Biocontainment Techniques and Applications for Yeast Biotechnology

Abstract

Biocontainment techniques for genetically modified yeasts (GMYs) are pivotal due to the importance of these organisms for biotechnological processes and also due to the design of new yeast strains by using synthetic biology tools and technologies. Due to the large genetic modifications that many yeast strains display, it is highly desirable to avoid the leakage of GMY cells into natural environments and, consequently, the spread of synthetic genes and circuits by horizontal or vertical gene transfer mechanisms within the microorganisms. Moreover, it is also desirable to avoid patented yeast gene technologies spreading outside the production facility. In this review, the different biocontainment technologies currently available for GMYs were evaluated. Interestingly, uniplex-type biocontainment approaches (UTBAs), which rely on nutrient auxotrophies induced by gene mutation or deletion or the expression of the simple kill switches apparatus, are still the major biocontainment approaches in use with GMY. While bacteria such as Escherichia coli account for advanced biocontainment technologies based on synthetic biology and multiplex-type biocontainment approaches (MTBAs), GMYs are distant from this scenario due to many reasons. Thus, a comparison of different UTBAs and MTBAs applied for GMY and genetically engineered microorganisms (GEMs) was made, indicating the major advances of biocontainment techniques for GMYs.

1. Introduction

Genetically engineered or modified microorganisms (GEMs/GMMs) are the workhorse for basic and applied research as well as in industry. GEMs are employed in the food industry to improve protein synthesis or to generate small molecules that impact the nutritional value of a food, such as flavor enhancers, oligosaccharides, vitamins, and amino acids [1]. GEMs are also employed for food enzyme production, including enzymes such as lactase, amylase, proteases, and phospholipases [1,2]. Another biotechnological field that benefits from the application of GEMS is bioremediation, which allows for the removal of pollutants (e.g., heavy metals) from water and soil [3]; finally, different GEMs species are employed for the production of clinically important peptides/proteins (e.g., insulin) [4], vaccines [5], biofertilizers [6,7], biocontrol [8], and biofuels [9].

Considering eukaryotic GEMs, the yeasts constitute an important group of microorganisms with many industrial applications. Yeasts are used as a cell factory for the production of chemicals and biologicals due to their ability to grow on inexpensive culture media and them having well-established fermentative technology [10]. One of the most popular yeasts employed as a GEM is Saccharomyces cerevisiae. S. cerevisiae has many different biotechnology applications, from the production of traditional fermented foods [11,12] to cell factories for the synthesis of chemicals and pharmaceuticals, such as bioethanol, propanol, butanol, artemisinic acid, and insulin precursor [10,13,14]. In addition to its biotechnological importance, S. cerevisiae is considered an eukaryotic model organism for basic and applied research [15,16]. Other yeast species that have biotechnological importance include the halophilic/halotolerant yeast species such as Debaryomyces hansenii, which can metabolize different types of carbon sources and synthesize high levels of lipids, xylitol, and flavonoids, making it an attractive model for metabolic engineering [17,18]. Methylotrophic yeasts (e.g., Pichia pastoris and Ogataea polymorpha) are very efficient in producing heterologous protein on an industrial scale, with many strains and molecular tools available for genetic/synthetic biology engineering [19,20]. Other examples of non-Saccharomyces species with industrial importance include Arxula adeninivorans and Yarrowia lipolytica for the production of therapeutic heterologous proteins [21]. In addition, yeast hybrid strains/chassis from the Saccharomyces sensu stricto complex for industrial purposes are gaining industrial importance [22]. Finally, advances in synthetic biology are leading to the generation of “synthetic microorganisms” by using the so-called “bottom-up approach”, where isolated and well characterized biochemical components are modularly assembled in order to design artificial cells with specific phenotypes [23]. Another synthetic biology approach is the “top-down method”, where a microbial genome is reduced to its essential genes, allowing the factory of any desired DNA sequence to “tailor-make” a specific phenotype [24]. The top-down approach is currently being applied by the international Synthetic Yeast Genome project (known as the “Yeast 2.0” or “Sc2.0” project), whose major objectives are to redesign all chromosomes of S. cerevisiae and generate the first synthetic eukaryote [24,25].

