Demirci Ebru, Arentshorst Mark, Yilmaz Baran, Swinkels Aram, Reid Ian D, Visser Jaap, Tsang Adrian, Ram Arthur F J
Institute of Biology Leiden, Microbial Sciences, Leiden University, Leiden, Netherlands.
Centre for Structural and Functional Genomics, Concordia University, Montreal, QC, Canada.
Front Genet. 2021 Apr 20;12:666684. doi: 10.3389/fgene.2021.666684. eCollection 2021.
is an important filamentous fungus in industrial biotechnology for the production of citric acid and enzymes. In the late 1980s, the N400/NRRL3 strain was selected for both fundamental and applied studies in relation to several processes including gluconic acid and protein production. To facilitate handling of , the N400 wild-type strain was UV mutagenized in two consecutive rounds to generate N401 and N402. N402 was used as a reference laboratory strain and exhibits the phenotypes with reduced conidiophore stalk length and reduced radial growth. The conidiophore stalk length and radial growth of strain N400 were determined and compared to N401 and N402. The length of N400 conidiophore stalks (2.52 ± 0.40 mm) was reduced in N401 and N402 to 0.66 ± 0.14 mm and 0.34 ± 0.06 mm, respectively. Whereas N400 reached a colony diameter of 6.7 ± 0.2 cm after 7 days, N401 and N402 displayed reduced radial growth phenotype (4.3 ± 0.1 and 4.1 ± 0.1, respectively). To identify the mutations (dubbed and ) responsible for the phenotypes of N401 and N402, the genomes were sequenced and compared to the N400 genome sequence. A parasexual cross was performed between N400 and N402 derivatives to isolate segregants which allowed cosegregation analysis of single nucleotide polymorphisms and insertions and deletions among the segregants. The shorter conidiophore stalk and reduced radial growth in N401 () was found to be caused by a 9-kb deletion on chromosome III and was further narrowed down to a truncation of NRRL3_03857 which encodes a kinesin-like protein homologous to the UncA protein. The mutation responsible for the further shortening of conidiophore stalks in N402 () was found to be caused by a missense mutation on chromosome V in a hitherto unstudied C2H2 transcription factor encoded by the gene NRRL3_06646. The importance of these two genes in relation to conidiophore stalk length and radial growth was confirmed by single and double gene deletion studies. The mutations in the laboratory strain N402 should be taken into consideration when studying phenotypes in the N402 background.
是工业生物技术中用于生产柠檬酸和酶的重要丝状真菌。在20世纪80年代后期，选择了N400/NRRL3菌株用于与包括葡萄糖酸和蛋白质生产在内的多个过程相关的基础和应用研究。为了便于处理，N400野生型菌株连续两轮进行紫外线诱变以产生N401和N402。N402用作参考实验室菌株，表现出分生孢子梗长度缩短和径向生长减少的表型。测定了N400菌株的分生孢子梗长度和径向生长，并与N401和N402进行比较。N400分生孢子梗的长度（2.52±0.40毫米）在N401和N402中分别减少到0.66±0.14毫米和0.34±0.06毫米。虽然N400在7天后达到了6.7±0.2厘米的菌落直径，但N401和N402表现出径向生长减少的表型（分别为4.3±0.1和4.1±0.1）。为了鉴定导致N401和N402表型的突变（分别称为 和 ），对基因组进行了测序并与N400基因组序列进行比较。在N400和N402衍生物之间进行了准性杂交以分离分离子，这允许对分离子之间的单核苷酸多态性以及插入和缺失进行共分离分析。发现N401（ ）中较短的分生孢子梗和减少的径向生长是由III号染色体上的9-kb缺失引起的，并进一步缩小到编码与UncA蛋白同源的驱动蛋白样蛋白的NRRL3_03857的截短。发现导致N402（ ）中分生孢子梗进一步缩短的突变是由V号染色体上一个由NRRL3_06646基因编码的迄今未研究的C2H2转录因子中的错义突变引起的。通过单基因和双基因缺失研究证实了这两个基因与分生孢子梗长度和径向生长的相关性。在研究N402背景下的表型时应考虑实验室菌株N402中的突变。
