Gene_locus Report for: fusox-CUT

Secreted-in-xylem (SIX) proteins of the vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici are secreted during infection of tomato and function in virulence or avirulence. F. oxysporum formae speciales have specific host ranges but the roles of SIX proteins in diverse hosts are unknown. We identified homologs of F. oxysporum f. sp. lycopersici SIX1, SIX4, SIX8, and SIX9 in the genome of Arabidopsis infecting isolate Fo5176. A SIX4 homolog (termed Fo5176-SIX4) differed from that of F. oxysporum f. sp. lycopersici (Fol-SIX4) by only two amino acids, and its expression was induced during infection of Arabidopsis. Transgenic Arabidopsis plants constitutively expressing Fo5176-SIX4 had increased disease symptoms with Fo5176. Conversely, Fo5176-SIX4 gene knock-out mutants (Deltasix4) had significantly reduced virulence on Arabidopsis, and this was associated with reduced fungal biomass and host jasmonate-mediated gene expression, the latter known to be essential for host symptom development. Full virulence was restored by complementation of Deltasix4 mutants with either Fo5176-SIX4 or Fol-SIX4. Thus, Fo5176-SIX4 contributes quantitatively to virulence on Arabidopsis whereas, in tomato, Fol-SIX4 acts in host specificity as both an avirulence protein and a suppressor of other race-specific resistances. The strong sequence conservation for SIX4 in F. oxysporum f. sp. lycopersici and Fo5176 suggests a recent common origin.

Cutinolytic enzymes are secreted by fungal pathogens attacking the aerial parts of the plant, to facilitate penetration of the outermost cuticular barrier of the host. The role of cutinases in soil-borne root pathogens has not been studied thus far. Here we report the characterization of the zinc finger transcription factor Ctf1 from the vascular wilt fungus Fusarium oxysporum, a functional orthologue of CTF1alpha that controls expression of cutinase genes and virulence in the pea stem pathogen Fusarium solani f. sp. pisi. Mutants carrying a Deltactf1 loss-of-function allele grown on inducing substrates failed to activate extracellular cutinolytic activity and expression of the cut1 and lip1 genes, encoding a putative cutinase and lipase, respectively, whereas strains harbouring a ctf1(C) allele in which the ctf1 coding region was fused to the strong constitutive Aspergillus nidulans gpdA promoter showed increased induction of cutinase activity and gene expression. These results suggest that F. oxysporum Ctf1 mediates expression of genes involved in fatty acid hydrolysis. However, expression of lip1 during root infection was not dependent on Ctf1, and virulence of the ctf1 mutants on tomato plants and fruits was indistinguishable from that of the wild-type. Thus, in contrast to the stem pathogen F. solani, Ctf1 is not essential for virulence in the root pathogen F. oxysporum.

The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of di-(2-ethylhexyl)-phthalate (DEHP) was investigated. The DEHP-degradation rate of fungal cutinase was surprisingly high, i.e. almost 70% of the initial DEHP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DEHP disappeared within the initial 15 min. With the yeast esterase, despite the same concentration, more than 85% of the DEHP remained even after 3 days of treatment. During the enzymatic degradation of DEHP, several DEHP-derived compounds were detected and time-course changes in composition were also monitored. During degradation with fungal cutinase, most DEHP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. In the degradation by yeast esterase, two organic chemicals were produced from DEHP: IBF and an unidentified compound (X). The final chemical composition after 3 days was significantly dependent on the enzyme used. Fungal cutinase produced IBF as a major degradation compound. However, in the DEHP degradation by yeast esterase, compound X was produced in abundance in addition to IBF. The toxic effects of the final degradation products were investigated, using various recombinant bioluminescent bacteria and, as a result, the degradation products from yeast esterase were shown to contain a toxic hazard, causing oxidative stress and damage to protein synthesis.

