Genomics and metabolic reconstructions

First published in 2002 and revisited in 2016, the full genome sequence of P. putida KT2440 has shed light on the diverse transport and metabolic systems (Belda et al. 2016; Nelson et al. 2002). Moreover, the pangenome of P. putida has been studied and revealed 3386 conserved genes belonging to the core genome and comprising genes related to ED and pentose phosphate (PP) pathway, proline and arginine metabolism, aromatic compound degradation, as well as a vast collection of nutrient transporters (Udaondo et al. 2016). Notably, besides sharing 85% of the coding regions with Pseudomonas aeruginosa, undesirable biotechnological traits, i.e., key virulence factors, exotoxins and type III secretion systems, are lacking in P. putida (Udaondo et al. 2016). Since 2010, the number of published genome sequences of P. putida has significantly increased (Fig. ​(Fig.2,2, Tables S1 and S2). Up to now, 28 complete genomes and 88 draft P. putida genomes can be accessed via the Pseudomonas genome DB (pseudomonas.com), allowing for the identification of new features for industrial application.

Reconstructed from sequence information, several genome-scale metabolic models (GSMM) are available for P. putida (Belda et al. 2016; Henry et al. 2010; Nogales et al. 2008; Oberhardt et al. 2011; Puchałka et al. 2008; Sohn et al. 2010; Yuan et al. 2017). Very recently, the GSMM iJN1462 of P. putida KT2440 was published (Nogales et al. 2020), which is comparable to high-quality Escherichia coli models in size and level of detail. With the increasing number of P. putida genomes available, these models can facilitate the identification of strains and enzymes most suitable for the respective industrial application and drive the design of superior producers (Buschke et al. 2013; Choi et al. 2019; Poblete-Castro et al. 2013).

Transcriptomics and proteomics

As suggested by the elevated number of open-reading frames (450 in total), encoding transcriptional factors and 24 alternative sigma subunits of the RNA polymerase, gene expression in P. putida is tightly regulated at the transcriptional level (Martínez-Bueno et al. 2002). Transcriptomic profiles of P. putida KT2440 grown on different substrates have been studied in response to environmental perturbations (glucose, fructose, succinate, citrate, ferulic acid, serine, and glycerol) (D'Arrigo et al. 2019; Kim et al. 2013; Nikel et al. 2014), as well as profiles during the shift between alternative carbon sources (Sudarsan et al. 2014). Furthermore, recent transcriptomic studies under osmotic, oxidative, and imipenem stress (Bojanovič et al. 2017) and increased heavy metal concentrations (Peng et al. 2018) added knowledge about the stress response mechanisms in P. putida. Notably, during exposure to solvents, a general stress response initiated the expression of molecular chaperons and other stress resistance proteins during exposure to solvents, which is favorable for biocatalysis in a hydrophobic milieu (Dominguez-Cuevas et al. 2006). More latterly, the transcriptional response during nitrogen-limited mcl-PHAs synthesis on gluconate (de novo fatty acid synthesis pathway) and oleic acid (β-oxidation pathway) as the sole carbon sources was studied. Several genes were suggested as possible targets for strain improvement towards more efficient production (Mozejko-Ciesielska et al. 2018). Moreover, by characterizing laboratory evolved P. putida KT2440, utilizing genome re-sequencing and qRT-PCR, the metabolic and transcriptional regulatory bases of ethylene glycol metabolism in P. putida were uncovered (Li et al. 2019). Another interesting recent study mimicked repeated glucose shortage under large-scale heterogeneously mixed fermentation to investigate the metabolic and transcriptional response of P. putida KT2440 to starvation (Ankenbauer et al. 2020). A transcriptional regulation program linked to the intracellular pool of 3-hydroxyalkanoates, precursors of PHAs, was identified to be probably associated with accession of cellular PHA, amino acids, and glycogen to quickly restore ATP levels and the adenylate energy charge, a desired feature for large-scale bioreactors. In addition, 20 repeatedly upregulated ATP-consuming genes were promoted as possible technical targets. These genes are likely to be associated with long-term cellular programs and are superfluous in a bioreactor environment where glucose starvation is temporary (Ankenbauer et al. 2020).

