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The present study examined the regulatory and metabolic response of the aromatic degrader Pseudomonas putida F1 and its tod operon, controlling toluene degradation, to fluorinated aromatic and aliphatic compounds. The tod operon is upregulated by inducer binding to the TodS sensing domain of a two-component regulator. The induced enzymes include toluene dioxygenase that initiates catabolic assimilation of benzenoid hydrocarbons. Toluene dioxygenase was shown to oxidize 6-fluoroindole to a meta-stable fluorescent product, 6-fluoroindoxyl. The fluorescent output allowed monitoring relative levels of tod operon induction in whole cells using microtiter well plates. Mono- and polyfluorinated aromatic compounds were shown to induce toluene dioxygenase, in some cases to a greater extent than compounds serving as growth substrates. Compounds that are oxidized by toluene dioxygenase and undergoing defluorination were shown to induce their own metabolism. 1,2,4-Trifluorobenzene caused significant induction and computational modelling indicated productive binding to the TodS sensor domain of the TodST regulator. Toluene dioxygenase also showed preferential binding of 1,2,4-trifluorobenzene such that defluorination was favoured. Fluorinated aliphatic compounds were shown to induce toluene dioxygenase. An aliphatic ether with seven fluorine atoms, 1,1,1,2-tetrafluoro-2-trifluoromethoxy-4-iodobutane (TTIB), was an excellent inducer of toluene dioxygenase activity and shown to undergo transformation in cultures of P. putida F1.

Lignin is a largely untapped source for the bioproduction of value-added chemicals. Pseudomonas putida KT2440 has emerged as a strong candidate for bioprocessing of lignin feedstocks due to its resistance to several industrial solvents, broad metabolic capabilities, and genetic amenability. Here we demonstrate the engineering of P. putida for the ability to metabolize syringic acid, one of the major products that comes from the breakdown of the syringyl component of lignin. The rational design was first applied for the construction of strain Sy-1 by overexpressing a native vanillate demethylase. Subsequent adaptive laboratory evolution (ALE) led to the generation of mutations that achieved robust growth on syringic acid as a sole carbon source. The best mutant showed a 30% increase in the growth rate over the original engineered strain. Genomic sequencing revealed multiple mutations repeated in separate evolved replicates. Reverse engineering of mutations identified in agmR, gbdR, fleQ, and the intergenic region of gstB and yadG into the parental strain recaptured the improved growth of the evolved strains to varied extent. These findings thus reveal the ability of P. putida to utilize lignin more fully as a feedstock and make it a more economically viable chassis for chemical production.

Assistive eco-physiological traits are necessary for microbes to adapt and colonize at polluted niches, enabling efficient clean-up. To demarcate species distinctiveness and eco-physiological traits of aromatic compounds metabolizing Pseudomonas sp. CSV86

Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two-component system regulators CbrAB, NtrBC and PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C-P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2-aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant-growth-promoting rhizobacterium Pseudomonas putida BIRD-1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR-type regulator, termed AepR, upstream of the 2AEP transaminase-phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate-specific regulators in phosphonate metabolism. [Abstract copyright: © 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.]

Type VI secretion systems (T6SS) are specific antibacterial weapons employed by diverse bacteria to protect themselves from competitors. Pseudomonas putida KT2440 possesses a functional T6SS (K1-T6SS) and exhibits antibacterial activity towards a broad range of bacteria. Here we found that the Wsp signal transduction system regulated K1-T6SS expression via synthesizing the second messenger cyclic di-GMP (c-di-GMP), thus mediating antibacterial activity in P. putida. High-level c-di-GMP produced by Wsp system repressed the transcription of K1-T6SS genes in structural operon and vgrG1 operon. Transcriptional regulator FleQ and ATPase FleN functioned as repressors in the Wsp system-modulated K1-T6SS transcription. However, FleQ and FleN functioned as activators in biofilm formation, and Wsp system promoted biofilm formation largely in a FleQ/FleN-dependent manner. Furthermore, FleQ-FleN complex bound directly to the promoter of K1-T6SS structural operon in vitro, and c-di-GMP promoted the binding. Besides, P. putida biofilm cells showed higher c-di-GMP levels and lower antibacterial activity than planktonic cells. Overall, our findings reveal a mechanism by which Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ-FleN, and demonstrate the relationship between plankton/biofilm lifestyles and antibacterial activity in P. putida.

Summary 5‐Ketofructose (5‐KF) is a promising low‐calorie natural sweetener with the potential to reduce health problems caused by excessive sugar consumption. It is formed by periplasmic oxidation of fructose by fructose dehydrogenase (Fdh) of Gluconobacter japonicus, a membrane‐bound three‐subunit enzyme containing FAD and three haemes c as prosthetic groups. This study aimed at establishing Pseudomonas putida KT2440 as a new cell factory for 5‐KF production, as this host offers a number of advantages compared with the established host Gluconobacter oxydans. Genomic expression of the fdhSCL genes from G. japonicus enabled synthesis of functional Fdh in P. putida and successful oxidation of fructose to 5‐KF. In a batch fermentation, 129 g l−1 5‐KF were formed from 150 g l−1 fructose within 23 h, corresponding to a space‐time yield of 5.6 g l−1 h−1. Besides fructose, also sucrose could be used as substrate for 5‐KF production by plasmid‐based expression of the invertase gene inv1417 from G. japonicus. In a bioreactor cultivation with pulsed sucrose feeding, 144 g 5‐KF were produced from 358 g sucrose within 48 h. These results demonstrate that P. putida is an attractive host for 5‐KF production., 5‐Ketofructose is a promising candidate to serve as a non‐nutritive sweetener. It is formed by periplasmic oxidation of fructose with a membrane‐associated fructose dehydrogenase FdhSCL from Gluconobacter japonicus that involves an FAD‐containing subunit and a trihaem cytochrome c. In this study, we demonstrate that Pseudomonas putida KT2440 can functionally express the fdhSCL genes and serve as an efficient producer of 5‐ketofructose from fructose, but also from sucrose, which is split by a periplasmic invertase also derived from G. japonicus.