Plastics are highly durable and widely used materials. Current methodologies of plastic degradation, elimination, and recycling are flawed. In recent years, biodegradation (the usage of microorganisms for material recycling) has grown as a valid alternative to previously used methods. The evolution of bioengineering techniques and the discovery of novel microorganisms and enzymes with degradation ability have been key. One of the most produced plastics is PET, a long chain polymer of terephthalic acid (TPA) and ethylene glycol (EG) repeating monomers. Many enzymes with PET degradation activity have been discovered, characterized, and engineered in the last few years. However, classification and integrated knowledge of these enzymes are not trivial. Therefore, in this work we present a summary of currently known PET degrading enzymes, focusing on their structural and activity characteristics, and summarizing engineering efforts to improve activity. Although several high potential enzymes have been discovered, further efforts to improve activity and thermal stability are necessary.

BACKGROUND: Cutinases are serine hydrolases that degrade cutin, a polyester of fatty acids that is the main component of plant cuticle. These biocatalysts have recently attracted increased biotechnological interest due to their potential to modify and degrade polyethylene terephthalate (PET), as well as other synthetic polymers.
METHODS: A cutinase from the mesophilic fungus Fusarium oxysporum, named FoCut5a, was expressed either in the cytoplasm or periplasm of Escherichia coli BL21. Its X-ray structure was determined to 1.9A resolution using molecular replacement. The activity of the recombinant enzyme was tested on a variety of synthetic esters and polyester analogues.
RESULTS: The highest production of recombinant FoCut5a was achieved using periplasmic expression at 16 degrees C. Its crystal structure is highly similar to previously determined Fusarium solani cutinase structure. However, a more detailed comparison of the surface properties and amino acid interactions revealed differences with potential impact on the biochemical properties of the two enzymes. FoCut5a showed maximum activity at 40 degrees C and pH8.0, while it was active on three p-nitrophenyl synthetic esters of aliphatic acids (C2, C4, C12), with the highest catalytic efficiency for the hydrolysis of the butyl ester. The recombinant cutinase was also found capable of hydrolyzing PET model substrates and synthetic polymers.
CONCLUSIONS: The present work is the first reported expression and crystal structure determination of a functional cutinase from the mesophilic fungus F. oxysporum with potential application in surface modification of PET synthetic polymers. GENERAL SIGNIFICANCE: FoCut5a could be used as a biocatalyst in industrial applications for the environmentally-friendly treatment of synthetic polymers.

Secreted-in-xylem (SIX) proteins of the vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici are secreted during infection of tomato and function in virulence or avirulence. F. oxysporum formae speciales have specific host ranges but the roles of SIX proteins in diverse hosts are unknown. We identified homologs of F. oxysporum f. sp. lycopersici SIX1, SIX4, SIX8, and SIX9 in the genome of Arabidopsis infecting isolate Fo5176. A SIX4 homolog (termed Fo5176-SIX4) differed from that of F. oxysporum f. sp. lycopersici (Fol-SIX4) by only two amino acids, and its expression was induced during infection of Arabidopsis. Transgenic Arabidopsis plants constitutively expressing Fo5176-SIX4 had increased disease symptoms with Fo5176. Conversely, Fo5176-SIX4 gene knock-out mutants (Deltasix4) had significantly reduced virulence on Arabidopsis, and this was associated with reduced fungal biomass and host jasmonate-mediated gene expression, the latter known to be essential for host symptom development. Full virulence was restored by complementation of Deltasix4 mutants with either Fo5176-SIX4 or Fol-SIX4. Thus, Fo5176-SIX4 contributes quantitatively to virulence on Arabidopsis whereas, in tomato, Fol-SIX4 acts in host specificity as both an avirulence protein and a suppressor of other race-specific resistances. The strong sequence conservation for SIX4 in F. oxysporum f. sp. lycopersici and Fo5176 suggests a recent common origin.

