Pseudomonas putida KT2440: the long journey of a soil-dweller to become a synthetic biology chassis

ABSTRACT

Although members of the genus Pseudomonas share specific morphological, metabolic, and genomic traits, the diversity of niches and lifestyles adopted by the family members is vast. One species of the group, Pseudomonas putida, thrives as a colonizer of plant roots and frequently inhabits soils polluted with various types of chemical waste. Owing to a combination of historical contingencies and inherent qualities, a particular strain, P. putida KT2440, emerged time ago as an archetype of an environmental microorganism amenable to recombinant DNA technologies, which was also capable of catabolizing chemical pollutants. Later, the same bacterium progressed as a reliable platform for programming traits and activities in various biotechnological applications. This article summarizes the stepwise upgrading of P. putida KT2440 from being a system for fundamental studies on the biodegradation of aromatic compounds (especially when harboring the TOL plasmid pWW0) to its adoption as a chassis of choice in metabolic engineering and synthetic biology. Although there are remaining uncertainties about the taxonomic classification of KT2440, advanced genome editing capabilities allow us to tailor its genetic makeup to meet specific needs. This makes its traditional categorization somewhat less important, while also increasing the strain’s overall value for contemporary industrial and environmental uses.

INTRODUCTION

Contemporary microbiology seems to be the battleground of at least three distinct narratives of the way we interplay with bacteria. The first (and quite predominant) version is presenting them as the causative agents of a plethora of infectious diseases that need to be confronted with new antimicrobial therapies, inhibition of horizontal gene transfer, and a deeper understanding of virulence (1). Second comes the chronicle of microbiomes as the ultimate support of basic life cycles at all scales from the human gut (2) to global geochemical processes (3). Note that this second perspective is way more benevolent in presenting microorganisms not as enemies, but as welcome companions of our transit through this planet (4). Finally, a third approach represents bacteria as the key actors in many biotechnological processes with a clear industrial and environmental interest (5). Not infrequently, the same or similar classes of microbes appear in each of these categories as the main characters of very different stories. The word Pseudomonas as a search query in PubMed yields more than 140,000 entries (https://shorturl.at/bfnoL), with around half of them relating to their condition as a pathogen of humans, animals, and plants. About one-third of the current literature on Pseudomonas deals, instead, with the ability of different species and strains to remove environmental pollutants, promoting plant growth or both—something generally considered worthy from a human perspective. The rest of the Pseudomonas literature is split in many other narrative lines, with a growing trend to present some strains of the genus as suitable platforms for metabolic engineering of new pathways, particularly those involving demanding redox transactions. In summary, the same type of bacteria can elicit negative or positive perceptions depending on the context. Not infrequently, virulent Pseudomonas strains may also be good degraders of environmental pollutants (6, 7) and plant colonizers (8). Yet, at the basis of being a pathogen, degrading pollutants or hosting harsh reactions lays the same basic metabolic network shared by virtually all Pseudomonas. The biochemistry of the genus seems to favor the generation of NAD(P)H, the main metabolic currency for combatting oxidative stress, over production of ATP, i.e., the metabolic architecture has evolved toward stress endurance rather than favoring the fastest growth (9–11). On this background, the species called P. putida has acquired considerable attention because a good number of its isolates seem to keep many of the desirable properties of the Pseudomonads (i.e., rapid growth, robust and diverse metabolism, stress resistance, and amenability to genetic programming) while minimizing other downsides typically associated with the genus, e.g., the presence of virulence determinants. To be sure, many P. putida strains are also opportunistic pathogens (12), but they are way below in the ranking of infectious agents (e.g., P. aeruginosa). Furthermore, even marginal traits that could be of concern in natural isolates can be deliberately erased from the genome, producing variants altogether safe for biotechnological uses, including environmental release. But what makes this species a special microorganism for these purposes, specifically such an attractive host for metabolic engineering—whether for biotransformations or for bioremediation?