Despite the importance of genetically modified yeasts (GMYs), there are concerns that the widespread use of GMYs could lead to a potential exchange of modified DNA molecules with other microorganisms in an ecosystem [26,27]. These concerns can be potentialized by considering that yeasts can exchange parts of their genomes with other microorganisms by interkingdom horizontal gene transfer [28], resulting in the widespread use of a transgene and/or a synthetic DNA molecule (Figure 1). Thus, highly effective biocontainment/safeguard strategies are needed to keep GMYs restricted to a laboratory and/or industrial environments (defined here as “production facilities”) [27,29] and restrict the “escape frequency” of GMYs. The escape frequency is the consequence of a combination of different molecular mechanisms (e.g., mutagenesis, gene loss, recombination) as well as environmental/evolutionary processes that result in the spread of GMY/GEM into the environment (Figure 1). The National Institutes of Health (NIH) guidelines for research involving recombinant or synthetic nucleic acid molecules recommend an escape frequency of 1 cell per 10⁸ cells as the standard [30].

Another major issue regarding the massive use of GMY/GEM is that the simple physico-chemical inactivation of cell biomass (e.g., by heat and/or pH treatment) does not avoid the DNA stability/persistence in the environment after its release from the cell (Figure 1). Data gathered from the PCR analysis of soil fertilized with the waste product of industrial GMY/GEM fermentations showed the presence of genes related to antibiotic resistance commonly found in synthetic plasmids [31]. Additionally, engineered DNA molecules can be detected in soil several days or months after its introduction due to the intrinsic high stability of DNA molecules [32,33]. Interestingly, none of these studies observed the horizontal transfer of released engineered DNA molecules to the native soil microbiomes; however, the chance of horizontal gene transfer from released synthetic DNA molecules in the environment cannot be excluded, especially if those molecules confer a phenotype advantage to the native microbiome.

Different molecular approaches can be employed for GMY biocontainments, including auxotrophic mutations, synthetic molecular mechanisms (xenobiology), and kill switches based on the expression of toxin-antitoxin [27,34] or exo-/endonucleases [29,35] (Figure 1). However, all of these approaches are not failproof, since molecular and evolutive mechanisms lead to the inactivation of biocontainment devices [36] (Figure 1). This is especially true when a biocontainment approach makes use of a single component/device (defined in this work as a “uniplex-type biocontainment approach” or UTBA;

Table 1. Advantages and disadvantages of different biocontainment approaches for genetically modified yeasts.

Disadvantages	Advantages	Biocontainment
Can be inactivated by molecular and evolutive mechanisms. Presence of metabolites from other organisms can surpass biocontainment. Potential horizontal gene transfer events can surpass biocontainment mechanisms.	Can be easy to implement for different yeast species. Some approaches were extensively tested outside production facilities. Orthogonal components diminish the impact of the biocontainment apparatus on cellular physiology. Can employ synthetic small molecules or DNA parts to prevent biocontainment escape. Some UTBAs can be combined into an MTBA.	Uniplex-type approach (UTBA)
Complex design and implementation. Lower cell fitness and promotes the selection of inactive biocontainment devices. Not extensively tested outside production facilities.	Designed with redundant or safeguard mechanisms for mutation tolerance. Orthogonal circuits that respond to specific environmental inputs to induce cell death outside the production facility. Can combine molecular mechanisms targeting different cellular components. Can apply synthetic molecules or biological components.	Multiplex-type approach (MTBA)

and Figure 1), such as natural auxotrophic markers. On the other hand, the combination of different biocontainment approaches (“multiplex-type biocontainment approach” or MTBA; Table 1) reduces its potential escape frequency at the expense of low cell fitness (Table 1) [27,34]. Paradoxically, the reduction in cell fitness increases the frequency of inactivated biocontainment devices [37]. Thus, an effective biocontainment strategy should consider different escape mechanisms, including mutagenic drift, the environmental supplementation of nutrients and/or essential molecules, and horizontal gene transfer (HGT) [38].

