SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes.

The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications. In particular, SEVA 4.0 includes (i) a sub-collection of plasmids for easing the composition of multiple DNA segments with MoClo/Golden Gate technology, (ii) vectors for Gram-positive bacteria and yeast and [iii] off-the-shelf constructs with built-in functionalities. A growing collection of plasmids that capture part of the standard-but not its entirety-has been compiled also into the DB and repository as a separate corpus (SEVAsib) because of its value as a resource for constructing and deploying phenotypes of interest. Maintenance and curation of the DB were accompanied by dedicated diffusion and communication channels that make the SEVA platform a popular resource for genetic analyses, genome editing and bioengineering of a large number of microorganisms.

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes.

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways for a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Building on this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-informed engineering approach. A growth-coupling strategy, driven by AcCoA demand, allowed for direct selection of an alternative sugar assimilation route-the phosphoketolase (PKT) shunt from bifidobacteria. Adaptive laboratory evolution forced the synthetic P. putida auxotroph to rewire its metabolic network to restore C2 prototrophy via the PKT shunt. Large-scale structural chromosome rearrangements were identified as possible mechanisms for adjusting the network-wide proteome profile, resulting in improved PKT-dependent growth phenotypes. 13C-based metabolic flux analysis revealed an even split between the native Entner-Doudoroff pathway and the synthetic PKT bypass for glucose processing, leading to enhanced carbon conservation. These results demonstrate that the P. putida metabolism can be radically rewired to incorporate a synthetic C2 metabolism, creating novel network connectivities and highlighting the importance of unconventional engineering strategies to support efficient microbial production.

Anaerobic glucose uptake in Pseudomonas putida KT2440 in a bioelectrochemical system.

Providing an anodic potential in a bio-electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox imbalances. In this setup, the bacteria could be exploited to produce chemicals via oxidative pathways at high yield. However, the absence of anaerobic growth and low carbon turnover rates remain as obstacles for the application of such an electro-fermentation technology. Growth and carbon turnover start with carbon uptake into the periplasm and cytosol. P. putida KT2440 has three native transporting systems for glucose, each differing in energy and redox demand. This architecture previously led to the hypothesis that internal redox and energy constraints ultimately limit cytoplasmic carbon utilization in a bio-electrochemical system. However, it remains largely unclear which uptake route is predominantly used by P. putida under electro-fermentative conditions. To elucidate this, we created three gene deletion mutants of P. putida KT2440, forcing the cells to exclusively utilize one of the routes. When grown in a bio-electrochemical system, the pathway mutants were heavily affected in terms of sugar consumption, current output and product formation. Surprisingly, however, we found that about half of the acetate formed in the cytoplasm originated from carbon that was put into the system via the inoculation biomass, while the other half came from the consumption of substrate. The deletion of individual sugar uptake routes did not alter significantly the secreted acetate concentrations among different strains even with different carbon sources. This means that the stoichiometry of the sugar uptake routes is not a limiting factor during electro-fermentation and that the low rates might be caused by other reasons, for example energy limitations or a yet-to-be-identified oxygen-dependent regulatory mechanism.

Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440.

IMPORTANCE: Investigating fundamental aspects of metabolism is vital for advancing our understanding of the diverse biochemical capabilities and biotechnological applications of bacteria. The origin of the essential thymidylate kinase function in the model bacterium Pseudomonas putida KT2440, seemingly interrupted due to the presence of a large genomic island that disrupts the cognate gene, eluded a satisfactory explanation thus far. This is a first-case example of an essential metabolic function, likely acquired by horizontal gene transfer, which "landed" in a locus encoding the same activity. As such, foreign DNA encoding an essential dNMPK could immediately adjust to the recipient host-instead of long-term accommodation and adaptation. Understanding how these functions evolve is a major biological question, and the work presented here is a decisive step toward this direction. Furthermore, identifying essential and accessory genes facilitates removing those deemed irrelevant in industrial settings-yielding genome-reduced cell factories with enhanced properties and genetic stability.

Core and auxiliary functions of one-carbon metabolism in Pseudomonas putida exposed by a systems-level analysis of transcriptional and physiological responses.