Leaving behind the stigma of being a Pseudomonas

On a first glance, P. putida could be just yet another Gram-negative, saprotrophic, and mesophilic bacterium that happens to be a frequent member of the soil and/or root-associated microbiome (13). But there are circumstances that soon captured the attention of researchers in view of possible biotechnological exploitation. To start with, while still being a mesophilic bacterium, P. putida strains thrive comfortably in sites exposed to meteorological inclemencies and frequently polluted by waste chemicals, metals, solvents, and oil-derived products, many of which are serious stressors (10). This indicated that such bacteria evolved to endure a range of physicochemical insults that not only appear in natural settings, but they also resemble conditions found in bioreactors and industrial processes (14, 15). A second feature of P. putida was leveraged by the time of the onset of recombinant DNA technology—the amenability of many of its strains to genetic engineering and their safety as hosts of plasmid vectors (16, 17). Such a safety has been later substantiated when whole DNA sequencing showed a virtual lack of virulence determinants in strains typically used in biotechnological applications (18). The reported cases of infection by P. putida are mostly limited to specific antibiotic-resistant strains in patients undergoing invasive procedures or immunocompromised hosts (19), not much different from other bacteria generally considered harmless. Note, however, that the GRAS (generally recognized as safe) status that was often attributed to strains such as KT2440 (see below) could not be verified, as critical tests on effects of ingestion have never been reported (20)—although they may have accidentally happened. The third feature that rendered P. putida appealing includes its competence for extending its metabolic networks towards unusual, often xenobiotic, organic compounds, many of which become palatable carbon and energy sources (21). The main carriers of biodegradative activities in soil are often slow-growers and non-culturable species (22). In contrast, while P. putida strains may not be the main catalysts for the removal of chemical pollutants in situ, their generally fast growth makes them more appealing for laboratory studies and further development as biotechnological platforms. On these bases, it is little wonder that P. putida strains depart from the the guild of the so-called model organisms (of which Escherichia coli is the main star) and it joins instead that of robust bacterial workhorses for human exploitation. While still being a Pseudomonas (and, thus, bearing the stigma of belonging to a bacterial family populated by pathogens), these features set P. putida apart as a particularly noteworthy microorganism for industrial and environmental uses (14). That P. putida exhibits a remarkable ability to both withstand and metabolize toxic chemicals—in the form of substrates, intermediates, or products—is particularly valuable in processes that involve raw feedstocks, e.g., biomass hydrolysates, containing chemical inhibitors that would typically prevent the growth of most organisms.

Biography of P. putida KT2440

Out of the different isolates of P. putida that have been studied and employed in biotechnological scenarios, the one called KT2440 [KT, after Kenneth Timmis, in whose Laboratory the strain was first generated (23)] has been the focus of considerable attention in the last decade (Fig. 1). Not just as one more Gram-negative host of recombinant DNA constructs in an environmental bacterium, but as a veritable chassis for advanced Synthetic Biology (SynBio)-based bioengineering endeavors (10, 24). SynBio is a view of living systems through the lens of engineering for both gaining a fundamental understanding of biological systems and their rational programming for new-to-nature functions and activitities. To an extent, SynBio is a natural follow up of Genetic Engineering—whereby the term engineering is not a loose analogy but an actual methodology and interpretive frame of live objects (25). In this context, it cannot come as a surprise that members of the P. putida group have emerged as the starting point for the forward design of all-programmable, whole-cell catalysts able to push the boundaries of extant biochemistry. But why is strain KT2440 at the spotlight?

Fig 1.

Morphological characteristics of P. putida KT2440. (A) Cells are typically rod-shaped and characteristically show 5–7 flagella in one of their poles (26). Reproduced with permission from reference (27). Such a morphology can be genetically changed for the sake of delivering catalytic activities in different physical forms. Panels (B) and (C) show, for instance, transmission microscope images of cells deleted from the minCDE gene cluster, which lead to asymmetrical divisions and production of DNA-free minicells. Some of these minicells may inherit the polar flagella structure (Photo credit: Max Stammnitz.)