The purpose of this review manuscript is to focus on different UTBA and MTBA strategies for GMY biocontainment and on what approaches have been or have not been applied for industrially important GMYs.

2. Uniplex-Type Biocontainment Approaches

2.1. Natural Auxotrophic Markers

Natural auxotrophy (Figure 1) is a simple and widespread UTBA technique commonly applied to prevent the release and proliferation of GMYs outside the production facility. In general terms, natural auxotrophy can be defined as a nutritional deficiency induced by mutated genes resulting in a GMY strain that depends on the addition of the nutrient on its growth media [39]. The yeast S. cerevisiae has been used as a model for auxotrophic markers due to the facility of inducing deletion and/or point mutations in different genes associated with nutrient metabolism [39]. In this sense, genes linked to the metabolism of amino acids such as L-histidine, L-leucine, L-tryptophan, and L-methionine [39] and nitrogen bases (e.g., adenine and uracil) [40] have been used as auxotrophic markers for decades.

Considering nitrogen base auxotrophies, the allele ade2-1, which contains a nonsense mutation (Glu64STOP) [41], is widely found in different laboratorial S. cerevisiae strains and confers adenine dependence. A major phenotypic characteristic of ade2-1 is the development of a red ochre color due to the intracellular accumulation of oxidized adenine-associated metabolic intermediates [40,42], a phenotype that can be useful for red–white colony screening [43], redox biology [44], and drug discovery [40].

The depletion of adenine reserves in yeast cells increases trehalose synthesis and leads to cell cycle arrest, recapitulating the protective effects observed for desiccation stress tolerance. As a consequence, the adenine-deficient cells become viable for a longer period of time and evade biocontainment [42]. Complementally, it was observed that adenine auxotrophy increases the mutagenesis rate in yeast cells [45], a condition that is observed for other auxotrophic markers such as leu2 and/or lys2 alleles. Yeast cells carrying leu2 and/or lys2 alleles, when subjected to L-lysine or L-leucine starvation, display an increase in the number of respiratory deficient cells (rho⁻ cells) due to the accumulation of mutations in the mitochondrial genome, a condition termed “adaptive mutation” [46]. Additionally, adaptive mutations are linked to auxotrophy marker reversion to the wild-type state, as observed for the his4 marker in yeast under selective pressure [47].

In order to avoid the reversion of auxotrophic markers, a series of deletion gene markers have been generated for S. cerevisiae [48], as well as for other non-Saccharomyces species, such as Kluyveromyces lactis [49]. However, it has been observed in some non-Saccharomyces species that auxotrophic gene deletion leads to a bradytroph/leak auxotroph phenotype, such as those observed for PHA2 in Pichia pastoris, which is linked to L-phenylalanine biosynthesis [50]. In this case, the auxotrophic phe⁻ cells are able to survive under L-phenylalanine starvation due to alternative L-phenylalanine biosynthesis mechanisms [50].

Considering the impact of auxotrophy in yeast metabolism, the effect of adaptive mutation in the selection of prototrophic cells, and bradytrophy due to the presence of poorly described biochemical pathways in non-Saccharomyces species, natural auxotrophy per se has strong limitations regarding its use as a biocontainment strategy. In addition, the presence of other microorganisms in the environment and/or production facility (Figure 1) can provide the auxotrophy-dependent nutrients for GMY mutants, bypassing the auxotrophy requirement [51].

A solution for overcoming the natural auxotrophies limitations is the use of synthetic amino acids and nitrogen bases that are not available outside the production facility and cannot be biologically supplied. This approach, termed “xenobiology” or “synthetic auxotrophies” (Figure 1), makes use of top-down synthetic biology techniques for engineering GMYs and creating new biocontainment strategies [35]. Another approach to circumventing the limitations of natural auxotrophies is the use of a so-called “conditional gene essentiality” (Figure 1), which employs promoter engineering to modify genes linked to nutrient metabolism and strictly regulate their expression by using synthetic molecules and/or orthogonal RNA polymerases [52].

