Integrativna ekspresija gena za glukoamilazu u soju pivskoga kvasca Saccharomyces pastorianus

The recombinant brewer’s yeast Saccharomyces pastorianus strain was constructed byintroducing the ilv2:GLA fragment released from pMGI6, carrying glucoamylase gene (GLA) and using the yeast α-acetolactate synthase gene (ILV2) as the recombination sequence. The strain was able to utilise starch as the sole carbon source, its glucoamylase activity was 6.3 U/mL and its α-acetolactate synthase activity was lowered by 33.3 %. The introduced GLA gene was integrated at the recipient genomic ILV2 gene, one copy of ILV2 gene was disrupted and the other copy remained intact. Primary wort fermentation test confirmed that the diacetyl and residual sugar concentration in the wort fermented by the recombinant strain were reduced by 65.6 and 34.2 % respectively, compared to that of the recipient strain. Under industrial operating conditions, the maturation time of beer fermented by the recombinant strain was reduced from 7 to 4 days, there were no significant differences in the appearance and mouthfeel, and the beer satisfied the high quality demands. That is why the strain could be used in beer production safely.Uvođenjem ilv2:GLA isječka iz pMGI6, kao nosioca gena za glukoamilazu (GLA) i uporabom rekombinacijske sekvencije gena za α-acetolaktat sintazu (ILV2), konstruiran je rekombinantni soj pivskoga kvasca Saccharomyces pastorianus. Utvrđeno je da soj može koristiti škrob kao jedini izvor ugljika, da aktivnost glukoamilaze iznosi 6,3 U/mL, a da je aktivnost α-acetolaktat sintaze smanjena za 33,3 %. Uvedeni gen GLA, integriran u gen primatelja ILV2, raskinuo je jednu kopiju gena ILV2, dok je druga kopija ostala nepromijenjena. Prvi test fermentacije sladovine potvrdio je da je fermentacijom s pomoću rekombinantnog soja koncentracija diacetila smanjena za 65,6 %, a koncentracija ostatka šećera za 34,2 % u usporedbi sa sojem primatelja. U industrijskim uvjetima zrenje piva fermentiranog s pomoću rekombinantnog soja smanjeno je sa 7 na 4 dana, pri čemu nema značajne razlike u izgledu i okusu, te je ono zadovoljilo visoke zahtjeve. Stoga se ovaj soj može sigurno upotrijebiti u proizvodnji piva

Integrativna ekspresija gena za glukoamilazu u soju pivskoga kvasca Saccharomyces pastorianus

The recombinant brewer’s yeast Saccharomyces pastorianus strain was constructed byintroducing the ilv2:GLA fragment released from pMGI6, carrying glucoamylase gene (GLA) and using the yeast α-acetolactate synthase gene (ILV2) as the recombination sequence. The strain was able to utilise starch as the sole carbon source, its glucoamylase activity was 6.3 U/mL and its α-acetolactate synthase activity was lowered by 33.3 %. The introduced GLA gene was integrated at the recipient genomic ILV2 gene, one copy of ILV2 gene was disrupted and the other copy remained intact. Primary wort fermentation test confirmed that the diacetyl and residual sugar concentration in the wort fermented by the recombinant strain were reduced by 65.6 and 34.2 % respectively, compared to that of the recipient strain. Under industrial operating conditions, the maturation time of beer fermented by the recombinant strain was reduced from 7 to 4 days, there were no significant differences in the appearance and mouthfeel, and the beer satisfied the high quality demands. That is why the strain could be used in beer production safely.Uvođenjem ilv2:GLA isječka iz pMGI6, kao nosioca gena za glukoamilazu (GLA) i uporabom rekombinacijske sekvencije gena za α-acetolaktat sintazu (ILV2), konstruiran je rekombinantni soj pivskoga kvasca Saccharomyces pastorianus. Utvrđeno je da soj može koristiti škrob kao jedini izvor ugljika, da aktivnost glukoamilaze iznosi 6,3 U/mL, a da je aktivnost α-acetolaktat sintaze smanjena za 33,3 %. Uvedeni gen GLA, integriran u gen primatelja ILV2, raskinuo je jednu kopiju gena ILV2, dok je druga kopija ostala nepromijenjena. Prvi test fermentacije sladovine potvrdio je da je fermentacijom s pomoću rekombinantnog soja koncentracija diacetila smanjena za 65,6 %, a koncentracija ostatka šećera za 34,2 % u usporedbi sa sojem primatelja. U industrijskim uvjetima zrenje piva fermentiranog s pomoću rekombinantnog soja smanjeno je sa 7 na 4 dana, pri čemu nema značajne razlike u izgledu i okusu, te je ono zadovoljilo visoke zahtjeve. Stoga se ovaj soj može sigurno upotrijebiti u proizvodnji piva