In reality, P. putida KT2440 is not a forthright environmental isolate itself, but a laboratory-generated plasmid-less derivative of strain P. putida mt-2. This specimen was first identified in Japan when looking for soil bacteria able to grow on some aromatic compounds, e.g., m-toluate, and displaying catechol 2,3-dioxygenase activity, the first crystallized enzyme among oxygenases (28, 29). Note that, at that point, the taxonomy of the isolate was not entirely settled (30; see below). Some 10 years later, the degradation ability of strain mt-2 was traced to the presence of a large catabolic plasmid, called pWW0 (31, 32). This plasmid endows the host cells the competence to catabolize not only m-toluate but also toluene, m-xylene, and other aromatic compounds. A historical account of the origins and whereabouts of mt-2 strain has been described by Nakazawa (29). By now, the literature contains >200 papers on the structure of the pWW0 plasmid (and similar ones; 33), the enzymology and genetics of the aromatic degradation degradation process, its regulation, its interplay with the metabolic network of the host, and many other details of the process (34). These still-ongoing studies make the catabolic capacity of P. putida mt-2 perhaps the most intensively studied case of bacterial biodegradation of aromatic chemicals. But, as indicated, the critical moment of our story (perhaps not realized at the time) took place when the P. putida mt-2 strain was cured of the pWW0 plasmid in Kenneth Timmis’ Laboratory, thereby generating variant KT2440 (23). Note that the same or very similar strains were also cured of the plasmid by other research teams (e.g., in Peter Williams’ group), thus originating virtually isogenic equivalents to KT2440, e.g., the one called to this day PaW85 (35). For quite a long time, strain KT2440 was leveraged as a Gram-negative host of recombinant DNA constructs (16), in particular, for pathways that could not be expressed well in E. coli (36), as well as an excellent biofilm-builder (37) and plant growth-promoting root colonizer (38). Also, the same strain became a superb model for molecular dissection of the intricacies of nutrient choice (i.e., catabolite repression) in environmental bacteria (39, 40). Yet, the authentic boost to become a dependable SynBio and metabolic engineering chassis started only after completing the sequence of its genome (41, 42). This not only exposed a range of new and appealing properties (13), but also opened a way to exploit them with a whole range of growingly available molecular tools for deep genome editing.

THE METABOLIC POWERHOUSE OF KT2440

Availability of the genome sequence, development of metabolic models, and experimental validation of many key predictions have provided in recent years a solid mechanistic foundation to what in the past was mostly a series of phenomenological observations (43–45). A good deal of the properties that make P. putida attractive as a metabolic engineering platform stems from the high rates of NADPH regeneration when growing on hexoses, as glucose is converted into glyceraldehyde-3-phosphate and pyruvate via the Entner-Doudoroff (ED) pathway (Fig. 2). Part of these trioses-phosphate is recycled back to the pool of hexoses-phosphate, restoring one molecule of NADPH in the process via a combination of activities of the ED pathway, the pentose phosphate pathway, and an incomplete Embden-Meyerhof-Parnas route—collectively termed EDEMP cycle (46; Fig. 2). This central metabolic architecture has been shown to boost expression of heterologous pathways (including new-to-nature biosynthetic routes) and providing an efficient source of reducing power that maintains a high level of tolerance to stressful conditions (36). Furthermore, and in connection to its origin as a soil-dwelling bacterium, KT2440 can both thrive in the rhizosphere and degrade a range of aromatic compounds derived from lignin degradation, e.g., benzoate, 4-hydroxybenzoate, p-coumarate, caffeate, and vanillate (47, 48). These metabolic features render P. putida KT2440 an important asset for bioprocesses dependent on biomass-derived feedstocks. Furthermore, the versatile metabolism in this bacterium enables the consumption of a wide range of structurally diverse compounds, a feature that can be further expanded by engineering novel reactions for synthetic assimilation, e.g., for one-carbon substrates (49). The default redox activities of P. putida can be further fortified by interfacing cells with bioelectrochemical hardware by means of redox mediators (50). These improve performance for various applications ranging from biosensing to e-assisted biodegradation to e-assisted biocatalysis and CO₂ reduction (51, 52).

Fig 2.