2.2. Xenobiology and Synthetic Auxotrophies

Xenobiology focuses on the development of synthetic biological devices and systems that utilize non-canonical amino acids (ncAAs), nucleic acids with a non-standard sugar backbone (xeno-nucleic acids or XNA), and non-natural nitrogen base pairs for different purposes, including the design of synthetic metabolic processes (neo-metabolism) [53] and biocontainment [52]. Many of the xenobiology devices are designed by principle using orthogonality, where synthetic components (e.g., proteins, RNAs, DNAs, and small molecules) are engineered for a purposed function and will not interfere with the natural biochemistry of a host cell [53,54]. The orthogonality also ensures that these components will not be used by natural biological systems, making it useful as a biocontainment strategy [55,56].

Biocontainment-based xenobiology is mostly centered on the use of different ncAAs for protein synthesis for both GEM and GMY. For example, the application of genetic code expansion (GCE) or orthogonal translation systems (OTSs) techniques [57,58] has been used with success in Escherichia coli biocontainment [56].

In bacteria, GCE/OTS relies on an orthogonal pyrrolysyl-tRNA synthetase/tRNA^Pyl_CUA (PylRS/tRNA^Pyl_CUA) pair derived from Methanosarcina barkeri, M. mazei, or Methanocaldococcus jannaschii to incorporate ncAAs in proteins [59,60], in the reassignment of the UAG amber codon, a rare stop codon in both E. coli and S. cerevisiae [61], and in the deletion of the release factor 1 (RF1). Considering GCE for E. coli biocontainment, the genome of this bacterium was refactored by the introduction of a reassigned UAG codon into 22 essential genes together with a Methanocaldococcus jannaschii PylRS that is able to incorporate L-phenylalanine derivatives into tRNA^Pyl_CUA [56], generating a synthetic auxotrophy. Data from this work indicated that the synthetic E. coli auxotroph cells were dependent on the addition of L-phenylalanine derivatives; moreover, these synthetic auxotrophs have undetectable escape frequencies in both solid and liquid culture media [56]. Additional work related to the creation of E. coli synthetic auxotroph strains has been carried out [38], pointing to the feasibility of this biocontainment technique for bacteria.

The generation of yeast ncAAS-dependent synthetic auxotrophs was achieved with very limited success [58,62]. Yeasts are naturally able to incorporate ncAAs into proteins [61,63] and a GCE/OTS technique for S. cerevisiae was developed by Chin et al. [62]. In this work, the authors engineered orthogonal codons, anticodons, and tRNA synthetases, including an E. coli tyrosine-tRNA synthetase (TyrRS) and an amber suppressor tRNA^Tyr_CUA, to generate a library of TyrRS mutants that pair only with ncAAs [62]. Once this TyrRS library was transformed into an S. cerevisiae strain, five different ncAAs were incorporated into the human superoxide dismutase 1 protein (hSOD1) [62].

Other relevant works related to the development of GCE/OTS for S. cerevisiae and Pichia pastoris have been conducted by using the E. coli TyrRS/tRNA^Tyr_CUA or leucyl- (Leu)RS/tRNA^Leu5_CUA pairs for ncAA incorporation into proteins [60,64,65,66,67,68,69,70,71,72]. However, the use of GCE/OTS in yeast has some major challenges, including the low expression of tRNA^Pyl_CUA in yeast due to the absence of intragenic promoter sequences A- and B-boxes [58,60]; moreover, the eukaryotic release factor 1 (eRF1), codified in yeast by the SUP45 essential gene [73], composes the Sup45p-Sup35p complex that is necessary to end translation by binding into all three stop codons in yeast cells [74]. Comparatively, the E. coli RF1, which recognizes the UAG/UAA codons, can be deleted without major physiological impacts into the cell due to the functional superimposition with the release factor 2 (RF2) [75].

