Abstract:BackgroundAspergillus sojae has been an important filamentous fungus in Biotechnology due to its use in diverse fermentative processes for the production of various food products. Furthermore, this fungus is a common expression system for the production of enzymes and other metabolites. The availability of molecular genetic tools to explore its biology is thus of big interest. In this study, an Agrobacterium tumefaciens-mediated transformation (ATMT) system for A. sojae was developed and its applicability eval… Show more
“…1 ). This vector was derived from the pRM-eGFP vector (Mora-Lugo et al 2014 ) by subcloning a 1.7-kb synthetic cassette (ordered from Eurofins, Ebersberg, Germany) between Sal I and Pvu I restriction sites. This cassette contains vgb , which was codon usage optimized for Aspergillus species according to the CUTG database (GenBank) and fused to the 5′ end of the reporter egfp gene.…”
Section: Methodsmentioning
confidence: 99%
“…Accuracy of the plasmid sequence was examined by restriction enzyme digestion and sequencing analysis. The recombinant A. tumefaciens strain containing the pRM-vgb vector was used to transform A. sojae by the ATMT procedure as described by Mora-Lugo et al ( 2014 ). Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Mostly in bacterial and yeast bioprocesses, cell growth, bioremediation, and enhanced protein production have been improved within this so called “ vgb/ VHb technology” (Hofmann et al 2009 ; Kahraman et al 2011 ; Kahraman and Erenler 2012 ; Wu and Fu 2012 ; Zhu et al 2011 ). However, the potential of VHb has not been investigated in detail in filamentous fungi, mainly due to the availability of fewer genetic molecular tools to engineer them in comparison to bacterial and yeast hosts (Fleissner and Dersch 2010 ; Mora-Lugo et al 2014 ; Ward 2012 ).…”
Section: Introductionmentioning
confidence: 99%
“…The potential of some of these high-yield pectinase mutants has been further explored by measuring various pectinase activities including exo-/endo-polygalacturonase (exo-PG/endo-PG), exo-polymethylgalacturonase (exo-PMG), and pectin lyase using two different carbon sources in SSF (Mata-Gomez et al 2014 ). Recently, an Agrobacterium tumefaciens -mediated transformation (ATMT) method was described for A. sojae , and heterologous expression of the enhanced green fluorescent protein (EGFP) was demonstrated successfully (Mora-Lugo et al 2014 ). This study opens up new possibilities of protein expression studies in A. sojae and explore systematically strain improvement strategies for this fungus based on genetic molecular tools.…”
The biotechnological value of Aspergillus sojae ATCC 20235 (A. sojae) for production of pectinases in solid-state fermentation (SSF) has been demonstrated recently. However, a common drawback of fungal solid-state cultures is the poor diffusion of oxygen into the fungi that limits its growth and biological productivity. The bacterial Vitreoscilla hemoglobin (VHb) has favored the metabolism and productivities of various bacterial and yeast strains besides alleviating hypoxic conditions of its native host, but the use of VHb in filamentous fungi still remains poor explored. Based on the known effects of VHb, this study assessed its applicability to improve A. sojae performance in SSF. The VHb gene (vgb) under control of the constitutive Aspergillus nidulants gpdA promoter was introduced into the genome of A. sojae by Agrobacterium-mediated transformation. Successful fungal transformants were identified by fluorescence microscopy and polymerase chain reaction (PCR) analyses. In solid-state cultures, the content of protease, exo-polygalacturonase (exo-PG), and exo-polymethylgalacturonase (exo-PMG) of the transformed fungus (A. sojae vgb+) improved were 26, 60, and 44 % higher, respectively, in comparison to its parental strain (A. sojae wt). Similarly, biomass content was also 1.3 times higher in the transformant strain. No significant difference was observed in endo-polygalacturonase (endo-PG) content between both fungal strains, suggesting dissimilar effects of VHb towards different enzymatic productions. Overall, our results show that biomass, protease, and exo-pectinase content of A. sojae in SSF can be improved by transformation with VHb.