Genomic signatures of P. putida KT2440 traits. (A) Inspection of the KT2440 genome and phenotypic characterization exposes a number of what could be considered good and bad traits for biotechnological applications as shown in the list. Note that all of them can be either genetically fortified or erased for generating growingly improved chassis for synthetic biology and metabolic engineering. rDNA, recombinant DNA. (B) The metabolic core of strain KT2440 (shared by many other Pseudomonas) involves the merge of a set of reactions of the standard Embden–Meyerhof–Parnas (EMP) pathway with others of the alternative Entner-Doudoroff (ED) route, which are connected to the pentoses cycle and other key transformations. The most remarkable feature of this core (what can be considered the biochemical heart of the cells) is the active recycling of trioses-phosphate back to hexoses-phosphate. This metabolic architecture delivers more reducing power and less ATP than other typical metabolic networks, e.g., that of E. coli (11, 46).

How to become an industry-phillic SynBio chassis

Metabolic engineering and SynBio have advanced our ability to produce chemicals, including new-to-Nature compounds, to satisfy ever increasing societal demands (53). The selection of an appropriate microbial host is a key step of this process. In this sense, the development of SynBio tools and strategies for advanced metabolic engineering enables the incorporation of a number of non-traditional microorganisms as hosts for developing efficient microbial cell factories (54, 55). The list of the microbial species and strains that can be adopted for such purposes continues to expand as more tools for precise gene and genome manipulation become available. Yet, the implementation of microbial cell factories at a large scale is limited to a small number of bacterial species that have been selected for industrial exploitation (56). This state of affairs coincides with the interest in developing extensive microbial processes, elicited by increased concerns about climate change and the prospect of dwindling petroleum resources, that have shifted manufacturing toward bioproduction. The first step in selecting a candidate species for industrial applications is exploring whether the organism at stake (i) has been studied to provide enough basic knowledge on physiology and metabolism as the basis for designing culture media and bioprocesses; (ii) requires simple nutritional requirements, including readily-accessible carbon and nitrogen sources; (iii) has built-in resistance to physicochemical stress, (iv) can grow fast with efficient substrate(s) utilization, (v) is genetically tractable by using tools for targeted manipulations, and (vi) efficiently secretes metabolites and products of interest to facilitate downstream purification steps. While various organisms check all these requirements (which we suggest to be termed industriphilicity), P. putida KT2440, a versatile and robust bacterium, has progressively consolidated as one platform that optimally fulfills all these criteria (56), specially owing to its capacity to host novel, often harsh chemical processes (24).

Dealing with tough chemical reactions

Not surprisingly, the industrial applications of metabolically engineered P. putida are related to its inherent capacity of growing under stressful conditions and displaying a rich metabolism for the biosynthesis of a variety of value-added chemicals from renewable resources. For instance, P. putida has been successfully engineered to produce bioplastics, such as polyhydroxyalkanoates (PHAs), from various carbon sources. PHAs are naturally synthesized by P. putida under specific conditions, e.g., nitrogen limitation, and these polymers are harnessed as carbon and energy storage (57). Additionally, PHAs offer an eco-friendly alternative to conventional, oil-derived materials. The biodegradability of PHAs as well as their material properties, e.g., thermoplasticity, insolubility, and lack of toxicity, makes them good alternatives to fuel-based plastics for sustainable packaging and other industrial uses (58). Further engineering of the native PHA biosynthesis pathways has led to the production of novel materials, decorated with chemical groups and substituents that multiply their range of practical applications (59). The production of pharmaceuticals is another avenue where engineered P. putida strains are actively used for whole-cell biocatalysis. For many of such target compounds, the natural tolerance of this bacterium toward aromatic molecules and its ability to process these structures using mono- and di-oxygenases were exploited. Relevant examples of this sort include the biosynthesis of 3-methylcatechol, o-cresol, cis,cis-muconate, and styrene from renewable feedstocks (60, 61). Other value-added compounds that have been produced in P. putida are rhamnolipids, terpenoids, and a number of aromatic compounds (e.g., phenol, vanillate, and anthranilate) derived from the shikimate pathway (36). The unique metabolism of strain KT2440 can be likewise exploited for bioremediation purposes (21). Building on the wealth of native pathways for the breakdown of complex substrates, new reactions can be added to support biodegradation of recalcitrant environmental pollutants. Such engineering efforts have included the degradation of aromatic compounds which are notoriously difficult to handle metabolically, via ring-opening oxygenolytic reactions (62). Examples comprise tough industrial waste, e.g., polychlorinated biphenyls (PCBs; 63), 2,4-dichlorophenoxyacetic acid (2,4-D; 64), or 1,3-dichloroprop-1-ene (65). In some cases, the degradation of environmental pollutants could be connected to the biosynthesis of value-added compounds (66), thereby closing a virtuous trash-to-treasure cycle.

