te Biesebeke R, Record E, van Biezen N, Heerikhuisen M, Franken A, Punt P J, van den Hondel C A M J J
Wageningen Centre for Food Sciences, P.O. Box 557, 6700 AN, Wageningen, The Netherlands.
Appl Microbiol Biotechnol. 2005 Nov;69(1):44-50. doi: 10.1007/s00253-005-1968-4. Epub 2005 Oct 20.
To study the relation between the number of hyphal tips and protein secretion during growth on a solid substrate, we have constructed two mutant strains of Aspergillus oryzae with increased hyphal branching. We have analysed hydrolytic enzyme activities during growth on wheat kernels (WK) of A. oryzae strains carrying the disrupted allele of the pclA gene encoding a secretion pathway specific (KEX2-like) endo-protease and the disrupted allele of the pg/pi-tp gene encoding a phosphatidylglycerol/phosphatidylinositol transfer protein. The biomass levels produced by the pclA and pg/pi-tp disrupted strains on wheat-based solid media were similar as found for the wild-type strain. However, the pclA disrupted strain showed much more compact colony morphology than the other two strains. Sporulation of the pclA and pg/pi-tp disrupted strains occurred, respectively, 2 days and 1 day later, compared to the wild type during fermentation on ground WK. During surface growth, microscopic analysis revealed that the hyphal growth unit length (L (hgu)) of the pclA and pg/pi-tp disrupted strains was, on average, 50 and 74% of that of the wild-type strain. This implies that in both mutant strains, a higher branching frequency occurs than in the wild-type strain. Compared to the wild-type strain, the pclA and pg/pi-tp disrupted strains produced at least 50% more amylase, at least 100% more glucoamylase and at least 90% more protease activity levels after growth on WK. These results support the hypothesis that branching mutants with an increased branching frequency can improve the solid state fermentation process.
为了研究在固体基质上生长期间菌丝尖端数量与蛋白质分泌之间的关系，我们构建了两株具有增加的菌丝分支的米曲霉突变株。我们分析了携带编码分泌途径特异性（类KEX2）内切蛋白酶的pclA基因的破坏等位基因以及编码磷脂酰甘油/磷脂酰肌醇转移蛋白的pg/pi-tp基因的破坏等位基因的米曲霉菌株在小麦籽粒（WK）上生长期间的水解酶活性。pclA和pg/pi-tp破坏菌株在基于小麦的固体培养基上产生的生物量水平与野生型菌株相似。然而，pclA破坏菌株显示出比其他两株菌株更紧凑的菌落形态。在磨碎的WK上发酵期间，与野生型相比，pclA和pg/pi-tp破坏菌株的孢子形成分别延迟2天和1天。在表面生长期间，显微镜分析表明，pclA和pg/pi-tp破坏菌株的菌丝生长单位长度（L(hgu)）平均分别为野生型菌株的50%和74%。这意味着在这两种突变菌株中，分支频率都比野生型菌株更高。与野生型菌株相比，在WK上生长后，pclA和pg/pi-tp破坏菌株产生的淀粉酶至少多50%，糖化酶至少多100%，蛋白酶活性水平至少多90%。这些结果支持了这样的假设，即具有增加的分支频率的分支突变体可以改善固态发酵过程。