Proteomics have been used to analyze mcl-PHA biosynthesis (Fu et al. 2015; Możejko-Ciesielska and Mostek 2019; Nikodinovic-Runic et al. 2009; Poblete-Castro et al. 2012b). Moreover, studies on post-transcriptional regulatory events, with vanillin and butanol as the sole carbon source, reported an increased abundance of enzymes of the tricarboxylic acid (TCA) cycle, indicating that this cycle is the main pathway for energy production when glucose is unavailable to P. putida (Simon et al. 2014; Simon et al. 2015). In addition, an increased abundance of efflux pumps and a rearrangement of transporter patterns have been shown during toluene exposure (Wijte et al. 2011). Recently, shotgun proteomics identified the oxoprolinase-encoding oplBA genes, responsible for the undesired hydrolysis of valerolactam P. putida, leading to a more efficient production process (Thompson et al. 2019). Besides, application of proteomics analysis and genome re-sequencing enhanced the understanding of the plastic monomer 1,4-butanediol metabolism in P. putida KT2440 (Li et al. 2020).

Metabolomics and fluxomics

The cyclic operation of the EDEMP pathway in P. putida has been proven by the combination of isotope labeling experiment, followed by quenching, extraction, and LC-MS analysis of intracellular intermediates of the central metabolism, and in vitro enzyme assays (Nikel et al. 2015; Sasnow et al. 2016). Lately, a high-resolution flux map of glucose-grown P. putida KT2440 was obtained from parallel labeling experiments using [1-¹³C]-, [6-¹³C]- and 50% [¹³C₆]-glucose, and straightforward GC–MS analysis of hydrolyzed cell pellets (Kohlstedt and Wittmann 2019). By then, GC–MS-based flux studies in P. putida were extremely limited, due the unresolvable cyclic network using common flux approaches. This was circumvented by deriving the labeling patterns for G6P and F6P from an extended approach that additionally included labeling information from glycogen, lipopolysaccharides, and peptidoglycan. The novel strategy displays a breakthrough for systems biology studies of P. putida (Mendonca et al. 2020) and related strains (Dolan et al. 2020). At a large, metabolic flux analysis has been foremost conducted with glucose as the sole carbon source (Kohlstedt and Wittmann 2019; Nikel et al. 2015; Sasnow et al. 2016); thus, the cyclic operation of the EDEMP pathway was also demonstrated using glycerol (Beckers et al. 2016; Dolan et al. 2020) as the sole carbon source. Furthermore, these metabolic flux studies revealed an inactive glyoxylate shunt during growth on the glycolytic substrate glucose (Sasnow et al. 2016), whereas the glyoxylate shunt was found active during growth on the gluconeogenic substrate glycerol (Beckers et al. 2016) or acetate (Dolan et al. 2020). A recent study has taken up this topic by using ¹³C-metabolomics to elucidate a carbon portioning of glucose and benzoate within the metabolic network. Carbon atoms derived from glucose were cycled through the EDEMP and PP pathway, whereas benzoate was preferentially catabolized through the TCA cycle and glyoxylate shunt and the atoms derived from this substrate did not enter the EDEMP nor the PP pathway. This segregation was shown to sustain biosynthetic flux demands (Kukurugya et al. 2019) and appears an interesting feature for some bio-production processes, where a multi-substrate feeding strategy can be advantageous.