From native biochemistry to synthetic metabolism

Paired to the production of well-established molecules, the engineering of completely novel metabolic networks (i.e., synthetic metabolism) is gaining momentum in metabolic engineering (67). The motivation behind these efforts relates to the need to expand the number of nature of products in the biotechnological agenda. Accessing new-to-nature products through metabolic engineering is a necessity in a rapidly changing world in which the availability of oil-based resources is becoming critically limited. Redesigning the biochemical palate of bacteria is the way forward to provide access to these products, and a number of abiotic chemical elements in the periodic table are amenable to biologization. P. putida offers an ideal context for engineering these novel chemical reactions. One relevant example is the production of fluorochemicals, which involves highly reactive metabolic intermediates and final products (68). Fluorinated compounds are of special interest for biotechnology owing to their importance in our everyday life—ca. 25% of the most used pharmaceuticals contain a F atom that often enhances their mechanism of action and allows for improved uptake due to increased hydrophobicity. In addition, a plethora of compounds for the agrochemical industry, diagnostics, or material sciences is based on fluorochemistry. In a recent study, the biosynthesis of fluorometabolites was engineered in P. putida by expressing an optimized fluorinase from Archaea under the control of a fluoride-responsive riboswitch (69). In these engineered strains, the addition of mineral F^– to the medium triggers the expression of biofluorination genes, circumventing the need for expensive additional chemical inducers to afford fluorometabolite production.

Engineering P. putida for such novel reactions is not without challenges. The introduction of foreign metabolic pathways often disrupts the native flow of metabolites and cofactors in the cell, an issue that can be tackled by optimizing gene expression, enhancing the efficiency of biochemical pathways (e.g., by metabolic funneling), and improving the tolerance of P. putida to the produced compounds by adaptive laboratory evolution. Advances in SynBio and gene editing technologies (70, 71), e.g., the wealth of CRISPR/Cas9 tools (72), are opening new avenues for further enhancing the metabolic capabilities of this bacterium. Future research in this domain may focus on improving the efficiency and stability of engineered pathways in P. putida and exploring its potential in new areas, e.g., renewable energy applications.

Bioengineering beyond biochemistry

An emerging angle of metabolic engineering, enabled by the plethora of molecular tools available for P. putida since the onset of SynBio, is what could be called concurrent design. This strategy is directed not just to improve the reactions or the pathways of interest but also endowing cells with a physical form, physiological robusteness, and material properties that optimize the process as a whole, including upstream and downstream steps. This includes, for instance, conditional generation of biofilms, where immobilization of the bacterial cells can enhance their catalytic performance (73) and inducible flocculation (74). A landmark in this roadmap was the generation of strain P. putida EM42, which has been genomically erased of a number of chromosomally encoded functions (e.g., flagella, prophages, some transposons, exopolymeric components), resulting in a more stress-resistant phenotype (including thermal tolerance) and higher availability of reducing power, critical for supporting redox reactions and counteracting stress provoked by reactive oxygen species (75, 76). Another remarkable designed derivative is strain EM371 (74), which has been deliberately deleted of up to 23 chromosomal segments (that make ca. 5% of the genomic DNA), many of them involved in the production of bulky cell-surface structures (Fig. 3). Although surface display of heterologous proteins with autotransporters is certainly feasible in the non-edited strain (77), the surfome simplification born by EM371 results in a surface-naked strain that is optimal for ectopic presentation of adhesins and multiprotein assemblies (78). Furthermore, EM371 cells can be abstracted as spherocylinders that can be decorated on their surface with matching interaction partners at given locations of their physical shape. This quality enables the simulation and implementation of synthetic consortia formed by different strains that self-assemble in three dimensions following given compositional rules (74, 79). Finally, surface-naked P. putida eases the engineering of cell monolayers on solid surfaces coated with an antigen or molecular motif recognized by a genetically determined adhesin attached to the cell envelope (80).