In order to circumvent the limitations inherently associated with the implementation of a GCE/OST-based biocontainment in GMY, different approaches were applied, such as (i) prospecting new archeal PylRS with higher ncAA incorporation efficiency in S. cerevisiae [76], (ii) selecting yeast strains with specific mutations (e.g., yil014c-aΔ and alo1Δ) for increasing ncAAs incorporation into tRNA^Pyl_CUA [77], (iii) improving E. coli TyrRS and LeuRS with enhanced ncAAs polyspecificity and efficiency by using random mutagenesis and directed selection [78], (iv) the use of an OTS based on the recognition of quadruplet codons by an engineered orthogonal ribosome that allows for the expansion of the genetic code to 256 codons (this quadruplet codon-base OTS has been implemented more or less successfully in E. coli [58,79,80] and mammalian cells [81]), and (v) the use of synthetic/unnatural nitrogen base pairs (UBPs) to expand the genetic code and increase the repertoire of ncAAs that can be used for protein synthesis in E. coli [82].

There are different approaches when considering the use of UPBs and XNAs for xenobiology and GMY/GEM biocontainment [83]. In fact, it is expected that XNA technology could be efficient for GMY/GEM biocontainment since the building blocks of XNAs (e.g., nucleobases, sugar moieties and phosphate-modified groups) cannot be found in natural environments and the GMY/GEM cells should be able to incorporate these building blocks into new XNA polymers by the usage of specialized polymerization enzymes and transmembrane proteins that are able to take up these precursors [84,85]. Thus, UBPs and XNAs can be like a “genetic firewall” [84], where HGT events could be avoided regarding the restrained aspects of XNA technology.

The developments of XNA technologies follow two major mainstreams: (i) the use of unnatural nucleobases, sugar moieties, and phosphate-modified groups (XNA substrates) to incorporate into XNAs or hybrid XNA/DNA/RNA polymers by canonical DNA and/or RNA polymerases [55,86]; and (ii) the design of new DNA and/or RNA enzymes (XNAzymes) that are able to metabolize XNA polymers and/or substrates with an “alien” chemistry, such as threose nucleic acid (TNA), cyclohexenyl nucleic acid (CeNA), arabino nucleic acid (ANA), 2′-fluoro-arabino nucleic acid (FANA), glycol nucleic acid (GNA), and locked nucleic acid (LNA) [87,88,89,90,91,92,93,94]. In all cases, the XNAs should display orthogonality in vivo with little or, preferentially, no interaction with the canonical components of the DNA/RNA metabolism [95]. Unfortunately, many different XNA technologies were not implemented in vivo, which renders their usage for GMY/GEM biocontainment still far away from a technical and practical viewpoint [96]. Finally, both GCE/OTS and XNA technologies were not tested in an open and uncontrolled environment (Figure 1), making their behavior unpredictable for real biocontainment applications [26].

Another approach related to the induction of synthetic auxotrophies in E. coli is based on the selection of essential proteins whose structure and activity are dependent on the presence of small molecule ligands, such as the so called “synthetic auxotrophs based on ligand-dependent essential genes” (SLiDE) technique [97]. In this work, the authors were able to select five essential genes in E. coli by applying protein engineering and saturation mutagenesis and generate proteins dependent on benzothiazole. The authors related a very low escape frequency (<3 × 10⁻¹¹) under laboratory assays [97]. Similar works on bacterial synthetic auxotrophs have been conducted, including phosphite-dependent Synechococcus elongatus [98] and Pseudomonas putida [99]. In S. cerevisiae, a series of mutations in the CDC10 gene have been identified, which codify an essential septin protein that can be rescued in the presence of small molecules, such as guanidinium ion [100,101]. Although the authors of this work have not applied the CDC10 conditional mutants for biocontainment, the data may indicate new techniques based on chemical rescue for GMY biocontainment.

2.3. Conditional Gene Essentiality

The conditional essentiality is, similar to auxotrophy or xenobiology, a plethora of molecular techniques where essential genes that codify for proteins and enzymes needed for cell growth and maintenance are modified to have its expression regulated by external agents [52]. In this regard, the conditional gene essentiality has been applied for biocontainment in S. cerevisiae, E. coli, and other bacteria with a low escape frequency (Figure 1) [27,52]. In S. cerevisiae, conditional gene essentiality was employed to regulate the expression of histone genes [27], fatty acid synthetase, mitochondrial and cytoplasmic histidine tRNA synthetase (HisRS), RNA polymerase II subunit B, and interorganellar chaperones and GTPases related to vesicular transport and fusion [102]. These genes have their original regulatory sequences replaced by a combination of promoters whose activities are modulated by galactose and estradiol [27]. Moreover, the orthogonality is ensured in this biocontainment system by providing a “fail-safe” recombination-induced lethality mechanism based on small molecule-dependent Cre recombinase [27,103].

