Genetically modified filamentous fungi and uses thereof
a filamentous fungus, a technology of filamentous fungi, applied in the field of filamentous fungi, can solve the problems of complex cleaning equipment, low productivity, and heterogeneous culture, and achieve the effect of improving the properties of filamentous fungi in submerged environments and reducing mycelia adhesion
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example 1
Target Selection and Gene Deletion
[0152]Transcriptional data of biomass obtained from chemostat cultivations of a transcription factor mutant and wild type strain at two different pH conditions (pH 2.5 and 5.0) was analyzed, using ANOVA statistical testing, to determine genes subjected to differential transcriptional regulation. The regulatory effect of the pH was isolated and fold change ratio (LOG 2) was calculated. Empiric Bayesian statistics were used to moderate the standard errors within each gene, and Benjamini-Hochberg's method, to adjust for multitesting. A cut-off value of adjusted P<0.05 was set to assess statistical significance A summary of the data are found in table 1.
TABLE 1Significantly expressed hydrophobins.JGI IDFold change, pH Adj. P value1285304.4721.14E−0553462−4.9222.86E−05456833.5010.0047456851.1820.0074431841.7880.0257
[0153]From table 1, it is clear that hydrophobin 128530 and 45683 are progressively reacting to increasing pH. This response is likewise seen...
example 2
[0154]As gene deletion strategy, a bipartite gene knockout method was selected (Nielsen et al. 2006). The knockout substrate consisted of two-parts; one, contained a fragment upstream of the gene, fused to a truncate hygromycin marker (2 / 3 the marker gene, lacking the end). The second part contained a truncate hygromycin marker (2 / 3 the marker gene, lacking the start) fused to a fragment, downstream to of the gene. The knockout substrate was amplified individually by PCR and transformed into in A. niger by standard protoplast / PEG mediated transformation. The gene deletion was achieved by insertion of the substrate into A. niger's genome by means of homologous recombination.
example 3
[0155]The gene deletion was verified using PCR and tested for ectopic insertions by Southern analysis.
[0156]The mutant strain (delta 128530) was initially investigated in 500 mL shake flaks, without baffels. The flasks were filled with 100 mL of watman medium and inoculated to a final spore concentration of 2E09 spores / L, the agitation speed was 150 RPM and the temperature was controlled to 30° C. After 4 days photos were taken of the flasks, see FIG. 1.
[0157]Further investigation of the mutant was done in a 2 L Sartorius bioreactor with a working volume of 1.8 L. The temperature was maintained at 30° C. during the cultivation and pH was controlled by automatic addition of 2 M NaOH and 1 M HCl. Initial conditions in the bioreactor were pH: 3.0; stirring rate: 100 rpm; and aeration: 0.2 volumes of air per volume of fluid per minute (vvm). After germination, the stirring rate was gradually increased to 800 rpm and the air flow to 1 vvm. The pH was adjusted to 5.0 with addition of 2 M ...
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