Integrative Expression of Glucoamylase Gene in a Brewer’s Yeast Saccharomyces pastorianus Strain

The recombinant brewer’s yeast Saccharomyces pastorianus strain was constructed byintroducing the ilv2:GLA fragment released from pMGI6, carrying glucoamylase gene (GLA) and using the yeast α-acetolactate synthase gene (ILV2) as the recombination sequence. The strain was able to utilise starch as the sole carbon source, its glucoamylase activity was 6.3 U/mL and its α-acetolactate synthase activity was lowered by 33.3 %. The introduced GLA gene was integrated at the recipient genomic ILV2 gene, one copy of ILV2 gene was disrupted and the other copy remained intact. Primary wort fermentation test confirmed that the diacetyl and residual sugar concentration in the wort fermented by the recombinant strain were reduced by 65.6 and 34.2 % respectively, compared to that of the recipient strain. Under industrial operating conditions, the maturation time of beer fermented by the recombinant strain was reduced from 7 to 4 days, there were no significant differences in the appearance and mouthfeel, and the beer satisfied the high quality demands. That is why the strain could be used in beer production safely

Factors controlling mycotoxin contamination in maize and food in the Hebei province, China

International audienceAbstractMycotoxins contaminate maize and thus pose a serious economic and health risk. Despite the prevalence of mycotoxins in maize and other crops, the agricultural management system has not yet been fully implemented in China. Thereby, there is an urgent need to implement management programs to control mycotoxin contamination in maize and maize-based feed. Here, we conducted a survey on the traditional maize production and feed processing practices in the Hebei province. We analyzed the factors of fungal infection and subsequent mycotoxin contamination in maize and feed supply chain and management systems. Our results show that the stored maize samples from local feed mills were heavily contaminated with fumonisin, at 9638 μg/kg on the average, and deoxynivalenol, at 996.3 μg/kg on the average. We also found that more than 75.0 % of the farmers followed good agricultural practices, whereas only 38.1 % of the farmers cared for storage conditions. The main factors for mycotoxin contamination included less strict receiving and inspection criteria, inappropriate storage conditions, and poor processing practices. The critical control points were feed mill receiving, storage, and feed processing. Visual quality, moisture contents, and mycotoxin levels in maize and feed were used as monitoring parameters at critical control points. This is the first study to thoroughly explore traditional maize and feed manufacturing practices adopted by farmers

Gβ-like CpcB plays a crucial role for growth and development of Aspergillus nidulans and Aspergillus fumigatus.