The soil bacterium Pseudomonas putida is a robust biomanufacturing host that assimilates a broad range of substrates while efficiently coping with adverse environmental conditions. P. putida is equipped with functions related to one-carbon (C1) compounds (e.g. methanol, formaldehyde, and formate) oxidation-yet pathways to assimilate these carbon sources are largely absent. In this work, we adopted a systems-level approach to study the genetic and molecular basis of C1 metabolism in P. putida. RNA sequencing identified two oxidoreductases, encoded by PP_0256 and PP_4596, transcriptionally active in the presence of formate. Quantitative physiology of deletion mutants revealed growth defects at high formate concentrations, pointing to an important role of these oxidoreductases in C1 tolerance. Moreover, we describe a concerted detoxification process for methanol and formaldehyde, the C1 intermediates upstream formate. Alcohol oxidation to highly-reactive formaldehyde by PedEH and other broad-substrate-range dehydrogenases underpinned the (apparent) suboptimal methanol tolerance of P. putida. Formaldehyde was mostly processed by a glutathione-dependent mechanism encoded in the frmAC operon, and thiol-independent FdhAB and AldB-II overtook detoxification at high aldehyde concentrations. Deletion strains were constructed and characterized towards unveiling these biochemical mechanisms, underscoring the worth of P. putida for emergent biotechnological applications-e.g. engineering synthetic formatotrophy and methylotrophy. IMPORTANCE C1 substrates continue to attract interest in biotechnology, as their use is both cost-effective and ultimately expected to mitigate the impact of greenhouse gas emissions. However, our current understanding of bacterial C1 metabolism remains relatively limited in species that cannot grow on (i.e., assimilate) these substrates. Pseudomonas putida, a model Gram-negative environmental bacterium, constitutes a prime example of this sort. The biochemical pathways active in response to methanol, formaldehyde, and formate have been largely overlooked-although the ability of P. putida to process C1 molecules has been previously alluded to in the literature. By using a systems-level strategy, this study bridges such knowledge gap through the identification and characterization of mechanisms underlying methanol, formaldehyde, and formate detoxification-including hitherto unknown enzymes that act on these substrates. The results reported herein both expand our understanding of microbial metabolism and lay a solid foundation for engineering efforts toward valorizing C1 feedstocks.

Synthetic metabolism for biohalogenation.

The pressing need for novel bioproduction approaches faces a limitation in the number and type of molecules accessed through synthetic biology. Halogenation is widely used for tuning physicochemical properties of molecules and polymers, but traditional halogenation chemistry often lacks specificity and generates harmful by-products. Here, we pose that deploying synthetic metabolism tailored for biohalogenation represents an unique opportunity towards economically attractive and environmentally friendly organohalide production. On this background, we discuss growth-coupled selection of functional metabolic modules that harness the rich repertoire of biosynthetic and biodegradation capabilities of environmental bacteria for in vivo biohalogenation. By rationally combining these approaches, the chemical landscape of living cells can accommodate bioproduction of added-value organohalides which, as of today, are obtained by traditional chemistry.

Standardization of regulatory nodes for engineering heterologous gene expression: a feasibility study.

The potential of LacI/Ptrc , XylS/Pm , AlkS/PalkB , CprK/PDB3 and ChnR/PchnB regulatory nodes, recruited from both Gram-negative and Gram-positive bacteria, as the source of parts for formatting expression cargoes following the Standard European Vector Architecture (SEVA) has been examined. The five expression devices, which cover most known regulatory configurations in bacteria were assembled within exactly the same plasmid backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance was then analysed in an Escherichia coli strain of reference through the readout of a fluorescence reporter gene that contained strictly identical translation signal elements. This approach allowed us to describe and compare the cognate expression systems with quantitative detail. The constructs under scrutiny diverged considerably in their capacity, expression noise, inducibility and ON/OFF ratios. Inspection of such a variance exposed the different constraints that rule the optimal arrangement of functional DNA segments in each case. The data highlighted also the ease of standardizing inducer-responsive devices subject to transcriptional activation as compared to counterparts based on repressors. The study resulted in a defined collection of formatted expression cargoes lacking any cross talk while offering a panoply of choices to potential users and help interoperability of the specific constructs.

Combinatorial pathway balancing provides biosynthetic access to 2-fluoro-cis,cis-muconate in engineered Pseudomonas putida.