Summary Whole‐cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco‐efficient route to produce phenolic amines. Here, a novel cell growth‐based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp‐SpuC‐II and ATA from Chromobacterium violaceum (Cv‐ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp‐SpuC‐II and Cv‐ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94‐ and 92‐fold increased specific transaminase activity, respectively. Whole‐cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp‐SpuC‐II and Cv‐ATA, respectively. Still, amine production was limited by a substantial re‐assimilation of the product and formation of the by‐products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv‐ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re‐assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement., In this study, Pseudomonas putida was engineered for whole‐cell conversion of ferulic acid and vanillin to vanillylamine with ammonium as nitrogen source. Furthermore, a novel growth‐based screening method to select for recombinant vanillin transaminases was developed and used to identify SpuC‐II and Cv‐ATA as suitable enzymes for in vivo biocatalysis with P. putida.

Summary The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. A number of inducible promoters (P lac , P tac , P tetR/tetA and P bad ) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype., To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose.

Pseudomonas putida is a highly solvent-resistant microorganism and useful chassis for the production of value-added compounds from lignocellulosic residues, in particular aromatic compounds that are made from phenylalanine. The use of these agricultural residues requires a two-step treatment to release the components of the polysaccharides of cellulose and hemicellulose as monomeric sugars, the most abundant monomers being glucose and xylose. Pan-genomic studies have shown that Pseudomonas putida metabolizes glucose through three convergent pathways to yield 6-phosphogluconate and subsequently metabolizes it through the Entner–Doudoroff pathway, but the strains do not degrade xylose. The valorization of both sugars is critical from the point of view of economic viability of the process. For this reason, a P. putida strain was endowed with the ability to metabolize xylose via the xylose isomerase pathway, by incorporating heterologous catabolic genes that convert this C5 sugar into intermediates of the pentose phosphate cycle. In addition, the open reading frame T1E_2822, encoding glucose dehydrogenase, was knocked-out to avoid the production of the dead-end product xylonate. We generated a set of DOT-T1E-derived strains that metabolized glucose and xylose simultaneously in culture medium and that reached high cell density with generation times of around 100 min with glucose and around 300 min with xylose. The strains grew in 2G hydrolysates from diluted acid and steam explosion pretreated corn stover and sugarcane straw. During growth, the strains metabolized > 98% of glucose, > 96% xylose and > 85% acetic acid. In 2G hydrolysates P. putida 5PL, a DOT-T1E derivative strain that carries up to five independent mutations to avoid phenylalanine metabolism, accumulated this amino acid in the medium. We constructed P. putida 5PLΔgcd (xylABE) that produced up to 250 mg l of phenylalanine when grown in 2G pretreated corn stover or sugarcane straw. These results support as a proof of concept the potential of P. putida as a chassis for 2G processes., MINECO (Grant / Award Number:’RTI2018-094370-B-I00)

Pseudomonas sp. CSV86, an Indian soil isolate, degrades wide range of aromatic compounds like naphthalene, benzoate and phenylpropanoids, amongst others. Isolate displays the unique and novel property of preferential utilization of aromatics over glucose and co-metabolizes them with organic acids. Interestingly, as compared to other Pseudomonads, strain CSV86 harbours only high-affinity glucokinase pathway (and absence of low-affinity oxidative route) for glucose metabolism. Such lack of gluconate loop might be responsible for the novel phenotype of preferential utilization of aromatics. The genome analysis and comparative functional mining indicated a large genome (6.79 Mb) with significant enrichment of regulators, transporters as well as presence of various secondary metabolite production clusters, suggesting its eco-physiological and metabolic versatility. Strain harbours various integrative conjugative elements (ICEs) and genomic islands, probably acquired through horizontal gene transfer events, leading to genome mosaicity and plasticity. Naphthalene degradation genes are arranged as regulonic clusters and found to be part of ICECSV86nah . Various eco-physiological properties and absence of major pathogenicity and virulence factors (risk group-1) in CSV86 suggest it to be an ideal candidate for bioremediation. Further, strain can serve as an ideal chassis for metabolic engineering to degrade various xenobiotics preferentially over simple carbon sources for efficient remediation.

BACKGROUND AND OBJECTIVES Frozen plasma (FP) is thawed prior to transfusion and stored for ≤5 days at 1-6°C. The effect of temperature excursions on the quality and safety of thawed plasma during 5-day storage was determined. MATERIALS AND METHODS Four plasma units were pooled, split and stored at ≤-18°C for ≤90 days. Test units T30 and T60 were exposed to 20-24°C (room temperature [RT]) for 30 or 60 min, respectively, on days 0 and 2 of storage. Negative and positive control units remained refrigerated or at RT for 5 days, respectively. On Day 5, test units were exposed once to RT for 5 h. Quality assays included stability of coagulation factors FV, FVII, FVIII, fibrinogen and prothrombin time. Bacterial growth was performed in units inoculated with ~1 CFU/ml or ~100 CFU/ml of Serratia liquefaciens, Pseudomonas putida, Pseudomonas aeruginosa or Staphylococcus epidermidis on Day 0. RESULTS Testing results of all quality parameters were comparable between T30 and T60 units (p

Summary Understanding metabolism is fundamental to access and harness bacterial physiology. In most bacteria, nutrient utilization is hierarchically optimized according to their energetic potential and their availability in the environment to maximise growth rates. Low‐throughput methods have been largely used to characterize bacterial metabolic profiles. However, in‐depth analysis of large collections of strains across several conditions is challenging since high‐throughput approaches are still limited – especially for non‐traditional hosts. Here, we developed a high‐throughput dilution‐resolved cultivation method for metabolic footprinting of Pseudomonas putida and Pseudomonas aeruginosa. This method was benchmarked against a conventional low‐throughput time‐resolved cultivation approach using either a synthetic culture medium (where a single carbon source is present) for P. putida or a complex nutrient mixture for P. aeruginosa. Dynamic metabolic footprinting, either by sugar quantification or by targeted exo‐metabolomic analyses, revealed overlaps between the bacterial metabolic profiles irrespective of the cultivation strategy, suggesting a certain level of robustness and flexibility of the high‐throughput dilution‐resolved method. Cultivation of P. putida in microtiter plates imposed a metabolic constraint, dependent on oxygen availability, which altered the pattern of secreted metabolites at the level of sugar oxidation. Deep‐well plates, however, constituted an optimal cultivation set‐up yielding consistent and comparable metabolic profiles across conditions and strains. Altogether, the results illustrate the usefulness of this technological advance for high‐throughput analyses of bacterial metabolism for both biotechnological applications and automation purposes.