“…1 ). This vector was derived from the pRM-eGFP vector (Mora-Lugo et al 2014 ) by subcloning a 1.7-kb synthetic cassette (ordered from Eurofins, Ebersberg, Germany) between Sal I and Pvu I restriction sites. This cassette contains vgb , which was codon usage optimized for Aspergillus species according to the CUTG database (GenBank) and fused to the 5′ end of the reporter egfp gene.…”
Section: Methodsmentioning
confidence: 99%
“…Accuracy of the plasmid sequence was examined by restriction enzyme digestion and sequencing analysis. The recombinant A. tumefaciens strain containing the pRM-vgb vector was used to transform A. sojae by the ATMT procedure as described by Mora-Lugo et al ( 2014 ). Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Mostly in bacterial and yeast bioprocesses, cell growth, bioremediation, and enhanced protein production have been improved within this so called “ vgb/ VHb technology” (Hofmann et al 2009 ; Kahraman et al 2011 ; Kahraman and Erenler 2012 ; Wu and Fu 2012 ; Zhu et al 2011 ). However, the potential of VHb has not been investigated in detail in filamentous fungi, mainly due to the availability of fewer genetic molecular tools to engineer them in comparison to bacterial and yeast hosts (Fleissner and Dersch 2010 ; Mora-Lugo et al 2014 ; Ward 2012 ).…”
Section: Introductionmentioning
confidence: 99%
“…The potential of some of these high-yield pectinase mutants has been further explored by measuring various pectinase activities including exo-/endo-polygalacturonase (exo-PG/endo-PG), exo-polymethylgalacturonase (exo-PMG), and pectin lyase using two different carbon sources in SSF (Mata-Gomez et al 2014 ). Recently, an Agrobacterium tumefaciens -mediated transformation (ATMT) method was described for A. sojae , and heterologous expression of the enhanced green fluorescent protein (EGFP) was demonstrated successfully (Mora-Lugo et al 2014 ). This study opens up new possibilities of protein expression studies in A. sojae and explore systematically strain improvement strategies for this fungus based on genetic molecular tools.…”
The biotechnological value of Aspergillus sojae ATCC 20235 (A. sojae) for production of pectinases in solid-state fermentation (SSF) has been demonstrated recently. However, a common drawback of fungal solid-state cultures is the poor diffusion of oxygen into the fungi that limits its growth and biological productivity. The bacterial Vitreoscilla hemoglobin (VHb) has favored the metabolism and productivities of various bacterial and yeast strains besides alleviating hypoxic conditions of its native host, but the use of VHb in filamentous fungi still remains poor explored. Based on the known effects of VHb, this study assessed its applicability to improve A. sojae performance in SSF. The VHb gene (vgb) under control of the constitutive Aspergillus nidulants gpdA promoter was introduced into the genome of A. sojae by Agrobacterium-mediated transformation. Successful fungal transformants were identified by fluorescence microscopy and polymerase chain reaction (PCR) analyses. In solid-state cultures, the content of protease, exo-polygalacturonase (exo-PG), and exo-polymethylgalacturonase (exo-PMG) of the transformed fungus (A. sojae vgb+) improved were 26, 60, and 44 % higher, respectively, in comparison to its parental strain (A. sojae wt). Similarly, biomass content was also 1.3 times higher in the transformant strain. No significant difference was observed in endo-polygalacturonase (endo-PG) content between both fungal strains, suggesting dissimilar effects of VHb towards different enzymatic productions. Overall, our results show that biomass, protease, and exo-pectinase content of A. sojae in SSF can be improved by transformation with VHb.
“…ATMT is a popular approach for the genetic transformation of fungi because it has several advantages. Firstly, different types of tissue, such as conidia, mycelium or even fruiting bodies, can be transformed by ATMT (Mora-Lugo et al, 2014;Zheng et al, 2017). Thereby, it avoids the enzymatic treatment and tedious process of protoplast preparation.…”
Transferring DNA into cells is an essential research method for molecular cloning and gene function studies. As molecular biology and materials physics develop, more and more new transformation methods have been applied to mammalian cells. Some techniques have been successfully developed for several types of fungi, but their efficiencies are extremely low. To better study the functional genes of fungi, and to improve the characteristics of fungi in an easy, safe and reliable way, many investigations have been conducted to effectively develop such technologies for a wide variety of species and to increase the efficiency and reproducibility of genetic transformation. The objective of this paper is to review the latest development of transformation methods used for the genetic transformation of fungi, including several promising transformation approaches, together with their advantages and drawbacks, which may open up novel methods for fungi research.
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