van Leeuwe Tim M, Arentshorst Mark, Punt Peter J, Ram Arthur F J
Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, the Netherlands.
Dutch DNA Biotech, Hugo R Kruytgebouw 4-Noord, Padualaan 8, 3584 CH Utrecht, the Netherlands.
Gene X. 2020 Jan 28;5:100028. doi: 10.1016/j.gene.2020.100028. eCollection 2020 Dec.
Post-fermentation fungal biomass waste provides a viable source for chitin. Cell wall chitin of filamentous fungi, and in particular its de-acetylated derivative chitosan, has a wide range of commercial applications. Although the cell wall of filamentous fungi comprises 10-30% chitin, these yields are too low for cost-effective production. Therefore, we aimed to identify the genes involved in increased chitin deposition by screening a collection of UV-derived cell wall mutants in . This screen revealed a mutant strain (RD15.4#55) that showed a 30-40% increase in cell wall chitin compared to the wild type. In addition to the cell wall chitin phenotype, this strain also exhibited sensitivity to SDS and produces an unknown yellow pigment. Genome sequencing combined with classical genetic linkage analysis identified two mutated genes on chromosome VII that were linked with the mutant phenotype. Single gene knockouts and subsequent complementation analysis revealed that an 8 bp deletion in NRRL3_09595 is solely responsible for the associated phenotypes of RD15.4#55. The mutated gene, which was named (), encodes an orthologue of Bypass of (), a negative regulator of transcription elongation. We propose that this conserved fungal protein is involved in preventing cell wall integrity signaling under non-inducing conditions, where loss of function results in constitutive activation of the cell wall stress response pathway, and consequently leads to increased chitin content in the mutant cell wall.
发酵后真菌生物质废料是几丁质的一个可行来源。丝状真菌的细胞壁几丁质，尤其是其脱乙酰化衍生物壳聚糖，具有广泛的商业应用。尽管丝状真菌的细胞壁含有10%-30%的几丁质，但这些产量对于具有成本效益的生产来说太低了。因此，我们旨在通过筛选一系列紫外线诱导的细胞壁突变体来鉴定参与增加几丁质沉积的基因。该筛选揭示了一个突变菌株（RD15.4#55），与野生型相比，其细胞壁几丁质增加了30%-40%。除了细胞壁几丁质表型外，该菌株还对十二烷基硫酸钠敏感，并产生一种未知的黄色色素。基因组测序结合经典遗传连锁分析确定了七号染色体上的两个突变基因与突变表型相关。单基因敲除及后续互补分析表明，NRRL3_09595中的8bp缺失是RD15.4#55相关表型的唯一原因。这个突变基因被命名为（），编码转录延伸负调控因子（）的直系同源物。我们提出，这种保守的真菌蛋白在非诱导条件下参与防止细胞壁完整性信号传导，其功能丧失会导致细胞壁应激反应途径的组成性激活，从而导致突变体细胞壁中几丁质含量增加。