Summary The ability to directly modify native and established biofilms has enormous potential in understanding microbial ecology and application of biofilm in 'real‐world' systems. However, efficient genetic transformation of established biofilms at any scale remains challenging. In this study, we applied an ultrasound‐mediated DNA delivery (UDD) technique to introduce plasmid to established non‐competent biofilms in situ. Two different plasmids containing genes coding for superfolder green fluorescent protein (sfGFP) and the flavin synthesis pathway were introduced into established bacterial biofilms in microfluidic flow (transformation efficiency of 3.9 ± 0.3 × 10‐7 cells in biofilm) and microbial fuel cells (MFCs), respectively, both employing UDD. Gene expression and functional effects of genetically modified bacterial biofilms were observed, where some cells in UDD‐treated Pseudomonas putida UWC1 biofilms expressed sfGFP in flow cells and UDD‐treated Shewanella oneidensis MR‐1 biofilms generated significantly (P, In this study, we applied an ultrasound‐mediated DNA delivery (UDD) technique to introduce plasmids containing genes coding for superfolder green fluorescent protein (sfGFP) and the flavin synthesis pathway into established bacterial biofilms in microfluidic flow and microbial fuel cells (MFCs) respectively. Gene expression and functional effects of genetically modified bacterial biofilms were observed, where some cells in UDD‐treated Pseudomonas putida UWC1 biofilms expressed sfGFP in flow cells and UDD‐treated Shewanella oneidensis MR‐1 biofilms generated significantly greater (61%) bioelectricity production in MFC than a wild‐type control group. The effects of UDD were amplified in subsequent growth under selection pressure due to antibiotic resistance and metabolism enhancement. This is the first study to report on a potentially scalable direct genetic engineering method for established non‐competent biofilms, which can be exploited in enhancing their capability towards environmental, industrial and medical applications.

Summary Here, we present an improved whole‐cell biocatalysis system for the synthesis of heteroaromatic N‐oxides based on the production of a soluble di‐iron monooxygenase PmlABCDEF in Pseudomonas sp. MIL9 and Pseudomonas putida KT2440. The presented biocatalysis system performs under environmentally benign conditions, features a straightforward and inexpensive procedure and possesses a high substrate conversion and product yield. The capacity of gram‐scale production was reached in the simple shake‐flask cultivation. The template substrates (pyridine, pyrazine, 2‐aminopyrimidine) have been converted into pyridine‐1‐oxide, pyrazine‐1‐oxide and 2‐aminopyrimidine‐1‐oxide in product titres of 18.0, 19.1 and 18.3 g l‐1, respectively. To our knowledge, this is the highest reported productivity of aromatic N‐oxides using biocatalysis methods. Moreover, comparing to the chemical method of aromatic N‐oxides synthesis based on meta‐chloroperoxybenzoic acid, the developed approach is applicable for a regioselective oxidation that is an additional advantageous option in the preparation of the anticipated N‐oxides., In this study, we describe a new whole‐cell biocatalysis system for aromatic N‐oxides synthesis based on production of soluble di‐iron monooxygenase PmlABCDEF. Various pyridine, pyrazine, and pyrimidine derivatives could be converted into appropriate N‐oxides on a preparative scale in the simple shake‐flask cultivation. This improved synthesis method was used to yield specific pyrazine and pyrimidine oxidation products that were difficult to obtain by employing a standard mCPBA‐based technique.

Polycyclic aromatic hydrocarbons (PAHs) and dioxin-like compounds, including sulfur, nitrogen and oxygen heterocycles, are widespread and toxic environmental pollutants. A wide variety of microorganisms capable of growing with aromatic polycyclic compounds are essential for bioremediation of the contaminated sites and the Earth's carbon cycle. Here, cells of Pseudomonas putida B6-2 (ATCC BAA-2545) grown in the presence of biphenyl (BP) are able to simultaneously degrade PAHs and their derivatives, even when they are present as mixtures, and tolerate high concentrations of extremely toxic solvents. Genetic analysis of the 6.37 Mb genome of strain B6-2 reveals coexistence of gene clusters responsible for central catabolic systems of aromatic compounds and for solvent tolerance. We used functional transcriptomics and proteomics to identify the candidate genes associated with catabolism of BP and a mixture of BP, dibenzofuran, dibenzothiophene and carbazole. Moreover, we observed dynamic changes in transcriptional levels with BP, including in metabolic pathways of aromatic compounds, chemotaxis, efflux pumps and transporters potentially involved in adaptation to PAHs. This study on the highly versatile activities of strain B6-2 suggests it to be a potentially useful model for bioremediation of polluted sites and for investigation of biochemical, genetic and evolutionary aspects of Pseudomonas.

Two-component systems (TCSs) are predominant means by which bacteria sense and respond to environment signals. Genome of Pseudomonas putida contains dozens of putative TCS-encoding genes, but phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterized function and transcriptional regulation of a conserved P. putida TCS, named TarR-TarS. TarS (PP_0769) encodes a potential histidine kinase, and tarR (PP_0768) encodes a potential response regulator. Protein-protein interaction assay and phosphorylation assay confirmed that TarR-TarS was a functional TCS. Growth assay under antibiotics revealed that TarR-TarS positively regulated bacterial resistance to multiple antibiotics. Pull-down assay revealed that TarR directly interacted with PP_0800 (a hypothetical protein) and GroEL (the chaperonin). GroEL played a positive role in antibiotic resistance, while PP_0800 seemed to have no effect on antibiotic resistance. The regulator FleQ indirectly activated tarR-tarS transcription. However, the second messenger c-di-GMP antagonized FleQ activation to inhibit tarR-tarS transcription. The sigma factor FliA directly activated tarR-tarS transcription via a consensus motif. These findings reveal function and transcriptional regulation of TarR-TarS, and enrich knowledge regarding the relationship between c-di-GMP and antibiotic susceptibility in P. putida.

