Laura I. de Eugenio

The turnover of medium-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2442 and the fundamental role of PhaZ depolymerase for the metabolic balance

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by a wide range of bacteria, including Pseudomonads. These polymers are accumulated in the cytoplasm as carbon and energy storage materials when culture conditions are unbalanced and hence, they have been classically considered to act as sinks for carbon and reducing equivalents when nutrients are limited. Bacteria facing carbon excess and nutrient limitation store the extra carbon as PHAs through the PHA polymerase (PhaC). Thereafter, under starvation conditions, PHA depolymerase (PhaZ) degrades PHA and releases R-hydroxyalkanoic acids, which can be used as carbon and energy sources. To study the influence of a deficient PHA metabolism in the growth of Pseudomonas putida KT2442 we have constructed two mutant strains defective in PHA polymerase (phaC1)- and PHA depolymerase (phaZ)-coding genes respectively. By using these mutants we have demonstrated that PHAs play a fundamental role in balancing the stored carbon/biomass/number of cells as function of carbon availability, suggesting that PHA metabolism allows P. putida to adapt the carbon flux of hydroxyacyl-CoAs to cellular demand. Furthermore, we have established that the coordination of PHA synthesis and mobilization pathways configures a functional PHA turnover cycle in P. putida KT2442. Finally, a new strain able to secrete enantiomerically pure R-hydroxyalkanoic acids to the culture medium during cell growth has been engineering by redirecting the PHA cycle to biopolymer hydrolysis.

The Phosphotransferase System Formed by PtsP, PtsO, and PtsN Proteins Controls Production of Polyhydroxyalkanoates in Pseudomonas putida

The genome of Pseudomonas putida KT2440 encodes five proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system. Two of these (FruA and FruB) form a dedicated system for fructose intake, while enzyme I(Ntr) (EI(Ntr); encoded by ptsP), NPr (ptsO), and EII(Ntr) (ptsN) act in concert to control the intracellular accumulation of polyhydroxyalkanoates, a typical product of carbon overflow.

Biochemical Evidence That phaZ Gene Encodes a Specific Intracellular Medium Chain Length Polyhydroxyalkanoate Depolymerase in Pseudomonas putida KT2442 CHARACTERIZATION OF A PARADIGMATIC ENZYME

Polyhydroxyalkanoates (PHAs) can be catabolized by many microorganisms using intra- or extracellular PHA depolymerases. Most of our current knowledge of these intracellular enzyme-coding genes comes from the analysis of short chain length PHA depolymerases, whereas medium chain length PHA (mcl-PHA) intracellular depolymerization systems still remained to be characterized. The phaZ gene of some Pseudomonas putida strains has been identified only by mutagenesis and complementation techniques as putative intracellular mcl-PHA depolymerase. However, none of their corresponding encoded PhaZ enzymes have been characterized in depth. In this study the PhaZ depolymerase from P. putida KT2442 has been purified and biochemically characterized after its overexpression in Escherichia coli. To facilitate these studies we have developed a new and very sensitive radioactive method for detecting PHA hydrolysis in vitro. We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. We have modeled the three-dimensional structure of PhaZ complexed with a 3-hydroxyoctanoate dimer. Using this model, we found that the enzyme appears to be built up from a coreα/β hydrolase-type domain capped with a lid structure with an active site containing a catalytic triad buried near the connection between domains. All these data constitute the first biochemical characterization of PhaZ and allow us to propose this enzyme as the paradigmatic representative of intracellular endo/exo-mcl-PHA depolymerases.

Synthesis and Degradation of Polyhydroxyalkanoates

Abbreviations: PHA: Polyhydroxyalkanoate, GAPs: Granule associated proteins, scl-PHAs: Short-chain-length PHAs, mcl-PHAs: Medium-chainlength PHAs, PHB: Polyhydroxybutyrate, CoA: Coenzyme A, ACP: Acyl carrier protein, GFP: Green fluorescent protein, P(HB-co-HA): Poly(3hydroxybutyrate-co-3-hydroxyalkanoates), PHO: Poly-3-hydroxyoctanoate, PHV: Poly-3-hydroxyvalerate, SCP: single-cell-protein.

The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442.

The promoters of the pha gene cluster encoding the enzymes involved in the metabolism of polyhydroxyalkanoates (PHAs) in the model strain Pseudomonas putida KT2442 have been identified and compared. The pha locus is composed by five functional promoters upstream the phaC1, phaZ, phaC2, phaF and phaI genes (P(C1), P(Z), P(C2), P(F) and P(I) respectively). P(C1) and P(I) are the most active promoters of the pha cluster allowing the transcription of phaC1ZC2D and phaIF operons. All promoters with the sole exception of P(F) are carbon source-dependent. Their transcription profiles explain the simultaneous production of PHA depolymerase and synthases to maintain the metabolic balance and PHA turnover. Mutagenesis analyses demonstrated that PhaD, a TetR-like transcriptional regulator, behaves as a carbon source-dependent activator of the pha cluster. The phaD gene is mainly transcribed as part of the phaC1ZC2D transcription unit and controls its own transcription and that of phaIF operon. The ability of PhaD to bind the P(C1) and P(I) promoters was analysed by gel retardation and DNase I footprinting assays, demonstrating that PhaD interacts with a region of 25 bp at P(C1) promoter (named OPRc1) and a 29 bp region at P(I) promoter (named OPRi). These operators contain a single binding site formed by two inverted half sites of 6 bp separated by 8 bp which overlap the corresponding promoter boxes. The 3D model structure of PhaD activator predicts that the true effector might be a CoA-intermediate of fatty acid beta-oxidation.