Polyhydroxyalkanoates

Endopolymeric mcl-PHAs are naturally synthesized by P. putida as a carbon and energy storage compound under specific conditions, such as carbon excess during nutrient limitation (N, O₂, P, S) (Poblete-Castro et al. 2012b). The family of PHA polyesters is one of the best-known product classes studied in Pseudomonas species (Prieto et al. 2016). PHAs offer an excellent alternative for petroleum-based plastics, due to their biodegradability and competitive material properties, such as biocompatibility, lack of toxicity, insolubility, and thermostability (Mozejko-Ciesielska et al. 2019). The structural composition of PHAs can be adjusted by feeding of precursors (Wang et al. 2010), cultivation conditions, and strain engineering (Tripathi et al. 2013). The companies Kaneka (Japan), Telles (USA), Jiangsu Nantian (China), Tianjin GreenBio (China), Tepha (USA), DSM (The Netherlands), Biomer Biotechnology (Germany), Bio-on (Italy), Polyferm (Canada), and Biomatera (Canada) report to produce PHA polymers on an industrial scale using P. putida (Poltronieri and Kumar 2017). The global PHA market size is expected to grow from US$ 57 million in 2019 to US$ 98 by 2024, wherein the major constrain for growth is the cost competitiveness to conventional polymers (MarketsandMarkets™ 2019). The P. putida can produce mcl-PHAs from a broad spectrum of carbon sources, giving the opportunity to use cheap renewable feedstocks, such as crude glycerol from biodiesel production (Poblete-Castro et al. 2014), plant-derived fatty acids (Cerrone et al. 2014), food waste (Follonier et al. 2014), lignin-derived aromatics (Liu et al. 2017; Salvachúa et al. 2020a), and even non-degradable plastic waste, PET monomers (Kenny et al. 2008). In recent studies, the first production of PHAs from cellobiosan, levoglucosan (Linger et al. 2016), and cellobiose (Dvořák et al. 2020b) was reported. The P. putida EM42-expressing bglC from Thermobifida fusca, encoding β-glucosidase, accumulates mcl-PHA and concomitantly secretes xylonate, a platform chemical (Dvořák et al. 2020b). Such co-production of valuable bioproducts from low-cost substrates along with PHA represents a good opportunity to mitigate the overall PHA production costs (Li et al. 2017). Besides feedstock costs, downstream processing and product recovery is an important cost factor for the industrial production of intracellular compounds, which needs to be addressed to make the industrial application economical feasible. Some of the conventional methods include solvent extraction and chemical digestion. However, these methods are also marked with possible environmental drawbacks, high costs, or degradation of the polymer. Recently, a recovery of nearly 94% of the synthesized mcl-PHA after 3 h could be shown with cell disruption through a programmable cell lysis system in P. putida KT2440 engineered to respond to osmotic state (Poblete-Castro et al. 2020a). Taken together, recent achievements will pave the way to further reduce process costs at the level of raw material selection and downstream processing.

Alginates

Even though less studied, P. putida can produce exopolymeric alginates (Conti et al. 1994) and dehydration has been suggested as the general signal for the production of this polysaccharides (Chang et al. 2007). Alginate is a common additive to cosmetics and foods and has many medical applications. However, up to now, alginate production has been mainly studied in the pathogenic P. aeruginosa and there has been no commercial application associated to these efforts (Liu et al. 2019; Valentine et al. 2020).

Cis,cis-muconic acid

The arsenal of oxidoreductases, mono- and dioxygenases encoded in the genome of P. putida, enables this bacterium to degrade a variety of aromatic compounds found in the renewable feedstock lignin (Nogales et al. 2017). The aromatics are channeled through catabolic funneling via the β-ketoadipate (β-KA) pathway converging in only a few central intermediates (i.e., catechol, protocatechuate (PCA)), which are ring-cleaved and further converted into TCA cycle intermediates. One intermediate of this pathway, cis,cis-muconic acid (MA), is a very promising molecule, as it can be used as a starting material for the synthesis of value-added chemicals, such as caprolactam, terephthalic and adipic acid, as well as a bulk chemical in polymer reactions for the production of muconic homo- and copolymers (Khalil et al. 2020). Upon disruption of the degradation route at the level of muconate cycloisomerase (CatB), a stoichiometric conversion of aromatic substrates into MA can be achieved (van Duuren et al. 2011). Several studies reported successful MA production using P. putida KT2440 (Johnson et al. 2017; Johnson et al. 2016; Kohlstedt et al. 2018; Salvachúa et al. 2018; Sonoki et al. 2018; van Duuren et al. 2012; van Duuren et al. 2011; Vardon et al. 2015). In a landmark study, the biotransformation of a broad range of aromatic monomers, namely protocatechuate, ferulate, coniferyl alcohol, vanillin, caffeate, p-coumarate, 4-hydroxybenzoate (feeding into the PCA branch) and catechol, benzoate, and phenol (fueling the catechol branch), into MA was demonstrated. For this purpose, the PCA branch of β-KA pathway was connected to the catechol branch by heterologous expression of PCA decarboxylase encoded by the aroY gene from Enterobacter cloacae, with a simultaneous disruption of the natural PCA degradation route catalyzed by protocatechuate 3,4-dioxygenase (PcaHG). As a result and important proof of concept, 0.7 g L⁻¹ MA was produced from alkaline pretreated liquor—a waste stream from bioethanol production (Vardon et al. 2015). However, for some aromatic monomers tested, accumulation of intermediates was observed, indicating an inefficient conversion to MA (Vardon et al. 2015). Subsequently, improvement of MA production efficiency from aromatics of the PCA branch was achieved by increasing PCA decarboxylase activity via co-expression of accessory proteins ecdBD from E. cloacae (Johnson et al. 2016) and by tackling this bottleneck on the regulatory level by eliminating CCR (Johnson et al. 2017). Since all lignin-derived aromatics eventually merge at the level of the highly toxic intermediate catechol, this node is of interest as metabolic engineering target. In a seminal study, during processing of toxic catechol, cellular energy limitation was identified as a major challenge (Kohlstedt et al. 2018). To overcome this drawback on the operation side, implemented specific feed pauses during fed-batch fermentation allowed the regeneration of cellular energy levels and a final MA titer of 64.2 g L⁻¹ from catechol was obtained, exceeding previously reported values by more than tenfold. A second strategy reported in this study created superior KT2440 cell factories. Several rounds of metabolic engineering resulted in an improved platform strain for catechol conversion: increased catechol 1,2-dioxygenase activity by implementing a copy of the catA2 gene (natively directly downstream of the catA gene under control of the native P_cat promoter). Thereby, a strongly improved tolerance to catechol and a higher catechol degradation rate could be achieved. The developed process was transferred to pilot-scale–producing MA at the kilogram scale with 98% purity. Subsequently, by heterologous expression of a phenolhydroxylase, the strain is now able to additionally convert phenol into MA and cresols into methylated derivatives of MA. The final strain produced 13 g L⁻¹ MA from a softwood lignin hydrolysate (Kohlstedt et al. 2018). Engineered strains for MA are no longer able to grow on aromatics; therefore, glucose is commonly used as the growth substrate. Recently, a study reported glucose-independent MA production using engineered KT2440. This strain was able to produce small amounts (20 mg L⁻¹) of MA from depolymerized softwood lignin (Sonoki et al. 2018). Moreover, MA production was also achieved using glucose as the sole substrate (Bentley et al. 2020; Johnson et al. 2019).

