Research Area of Gene Technology and Applied Biochemistry, Institute for Chemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1a/1665, A-1060 Wien, Austria.
Biotechnol Biofuels. 2012 Jan 2;5(1):1. doi: 10.1186/1754-6834-5-1.
The ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies.
Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414.
Using this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production.
食真菌菌，里氏木霉（Hypocrea jecorina 的无性型），是一种生物技术的骨干，也是目前最有效的纤维素酶生产者之一。虽然传统上通过随机诱变来改良菌株，但只有使用重组技术才能更好地了解纤维素酶的调控。
为了提高效率和高通量方法，我们在此提出了里氏木霉基因敲除的构建试剂盒。我们提供了使用 pyr4、amdS 和 hph 选择标记进行基因缺失的引物数据库。为了实现高通量基因敲除，我们构建了使用酵母介导重组的载体，然后通过孢子电穿孔转化缺乏非同源末端连接（NHEJ）的里氏木霉菌株。随后，通过与从亲本菌株 QM9414 衍生的有性能力的菌株杂交，消除了 NHEJ 缺陷。
使用这种策略和提供的材料，里氏木霉的高通量基因缺失变得可行。此外，通过有性发育的应用，可以有效地去除 NHEJ 缺陷，而无需额外的选择标记。对于通过杂交具有不同基因缺失的菌株构建多个突变体，也具有相同的优势，与转化方法相比，现在所需的手工时间和筛选工作量大大减少。因此，这个工具包可以极大地促进对里氏木霉资源的有效利用，用于纤维素酶表达，从而促进第二代生物燃料的生产。