2.4. Orthogonal DNA Replication and RNA Transcription

Another potential strategy for GMY biocontainment is to implement orthogonal DNA and RNA polymerases [51]. In yeast, an orthogonal DNA replication system derived from the Kluyveromyces lactis cytoplasmic plasmid pGKL1/2, named “OrthoRep”, has been applied for studies of the in vivo continuous evolution of target genes due to the high error-prone activity of DNA polymerase 1 (DNAP1) and DNA polymerase 2 (DNAP2), both codified by pGLK1/2 [104,105,106]. Similar in vitro works were also described by using the orthogonal DNA polymerase derived from bacteriophage phi29 to generate a self-contained synthetic transcription and translation-coupled DNA replication system [107,108,109]. Additionally, orthogonal DNA replication systems, including XNA polymerases and/or engineered DNA polymerases [110], are being developed for xenobiology applications. However, the use of different orthogonal DNA replication mechanisms as a biocontainment system for GMY/GEM was not reported until now, pointing to an unexplored research field (Figure 1).

Like orthogonal DNA replication systems, orthogonal RNA transcription systems are also underexplored in the context of GMY/GEM biocontainment. Some examples include orthogonal genic circuits based on the use of bacteriophage T7 RNA polymerase in bacteria and yeast for different biotechnological purposes [111,112]. However, no biocontainment applications using T7 RNA polymerase-based techniques for GMY/GEM were described until now (Figure 1).

2.5. Nuclease-Based Kill Switches

Kill switches are defined as gene circuits activated by specific environmental inputs, resulting in the expression of lethal genes that lead to cell death [113]. Different kill switch designs have been used for GEM biocontainment (Figure 1), including gene circuits that respond to environmental changes by activating/deactivating a type II toxin–antitoxin system CcdB/CcdA [113]. Besides this, specific and unspecific nucleases, auxotrophies, as well as type I toxin–antitoxin pairs have been employed to engineer kill switch circuits for biocontainment purposes, many of them in an MTBA format (Table 1) [34,56,114,115,116,117] in order to reduce the GEM escape frequency.

Considering nucleases for kill switch design, its usefulness for bacteria biocontainment has been shown. For example, the expression of the EcoRI endonuclease–EcoRI methylase pair combined with conditional gene essentiality in E. coli considerably lowers the escape frequency [34]. In this sense, combining EcoRI and mf-Lon protease in a strictly regulated gene circuit allowed for the development of the “Deadman” kill switch, which considerably lowered the escape frequency associated with a high genetic stability [114]. Other non-specific nucleases, such as nucA from Serratia marcescens [118] or nuclease A from Staphylococcus aureus, have been employed in different bacterial biocontainment projects [117].

In S. cerevisiae, a kill switch based on the conditional expression of S. marcescens nucA under the control of a glucose-repressed ADH2/GAPDH hybrid promoter has been proposed [119]. This work showed that, under non-repressible conditions, the nucA was efficiently expressed, leading to yeast cell death in both laboratorial and soil microcosm assays [119]. Interestingly, type II restriction enzymes have been expressed in yeast to study DNA damage and repair mechanisms [120,121,122]. Data from the expression of type II restriction enzymes in yeast cells point to a low survival and DNA damage tolerance, especially when blunt ends are induced by type II restriction enzymes (e.g., PvuII) [122]; however, the use of type II restriction enzymes as part of a kill switch mechanism in yeast cells for biocontainment purposes was not described until now.

In addition to conventional exo- and endonucleases, the CRISPR-associated nucleases (Cas) have been used in E. coli to design kill switches. These kill switch gene circuits make use of Cas3 [123] or Cas9 [124] and were successfully used to biocontain E. coli cells, both displaying an escape frequency ≤10⁻⁸ cells. A major advantage of the Cas-based kill switches is the use of guide RNAS (gRNAs) to select specific DNA sequence targets or microorganism strains, allowing for selectively eliminating the target strain from the microbiome [124,125]. Until now, no Cas-based kill switches were reported for GMY.