Adipic acid and nylon 66

To showcase the entire value chain from lignin to bio-based nylon, MA underwent hydrogenation to adipic acid, a precursor for commercial nylon-66 (Kohlstedt et al. 2018; Vardon et al. 2015; Vardon et al. 2016). The combined chemical and biochemical process is displayed in Fig. ​Fig.6.6. A recent limited life cycle assessment concluded the feasibility of the bio-based production of adipic acid from softwood lignin-derived aromatics based on P. putida as the whole-cell biocatalyst (van Duuren et al. 2020) and even possible offset credits were promoted for the bioethanol biorefinery, if the lignin in the their wastewater is no longer burnt but converted to the value-added product adipic acid instead (Corona et al. 2018). Purification of MA produced by P. putida from lignin model compounds by activated carbon treatment was demonstrated with a good recovery at > 97% purity. Subsequently, Pd/C-aided catalytic hydrogenation of MA yielded bio-based adipic acid (Vardon et al. 2015).

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Fig. 6.

Cascaded biochemical and chemical route for nylon-66 production using metabolically engineered P. putida. Lignin depolymerization, microbial conversion of aromatic monomers to MA, subsequent product recovery and purification, hydrogenation to adipic acid and final polycondensation with hexamethylenediamine (HMDA) to nylon-66 (Kohlstedt et al. 2018)

Recently, the feasibility of direct de novo synthesis of adipic acid from lignin model compounds was demonstrated using P. putida KT2440 (Niu et al. 2020). To this end, the pathway design profited from the C6 structure of the β-KA pathway intermediate 3-ketoadipoyl-CoA, i.e., there is no need to exploit a condensation reaction between an acetyl-CoA and a succinyl-CoA unit as previously reported for adipic acid biosynthesis using glucose- or glycerol-grown E. coli (Cheong et al. 2016; Yu et al. 2014; Zhao et al. 2018).

