Derntl Christian, Mach Robert L, Mach-Aigner Astrid R
Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorfer Strasse 1a, 1060 Vienna, Austria.
Biotechnol Biofuels. 2019 Sep 30;12:231. doi: 10.1186/s13068-019-1575-8. eCollection 2019.
The filamentous ascomycete is industrially used to produce cellulases and xylanases. Cost-effective production of cellulases is a bottleneck for biofuel production. Previously, different strain and process optimizations were deployed to enhance enzyme production rates. One approach is the overexpression of the main activator Xyr1 and a second is the construction of synthetic transcription factors. Notably, these genetic manipulations were introduced into strains bearing the wild-type gene and locus.
Here, we constructed a Xyr1-deficient strain expressing a non-functional truncated version of Xyr1. This strain was successfully used as platform strain for overexpression of Xyr1, which enhanced the cellulase and xylanase production rates under inducing conditions, with the exception of lactose-there the cellulase production was severely reduced. Further, we introduced fusion transcription factors consisting of the DNA-binding domain of Xyr1 and the transactivation domain of either Ypr1 or Ypr2 (regulators of the sorbicillinoid biosynthesis gene cluster). The fusion of Xyr1 and Ypr2 yielded a moderately transactivating transcription factor, whereas the fusion of Xyr1 and Ypr1 yielded a highly transactivating transcription factor that induced xylanases and cellulases nearly carbon source independently. Especially, high production levels of xylanases were achieved on glycerol.
During this study, we constructed a Xyr1-deficient strain that can be fully reconstituted, which makes it an ideal platform strain for Xyr1-related studies. The mere overexpression of Xyr1 turned out not to be a successful strategy for overall enhancement of the enzyme production rates. We gained new insights into the regulatory properties of transcription factors by constructing respective fusion proteins. The Xyr1-Ypr1-fusion transcription factor could induce xylanase production rates on glycerol to outstanding extents, and hence could be deployed in the future to utilize crude glycerol, the main co-product of the biodiesel production process.
丝状子囊菌在工业上用于生产纤维素酶和木聚糖酶。纤维素酶的经济高效生产是生物燃料生产的一个瓶颈。此前，人们采用了不同的菌株和工艺优化方法来提高酶的生产速率。一种方法是过表达主要激活因子Xyr1，另一种方法是构建合成转录因子。值得注意的是，这些基因操作被引入到携带野生型基因和位点的菌株中。
在此，我们构建了一个表达无功能截短版Xyr1的Xyr1缺陷菌株。该菌株成功用作过表达Xyr1的平台菌株，在诱导条件下提高了纤维素酶和木聚糖酶的生产速率，但乳糖除外——在乳糖条件下纤维素酶的产量严重降低。此外，我们引入了由Xyr1的DNA结合结构域和Ypr1或Ypr2（山梨素生物合成基因簇的调节因子）的反式激活结构域组成的融合转录因子。Xyr1和Ypr2的融合产生了一个中等反式激活的转录因子，而Xyr1和Ypr1的融合产生了一个高度反式激活的转录因子，该因子几乎不依赖碳源诱导木聚糖酶和纤维素酶。特别是，在甘油上实现了高水平的木聚糖酶生产。
在本研究中，我们构建了一个可完全重构的Xyr1缺陷菌株，这使其成为Xyr1相关研究的理想平台菌株。事实证明，单纯过表达Xyr1并不是全面提高酶生产速率的成功策略。通过构建相应的融合蛋白，我们对转录因子的调控特性有了新的认识。Xyr1-Ypr1融合转录因子可以在很大程度上诱导甘油上木聚糖酶的生产速率，因此未来可用于利用生物柴油生产过程中的主要副产物粗甘油。
