粗糙脉孢菌纤维素酶液体发酵优良形态突变体筛选

Improving cellulases production with Neurospora crassa by morphology mutants screening

丝状真菌被广泛地用于包括纤维素酶在内的工业酶生产过程。在液体深层发酵中,丝状真菌菌丝形态直接影响发酵液的流变特性,进而与目标酶蛋白产量存在着重要的关联。目前,针对丝状真菌工业酶液体发酵菌丝形态的研究依然是从传统的发酵工程学角度出发,对与发酵水平紧密相关的形态、粘度等性状相关基因的认识远远不够。为了挖掘深层发酵中对丝状真菌发酵产酶性能具有重要影响的形态发育相关基因,以粗糙脉孢菌Neurospora crassa单基因突变体库中的95株形态突变株为研究对象,在结晶纤维素为碳源的条件下进行筛选,探寻与野生型菌株蛋白产量有显著差异的突变株。同时,对这些突变株的内切-β-1,4-葡聚糖酶酶活、β-葡萄糖苷酶酶活、发酵液粘度和菌丝干重进行了测定,并观察了发酵液中突变株的菌丝形态。实验结果表明,与野生型菌株相比,突变株SZY32、SZY35、SZY39和SZY43发酵液中蛋白浓度显著降低,突变株SZY11、SZY63、SZY69和SZY87发酵液中蛋白浓度显著性提高。值得注意的是,突变株SZY11和SZY43发酵液菌丝体主要形态为菌球状,其发酵液粘度分别降低75%和50%,突变株SZY87在发酵液中呈长丝状,发酵液粘度显著升高至少2倍。这些与产酶水平相关的形态、粘度基因的获得将有助于丝状真菌纤维素酶等工业酶高产工程菌株的理性构建。

Filamentous fungi are widely used for large-scale production of cellulases. Morphological characteristics of mycelia under submerged condition are closely correlated with cellulases productivity. In order to find out the critical genes involved in the mycelial morphology development and cellulases production in liquid fermentation, 95 Neurospora crassa morphological mutants （named as SZY1-95） were screened for cellulases production. Compared with the wild type, cellulases production in four mutants SZY32, SZY35, SZY39 and SZY43 were significantly decreased, whereas mutants SZY63, SZY69, SZY87 and SZY11 produced much more cellulases than that of the wild type strain. Meanwhile, endo-beta-l,4-glucanase activity, beta-glucosidase activity, viscosity of broth and dry weight of these mutants were measured. The mycelial morphology of the mutants was also studied by microscope. Particularly, pellets were formed in mutant SZY11 and SZY43, whose viscosities were 25% and 50% of the wild type strain, respectively. Mutant SZY87 appeared long hyphae, and the viscosity of its broth was at least 2 folds of the wild type strain. These results indicate that a single gene deletion could influence the mycelial morphology in liquid fermentation, and increased the cellulases production. The low-viscosity related genes identified in our study will be the potential candidates for genetic modification of filamentous fungi.