2.6. Kill Switches Based on Type I and II Toxin–Antitoxin Systems

Another kill switch extensively applied in GEM biocontainment is based on type I and II toxin–antitoxin (TA) systems. These kill switch-based TA systems efficiently lead to controlled cell death outside the production facility and can be easily engineered in GEM/GYM cells (Figure 1). What makes the TA systems so attractive for biocontainment applications is their pleiotropy, which targets different molecular mechanisms, such as DNA and mRNA synthesis, the cell cycle, nucleotide synthesis, and protein translation [126,127].

The prokaryotic TA system is classified into seven types numbered from I to VII [128,129], which act in different cell mechanisms. For example, the type I toxin–antitoxin system prevents toxin RNA translation through the binding of sRNA [128], while the type II toxin–antitoxin system is based on the interaction of an endoribonuclease and an inhibitor, forming a stable complex [130]. Type III toxin–antitoxin systems consist of the inhibition of the toxin protein by an antitoxin RNA [131]. Type IV toxin–antitoxin systems are composed of two proteins that do not form a complex; instead, the antitoxin acts as an antagonist of the toxin at its cellular targets [132]. The type V toxin–antitoxin system differs from the others by having its antitoxin cleave the toxin mRNA [133]. Type VI is composed of an antitoxin-based proteolytic adapter that degrades the toxin-based protein [134], while type VII is composed of an antitoxin that enzymatically neutralizes the toxin by post-translational modification [129].

Type I and II are historically employed for biocontainment gene circuit design, especially for E. coli [135]. For example, the type I hok-sok pair has been applied for the design of the conditional suicide of plasmid-containing E. coli cells in phosphate- [136] or tryptophan-limited [135] environments. Additionally, type I TA systems have been used in synthetic biology projects related to GEM biocontainment [117]. Similarly, type II TA systems have been applied into kill switch circuits based on the use of the ccdB–ccdA pair regulated by a bi-stable cI/Cro memory switch (“essentializer” circuit) and by a cold-inducible promoter (“cryodeath” circuit) [113]. Finally, type II TA systems have been applied for the design of “plasmid addiction” systems to prevent the loss of plasmids under the production facility [137]

In addition to GEM, the use of type I and II TA systems was proven to be effective for GMY biocontainment, such as the expression of the relE–relB system in S. cerevisiae [138]. When not being neutralized by the RelB antitoxin, the RelE toxin inhibits protein synthesis by cleaving mRNA that are being translated on the ribosome [139]. When RelB is neutralizing RelE, it displaces the toxin’s C-terminal and forms the RelBE complex [140]. To express this TA system in S. cerevisiae, two recombinant plasmids were constructed, one containing the relE toxin gene under the control of the galactose-induced GAL1 promoter (pKP727) and another containing relE, regulated by the GAL1 promoter, and the relB antitoxin gene, under the control of the methionine-repressible MET25 promoter (pKP1006). Yeast cells transformed with the plasmid pKP727 showed visible growth inhibitory effects induced by the relE toxin; on the other hand, yeast transformed with the plasmid pKP1006 showed a higher growth rate. These data indicate that the toxic effects of relE can be minimized by the relB antitoxin in yeast [138]. Similar to the relE–relB system, the ribonuclease-associated toxin Kid (killing determinant) and its antitoxin Kis (killing suppressor) [141] have been used in S. cerevisiae as a potential biocontainment system. For this purpose, an expression system containing the antitoxin Kis controlled by the MET25 promoter and the Kid toxin controlled by the Cu²⁺-induced CUP1 promoter was cloned into the recombinant integrative plasmid pRS303. In the presence of both methionine and Cu²⁺, the expression of the Kis antitoxin is inhibited, while the expression of the Kid toxin is induced, leading to cell death [142].

