30].

To minimize potential risks, strategies include using non-antibiotic selection markers and avoiding gene transfer to indigenous organisms ^[31]. To resolve this problem, biocontainment techniques based on cellular circuits, inducible systems or auxotrophy have been developed ^[32]. The most promising technique for biocontainment of genes in a natural environment is through toxin-antitoxin systems. This protection system will secure the genetic material from horizontal gene transfer ^[33]. A toxin is encoded in a plasmid, while the antitoxin is encoded into the genome. Therefore, if a gene transfer occurs, the new host would die as it incorporated the plasmid with the toxin but does not possess the antitoxin ^[32]. Another strategy that can be used to protect the inserted genes is the use of conditional replication origins. The origin needs an initiator protein to replicate the plasmid, which is inserted into the chromosome of the host. In this way, the replication of the plasmid is blocked if it is transferred to another cell ^[33].

It must be taken into consideration that legal regulations play an important role when implementing these new alternatives, as well as the possible environmental risks it may involve. The good news is genetic engineering and synthetic biology offer to mitigate these biological risks through different biocontainment mechanisms. Therefore, based on the different bioinformatics studies and laboratory results obtained, it is important to carry out more in situ tests to analyze the behavior of GEMs in an uncontrolled environment, as well as their proper biomonitoring, in order to establish this alternative in bioremediation as one of the most effective and safe.

4. Conclusions

Petroleum-derived pollutants are highly toxic, creating serious and harmful consequences in any environment. Developing innovative, fast, safe, and cost-effective techniques for their elimination is of great importance. Bioremediation as a contaminant removal technique has been very successful, and although several microorganisms possess degradative capacities, optimizing these techniques is necessary due to the pollutant’s persistence. Over the years, advances in different areas of science have led to improvements in various degradation techniques. Knowledge in systems biology, molecular tools, and multiomics are the basis of synthetic biology, which creates a new era in bioremediation. Analysis, design, construction and fine-tuning of genetically and metabolically optimized microorganisms maximize toxic compounds degradation.

Creating biosensors to detect and monitor contaminants, understanding microbial dynamics to construct synthetic consortia, as well as creating new metabolic pathways or enzymatic enhancement, are some of the possibilities offered by synthetic biology. It is still necessary to carry out more in situ experiments to support different results obtained in laboratories, as well as establishing the necessary safety parameters for an engineered microorganism to enter the environment. The most important thing is that now it is possible to create ideal techniques to degrade persistent and harmful pollutants such as hydrocarbons. Even though some areas need further research, synthetic biology puts science on the right track. With these new tools at hand, bioremediation positions itself as one of the best and most effective pollutant removal process available today.

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