New tool for spreading proteins to the environment: Cry1Ab toxin immobilized to bioplastics

A new tool to provide an environmentally friendly way to deliver active proteins to the environment has been developed, based on the use of polyhydroxyalkanoate (PHA, bioplastic) granules. To illustrate this novel approach, a derived Cry1Ab insect-specific toxin protein was in vivo immobilized into PHA granules through the polypeptide tag BioF. The new toxin, named Fk-Bt1, was shown to be active against Sesamia nonagrioides (Lepidoptera: Noctuidae). The dose–mortality responses of the new toxin granule formulation (PFk-Bt1) and purified Cry1Ab have been compared, demonstrating the effectiveness of PFk-Bt1 and suggesting a common mode of action.

Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation.

ABSTRACT This paper reports on a novel β-xylosidase from the hemicellulolytic fungus Talaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu2+ tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, and Km and V max values of 0.17 mM and 52.0 U/mg, respectively, using commercial p-nitrophenyl-β-d-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.

Cell system engineering to produce extracellular polyhydroxyalkanoate depolymerase with targeted applications

Novel platforms based on the application of bacterial cell systems as factories for production of new bioproducts open avenues and dramatically expand the catalogue of existing biomaterials. Herein, we designed the strategy based on in vivo production of extracellular Pseudomonas fluorescens GK13 (PhaZGK13) depolymerase to degrade previously biosynthesized polyhydroxyalkanotes (PHAs) or to obtain 3-hydroxyalkanoic acids (HAs). With this aim, extracellular PhaZGK13 was produced in recombinant strains and the optimal conditions for controlled release of HAs and oligomers by growing cells were set up with a particle suspension of (14)C-labelled PHA, being maximal after 24h of incubation. Genetic modification of key factors involved in fatty acids metabolism revealed the influence of an active β-oxidation pathway on the extracellular degradation of PHA and subsequent HAs isolation. The highest HAs production was obtained using Pseudomonas putida KT2442 fadB mutant (0.27mg/mL) due to the reduced ability of this strain to metabolize the degradation products. The system was applied to produce new added value HAs harboring thioester groups in the side chain from the functionalized mcl-PHA, PHACOS. Remarkably, hydrolyzed PHACOS showed greater potential to inhibit Staphylococcus aureus(T) growth when compared to that of degradation products of non functionalized polyhydroxyoctanoate-co-hexanoate P(HO-co-HH).

Differential β-glucosidase expression as a function of carbon source availability in Talaromyces amestolkiae: a genomic and proteomic approach

BackgroundGenomic and proteomic analysis are potent tools for metabolic characterization of microorganisms. Although cellulose usually triggers cellulase production in cellulolytic fungi, the secretion of the different enzymes involved in polymer conversion is subjected to different factors, depending on growth conditions. These enzymes are key factors in biomass exploitation for second generation bioethanol production. Although highly effective commercial cocktails are available, they are usually deficient for β-glucosidase activity, and genera like Penicillium and Talaromyces are being explored for its production.ResultsThis article presents the description of Talaromyces amestolkiae as a cellulase-producer fungus that secretes high levels of β-glucosidase. β-1,4-endoglucanase, exoglucanase, and β-glucosidase activities were quantified in the presence of different carbon sources. Although the two first activities were only induced with cellulosic substrates, β-glucosidase levels were similar in all carbon sources tested. Sequencing and analysis of the genome of this fungus revealed multiple genes encoding β-glucosidases. Extracellular proteome analysis showed different induction patterns. In all conditions assayed, glycosyl hydrolases were the most abundant proteins in the supernatants, albeit the ratio of the diverse enzymes from this family depended on the carbon source. At least two different β-glucosidases have been identified in this work: one is induced by cellulose and the other one is carbon source-independent. The crudes induced by Avicel and glucose were independently used as supplements for saccharification of slurry from acid-catalyzed steam-exploded wheat straw, obtaining the highest yields of fermentable glucose using crudes induced by cellulose.ConclusionsThe genome of T. amestolkiae contains several genes encoding β-glucosidases and the fungus secretes high levels of this activity, regardless of the carbon source availability, although its production is repressed by glucose. Two main different β-glucosidases have been identified from proteomic shotgun analysis. One of them is produced under different carbon sources, while the other is induced in cellulosic substrates and is a good supplement to Celluclast in saccharification of pretreated wheat straw.

A novel CalB-type lipase discovered by fungal genomes mining

The fungus Pseudozyma antarctica produces a lipase (CalB) with broad substrate specificity, stability, high regio- and enantio-selectivity. It is active in non-aqueous organic solvents and at elevated temperatures. Hence, CalB is a robust biocatalyst for chemical conversions on an industrial scale. Here we report the in silico mining of public metagenomes and fungal genomes to discover novel lipases with high homology to CalB. The candidates were selected taking into account homology and conserved motifs criteria, as well as, phylogeny and 3D model analyses. The most promising candidate (PlicB) presented interesting structural properties. PlicB was expressed in a heterologous host, purified and partially characterized. Further experiments will allow finding novel catalytic properties with biotechnological interest.