Growth, development, virulence and secondary metabolism in fungi are governed by heterotrimeric G proteins (G proteins). A Gβ-like protein called Gib2 has been shown to function as an atypical Gβ in Gpa1-cAMP signaling in Cryptococcus neoformans. We found that the previously reported CpcB (cross pathway control B) protein is the ortholog of Gib2 in Aspergillus nidulans and Aspergillus fumigatus. In this report, we further characterize the roles of CpcB in governing growth, development and toxigenesis in the two aspergilli. The deletion of cpcB results in severely impaired cellular growth, delayed spore germination, and defective asexual sporulation (conidiation) in both aspergilli. Moreover, CpcB is necessary for proper expression of the key developmental activator brlA during initiation and progression of conidiation in A. nidulans and A. fumigatus. Somewhat in accordance with the previous study, the absence of cpcB results in the formation of fewer, but not micro-, cleistothecia in A. nidulans in the presence of wild type veA, an essential activator of sexual development. However, the cpcB deletion mutant cleistothecia contain no ascospores, validating that CpcB is required for progression and completion of sexual fruiting including ascosporogenesis. Furthermore, unlike the canonical GβSfaD, CpcB is not needed for the biosynthesis of the mycotoxin sterigmatocystin (ST) as the cpcB null mutant produced reduced amount of ST with unaltered STC gene expression. However, in A. fumigatus, the deletion of cpcB results in the blockage of gliotoxin (GT) production. Further genetic analyses in A. nidulans indicate that CpcB may play a central role in vegetative growth, which might be independent of FadA- and GanB-mediated signaling. A speculative model summarizing the roles of CpcB in conjunction with SfaD in A. nidulans is presented

Summary of <i>cpcB</i>.

<p>(A∼B) <i>cpcB</i> mRNA levels during the life cycle of <i>A. nidulans</i> (A) and <i>A. fumigatus</i> (B). Conidia (asexual spores) are indicated as C. The numbers indicate the time (hours) after incubation in liquid MMG (Vegetative), and on solid MMG under conditions inducing conidiation (Asexual) or sexual development (Sexual). The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of mRNA bands using ImageJ and each band was normalized by the amount of 18S rRNA (<i>cpcB</i>/18S rRNA) in the relevant lane. Ethidium bromide staining of ribosomal RNAs was used as a loading control. (C) Alignment of <i>A. nidulans</i> (Ani) CpcB, <i>A. fumigatus</i> (Afu) CpcB, <i>A. flavus</i> (Afl) CpcB with Gib2 of <i>C. neoformans var. grubii</i> (Cgr; accession: AY907679) and <i>C. neoformans var. neoformans</i> (Cne; accession: AY907680). ClustalW (<a href="http://align.genome.jp/" target="_blank">http://align.genome.jp/</a>) and BoxShade 3.21 (<a href="http://www.ch.embnet.org/software/BOX_form.html" target="_blank">http://www.ch.embnet.org/software/BOX_form.html</a>) were used for the alignment and presentation. (D) A phylogenetic tree of CpcB-like proteins identified in various fungal species including <i>A. clavatus</i>, <i>A. flavus</i>, <i>A. fumigatus</i>, <i>A. nidulans</i>, <i>A. niger</i>, <i>A. oryzae</i>, <i>A. terreus (A = Aspergillus)</i>, <i>C. neoformans</i>, <i>Neurospora crassa</i>, <i>Penicillium chrysogenum</i>, <i>Penicillium marneffei</i>, <i>Schizophyllum commune</i>, <i>Schizosaccharomyces japonicus</i> and <i>Talaromyces stipitatus</i>. The putative CpcB proteins were retrieved from NCBI BlastX (<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a>) using <i>A. nidulans</i> CpcB. A phylogenetic tree of 14 putative CpcB homologues was generated by the TreeTop software (<a href="http://genebee.msu.su/services/phtree_reduced.html" target="_blank">http://genebee.msu.su/services/phtree_reduced.html</a>) using the alignment data from ClustalW. The phylogenetic tree is constructed based on the matrix of pairwise distances between sequences. Numbers indicate the computed distances given the residue substitution weights from the alignment data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070355#pone.0070355-Brodskii1" target="_blank">[59]</a>.</p

Phenotype of the Δ<i>flbA</i>Δ<i>cpcB</i> and Δ<i>rgsA</i>Δ<i>cpcB</i> mutants.