Fig 3.

Deep editing of P. putida KT2440 for generation of a surface-naked variant. (A) Chromosomal location of the whole of directed deletions entered in the KT2440 genome for eliminating expression of bulky surface structures, e.g., flagella, fimbriae, components of the lipopolysaccharide, genomic parasites (prophages, transposons), and other instability determinants (74). As sketched in the cartoon of panel (B), the result is a strain whose envelope has been erased of bulky protuberances, deleted of prophages (genomic segments in color), and endowed with a smooth surface that optimises either display or ectopic expression of adhesins and other functional proteins.

Whether such deeply engineered catalysts are destined for use in industrial or environmental settings raises questions on their propagation beyond the laboratory. This framework is important for the sake of both intellectual property protection and liability in case any damage results from accidental or deliberate release. Given that a 100% certainty of containment may not be altogether feasible (81), a current, more realistic trend (82) is shifting the emphasis from restraint to traceability with genomic barcodes that provide durable unique identifiers connected to digital twins (83). In a further screw turn, it is possible to entertain P. putida-derived catalysts entirely devoid of DNA while keeping the full biochemical power of living cells. This question has been recently approached by either dissolution of the chromosome upon conditional expression of a nuclease (84) or through generation of DNA-free minicells (Fig. 1) that maintain its physiological vigor and enzymatic capabilities for at least 2 weeks (85). Finally, the recent discovery of enzyme-enriched vesicles released by KT2440 strain when exposed to polymeric lignin (86) opens new opportunities to program this bacterium for complex in situ and ex situ operations. Whether focused on the reactions proper or in the way such transformations are delivered to the substrates, the SynBio-based developments discussed above denote a growing applicability of KT2440, its siblings, and their engineered descendants as eco-friendly alternatives to many of the catalysts habitually employed in the chemical industry.

THE EVOLVING (AND PROBLEMATIC) TAXONOMY OF STRAIN KT2440

As mentioned before, the complete genome sequence of KT2440 strain was the turning point for establishing this bacterium as an exceptional metabolic engineering chassis. The first version of the P. putida KT2440 genome was published early in this century based on the classical strategy of shotgun Sanger sequencing of large- and small-insert libraries combined with primer walking (41). A revisited version of the P. putida KT2440 genome using Illumina sequencing technology was published in 2016 (42), including the identification of 242 new protein-coding genes and functional re-annotation of 1,548 genes, thereby allowing the development of much improved metabolic models. A comparative study of both complete and partial genomic sequences of strains KT2440 and mt-2 available in databases exposes some interesting details. For instance, the chromosomal DNA of the ancestral P. putida mt-2 strain included in GenBank under accession number CP136395 is 1,566 bp larger than the previously reported KT2440 genomes. Also, the mt-2 sequence and later versions of the KT2440 sequence correct the earlier frameshift attributed to the pckA gene (encoding phosphoenolpyruvate carboxykinase), which was argued by Belda et al. (42) to block gluconeogenesis. It is still unclear whether such a consequential difference is the result of a sequencing error or one more asset of the adaptive metabolic arsenal of the strains, which may shift between two major biochemical regimes through a sort of phase-variation mechanism. Note, however, that up to that point, the identity of mt-2 and KT2440 as strains of the species P. putida was generally accepted.