The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. Many bacteria alter their PG chemistry as a strategy to adapt their cell wall to external challenges. Therefore, identifying these environmental cues is important to better understand the interplay between microbes and their habitat. Here we used the soil bacterium Pseudomonas putida to uncover cell wall modulators from plant extracts and found canavanine (CAN), a non-proteinogenic amino acid. We demonstrated that cell wall chemical editing by CAN is licensed by P. putida BSAR, a broad-spectrum racemase which catalyzes production of DL-CAN from L-CAN, which is produced by many legumes. Importantly, D-CAN diffuses to the extracellular milieu thereby having a potential impact on other organisms inhabiting the same niche. Our results show that D-CAN alters dramatically the PG structure of Rhizobiales (e.g. Agrobacterium tumefaciens, Sinorhizobium meliloti), impairing PG crosslinkage and cell division. Using A. tumefaciens we demonstrated that the detrimental effect of D-CAN is suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin binding protein 3a. Collectively, this work highlights the role of amino acid racemization in cell wall chemical editing and fitness. This article is protected by copyright. All rights reserved.

Summary Propionic acid (PA) has been widely used as a food preservative and chemical intermediate in the agricultural and pharmaceutical industries. Environmental and friendly biotechnological production of PA from biomass has been considered as an alternative to the traditional petrochemical route. However, because PA is a strong inhibitor of cell growth, the biotechnological host should be not only able to produce the compound but the host should be robust. In this study, we identified key PA tolerance factors in Pseudomonas putida KT2440 strain by comparative transcriptional analysis in the presence or absence of PA stress. The identified major facilitator superfamily (MFS) transporter gene cluster of PP_1271, PP_1272 and PP_1273 was experimentally verified to be involved in PA tolerance in P. putida strains. Overexpression of this cluster improved tolerance to PA in a PA producing strain, what is useful to further engineer this robust platform not only for PA synthesis but for the production of other weak acids., In this study, one major facilitator superfamily transporter (MFS2, locus tags of PP_1271‐PP_1273), but not MFS1 (locus tag of PP_0503), is beneficial to propionate tolerance in Pseudomonas putida strain KT2440. Thus, this study revealed the key genes responsible for PA tolerance in P. putida strains and provided the useful information to further engineer robust strains for PA production.

Rhamnlopids are biosurfactants with a wide range of industrial applications. The understanding of their biosynthesis and the genetic regulation of their production have impacted the development of strains that are suitable for industrial production of these biosurfactants., Summary Rhamnolipids are biosurfactants with a wide range of industrial applications that entered into the market a decade ago. They are naturally produced by Pseudomonas aeruginosa and some Burkholderia species. Occasionally, some strains of different bacterial species, like Pseudomonas chlororaphis NRRL B‐30761, which have acquired RL‐producing ability by horizontal gene transfer, have been described. P. aeruginosa, the ubiquitous opportunistic pathogenic bacterium, is the best rhamnolipids producer, but Pseudomonas putida has been used as heterologous host for the production of this biosurfactant with relatively good yields. The molecular genetics of rhamnolipids production by P. aeruginosa has been widely studied not only due to the interest in developing overproducing strains, but because it is coordinately regulated with the expression of different virulence‐related traits by the quorum‐sensing response. Here, we highlight how the research of the molecular mechanisms involved in rhamnolipid production have impacted the development of strains that are suitable for industrial production of this biosurfactant, as well as some perspectives to improve these industrial useful strains.

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products ("normal" and "abnormal") when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (kcat /KM ) 8.9-fold to 484 s-1 mM-1 for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMOC308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L-1 d-1 . This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.

Degradation of the fungicide iprodione by the Paenarthrobacter sp. strain YJN-5 is initiated via hydrolysis of its N1 amide bond to form N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine. In this study, another iprodione-degrading strain, Paenarthrobacter sp. YJN-D, which harbours the same metabolic pathway as strain YJN-5 was isolated and characterized. The genes that encode the conserved iprodione catabolic pathway were identified based on comparative analysis of the genomes of the two iprodione-degrading Paenarthrobacter sp. and subsequent experimental validation. These genes include an amidase gene, ipaH (previously reported in AEM e01150-18); a deacetylase gene, ddaH, which is responsible for hydantoin ring cleavage of N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine, and a hydrolase gene, duaH, which is responsible for cleavage of the urea side chain of (3,5-dichlorophenylurea)acetic acid, thus yielding 3,5-dichloroaniline as the end product. These iprodione-catabolic genes are distributed on three plasmids in strain YJN-5 and are highly conserved between the two iprodione-degrading Paenarthrobacter strains. However, only the ipaH gene is flanked by a mobile genetic element. Two iprodione degradation cassettes bearing ipaH-ddaH-duaH were constructed and expressed in strains Pseudomonas putida KT2440 and Bacillus subtilis SCK6 respectively. Our findings enhance the current understanding of the microbial degradation mechanism of iprodione.

The role of archetypal ribonucleases (RNases) in the physiology and stress endurance of the soil bacterium and metabolic engineering platform Pseudomonas putida KT2440 has been inspected. To this end, variants of this strain lacking each of the most important RNases were constructed. Each mutant lacked either one exoribonuclease (PNPase, RNase R) or one endoribonuclease (RNase E, RNase III, RNase G). The global physiological and metabolic costs of the absence of each of these enzymes were then analysed in terms of growth, motility and morphology. The effects of different oxidative chemicals that mimic the stresses endured by this microorganism in its natural habitats were studied as well. The results highlighted that each ribonuclease is specifically related with different traits of the environmental lifestyle that distinctively characterizes this microorganism. Interestingly, the physiological responses of P. putida to the absence of each enzyme diverged significantly from those known previously in Escherichia coli. This exposed not only species-specific regulatory functions for otherwise known RNase activities but also expanded the panoply of post-transcriptional adaptation devices that P. putida can make use of for facing hostile environments.