<p>Photographs of colonies of WT (FGSC4), Δ<i>cpcB</i> (RJMP1.59-8), Δ<i>flbA</i> (QK1), and Δ<i>flbA</i>Δ<i>cpcB</i> (QK3) strains (A); and Δ<i>rgsA</i> (QK2) and Δ<i>rgsA</i>Δ<i>cpcB</i> (QK4) strains (B) grown on solid MMG for 3 days are shown. The two left panels show the point-inoculated strains (top and bottom), and the two right panels show close-up views of single and double mutant colonies shown in the left panels. Photomicrographs were taken by Zeiss Axioplan 2 stereomicroscope. Note the highly restricted growth of the double mutants (A and B), and the lack of conidiation in the Δ<i>flbA</i> and Δ<i>flbA</i> Δ<i>cpcB</i> mutant (A).</p

Requirement of CpcB for sexual development in <i>A. nidulans</i>.

<p>(A) Photographs of the point-inoculated colonies of WT (TNJ36), Δ<i>cpcB</i> (RJMP1.59-8) and complemented (C’; RJMP1.59-8C) strains of <i>A. nidulans</i> grown on MMG for 7 days (top panels), and the close-up views of the colonies (bottom panels) captured by a Zeiss Axioplan 2 stereomicroscope are shown. Cleistothecia are marked as CT. (B) Morphology of cleistothecia formed by the three strains. Images were captured by a Zeiss Axioplan 2 stereomicroscope. (C) Quantitative analyses of cleistothecia (CLS) per cm² produced by the three strains grown on MMG for 7 days. (D) Northern blot analyses for levels of <i>veA</i> and <i>vos</i>A transcripts after sexual developmental induction of <i>A. nidulans</i> WT (FGSC4) and Δ<i>cpcB</i> (RJMP1.59-8) strains. The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of RNA bands using ImageJ and normalized by the amounts of 18S rRNA (<i>veA</i>/18S rRNA or <i>vosA</i>/18S rRNA) in each sample. Ethidium bromide staining of ribosomal RNAs was used as a loading control. Numbers indicate the time post sexual developmental induction.</p

Requirement of CpcB for proper growth and conidiation in <i>A. nidulans</i> and <i>A. fumigatus</i>.

<p>(A) Photographs of the point-inoculated colonies of WT control (TNJ36), Δ<i>cpcB</i> (RJMP1.59-8) and complemented (C’; RJMP1.59-8C) strains of <i>A. nidulans</i> grown on solid MMG for 4 days (top panels) and the close-up views of the colonies (bottom panels) are shown in left panel. Photographs of the colonies of WT (AF293), Δ<i>AfucpcB</i> (Af293.1-7) and complemented (C’; Af293.1-7C) strains of <i>A. fumigatus</i> grown on solid MM+0.5%YE for 4 days (top panels), and the close-up views of the colonies (bottom panels) are shown in right panel. (B) Quantitative analyses of the diameter of colonies and conidiation levels in the designated strains. (C) Northern blot analyses for levels of <i>brl</i>A transcript in the WT (FGSC4) and Δ<i>AnicpcB</i> (RJMP1.59-8) strains of <i>A. nidulans</i>, and in WT (Af293) and Δ<i>AfucpcB</i> (Af293.1-7) strains of <i>A. fumigatus.</i> The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of mRNA bands using ImageJ and normalized by the amounts of 18S rRNA (<i>brlA</i>/18S rRNA) in each sample. Ethidium bromide staining of rRNA was used as a loading control.</p

The roles of CpcB in conidial germination and hyphal growth.

<p>(A∼B) Quantitative analyses of conidial germination and hyphal growth post conidial germination of WT (filled bar; TNJ36), Δ<i>cpcB</i> (blank bar; RJMP1.59-8) and complemented (C) (shaded bar; RJMP1.59-8C) <i>A. nidulans</i> strains on MMG (A); and of WT (AF293), Δ<i>AfucpcB</i> (Af293.1-7) and complemented (C’; Af293.1-7C) <i>A. fumigates</i> strains on MMG (B). (C) Photographs of the three <i>A. nidulans</i> strains grown on solid MMG for 1 day (top panels) and the close-up views (bottom panels). Note the lack of conidiophores in Δ<i>cpcB</i> strain. (D) Photographs of the three <i>A. fumigates</i> strains of grown on solid MMG for 1 day (top panels), and the close-up views (bottom panels). Note the differences in the size and number of conidiophores.</p