Current taxonomic credentials of strain KT2440

The mt-2 strain was initially identified as P. arvilla (28) and subsequently reclassified as P. putida (29). The high heterogeneity of P. putida strains led to their preliminary subdivision into two main biovars, A and B, based on the G + C content and differentiated by a suite of phenotypic traits (87). The type strain of P. putida ATCC 12633^T (DSM 291^T) was considered a member of biovar A, as was the case also with KT2440 (87). However, as early as 2002, taxonomic affiliation of strain KT2440 was questioned by using DNA–DNA hybridization (DDH) analysis between the chromosomes KT2440 and ATCC 12633^T (30). The two strains showed only 50.5% of genomic DNA similarity, way apart of the >70% generally considered by that time to be the reference for species identity (88). In addition, phenotypic data showed that KT2440 and ATCC 12633^T strains were not closely related (30). Average nucleotide identity (ANI) based on BLAST+ (ANIb) of the KT2440 genome showed values <91% when KT2440 was compared to other available P. putida genomes, indicating that this specimen might be considered a representative of a different Pseudomonas species not yet described at that time (87). In 2019, a phylogenetic study of 95 strains of the P. putida group involving multilocus sequence analysis (MLSA), ANI determination, in silico DDH and comparative phenotyping, suggested the existence of four new species branching out of P. putida group, i.e., P. alloputida, P. inefficax, P. persica, and P. shirazica. Based on this taxonomic classification, strain KT2440 was proposed to belong to the P. alloputida species, whose type strain is Kh7 (LMG 29756^T) isolated from bean rhizosphere in Iran (89). The values reported for the comparison of Kh7 and KT2440 genomes were 76.8% for in silico DDH and 97.3% for ANI, but no figures were disclosed for the resemblance of mt-2 versus Kh7 genomes in that study. However, analyses done for this article using Genome-to-Genome Distance Calculator [GGDC (90) and JSpeciesWS (91)] revealed values of 76.8% for in silico DDH and 96.6% for ANIb, respectively, thereby modifying the preliminary results obtained with the plasmid-less derivative.

What species KT2440 is, requires a better closure

While past and more recent evidence suggests that (with the newest taxonomic criteria) KT2440 is not a typical P. putida strain, we disagree with what we consider a hasty proposal to include it in a new class named alloputida. First, it should be noted that the genome of the reference Kh7 strain available for genomic comparisons is incomplete at the time of writing this article. In fact, the sequence is highly partitioned in 429 contigs (GenBank accession number OLKK01000000.1). A complete, still awaited version of the type strain genome could provide different values of genomic similarities. But even in the case that strain KT2440 were indeed not a typical P. putida, we consider the new name to be particularly confusing and misleading. The etymology of the word for the proposed P. alloputida species, allos (ancient Greek ἄλλος), means exactly other (89). Therefore, alloputida means just other putida something that, in our view, adds no new insight to the original name. Instead, it conceals the wide range of metabolic and genetic qualities exhibited by the most studied strain of the group, which certainly warrants a more recognizable designation. A more descriptive name for the species, highlighting their biotechnological value and versatile and cosmopolitan nature, widely recognized by the users, would make a better fit as a proper taxonomic affiliation and the remarkable journey of strain KT2440 as a model microorganism and SynBio chassis. While these issues are clarified and validated through community involvement, we advocate keeping the designation P. putida KT2440 for the plasmid-less derivative of mt-2 strain. But, the taxonomic questions do not stop there, as there is (and will be) a growing divergence between the genomic complement of many derivatives of KT2440 and the ancestral isolate. Such engineered differences would reach, in some cases, the ~5% threshold, generally considered to be the limit between bacterial species (92). In fact, future taxonomic standards will have to incorporate criteria for heavily genome-edited strains bearing not only significant sequence divergences in DNA shared with reference strains but also major differences in core metabolic and physiological functions (93).

