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Efficient cloning system for construction of gene silencing vectors in Aspergillus niger.

ABSTRACT: An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two semi-quantitative plate assays testing different activities for xylan degradation. Upon induction on D-xylose, transcript levels of xlnR were decreased in the xlnR-silenced strains, compared to a wild-type background. Under these conditions, the transcript levels of xyrA and xynB (two genes regulated by XlnR) were also decreased for these xlnR-silenced strains. These results indicate that the newly developed system for rapid generation of silencing vectors is an effective tool for A. niger, and this can be used to generate strains with a tailored spectrum of enzyme activities or product formation by silencing specific genes encoding, e.g., regulators such as XlnR.

SUBMITTER: Oliveira JM

PROVIDER: S-EPMC7420921 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Efficient cloning system for construction of gene silencing vectors in Aspergillus niger.

Oliveira José Miguel JM van der Veen Douwe D de Graaff Leo H LH Qin Ling L

Applied microbiology and biotechnology 20080815 5

An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two ...[more]

PMID: 18704394

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Project description:UNLABELLED:Palm oil obtained from E. guineensis Jacq. Tenera is known to have about 44% of palmitic acid (C16:0). Palmitoyl-Acyl Carrier Protein Thioesterase (PATE) is one of the key enzymes involved in plastidial fatty acid biosynthesis; and it determines the level of the C16:0 assimilation in oilseeds. This enzyme's activity in oil palm is responsible for high (> 44 % in E. guineensis Jacq. Tenera and 25 % in E. oleifera) content of C16:0 in its oil. By post-transcriptional PATE gene silencing, C16:0 content can be minimized for nutritional value improvement of the palm oil. The objective of this study was the construction of novel transformation vectors for PATE gene silencing. Six different transformation vectors targeted against PATE gene were constructed using 619 bp long PATE gene (5' region) fragment (from GenBank AF507115). In one set of three transformation vectors, PATE gene fragment was fused with CaMV 35S promoter in antisense, intron-spliced inverted repeat (ISIR), and inverted repeat (IR) orientations to generate antisense mRNA and hair-pin RNAs (hpRNA). In another set of three transformation vectors with same design, CaMV 35S was replaced with Oil palm mesocarp tissue-specific promoter (MSP). The expression cassette of antisense, ISIR, and IR of PATE gene fragments were constructed in primary cloning vector, pHANNIBAL or its derivative/s. Finally, all 6 expression cassettes were sub-cloned into pCAMBIA 1301 which contains the Hygromycinr and the GUS reporter genes for transformant selection and transformation detection respectively. The results of the RE analyses of the constructs and sequence analyses of PATE and MSP shows and confirms the orientation, size and locations of all the components from constructs. We hypothesize that 4 (pISIRPATE-PC, pIRPATE-PC, pMISIRPATE-PC and pMIRPATE-PC) out of 6 transformation vectors constructed in this study will be efficient and effective in palmitoyl-ACP thioesterase gene silencing in oil palm. ABBREVIATIONS:antiPATE - Antisense Palmitoyl-acyl carrier protein thioesterase, BCV - Binary cloning vector, cDNA - Complementary deoxyribonucleic acid, hpRNA - hair-pin RNA, ihpRNA - intron containing hair-pin RNA, IR - inverted repeat, ISIR - intron-spliced inverted repeat, MCS - Multiple cloning site, MSP - Oil palm mesocarp tissue-specific promoter, nt - Nucleotide/s, PATE - Palmitoyl-acyl carrier protein thioesterase, PCR - Polymerase chain reaction, PCV - Primary cloning vector, pDNA - Plasmid deoxyribonucleic acid, PTGS - Post-transcriptional gene silencing, RE - Restriction enzyme.

Project description:BACKGROUND:The lifestyle of filamentous fungi depends on the secretion of hydrolytic enzymes into the surrounding medium, which degrade polymeric substances into monomers that are then taken up to sustain metabolism. This feature has been exploited in biotechnology to establish platform strains with high secretory capacity including Aspergillus niger. The accepted paradigm is that proteins become mainly secreted at the tips of fungal hyphae. However, it is still a matter of debate if the amount of growing hyphal tips in filamentous fungi correlates with an increase in secretion, with previous studies showing either a positive or no correlation. RESULTS:Here, we followed a systematic approach to study protein secretion in A. niger. First, we put the glaA gene encoding for glucoamylase (GlaA), the most abundant secreted protein of A. niger, under control of the tunable Tet-on system. Regulation of glaA gene expression by omitting or adding the inducer doxycycline to cultivation media allowed us to study the effect of glaA under- or overexpression in the same isolate. By inducing glaA expression in a fluorescently tagged v-SNARE reporter strain expressing GFP-SncA, we could demonstrate that the amount of post-Golgi carriers indeed depends on and correlates with glaA gene expression. By deleting the racA gene, encoding the Rho-GTPase RacA in this isolate, we generated a strain which is identical to the parental strain with respect to biomass formation but produces about 20% more hyphal tips. This hyperbranching phenotype caused a more compact macromorphology in shake flask cultivations. When ensuring continuous high-level expression of glaA by repeated addition of doxycycline, this hyperbranching strain secreted up to four times more GlaA into the culture medium compared to its parental strain. CONCLUSION:The data obtained in this study strongly indicate that A. niger responds to forced transcription of secretory enzymes with increased formation of post-Golgi carriers to efficiently accommodate the incoming cargo load. This physiological adaptation can be rationally exploited to generate hypersecretion platforms based on a hyperbranching phenotype. We propose that a racA deletion background serves as an excellent chassis for such hypersecretion strains.

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Efficient cloning system for construction of gene silencing vectors in Aspergillus niger.

Publications

Efficient cloning system for construction of gene silencing vectors in Aspergillus niger.

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