The activity of individual microorganisms can be measured within environmental samples by detecting uptake of isotope-labelled substrates using nano-scale secondary ion mass spectrometry (nanoSIMS). Recent studies have demonstrated that sample preparation can decrease 13 C and 15 N enrichment in bacterial cells, resulting in underestimates of activity. Here, we explore this effect with a variety of preparation types, microbial lineages and isotope labels to determine its consistency and therefore potential for correction. Specifically, we investigated the impact of different protocols for fixation, nucleic acid staining and catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) on >14 500 archaeal and bacterial cells (Methanosarcina acetivorans, Sulfolobus acidocaldarius and Pseudomonas putida) enriched in 13 C, 15 N, 18 O, 2 H and/or 34 S. We found these methods decrease isotope enrichments by up to 80% - much more than previously reported - and that the effect varies by taxa, growth phase, isotope label and applied protocol. We make recommendations for how to account for this effect experimentally and analytically. We also re-evaluate published nanoSIMS datasets and revise estimated microbial turnover times in the marine subsurface and nitrogen fixation rates in pelagic unicellular cyanobacteria. When sample preparation is accounted for, cell-specific rates increase and are more consistent with modelled and bulk rates.

The coexistence of multiple homologous resistance-nodulation-division (RND) efflux pumps in bacteria is frequently described with overlapping substrate profiles. However, it is unclear how bacteria balance their transcription in response to the changing environment. Here, we characterized a repressor, SrpR, in Pseudomonas putida B6-2 (DSM 28064), whose coding gene is adjacent to srpS that encodes the local repressor of the RND-type efflux pump SrpABC gene cluster. SrpR was demonstrated as a specific repressor of another RND efflux pump gene cluster ttgABC that is locally repressed by TtgR. SrpR was found to be capable of binding to the ttgABC operator with a higher affinity (KD , 138.0 nM) compared to TtgR (KD , 15.4 μM). EMSA and β-galactosidase assays were performed to survey possible effectors of SrpR with 35 available chemicals being tested. Only 2,3,4-trichlorophenol was identified as an effector of SrpR. A regulation model was then proposed, representing a novel strategy for balancing the efflux systems with partially overlapping substrate profiles. This study highlights sophisticated interactions among the RND efflux pumps in a Pseudomonas strain, which may endow bacteria with certain advantages in a fluctuant environment.

Widespread antibiotic resistance genes (ARGs) have caused critical threats to public health on a global scale. Soil, composed mainly of minerals, acts as a source of resistance determinants, playing a considerable role in the development and dissemination of ARGs. The great abundance of ARGs in the soil environment raises concern about the effect of minerals on the spread of ARGs. Herein, the horizontal transfer of a model plasmid pMP2463 containing ARGs from Escherichia coli S17‐1 to Pseudomonas putida KT2440 was monitored following exposure to four common soil minerals, namely, kaolinite, montmorillonite, goethite and birnessite. Birnessite resulted in concentration‐dependent increases in conjugative transfer of plasmid pMP2463 by 1.3–4.3 fold, compared with that in the control group. However, no obvious laws were found in the change of conjugative transfer rate at different concentrations of kaolinite and montmorillonite. As for goethite, the conjugative transfer rate increased firstly and then decreased as the concentration increased. The possible mechanisms underlying birnessite‐induced conjugative transfer of plasmid were explored; birnessite is capable of initiating intracellular reactive oxygen species (ROS) formation, inducing the oxidative stress response. Additionally, birnessite notably facilitated the mRNA expression of the outer membrane protein genes, which contributed to cellular membrane pore formation and horizontal gene transfer, and altered the mRNA expression of conjugative‐related genes that are responsible for conjugative transfer of mobile genetic elements between bacteria. This study triggers questions regarding the potential role of soil minerals in the global dissemination of antimicrobial resistance and provides insights into the interactions between bacteria and minerals in the natural soil environment. HIGHLIGHTS: The effect of soil minerals on conjugative transfer of ARGs was explored. Birnessite induced the concentration‐dependent increase of conjugative transfer. Mechanisms underlying ROS formation, membrane permeability increase, gene expression alteration were revealed. Our results provide evidence for the effect of birnessite on antibiotic resistance dissemination.

Mediator‐based extracellular electron transfer (EET) pathways can balance the redox metabolism of microbes. However, such electro‐biosynthesis processes are constrained by the unknown underlying EET mechanisms. In this paper, Pseudomonas putida was studied to systematically investigate its EET pathway to transition metal complexes (i. e., [Fe(CN)6]3−/4− and [Co(bpy)3]3+/2+; bpy=2,2′‐bipyridyl) under anaerobic conditions. Comparative proteomics showed the aerobic respiratory components were upregulated in a bioelectrochemical system without oxygen, suggesting their potential contribution to EET. Further tests found inhibiting cytochrome c oxidase activity by NaN3 and NADH dehydrogenase by rotenone did not significantly change the current output. However, the EET pathway was completely blocked, while cytochrome c reductase activity was inhibited by antimycin A. Although it cannot be excluded that cytochrome c and the periplasmic subunit of cytochrome c oxidase donate electrons to the transition metal complexes, these results strongly demonstrate that cytochrome c reductase is a key complex for the EET pathway., Liquid wires for biohybrid systems: The electron transfer pathway between redox metal‐complexes and biological respiratory proteins is systematically investigated and revealed. Such a pathway can be readily applied to many non‐electrogenic microorganisms and thus expand the application of microbial electrochemical technology in many fields such as industrial biotechnology and biosensors.

Pseudomonas isolates from tropical environments have been underexplored and may form an untapped reservoir of interesting secondary metabolites. In this study, we compared Pseudomonas and cyclic lipopeptide (CLP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. Compared with other soils, Boteva andosols were characterized by high silt, organic matter, nitrogen and calcium. Besides, the cocoyam rhizosphere at Boteva was characterized by strains belonging mainly to the P. koreensis and P. putida (sub)groups, with representations in the P. fluorescens, P. chlororaphis, P. jessenii and P. asplenii (sub)groups. In contrast, P. putida isolates were prominent in conducive soils. Regarding CLP diversity, Boteva was characterized by strains producing 11 different CLP types with cocoyamide A producers, belonging to the P. koreensis group, being the most abundant. However, putisolvin III-V producers were the most dominant in the rhizosphere of conducive soils in both Cameroon and Nigeria. Furthermore, we elucidated the chemical structure of putisolvin derivatives-putisolvin III-V, and described its biosynthetic gene cluster. We show that high Pseudomonas and metabolic diversity may be driven by microbial competition, which likely contributes to soil suppressiveness to CRRD.