2,5-Furandicarboxylic acid

The important platform chemical hydroxymethyl furfuraldehyde (HMF) can be derived from mono- and polysaccharides and pre-treated biomass (Dutta et al. 2012). Further catalytic oxidation of HMF yields the building block 2,5-furandicarboxylic acid (FDCA), which has potential applications in the production of plasticizers, polyamides, and polyesters (Corbion 2020; Sousa et al. 2015). One polymer of particular interest is polyethylene furanoate (PEF), a copolymer of ethylene glycol and FDCA, which represents an ideal substituent for polyethylene terephthalate (PET) in packaging, due to its exceptional thermal and superior barrier properties (Sousa et al. 2015). It may also serve as precursor for poly(1,4-cyclohexanedimethylene furandicarboxylate (PCF) (Wang et al. 2018). The global FDCA market is growing with an assessment at US$ 243.9 million in 2020 and is expected increase to US$ 321.3 million by the end of 2026 (QY Research 2020). Companies, such as Stora Enso (Stora Enso 2019), Novamont (Novamont 2019), and Avantium (2023: 5000 tons/year) (Avantium 2020) are currently entering the market with the construction of (pilot) plants for the production of biomass-derived FDCA. Whereas, chemocatalysis is the predominant route for the production of FDCA, which exhibits some disadvantages, such as costly metal catalysts, utilization of organic solvents, and the requirement of high temperature and pressure (Yuan et al. 2020). However, biocatalytic approaches are hindered by the toxic biological effects of furan aldehydes, attributable to ROS-induced oxidative stress (Allen et al. 2010; Almeida et al. 2009). Engineered P. putida KT2440 was able to grow on HMF and furfural as the sole carbon source after heterologous expression of the catabolic HMF and furfural gene clusters from Burkholderia phytofirmans (Guarnieri et al. 2017). For biotransformation of HMF to FDCA, P. putida S12 was chosen as a host, due to its natural tolerance to chemical stressors and the availability of endogenous aldehyde dehydrogenases to oxidize HMF to its corresponding, less toxic carboxylic acid (Koopman et al. 2010). By episomally expressing the oxidoreductase encoding hmfH gene from Cupriavidus basilensis HMF14, the efficient production of FDCA was reported for the first time, using a whole-cell transformation (Koopman et al. 2010). Subsequently, FDCA production was optimized by episomal coexpression of hmfH, aldehyde hydrogenase adh and hmfT1 transporter gene from C. basilensis HMF14 in P. putida, resulting in high, reproducible production rates (Wierckx et al. 2012). Recently, chromosomal co-integration of hmfH and hmfT1 in P. putida S12 was demonstrated by coupling λ-Red-mediated recombineering with CRISPR/Cas9 technology (Pham et al. 2020). Very promising, application of the patent dealing with FDCA production from HMF, using P. putida is filed by a subsidiary of Corbion, a company which is active in the industrial production of PEF derived from FDCA (Corbion ; Wierckx et al. 2012).

Aromatics

The natural tolerance and ability to metabolize aromatics have been exploited for the region-selective bioproduction of 3-methylcatechol (Hüsken et al. 2001), the bioproduction of o-cresol from toluene in a two-phase 1-octanol/water system (Faizal et al. 2006), as well as epoxidation of styrene in a water/octanol two-liquid phase system (Blank et al. 2008). Other produced building blocks include pyruvate and lactate from p-coumarate and benzoate (Johnson and Beckham 2015). Moreover, the bioconversion of ferulic acid to the flavor agent vanillin was achieved with resting cells (Graf and Altenbuchner 2014). Interestingly, in this study, the sole deletion of the vanillin dehydrogenase gene (vdh) did not suppress degradation of the later and one hitherto unknown molybdate-dependent oxidoreductase was found to probably complement the vdh inactivation. The natural tolerance of P. putida towards aromatic compounds has not only been exploited for bioconversions and -transformations, but also for de novo synthesis of aromatic compounds originating from shikimate pathway intermediates. Using the solvent-tolerant P. putida S12 strain production of cinnamic acid (Nijkamp et al. 2005), phenol in a biphasic fed batch cultivation system with octanol (Wierckx et al. 2005) and p-hydroxystyrene in a water/1-decanol phase (Verhoef et al. 2009) were reported. Anthranilate, p-coumaric acid, and para-hydroxybenzoic acid were synthesized from glucose by KT2440-derived strains (Calero et al. 2016; Kuepper et al. 2015; Yu et al. 2016).

