Efficient production of soluble recombinant single chain Fv fragments by a <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strain KT2440 cell factory

Background  

Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs.     

Disruption of β-oxidation pathway in Pseudomonas putida KT2442 to produce new functionalized PHAs with thioester groups

This work describes the generation of novel PHAs (named PHACOS) with a new monomer composition containing thioester groups in the side chain, which confers new properties and made them suitable for chemical modifications after their biosynthesis. We have analyzed the PHACOS production abilities of the wild-type strain Pseudomonas putida KT2442 vs. its derived strain P. putida KT42FadB, mutated in the fadB gene from the central metabolic β-oxidation pathway involved in the synthesis of medium-chain-length PHA (mcl-PHA). Different fermentation strategies based on one- or two-stage cultures have been tested resulting in PHACOS with different monomer composition. Using decanoic acid as inducer of the growth and polymer synthesis and 6-acetylthiohexanoic acid as PHA precursor in a two-stage strategy, the maximum yield was obtained by culturing the strain KT42FadB. Nuclear magnetic resonance and gas chromatography coupled to mass spectrometry showed that polymers obtained from the wild-type and KT42FadB strains, included 6-acetylthio-3-hydroxyhexanoic acid (OH-6ATH) and the shorter derivative 4-acetylthio-3-hydroxybutanoic acid (OH-4ATB) in their composition, although in different ratios. While the polymer obtained from KT42FadB strain contained mainly OH-6ATH monomer units, mcl-PHA produced by the wild-type strain contained OH-6ATH and OH-4ATB. Furthermore, polyesters showed differences in the OH-alkyl derivates moiety. The strain KT42FadB overproduced PHACOS when compared to the production rate of the control strain in one- and two-stage cultures. Thermal properties obtained by differential scanning calorimetry indicated that both polymers have different glass transition temperatures related to their composition.     

Production and characterization of medium-chain-length polyhydroxyalkanoate with high 3-hydroxytetradecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442

Medium-chain-length polyhydroxyalkanoates (mcl-PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate, 3-hydroxydodecanoate, and high-content 3-hydroxytetradecanoate (HTD) was produced by knockout mutant Pseudomonas putida KT2442 termed P. putida KTOY06. When grown on 6 to14 g/L single-carbon-source tetradecanoic acid, P. putida KTOY06, which β-oxidation pathway was weakened by deleting genes of 3-ketoacyl-coenzyme A (CoA) thiolase (fadA) and 3-hydroxyacyl-CoA dehydrogenase (fadB), for the first time, produced several mcl-PHA including 31 to 49 mol% HTD as a major monomer. HHx contents in these mcl-PHAs remained approximately constant at less than 3 mol%. In addition, large amounts of oligo-HTD were detected in cells, indicating the limited ability of P. putida KTOY06 in polymerizing long-chain-length 3-hydroxyalkanoates. The mcl-PHA containing high HTD monomer contents was found to have both higher crystallinity and improved tensile strength compared with that of typical mcl-PHA.     

Growth of engineered Pseudomonas putida KT2440 on glucose,xylose, and arabinose: Hemicellulose hydrolysates and their major sugars as sustainable carbon sources

Lignocellulosic biomass is the most abundant bioresource on earth containing polymers mainly consisting of d ‐glucose, d ‐xylose, l ‐arabinose, and further sugars. In order to establish this alternative feedstock apart from applications in food, we engineered Pseudomonas putida KT2440 as microbial biocatalyst for the utilization of xylose and arabinose in addition to glucose as sole carbon sources. The d ‐xylose‐metabolizing strain P. putida KT2440_xylAB and l ‐arabinose‐metabolizing strain P. putida KT2440_araBAD were constructed by introducing respective operons from Escherichia coli. Surprisingly, we found out that both recombinant strains were able to grow on xylose as well as arabinose with high cell densities and growth rates comparable to glucose. In addition, the growth characteristics on various mixtures of glucose, xylose, and arabinose were investigated, which demonstrated the efficient co‐utilization of hexose and pentose sugars. Finally, the possibility of using lignocellulose hydrolysate as substrate for the two recombinant strains was verified. The recombinant P. putida KT2440 strains presented here as flexible microbial biocatalysts to convert lignocellulosic sugars will undoubtedly contribute to the economic feasibility of the production of valuable compounds derived from renewable feedstock.     

