Lichius Alexander, Bidard Frédérique, Buchholz Franziska, Le Crom Stéphane, Martin Joel, Schackwitz Wendy, Austerlitz Tina, Grigoriev Igor V, Baker Scott E, Margeot Antoine, Seiboth Bernhard, Kubicek Christian P
Research Division Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, A-1060, Vienna, Austria.
IFP Energies Nouvelles, 1-4 Avenue de Bois-Préau, 92852, Rueil-Malmaison, France.
BMC Genomics. 2015 Apr 20;16(1):326. doi: 10.1186/s12864-015-1526-0.
Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a.
In QM9136, we detected a surprisingly low number of mutagenic events in the promoter and coding regions of genes, i.e. only eight indels and six single nucleotide variants. One of these indels led to a frame-shift in the Zn₂Cys₆ transcription factor XYR1, the general regulator of cellulase and xylanase expression, and resulted in its C-terminal truncation by 140 amino acids. Retransformation of strain QM9136 with the wild-type xyr1 allele fully recovered the ability to produce cellulases, and is thus the reason for the cellulase-negative phenotype. Introduction of an engineered xyr1 allele containing the truncating point mutation into the moderate producer T. reesei QM9414 rendered this strain also cellulase-negative. The correspondingly truncated XYR1 protein was still able to enter the nucleus, but failed to be expressed over the basal constitutive level.
The missing 140 C-terminal amino acids of XYR1 are therefore responsible for its previously observed auto-regulation which is essential for cellulases to be expressed. Our data present a working example of the use of genome sequencing leading to a functional explanation of the QM9136 cellulase-negative phenotype.
里氏木霉是将生物质水解为单糖所需的纤维素酶和半纤维素酶的主要工业来源，这些单糖随后可用于生物燃料生产和生物精炼厂。当今使用的高产菌株是通过经典诱变产生的。作为该过程的副产物，产生了一些无法产生纤维素酶的突变体。为了确定导致这种无能的突变，我们对其中一个菌株QM9136的基因组进行了测序，并将其与它的亲本里氏木霉QM6a的基因组进行了比较。
在QM9136中，我们在基因的启动子和编码区检测到数量惊人少的诱变事件，即只有8个插入缺失和6个单核苷酸变体。其中一个插入缺失导致Zn₂Cys₆转录因子XYR1发生移码，XYR1是纤维素酶和木聚糖酶表达的一般调节因子，并导致其C末端截短140个氨基酸。用野生型xyr1等位基因对菌株QM9136进行再转化完全恢复了产生纤维素酶的能力，因此这就是纤维素酶阴性表型的原因。将含有截短点突变的工程化xyr1等位基因导入中度生产菌株里氏木霉QM9414也使该菌株变为纤维素酶阴性。相应截短的XYR1蛋白仍能够进入细胞核，但未能在基础组成水平上表达。
因此，XYR1缺失的140个C末端氨基酸是其先前观察到的自我调节的原因，而这种自我调节对于纤维素酶的表达至关重要。我们的数据展示了一个使用基因组测序的实例，该实例对QM9136纤维素酶阴性表型进行了功能解释。