Outlook

Since the initial endeavors of the mid-1980s aimed at employing strains of P. putida and related species as agents for environmental bioremediation, plant growth promotion, and whole-cell catalysis, strain KT2440 has firmly established itself as a favored platform for metabolic engineering guided by systems and SynBio. While the strain is by itself well-suited for many biotechnological operations, there remains ample opportunity to introduce additional desirable traits, facilitated by the growing number of dedicated genetic tools available for this purpose. Such tools have already increased its safety, predictability, and programmability of both biotransformations of interest and the material format in which catalysis is delivered to its targets. In this respect, we argue that the journey of strain KT2440 from being a soil dweller to become a recognizable SynBio chassis showcases each of the steps that a naturally ocurring isolate must follow to become a valuable biotechnological asset for the 4th Industrial Revolution (56, 94). Two clear perspectives emerge from this. One is the already ongoing drive to push the boundaries of exant metabolism towards new elements of the periodic table which—such as fluorine—have been traditionally considered mostly alien to life (68) incorporating also new-to-nature enzymes and non-standard amino acids (something currently available, to an extent, for E. coli and a few other hosts) (95). The other is the resurfacing and upgrading of ideas that were widespread during the 1980s, but never realized, about engineering live agents destined for environmental remediation of extensive antropogenenic emissions. The first wave of such attempts did not get very far because of lacking success stories, too primitive technologies, dearth of sufficient ecological knowledge, and generally negative public perception (96). But the ramping consequences of climate change advise revisiting such approaches and even applying them to a much larger scale (97). In each of these two fronts, strain KT2440 can have a role in showcasing all possibilities of leveraging microbial activities for human benefit.

Biographies

Víctor de Lorenzo is a Chemist by training and holds a position of Research Professor in the Spanish National Research Council (CSIC) in Madrid, where he eventually settled after working at the Pasteur Institute, the University of California in Berkeley, the University of Geneva, and the Gesellschaft für Biotechnologische Forschung (GBF) in Braunschweig. He currently heads the Laboratory of Environmental Synthetic Biology at the National Center for Biotechnology in Madrid. He specializes in molecular biology and genetic engineering of soil microorganisms (particularly Pseudomonas putida) as agents for the decontamination of sites damaged by anthropogenic emissions. At present, his work explores the interface between synthetic biology and global-scale environmental biotechnology and development of tools to foster such an interface. He is a member of the EMBO (European Molecular Biology Organization) and the American and European Academies of Microbiology

Danilo Pérez-Pantoja is a Full Professor at Universidad Tecnológica Metropolitana in Santiago, Chile. He graduated in Biochemistry from Universidad de Chile and earned his Ph.D. in Molecular Genetics and Microbiology from P. Universidad Católica de Chile, focusing on the genomics of biodegradative bacteria. He was awarded the Hermann Niemeyer Medal by the Chilean Society of Biochemistry as the best graduate student. In 2009, he moved to Spain to obtain an MSc in Bioinformatics and conduct post-doctoral research in Prof. de Lorenzo’s group at the National Center of Biotechnology, supported by a Marie Skłodowska-Curie Actions fellowship. His research focuses on the molecular mechanisms underlying the emergence of novel catabolic pathways towards xenobiotic compounds, or as described in a metaphorical view, how environmental bacteria conquest the chemical space. Back in Chile, he leads the Microbial Genomics and Biotechnology group, focusing on the biodegradation of emerging pollutants and metabolic engineering for lignin bioconversion.

Pablo I. Nikel, a native of Argentina, pursued a Ph.D. in Biotechnology and Molecular Biology in Buenos Aires, adapting signal transduction systems in E. coli for metabolic engineering. His journey then led him to the USA, where he joined Prof. George N. Bennett’s team at Rice University with support from the ASM. Pablo moved to Europe in 2010, becoming a post-doctoral fellow at Prof. de Lorenzo’s laboratory with funding from EMBO and the Marie Skłodowska-Curie Actions. He delved into the world of environmental bacteria, particularly Pseudomonas putida—a bacterial model that he has been associated with ever since. He currently heads the Systems Environmental Microbiology Group at DTU Biosustain (Denmark). His team is pioneering the implementation of synthetic metabolism to produce new-to-nature chemicals, pushing the boundaries of microbial biochemistry to obtain compounds traditionally made through conventional chemistry. Pablo was appointed Full Professor at the Technical University of Denmark in 2023.