Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

Background

The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD⁺ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD⁺. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.

Results

The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.

Conclusion

We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.     

Bioproduction of p-Hydroxystyrene from Glucose by the Solvent-Tolerant Bacterium Pseudomonas putida S12 in a Two-Phase Water-Decanol Fermentation

Two solvent-tolerant Pseudomonas putida S12 strains, originally designed for phenol and p-coumarate production, were engineered for efficient production of p-hydroxystyrene from glucose. This was established by introduction of the genes pal and pdc encoding l-phenylalanine/l-tyrosine ammonia lyase and p-coumaric acid decarboxylase, respectively. These enzymes allow the conversion of the central metabolite l-tyrosine into p-hydroxystyrene, via p-coumarate. Degradation of the p-coumarate intermediate was prevented by inactivating the fcs gene encoding feruloyl-coenzyme A synthetase. The best-performing strain was selected and cultivated in the fed-batch mode, resulting in the formation of 4.5 mM p-hydroxystyrene at a yield of 6.7% (C-mol of p-hydroxystyrene per C-mol of glucose) and a maximum volumetric productivity of 0.4 mM h⁻¹. At this concentration, growth and production were completely halted due to the toxicity of p-hydroxystyrene. Product toxicity was overcome by the application of a second phase of 1-decanol to extract p-hydroxystyrene during fed-batch cultivation. This resulted in a twofold increase of the maximum volumetric productivity (0.75 mM h⁻¹) and a final total p-hydroxystyrene concentration of 21 mM, which is a fourfold improvement compared to the single-phase fed-batch cultivation. The final concentration of p-hydroxystyrene in the water phase was 1.2 mM, while a concentration of 147 mM (17.6 g liter⁻¹) was obtained in the 1-decanol phase. Thus, a P. putida S12 strain producing the low-value compound phenol was successfully altered for the production of the toxic value-added compound p-hydroxystyrene.The demand for so called “green” production of chemicals is rapidly increasing due to the declining availability of fossil fuels and the urgency to reduce CO₂ emissions (10, 30). However, this bioproduction may be hindered by the toxicity of the product of interest, such as substituted aromatics, to the production host (1, 2, 12, 29). One way to cope with this product toxicity is to deploy solvent-tolerant microorganisms as biocatalysts (5, 28). Of special interest among these solvent-tolerant hosts are Pseudomonas putida strains that have been engineered to produce a variety of compounds such as p-hydroxybenzoate (25, 33), p-coumarate (19), and (S)-styrene oxide (22). In our laboratory, we study and employ the solvent-tolerant P. putida S12. This strain is well suited for the production of substituted aromatic chemicals (18, 19, 33, 38) thanks to its extreme solvent tolerance (5, 35) and metabolic versatility toward aromatics (14, 16, 34).An example of an industrially relevant but extremely toxic aromatic is p-hydroxystyrene (4-vinyl phenol) (23). This compound is widely used as a monomer for the production of various polymers that are applied in resins, inks, elastomers, and coatings. Ben-Bassat et al. (2, 3, 23) reported p-hydroxystyrene production from glucose in Escherichia coli. In this strain, phenylalanine/tyrosine ammonia lyase (PAL/TAL; encoded by pal) from Rhodotorula glutinis and p-coumaric acid decarboxylase (PDC; encoded by pdc) from Lactobacillus plantarum were introduced for the conversion of l-tyrosine into p-hydroxystyrene via p-coumarate. The maximum concentration of p-hydroxystyrene was limited to 3.3 mM due to the toxicity of the product to the E. coli host (3, 23). To alleviate product toxicity, a two-phase fermentation with 2-undecanone as the extractant was performed. This approach resulted in a modest 14.2 mM p-hydroxystyrene in the organic phase and 0.5 mM p-hydroxystyrene in the water phase (2). Toxicity-related adverse effects on p-hydroxystyrene production may also be avoided by dividing the whole process into three stages: production of l-tyrosine from glucose by E. coli, conversion of l-tyrosine into p-coumarate by immobilized PAL-overexpressing E. coli cells, and chemical decarboxylation of p-coumarate into p-hydroxystyrene (29).In this report, we address and strongly enhance the bio-based production of p-hydroxystyrene from glucose by employing the solvent-tolerant P. putida S12 as a host. Previously, two strains, P. putida S12 C3 (19) and P. putida S12 TPL3 (38), have been constructed for the production of the l-tyrosine-derived aromatics p-coumarate and phenol, respectively. These strains were highly optimized for aromatics production, resulting in a heavily increased metabolic flux toward l-tyrosine. Therefore, they are suitable platform strains for the production of other l-tyrosine-derived aromatics (33). The bifunctional enzyme PAL/TAL (EC 4.3.1.25) from Rhodosporidium toruloides and the enzyme PDC (EC 4.1.1.-) from L. plantarum were introduced into these strains to allow the conversion of l-tyrosine into p-hydroxystyrene (Fig. ​(Fig.1).1). These minor modifications resulted in an efficient biocatalyst for the production of the value-added compound p-hydroxystyrene from glucose.Open in a separate windowFIG. 1.Schematic overview of the biochemical pathway for p-hydroxystyrene production. TAL, tyrosine ammonia lyase; FCS, feruloyl-coenzyme A synthetase. The cross indicates the disruption of fcs, disabling p-coumarate degradation.     

Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae)

The utility of a nuclear protein-coding gene for reconstructing phylogenetic relationships within the family Culicidae was explored. Relationships among 13 species representing three subfamilies and nine genera of Culicidae were analyzed using a 762-bp fragment of coding sequence from the eye color gene, white. Outgroups for the study were two species from the sister group Chaoboridae. Sequences were determined from clone PCR products amplified from genomic DNA, and aligned following conceptual intron splicing and amino acid translation. Third codon positions were characterized by high levels of divergence and biased nucleotide composition, the intensity and direction of which varied among taxa. Equal weighting of all characters resulted in parsimony and neighboring-joining trees at odds with the generally accepted phylogenetic hypothesis based on morphology and rDNA sequences. The application of differential weighting schemes recovered the traditional hypothesis, in which the subfamily Anophelinae formed the basal clade. The subfamily Toxorhynchitinae occupied an intermediate position, and was a sister group to the subfamily Culicinae. Within Culicinae, the genera Sabethes and Tripteroides formed an ancestral clade, while the Culex-Deinocerites and Aedes- Haemagogus clades occupied increasingly derived positions in the molecular phylogeny. An intron present in the Culicinae- Toxorhynchitinae lineage and one outgroup taxon was absent in the basal Anophelinae lineage and the second outgroup taxon, suggesting that intron insertions or deletions may not always be reliable systematic characters.