Another TA system already tested in yeast is based on the ε-ζ (epsilon-zeta) genes from the gram-positive bacteria Streptococcus pyogenes [143]. The ε-ζ genes are organized in an operon together with the ω (omega) gene, which codify a repressor that modulates the transcription of the ζ toxin, the ε antitoxin, and its own [143]. This TA system was expressed in a yeast two-hybrid system without including the ω sequence, through which the toxicity of the ζ protein was shown as well as the efficiency of the ε protein in antagonizing its toxin [144]. Despite the efficiency of the ε-ζ pair in inducing yeast cell death, its use as a biocontainment system for GMY was not attempted until now.

Finally, it was observed that the expression of the chpK–chpI TA pair from Lepstospira interrogans in E. coli and S. cerevisiae modulates their cell growth [145,146]. However, the application of the chpK–chpI TA pair as a biocontainment system was not reported until now. In addition to S. cerevisiae, the expression of the endoribonuclease-associated toxin mazF from the E. coli mazEF module [147] in Pichia pastoris results in cell death and can be useful as a counter-selectable marker for genetic modification [148].

3. Multiplex-Type Biocontainment Approaches

Multiplex-type biocontainment approaches (MTBA) (Table 1) have several advantages over UTBAs. First, the combined use of different molecular mechanisms can reduce the selective evolutionary pressure observed for the majority of UTBAs that results in mutagenic drift, HGT events, and small molecule supplementation by an ecologic niche that inactivates the biocontainment-associated gene or gene circuits [38]. Interestingly, an early work on E. coli showed that duplicating the biocontainment-associated gene could lower the mutational events that lead to biocontainment inactivation [149]. The same authors suggest that the use of multiple copies of biocontainment-associated genes, with different regulatory mechanisms and independent molecular targets, could also diminish the populational evolutionary pressure to negatively select mutated, non-functional biocontainment circuits [149]. In this sense, the use of synthetic biology approaches, such as redesigning the organism genome by incorporating expanded genetic code alphabets, the application of orthogonal DNA/RNA devices and/or systems, or XNA technologies, could be a solution for reducing the evolutionary pressure to inactivate the biocontainment in GMY/GEM populations [27,52,53,54,83,84]. Unfortunately, such approaches are far away for direct biocontainment applications in eukaryotic cells, especially yeasts. Even for prokaryotes, these technologies are mainly restricted to E. coli, and many of them were never tested in non-laboratorial conditions. Even considering the production facility environment, it has been reported that synthetic transcriptional circuits based on the Boolean gates “AND” and “NOR” have altered responses in laboratory-scale fermentations, negatively impacting both biomass production and gene circuit expression [150]. However, some successful applications of multiplex technologies in biocontainment were already described, which include the “Deadman” and “Passcode” kill switches in E. coli [114] and the transcriptional and recombinational control of essential genes in S. cerevisiae [27]. Another approach is the use of overlapping “safeguards” composed of engineered riboregulators that control the expression of essential genes and engineered nucleases that cleave the E. coli genome in the absence of exogenously supplied synthetic small molecules [34]. These examples reinforce the idea that redundancy combined with the use of different molecular approaches are crucial factors for the design of a resilient biocontainment technology.

4. Conclusions and Perspectives

More than ever, the development of new biocontainment technologies for GMY is imperative, facing the availability of synthetic biology tools and/or fermentation technologies. The data gathered so far showed that the technologies available for GMY biocontainment are far away from what is available for GEMs (e.g., E. coli). While technologies such as xenobiology and MTBAs are available for E. coli, the same is not true for yeasts that mainly rely on natural auxotrophies or other non-redundant UTBAs for biocontainment. The major factors that contribute to the lack of new GMY-linked biocontainment technologies are the complexity of the eukaryotic genome, the intracellular compartmentalization and biochemical specialization of organelles, as well as natural mechanisms that increase the genetic diversity of the yeast population, such as meiosis or even non-homologous recombination mechanisms, which lead to the inactivation of biocontainment circuits. However, with the advances in top-down synthetic biology approaches for eukaryote genomes (e.g., “Yeast 2.0”), it is expected that technologies such as xenobiology and genetic code refactoring will become available for the development of different UTBAs and MTBAs for GMY.