This work describes the effects of the cell surface display of a synthetic phytochelatin in the highly metal tolerant bacterium Cupriavidus metallidurans CH34. The EC20sp synthetic phytochelatin gene was fused between the coding sequences of the signal peptide (SS) and of the autotransporter β-domain of the Neisseria gonorrhoeae IgA protease precursor (IgAβ), which successfully targeted the hybrid protein toward the C. metallidurans outer membrane. The expression of the SS-EC20sp-IgAβ gene fusion was driven by a modified version of the Bacillus subtilis mrgA promoter showing high level basal gene expression that is further enhanced by metal presence in C. metallidurans. The recombinant strain showed increased ability to immobilize Pb 2+ , Zn 2+ , Cu 2+ , Cd 2+ , Mn 2+ , and Ni 2+ ions from the external medium when compared to the control strain. To ensure plasmid stability and biological containment, the MOB region of the plasmid was replaced by the E. coli hok/sok coding sequence.
Aims: The aim of this study was to obtain improved strains of pectinolytic yeasts adapted to the conditions of an industrial fermentation process, which was continuously operated to convert citrus molasses into ethanol.
Methods and Results: The starter yeast of the industrial fermentation process was a commercial baker's yeast, which was capable of growing without forming any secretion halo of pectinase activity on solid medium. Nevertheless, isolates showing secretion of pectinolytic activity on plates were obtained from the fermentation process. The secretion of pectin‐degrading activity by isolates on plates was repressed by galactose and improved as the result of colony aging on polygalacturonic acid plates at 30°C. Liquefaction of polygalacturonate gels as well as the splitting of the pectin‐degrading activity into a wall‐linked and a supernatant fraction were also observed when the starter yeast was propagated under agitation in liquid medium containing pectin.
Conclusions: Isolates capable of secreting pectinolytic activity on plates were predominant at the end of the citrus molasses fermentation. Nevertheless, the sizes of the secretion haloes on plates were not necessarily an indication of the levels of pectinolytic activity secreted in the liquid medium.
Significance and Impact of the Study: Improved pectinolytic strains of Saccharomyces can be used as a source of pectinases for a variety of applications. This organism also participates in plant deterioration processes.
A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation.
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