Biotransformation of d-xylose to d-xylonate coupled to medium-chain-length polyhydroxyalkanoate production in cellobiose-grown Pseudomonas putida EM42

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 Pseudomonas putida EM42 to simultaneously valorize d -xylose and d -cellobiose – two important lignocellulosic carbohydrates – by converting them into the platform chemical d -xylonate 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 a high yield. Xylose oxidation was subsequently coupled to the growth of P. putida with cytoplasmic β-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 xylonate, identifies cellobiose as a new substrate for mcl-PHA production, and proposes a fresh strategy for the simultaneous valorization of xylose and cellobiose.     

A versatile Pseudomonas putida KT2440 with new ability: selective oxidation of 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furancarboxylic acid

Currently, biotransformation of 5-hydroxymethylfurfural (HMF) into a series of high-value bio-based platform chemicals is massively studied. In this study, selective biooxidation of HMF to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) by Pseudomonas putida KT2440 with superior titer, yield, and productivity was reported. The biocatalytic performances of P. putida KT2440 were optimized separately. Under optimal conditions, 100% yield of HMFCA was obtained when HMF concentration was less than 150 mM, while the maximum concentration of 155 mM was achieved from 160 mM HMF in 12 h. P. putida KT2440 was highly tolerate to HMF, up to 190 mM. Besides, it was capable of selective oxidation of other furan aldehydes to the corresponding carboxylic acids with good yield of 100%. This study further demonstrates the potential of P. putida KT2440 as a biocatalyst for biomass conversion, as this strain has been proved the capacity to convert and utilize many kinds of biomass-derived sugars and ligin-derived aromatic compounds.

     

Biosynthesis of zeaxanthin in recombinant Pseudomonas putida

Pseudomonas putida KT2440 strain was investigated for biosynthesis of the valuable xanthophyll zeaxanthin. A new plasmid was constructed harboring five carotenogenic genes from Pantoea ananatis and three genes from Escherichia coli under control of an l-rhamnose-inducible promoter. Pseudomonas putida KT2440 wild type hardly tolerated the plasmids for carotenoid production. Mating experiments with E. coli S17-1 strains revealed that the carotenoid products are toxic to the Pseudomonas putida cells. Several carotenoid-tolerant transposon mutants could be isolated, and different gene targets for relief of carotenoid toxicity were identified. After optimization of cultivation conditions and product processing, 51 mg/l zeaxanthin could be produced, corresponding to a product yield of 7 mg zeaxanthin per gram cell dry weight. The effect of various additives on production of hydrophobic zeaxanthin was investigated as well. Particularly, the addition of lecithin during cell cultivation increased volumetric productivity of Pseudomonas putida by a factor of 4.7 (51 mg/l vs. 239 mg/l).     

Protocols for RecET-based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

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.     

Utilization of <Emphasis Type="Italic">fadA</Emphasis> knockout mutant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> for overproduction of medium chain-length-polyhydroxyalkanoate

The fadBA operon in the fatty acid β-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA). Succinate at 150 mg l⁻¹ stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l⁻¹ with a cell dry weight of 10.3 g l⁻¹.     

Unravelling the thioesterases responsible for propionate formation in engineered Pseudomonas putida KT2440

Pseudomonas putida KT2440 is becoming a new robust metabolic chassis for biotechnological applications, due to its metabolic versatility, low nutritional requirements and biosafety status. We have previously engineered P. putida KT2440 to be an efficient propionate producer from L-threonine, although the internal enzymes converting propionyl-CoA to propionate are not clear. In this study, we thoroughly investigated 13 genes annotated as potential thioesterases in the KT2440 mutant. One thioesterase encoded by locus tag PP_4975 was verified to be the major contributor to propionate production in vivo. Deletion of PP_4975 significantly decreased propionate production, whereas the performance was fully restored by gene complement. Compared with thioesterase HiYciA from Haemophilus influenza, thioesterase PP_4975 showed a faster substrate conversion rate in vitro. Thus, this study expands our knowledge on acyl-CoA thioesterases in P. putida KT2440 and may also reveal a new target for further engineering the strain to improve propionate production performance.     

Antimicrobial activities of commercial nanoparticles against an environmental soil microbe, Pseudomonas putida KT2440

Background  

The release of heavy metal-containing nanoparticles (NP) into the environment may be harmful to the efficacy of beneficial microbes that function in element cycling, pollutant degradation and plant growth. Nanoparticles of Ag, CuO and ZnO are of interest as antimicrobials against pathogenic bacteria. We demonstrate here their antimicrobial activity against the beneficial soil microbe, Pseudomonas putida KT2440.     