Summary In contrast to the current paradigm of using microbial mono‐cultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed‐species consortia to perform functions that are difficult to programme into individual strains. In this work, we developed a synthetic microbial consortium composed of two genetically engineered microbes, a cyanobacterium (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). These microbial species specialize in the co‐culture: cyanobacteria fix CO2 through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose and to degrade the environmental pollutant 2,4‐dinitrotoluene (2,4‐DNT). By encapsulating S. elongatus within a barium–alginate hydrogel, cyanobacterial cells were protected from the toxic effects of 2,4‐DNT, enhancing the performance of the co‐culture. The synthetic consortium was able to convert 2,4‐DNT with light and CO2 as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrose‐dependent accumulation of polyhydroxyalkanoate, an added‐value biopolymer, in the engineered P. putida strain. Altogether, the synthetic consortium displayed the capacity to remediate the industrial pollutant 2,4‐DNT while simultaneously synthesizing biopolymers using light and CO2 as the primary inputs., This work describes the development of a synthetic microbial consortium composed of a cyanobacterium engineered to use light to fix inorganic carbon, and a heterotrophic bacterium engineered to degrade the environmental pollutant, 2,4‐dinitrotoluene. Together, the synthetic consortium displayed the capacity to remediate the industrial compound 2,4‐DNT while simultaneously synthesizing biopolymers using light and carbon dioxide as the primary inputs.

Summary Transforming petrochemical processes into bioprocesses has become an important goal of sustainable development. The chemical synthesis of 2,5‐furandicarboxylic acid (FDCA) from 5‐hydroxymethylfurfural (HMF) is expensive and environmentally unfavourable. The study aims to investigate a whole‐cell biocatalyst for efficient biotransformation of HMF to FDCA. For the first time, a genetically engineered Pseudomonas putida S12 strain expressing 5‐hydroxymethylfurfural oxidase (HMFO) was developed for the biocatalytic conversion of HMF to FDCA. This whole‐cell biocatalyst produced 35.7 mM FDCA from 50 mM HMF in 24 h without notable inhibition. However, when the initial HMF concentration was elevated to 100 mM, remarkable inhibition on FDCA production was observed, resulting in a reduction of FDCA yield to 42%. We solve this substrate inhibition difficulty by increasing the inoculum density. Subsequently, we used a fed‐batch strategy by maintaining low HMF concentration in the culture to maximize the final FDCA titre. Using this approach, 545 mM of FDCA was accumulatively produced after 72 hs, which is the highest production rate per unit mass of cells to the best of our knowledge., For the first time, a genetically engineered Pseudomonas putida S12 strain expressing 5‐hydroxymethylfurfural oxidase (HMFO) was developed for the biocatalytic conversion of HMF to FDCA. This whole‐cell biocatalyst produced 35.7 mM FDCA from 50 mM HMF in 24 h without notable inhibition. Using Fed‐batch approach, 545 mM of FDCA was accumulatively produced after 72 h, which is the highest production rate per unit mass of cells to the best of our knowledge.

Co-production of two or more desirable compounds from low-cost substrates by a single microbial catalyst could greatly improve the economic competitiveness of many biotechnological processes. However, reports demonstrating the adoption of such co-production strategy are still scarce. In this study, the ability of genome-edited strain Psudomonas putida EM42 to simultaneously valorise D-xylose and D-cellobiose - two important lignocellulosic carbohydrates - by converting them into the platform chemical D-xylonic acid and medium chain length polyhydroxyalkanoates, respectively, was investigated. Biotransformation experiments performed with P. putida resting cells showed that promiscuous periplasmic glucose oxidation route can efficiently generate extracellular xylonate with high yield reaching 0.97 g per g of supplied xylose. Xylose oxidation was subsequently coupled to the growth of P. putida with cytoplasmic beta-glucosidase BglC from Thermobifida fusca on D-cellobiose. This disaccharide turned out to be a better co-substrate for xylose-to-xylonate biotransformation than monomeric glucose. This was because unlike glucose, cellobiose did not block oxidation of the pentose by periplasmic glucose dehydrogenase Gcd, but, similarly to glucose, it was a suitable substrate for polyhydroxyalkanoate formation in P. putida. Co-production of extracellular xylose-born xylonate and intracellular cellobiose-born medium chain length polyhydroxyalkanoates was established in proof-of-concept experiments with P. putida grown on the disaccharide. This study highlights the potential of P. putida EM42 as a microbial platform for the production of xylonic acid, identifies cellobiose as a new substrate for mcl-PHA production, and proposes a fresh strategy for the simultaneous valorisation of xylose and cellobiose.

Aliphatic synthetic intermediates with high added value are generally produced from alkane sources (e.g., petroleum) by inert carbon-hydrogen (C-H) bond activation using classical chemical methods (i.e. high temperature, rare metals). As an alternative approach for these reactions, alkane monooxygenase from Pseudomonas putida (alkB) is able to catalyze the difficult terminal oxyfunctionalization of alkanes selectively and under mild conditions. Herein, we report an electrosynthetic system using an alkB biocathode which produces alcohols, epoxides, and sulfoxides through bioelectrochemical hydroxylation, epoxidation, sulfoxidation, and demethylation. The capacity of the alkB binding pocket to protect internal functional groups is also demonstrated. By coupling our alkB biocathode with a hydrogenase bioanode and using H2 as a clean fuel source, we have developed and characterized a series of enzymatic fuel cells capable of oxyfunctionalization while simultaneously producing electricity.

Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engineered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octylnonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer–Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g−1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.

C-S bond formation reactions are widely distributed in the biosynthesis of biologically active molecules, and thus have received much attention over the past decades. Herein, we report intramolecular C-S bond formation by a P450 monooxygenase, TleB, which normally catalyzes a C-N bond formation in teleocidin biosynthesis. Based on the proposed reaction mechanism of TleB, a thiol-substituted substrate analogue was synthesized and tested in the enzyme reaction, which afforded the unprecedented sulfur-containing thio-indolactam V, in addition to an unusual indole-fused 6/5/8-tricyclic product whose structure was determined by the crystalline sponge method. Interestingly, conformational analysis revealed that the SOFA conformation is stable in thio-indolactam V, in sharp contrast to the major TWIST form in indolactam V, resulting in differences in their biological activities.