The wealth of bio-based building blocks produced by engineered microorganisms seldom include halogen atoms. Muconate is a platform chemical with a number of industrial applications that could be broadened by introducing fluorine atoms to tune its physicochemical properties. The soil bacterium Pseudomonas putida naturally assimilates benzoate via the ortho-cleavage pathway with cis,cis-muconate as intermediate. Here, we harnessed the native enzymatic machinery (encoded within the ben and cat gene clusters) to provide catalytic access to 2-fluoro-cis,cis-muconate (2-FMA) from fluorinated benzoates. The reactions in this pathway are highly imbalanced, leading to accumulation of toxic intermediates and limited substrate conversion. By disentangling regulatory patterns of ben and cat in response to fluorinated effectors, metabolic activities were adjusted to favor 2-FMA biosynthesis. After implementing this combinatorial approach, engineered P. putida converted 3-fluorobenzoate to 2-FMA at the maximum theoretical yield. Hence, this study illustrates how synthetic biology can expand the diversity of nature's biochemical catalysis.

Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids.

Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ Red recombineering), yet most of them rely on the use of auxiliary plasmids, which have to be cured after the editing procedure. Temperature-sensitive replicons, counter-selectable markers or repeated passaging of plasmid-bearing cells have been traditionally employed to circumvent this hurdle. While these protocols work reasonably well in some bacteria, they are not applicable for other species or are time consuming and laborious. Here, we present a fast and versatile protocol of fluorescent marker-assisted genome editing in Pseudomonas putida, followed by clean curing of auxiliary plasmids through user-controlled plasmid replication. One fluorescent marker facilitates identification of genome-edited colonies, while the second reporter enables detection of plasmid-free bacterial clones. Not only is this protocol the fastest available for Pseudomonas species, but it can be easily adapted to any type of genome modifications, including sequence deletions, insertions, and replacements. Graphical abstract: Rapid genome engineering of Pseudomonas with curable plasmids.

Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection.

Pseudomonas species have become reliable platforms for bioproduction due to their capability to tolerate harsh conditions imposed by large-scale bioprocesses and their remarkable resistance to diverse physicochemical stresses. The last few years have brought forth a variety of synthetic biology tools for the genetic manipulation of pseudomonads, but most of them are either applicable only to obtain certain types of mutations, lack efficiency, or are not easily accessible to be used in different Pseudomonas species (e.g. natural isolates). In this work, we describe a versatile, robust and user-friendly procedure that facilitates virtually any kind of genomic manipulation in Pseudomonas species in 3-5 days. The protocol presented here is based on DNA recombination forced by double-stranded DNA cuts (through the activity of the I-SceI homing meganuclease from yeast) followed by highly efficient counterselection of mutants (aided by a synthetic CRISPR-Cas9 device). The individual parts of the genome engineering toolbox, tailored for knocking genes in and out, have been standardized to enable portability and easy exchange of functional gene modules as needed. The applicability of the procedure is illustrated both by eliminating selected genomic regions in the platform strain P. putida KT2440 (including difficult-to-delete genes) and by integrating different reporter genes (comprising novel variants of fluorescent proteins) into a defined landing site in the target chromosome.

Synthetic control of plasmid replication enables target- and self-curing of vectors and expedites genome engineering of Pseudomonas putida.

Genome engineering of non-conventional microorganisms calls for the development of dedicated synthetic biology tools. Pseudomonas putida is a Gram-negative, non-pathogenic soil bacterium widely used for metabolic engineering owing to its versatile metabolism and high levels of tolerance to different types of stress. Genome editing of P. putida largely relies on homologous recombination events, assisted by helper plasmid-based expression of genes encoding DNA modifying enzymes. Plasmid curing from selected isolates is the most tedious and time-consuming step of this procedure, and implementing commonly used methods to this end in P. putida (e.g. temperature-sensitive replicons) is often impractical. To tackle this issue, we have developed a toolbox for both target- and self-curing of plasmid DNA in Pseudomonas species. Our method enables plasmid-curing in a simple cultivation step by combining in vivo digestion of vectors by the I-SceI homing nuclease with synthetic control of plasmid replication, triggered by the addition of a cheap chemical inducer (3-methylbenzoate) to the medium. The system displays an efficiency of vector curing >90% and the screening of plasmid-free clones is greatly facilitated by the use of fluorescent markers that can be selected according to the application intended. Furthermore, quick genome engineering of P. putida using self-curing plasmids is demonstrated through genome reduction of the platform strain EM42 by eliminating all genes encoding ß-lactamases, the catabolic ben gene cluster, and the pyoverdine synthesis machinery. Physiological characterization of the resulting streamlined strain, P. putida SEM10, revealed advantageous features that could be exploited for metabolic engineering.

Engineering Pseudomonas putida KT2440 for the production of isobutanol.

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedback-resistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical YIso/S. Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non-growing conditions.