Cutinolytic enzymes are secreted by fungal pathogens attacking the aerial parts of the plant, to facilitate penetration of the outermost cuticular barrier of the host. The role of cutinases in soil-borne root pathogens has not been studied thus far. Here we report the characterization of the zinc finger transcription factor Ctf1 from the vascular wilt fungus Fusarium oxysporum, a functional orthologue of CTF1alpha that controls expression of cutinase genes and virulence in the pea stem pathogen Fusarium solani f. sp. pisi. Mutants carrying a Deltactf1 loss-of-function allele grown on inducing substrates failed to activate extracellular cutinolytic activity and expression of the cut1 and lip1 genes, encoding a putative cutinase and lipase, respectively, whereas strains harbouring a ctf1(C) allele in which the ctf1 coding region was fused to the strong constitutive Aspergillus nidulans gpdA promoter showed increased induction of cutinase activity and gene expression. These results suggest that F. oxysporum Ctf1 mediates expression of genes involved in fatty acid hydrolysis. However, expression of lip1 during root infection was not dependent on Ctf1, and virulence of the ctf1 mutants on tomato plants and fruits was indistinguishable from that of the wild-type. Thus, in contrast to the stem pathogen F. solani, Ctf1 is not essential for virulence in the root pathogen F. oxysporum.

The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of di-(2-ethylhexyl)-phthalate (DEHP) was investigated. The DEHP-degradation rate of fungal cutinase was surprisingly high, i.e. almost 70% of the initial DEHP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DEHP disappeared within the initial 15 min. With the yeast esterase, despite the same concentration, more than 85% of the DEHP remained even after 3 days of treatment. During the enzymatic degradation of DEHP, several DEHP-derived compounds were detected and time-course changes in composition were also monitored. During degradation with fungal cutinase, most DEHP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. In the degradation by yeast esterase, two organic chemicals were produced from DEHP: IBF and an unidentified compound (X). The final chemical composition after 3 days was significantly dependent on the enzyme used. Fungal cutinase produced IBF as a major degradation compound. However, in the DEHP degradation by yeast esterase, compound X was produced in abundance in addition to IBF. The toxic effects of the final degradation products were investigated, using various recombinant bioluminescent bacteria and, as a result, the degradation products from yeast esterase were shown to contain a toxic hazard, causing oxidative stress and damage to protein synthesis.

In the mid-1970s, information technology and recombinant DNA technology were considered as the breakthrough technologies of the final quarter of the 20th century. Now, about 25 years later, information technology has penetrated deeply into our society and nearly everyone uses this technology. Compared to the formidable success of information technology, the progress in the commercialization of recombinant DNA technology is moderate, even when taking into account that all that is related to the technological application of biological sciences needs extensive safety testing. However, there are signs that the speed of this commercialization will increase in the first decade of the 21st century. Moreover, new breakthroughs in our understanding of the complete genetic make up of eukaryotes will contribute to this increase in speed. An important aspect of the commercialization of this technology is the development of cells as factories for the production of valuable and/or useful molecules. Lower eukaryotes, such as yeasts and molds, are the most promising candidates to become the factories of the future, but at present these factories still contains a lot of process lines that may be superfluous under the well controlled conditions in fermentors. On the other hand, the speed and yield of these cellular production lines can be increased by eliminating the rate-determining steps of these process lines. In this contribution to the European Union symposium from Cell to Factory, some steps in the improvement of S. cerevisiae as cell factories for (heterologous) hydrophobic molecules are presented.

This study focuses on the different efficiencies of secretion of two fungal cutinases by Saccharomyces cerevisiae, a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028). Both cutinases are placed under control of the GAL7 promoter, by which the expression levels can be regulated. Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level. Pulse-chase experiments revealed that whereas CY000 cutinase is secreted, CY028 is irreversibly retained in the cell. Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic reticulum (ER). The increase of wild-type cutinase expression did not result in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase. Immunoprecipitation studies showed that in contrast to the wild-type cutinase, the hydrophobic mutant cutinase interacts with BiP. These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.