Summary Pseudomonas putida has emerged as a promising host for the production of chemicals and materials thanks to its metabolic versatility and cellular robustness. In particular, P. putida KT2440 has been officially classified as a generally recognized as safe (GRAS) strain, which makes it suitable for the production of compounds that humans directly consume, including secondary metabolites of high importance. Although various tools and strategies have been developed to facilitate metabolic engineering of P. putida, modification of large genes/clusters essential for heterologous expression of natural products with large biosynthetic gene clusters (BGCs) has not been straightforward. Recently, we reported a RecET‐based markerless recombineering system for engineering P. putida and demonstrated deletion of multiple regions as large as 101.7 kb throughout the chromosome by single rounds of recombineering. In addition, development of a donor plasmid system allowed successful markerless integration of heterologous BGCs to P. putida chromosome using the recombineering system with examples of – but not limited to – integrating multiple heterologous BGCs as large as 7.4 kb to the chromosome of P. putida KT2440. In response to the increasing interest in our markerless recombineering system, here we provide detailed protocols for markerless gene knockout and integration for the genome engineering of P. putida and related species of high industrial importance., Pseudomonas putida is a promising engineering host for the production of chemicals including secondary metabolites, but engineering tools for integration of large biosynthetic clusters of such valuable compounds to P. putida chromosome have been time‐consuming or uncontrollable. Recently, we reported a RecET‐based markerless recombineering system for P. putida and demonstrated knocking out chromosomal regions as large as 101.7 kb and integration and subsequent successful expression of multiple heterologous biosynthetic genes and clusters in P. putida. Detailed protocols for the markerless recombineering system for P. putida is provided.

Summary Targeted gene regulation is indispensable for reprogramming a cellular network to modulate a microbial phenotype. Here, we adopted the type II CRISPR interference (CRISPRi) system for simple and efficient regulation of target genes in Pseudomonas putida KT2440. A single CRISPRi plasmid was generated to express a nuclease‐deficient Cas9 gene and a designed single guide RNA, under control of l‐rhamnose‐inducible Prha BAD and the constitutive Biobrick J23119 promoter respectively. Two target genes were selected to probe the CRISPRi‐mediated gene regulation: exogenous green fluorescent protein on the multicopy plasmid and endogenous glpR on the P. putida KT2440 chromosome, encoding GlpR, a transcriptional regulator that represses expression of the glpFKRD gene cluster for glycerol utilization. The CRISPRi system successfully repressed the two target genes, as evidenced by a reduction in the fluorescence intensity and the lag phase of P. putida KT2440 cell growth on glycerol. Furthermore, CRISPRi‐mediated repression of glpR improved both the cell growth and glycerol utilization, resulting in the enhanced production of mevalonate in an engineered P. putida KT2440 harbouring heterologous genes for the mevalonate pathway. CRISPRi is expected to become a robust tool to reprogram P. putida KT2440 for the development of microbial cell factories producing industrially valuable products., We present an l‐rhamnose‐inducible single‐plasmid CRISPRi system for achieving the simple and efficient regulation of target genes in Pseudomonas putida KT2440. This regulatable CRISPRi system was able to control the expression of exogenous and endogenous genes in P. putida KT2440. We further provide examples of its application for metabolic flux alteration to enhance the production of mevalonate (MVA), a key intermediate metabolite for the biosynthesis of a myriad of terpenoids.

Summary Using agricultural wastes as a substrate for biotechnological processes is of great interest in industrial biotechnology. A prerequisite for using these wastes is the ability of the industrially relevant microorganisms to metabolize the sugars present therein. Therefore, many metabolic engineering approaches are directed towards widening the substrate spectrum of the workhorses of industrial biotechnology like Escherichia coli, yeast or Pseudomonas putida. For instance, neither xylose or arabinose from cellulosic residues, nor sucrose, the main sugar in waste molasses, can be metabolized by most E. coli and P. putida wild types. We evaluated a new, so far uncharacterized gene cluster for sucrose metabolism from Pseudomonas protegens Pf‐5 and showed that it enables P. putida to grow on sucrose as the sole carbon and energy source. Even when integrated into the genome of P. putida, the resulting strain grew on sucrose at rates similar to the rate of the wild type on glucose – making it the fastest growing, plasmid‐free P. putida strain known so far using sucrose as substrate. Next, we elucidated the role of the porin, an orthologue of the sucrose porin ScrY, in the gene cluster and found that in P. putida, a porin is needed for sucrose transport across the outer membrane. Consequently, native porins were not sufficient to allow unlimited growth on sucrose. Therefore, we concluded that the outer membrane can be a considerable barrier for substrate transport, depending on strain, genotype and culture conditions, all of which should be taken into account in metabolic engineering approaches. We additionally showed the potential of the engineered P. putida strains by growing them on molasses with efficiencies twice as high as obtained with the wild‐type P. putida. This can be seen as a further step towards the production of low‐value chemicals and biofuels with P. putida from alternative and more affordable substrates in the future., The article deals with the evaluation of a new, so far uncharacterized gene cluster from Pseudomonas protegens Pf‐5 to confer sucrose metabolism to P. putida. This metabolic engineering approach resulted in the fastest growing, plasmid‐free P. putida strain known so far using sucrose as substrate, unlocking new cheap substrates like molasses. Additionally, we could show that the outer membrane presents a considerable barrier for sucrose transport, as the native, constitutively expressed porins are not sufficient to allow unlimited growth on sucrose. We discuss the importance of considering the outer membrane barrier for metabolic engineering approaches.