Biosurfactants

Rhamnolipids are biodegradable and low toxic biosurfactants. The activity of biosurfactants enhances the solubility of hydrophobic molecules in water by decreasing the surface tension (Rehm 2009). Potential applications can be found in food industry, cosmetics, cleaning agents, biocontrol, and soil remediation (Fracchia et al. 2014; Loeschcke and Thies 2015). The P. putida is able to produce short-chain rhamnolipids after heterologous integration of the rhlAB(C) operon from P. aeruginosa (Tiso et al. 2017; Wittgens et al. 2011) and long-chain rhamnolipids after expression of rhl genes from Burkholderia glumae (Wittgens et al. 2018). Wherein, the chain length depends on the respective expressed rhl genes rather than the available 3-hydroxy fatty acids, a property which can be used for the production of tailor-made rhamnolipids (Wittgens et al. 2018). The chemical company Evonik Industries (Essen, Germany) announced the first industrial production of rhamnolipids in 2016 (Evonik Industries 2016), followed by an issued patent in 2017 for rhamnolipid production from n-butane by P. putida KT2440 (Thum et al. 2017).

Terpenoids

The high tolerance of P. putida against the toxicity of intermediates or terminal products has proven to be advantageous to produce terpenoids, one of the largest and structurally diverse groups of natural compounds. Taking the natural resistance into advantage, the successful de novo production of geranic acid, a monoterpenoid with several reported antibiotic activities and uses as flavor and fragrance agent, could be shown. This study also revealed a remarkably higher resistance of P. putida to more than 6-fold higher product concentrations in comparison to E. coli and Saccharomyces cerevisiae (Mi et al. 2014). Furthermore, oxy-functionalized derivatives of specific terpenoids are sometimes desired for their specific bioactivities. Naturally, such derivatives only occur in low amounts and both their isolation and their chemical synthesis are rather uneconomical. Thus, in vivo enzymatic transformation of toxic monoterpenoids by heterologously expressed cytochrome P450 monooxygenases as an alternative is under study. As an example, the conversion of limonene to perillic acid (Mirata et al. 2009; van Beilen et al. 2005) and the hydroxylation of 1,8-cineole (Mi et al. 2016) was achieved. In addition, another class of terpenoids, the carotenoids zeaxanthin and β-carotene, has been successfully produced in P. putida (Beuttler et al. 2011; Loeschcke et al. 2013). In this context, the astonishing metabolic plasticity of P. putida was demonstrated by reshaping the native, cyclical EDEMP pathway into a linear EMP pathway by heterologous expression of glycolytic genes from E. coli. This led to increased levels of PYR and G3P, both precursors of carotenoid production, resulting in 1.3-fold increase in carotenoid production compared with the parental strain (Sánchez-Pascuala et al. 2019). Further increase of precursor supply seems to be a crucial factor for efficient production.

Polyketides and non-ribosomal peptides

Polyketides (PKs) and non-ribosomal peptides (NRPs) display diverse groups of natural products that have commonly medically relevant activities. Both are assembled by condensation of simple carboxylic or amino acid building blocks. The produced polymers can be cyclized and decorated to form the final product. The PKs 2,4-diacetylphloroglucinol (Martinez et al. 2004) and the UV-protective pigment flaviolin (Gross et al. 2006a) were successfully produced in P. putida. An interesting PK/NRP hybrid compound is the antibiotic prodigiosin, due to its anticancer and immunosuppressant activities (Domröse et al. 2015). In E. coli prodigiosin transcription is inhibited (Danevčič et al. 2016). Yet, this is not the case for P. putida. The TREX expression system, which includes elements of transposon Tn5, enabled the random chromosomal integration of the pig gene cluster, derived from the native producer Serratia marcescens W83. Remarkably, a higher titer could be reached using a strong intrinsic chromosomal promoter (Domröse et al. 2015) compared with utilization of a synthetic T7 RNA polymerase-dependent promoter (Loeschcke et al. 2013). This study led to the discovery of rRNA promoters as strong native promoters for heterologous expression of biosynthetic gene clusters in P. putida (Domröse et al. 2019). Furthermore, through the expression of myxobacterial hybrid systems, the antibiotics myxochromide S (Stephan et al. 2006) and tubulysin (Chai et al. 2012) could be synthesized. Since many of the biosynthetic gene clusters required for the production of natural products are larger than 6 kb (Loeschcke and Thies 2015), sophisticated tools are needed for chromosomal integration. One such tool is the recent developed RecET-based markerless recombineering system for P. putida (Choi and Lee 2020). The site-specific integration of a 7.4-kb violacein cluster could be shown (Choi et al. 2018). Moreover, the first synthetic polyketide synthase-like pathway for the production of the omega-3 fatty acid docosahexaenoic acid (DHA) was successfully expressed in P. putida KT2440 (Gemperlein et al. 2016).