Development of genetic tools for heterologous protein expression in a pentose-utilizing environmental isolate of Pseudomonas putida

Pseudomonas putida has emerged as a promising host for the conversion of biomass-derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non-native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass-derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non-native genes was achieved by using chassis-independent recombinase-assisted genome engineering (CRAGE) for single-step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.     

Systemic resistance and lipoxygenase-related defence response induced in tomato by <Emphasis Type="Italic">Pseudomonas putida</Emphasis>strain BTP1

Background  

Previous studies showed the ability of Pseudomonas putida strain BTP1 to promote induced systemic resistance (ISR) in different host plants. Since ISR is long-lasting and not conducive for development of resistance of the targeted pathogen, this phenomenon can take part of disease control strategies. However, in spite of the numerous examples of ISR induced by PGPR in plants, only a few biochemical studies have associated the protective effect with specific host metabolic changes.     

Microbial production of 4-hydroxybutyrate,poly-4-hydroxybutyrate,and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by recombinant microorganisms

4-Hydroxybutyrate (4HB) was produced by Aeromonas hydrophila 4AK4, Escherichia coli S17-1, or Pseudomonas putida KT2442 harboring 1,3-propanediol dehydrogenase gene dhaT and aldehyde dehydrogenase gene aldD from P. putida KT2442 which are capable of transforming 1,4-butanediol (1,4-BD) to 4HB. 4HB containing fermentation broth was used for production of homopolymer poly-4-hydroxybutyrate [P(4HB)] and copolymers poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)]. Recombinant A. hydrophila 4AK4 harboring plasmid pZL-dhaT-aldD containing dhaT and aldD was the most effective 4HB producer, achieving approximately 4 g/l 4HB from 10 g/l 1,4-BD after 48 h of incubation. The strain produced over 10 g/l 4HB from 20 g/l 1,4-BD after 52 h of cultivation in a 6-L fermenter. Recombinant E. coli S17-1 grown on 4HB containing fermentation broth was found to accumulate 83 wt.% of intracellular P(4HB) in shake flask study. Recombinant Ralstonia eutropha H16 grew to over 6 g/l cell dry weight containing 49 wt.% P(3HB-13%4HB) after 72 h.     

In vivo blending of medium chain length polyhydroxy-alkanoates and polyhydroxybutyrate using recombinant Pseudomonas putida harboring phbCAB operon of Ralstonia eutropha

The in vivo blending of medium chain length polyhydroxyalkanoates (mcl-PHA) and polyhydroxybutyrate (PHB) was carried out using recombinant Pseudomonas putida after transforming the phbCAB operon of Ralstonia eutropha. The most suitable carbon sources for the production of mcl-PHA and PHB blends were identified to be octanoate and gluconate. The molar fractions of 3-hydroxyoctanoate and 3-hydroxybutyrate in the polymer blends were effectively modulated by controlling the mixing ratio of octanoate and gluconate, thereby producing a composition ranging from 95% mcl-PHA to 78% PHB.     

A polyhydroxyalkanoates bioprocess improvement case study based on four fed-batch feeding strategies

The modelling and optimization of a process for the production of the medium chain length polyhydroxyalkanoate (mcl-PHA) by the bacterium Pseudomonas putida KT2440 when fed a synthetic fatty acid mixture (SFAM) was investigated. Four novel feeding strategies were developed and tested using a constructed model and the optimum one implemented in further experiments. This strategy yielded a cell dry weight of 70.6 g l⁻¹ in 25 h containing 38% PHA using SFAM at 5 l scale. A phosphate starvation strategy was implemented to improve PHA content, and this yielded 94.1 g l⁻¹ in 25 h containing 56% PHA using SFAM at 5 l scale. The process was successfully operated at 20 l resulting in a cell dry weight of 91.2 g l⁻¹ containing 65% PHA at the end of a 25-h incubation.     

The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> becomes evident only in growing bacteria

Background  

We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida.     

Proteomic analysis of the response of the plant growth-promoting bacterium Pseudomonas putida UW4 to nickel stress

Background  

Plant growth-promoting bacteria can alleviate the inhibitory effects of various heavy metals on plant growth, via decreasing levels of stress-induced ethylene. However, little has been done to detect any mechanisms specific for heavy metal resistance of this kind of bacteria. Here, we investigate the response of the wild-type plant growth-promoting bacterium Pseudomonas putida UW4 to nickel stress using proteomic approaches. The mutant strain P. putida UW4/AcdS^-, lacking a functional 1-aminocyclopropane-1-carboxylic acid deaminase gene, was also assessed for its response to nickel stress.