14 p.-7 fig., Over the past few decades, considerable interest has been shown in developing nano- and microcarriers with biocompatible and biodegradable materials for medical and biotechnological applications. Microencapsulation is a technology capable of enhancing the survival rate of bacteria, providing stability in harsh environments. In the present paper, we developed a technology to encapsulate microorganisms within polyhydroxyalkanoate (PHA)-based microcapsules (MPs), employing a modified double emulsion solvent evaporation technique, with Pseudomonas putida KT2440 as a biotechnological model strain. The resulting MPs display a spherical morphology and an average particle size of 10 μm. The stability of the MPs was monitored under different conditions of storage and stress. The MPs remained stable for at least 24 days stored at 4°C in a water suspension. They exhibited greater tolerance to stress conditions; encapsulated cells remained viable for 2 h in alkaline solution and after 24 h of H2 O2 exposure at 10 and 20 mM. Results suggested the potential of MPs as a microcontainer of bacterial cells, even for biotechnological applications requiring high alkaline conditions and oxidative stress. We validated the potential applicability of the PHA-based microencapsulation method in other microorganisms by encapsulating the predatory bacterium Bdellovibrio bacteriovorus., The authors thank for the financial support from the Spanish Ministry of Science, Innovation and Universities (BIO2014-61515-EXP, BIO2017-83448-R) and the Community of Madrid (P2013/MIT2807, P2018/NMT4389).

The enzyme formaldehyde dehydrogenase (FalDH) from Pseudomonas putida is of particular interest for biotechnological applications as it catalyzes the oxidation of formaldehyde independent of glutathione. However, the consumption of a stoichiometric amount of nicotinamide adenine dinucleotide (NAD) can be challenging at the metabolic level as this may affect many other NAD-linked processes. A potential solution is to engineer FalDH to utilize non-natural cofactors. Here we devised FalDH variants to favor nicotinamide cytosine dinucleotide (NCD) by structure-guided modification of the binding pocket for the adenine moiety of NAD. Several mutants were obtained and the best one FalDH 9B2 had over 150-fold higher preference for NCD than NAD. Molecular docking analysis indicated that the cofactor binding pocket shrunk to better fit NCD, a smaller-sized cofactor. FalDH 9B2 together with other NCD-linked enzymes offer opportunities to assemble orthogonal pathways for biological conversion of C1 molecules.

Summary To meet the needs of synthetic biologists, DNA assembly methods have transformed from simple ‘cut‐and‐paste’ procedures to highly advanced, standardised assembly techniques. Implementing these standardised DNA assembly methods in biotechnological research conducted in non‐model hosts, including Pseudomonas putida and Pseudomonas aeruginosa, could greatly benefit reproducibility and predictability of experimental results. SEVAtile is a Type IIs‐based assembly approach, which enables the rapid and standardised assembly of genetic parts – or tiles – to create genetic circuits in the established SEVA‐vector backbone. Contrary to existing DNA assembly methods, SEVAtile is an easy and straightforward method, which is compatible with any vector, both SEVA‐ and non‐SEVA. To prove the efficiency of the SEVAtile method, a three‐vector system was successfully generated to independently co‐express three different proteins in P. putida and P. aeruginosa. More specifically, one of the vectors, pBGDes, enables genomic integration of assembled circuits in the Tn7 landing site, while self‐replicatory vectors pSTDesX and pSTDesR enable inducible expression from the XylS/Pm and RhaRS/PrhaB expression systems, respectively. Together, we hope these vector systems will support research in both the microbial SynBio and Pseudomonas field., SEVAtile is a Type IIs‐based assembly approach, which enables the rapid and standardized assembly of genetic parts – or tiles – to create genetic circuits in the established SEVA‐vector backbone. Contrary to existing DNA assembly methods, SEVAtile is an easy and straightforward method, which is compatible with any vector, both SEVA‐ and non‐SEVA. To prove the efficiency of the SEVAtile method, a three‐vector system was successfully generated to independently co‐express three different proteins in Pseudomonas putida and Pseudomonas aeruginosa.

Reactor design and feeding strategies are the key to successful trichloroethylene (TCE) treatment via cometabolism. An easy to maintain and operate laboratory scale packed‐bed biofilm reactor, featuring recycled gas and liquid effluent streams, was developed for continuous aerobic aqueous‐phase TCE biodegradation using Pseudomonas putida F1 as the biocatalyst and toluene as the primary substrate. The impacts of the influent toluene and TCE concentrations, flow rates, and recycle rates on TCE biodegradation were studied. Multivariate factors, including influent toluene and TCE concentrations, their interactions, and flow rates were further analyzed via a response surface model (RSM). Results showed that such bioreactor design eliminated oxygen limitation and removed or substantially reduced toluene competitive inhibition on TCE degradation. A TCE removal efficiency of >90% was achieved at an influent TCE concentration of ≤0.3 mg/L under an ˜8‐min mean hydraulic retention time. Toluene and TCE concentrations, in both linear and quadratic forms, were the most significant factors for TCE degradation, indicating both cell regenerating and inhibiting effects from toluene and a toxic effect from TCE. This RSM analysis has successfully identified an optimal influent toluene concentration path for each influent TCE concentration and provided insight for an extended TCE degradation at higher TCE concentrations.

Cyclic diguanylate (c-di-GMP) is a broadly conserved bacterial signalling molecule that modulates diverse cellular processes, such as biofilm formation, colony morphology and swimming motility. The intracellular level of c-di-GMP is controlled by diguanylate cyclases (DGCs) with GGDEF domain and phosphodiesterases (PDEs) with either EAL or HD-GYP domain. Pseudomonas putida KT2440 has a large group of genes on its genome encoding proteins with GGDEF/EAL/HD-GYP domains. However, phenotypic-genotypic correlation and c-di-GMP metabolism of these genes were largely unknown. Herein, by systematically constructing deletion mutants/overexpression strains of the 42 predicted c-di-GMP metabolism-related genes and analysing the phenotypes, we preliminarily revealed the role of each gene in biofilm formation, colony morphology and swimming motility. Subsequent results from protein sequence alignments and cellular c-di-GMP assessment indicated that 25 out of the 42 genes were likely to encode DGCs, nine genes were predicted to encode PDEs, four genes encoded bifunctional enzymes and the other four genes encoded enzymatically inactive proteins. This study offers a basic understanding of the roles of these 42 genes and can serve as a toolkit for investigators to further elucidate the functions of these GGDEF and EAL/HD-GYP domain-containing proteins.

The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE-encoding genes in its genome, but the phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c-di-GMP-metabolizing enzyme, GcsA. GcsA consists of two per-ARNT-sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ-dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c-di-GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c-di-GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c-di-GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c-di-GMP and cAMP in the regulation of the expression of GcsA in P. putida.