Biotechnology Labs, Astellas Pharma Inc., 5-2-3, Tokodai, Tsukuba, Ibaraki, 300-2698, Japan.
Technology Research Association of Highly Efficient Gene Design (TRAHED), 3-17-6 Hacchobori, Chuo-ku, Tokyo, 104-0032, Japan.
Appl Microbiol Biotechnol. 2018 Feb;102(3):1393-1405. doi: 10.1007/s00253-017-8685-7. Epub 2017 Dec 21.
In the production of useful microbial secondary metabolites, the breeding of strains is generally performed by random mutagenesis. However, because random mutagenesis introduces many mutations into genomic DNA, the causative mutations leading to increased productivity are mostly unknown. Therefore, although gene targeting is more efficient for breeding than random mutagenesis, it is difficult to apply. In this study, a wild-type strain and randomly mutagenized strains of fungal sp. No. 14919, a filamentous fungus producing the HMG-CoA reductase inhibitor polyketide FR901512, were subjected to point mutation analysis based on whole genome sequencing. Among the mutated genes found, mutation of the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) had a positive effect on increasing FR901512 productivity. By complementing the SCAP gene in the SCAP-mutated strain, productivity was decreased to the level of the SCAP-intact strain. Conversely, when either the SCAP or SREBP gene was deleted, the productivity was significantly increased. By genomic transcriptional analysis, the expression levels of three enzymes in the ergosterol biosynthesis pathway were shown to be decreased by SCAP mutation. These findings led to the hypothesis that raw materials of polyketides, such as acetyl-CoA and malonyl-CoA, became more available for FR901512 biosynthesis due to depression in sterol biosynthesis caused by knockout of the SREBP system. This mechanism was confirmed in Aspergillus terreus producing the polyketide lovastatin, which is structurally similar to FR901512. Thus, knockout of the SREBP system should be considered significant for increasing the productivities of polyketides, such as HMG-CoA reductase inhibitors, by filamentous fungi.
在有用的微生物次生代谢产物的生产中,通常通过随机诱变来培育菌株。然而,由于随机诱变会将许多突变引入基因组 DNA 中,因此导致生产力提高的因果突变大多是未知的。因此,虽然基因靶向比随机诱变更有利于培育,但很难应用。在这项研究中,对产生 HMG-CoA 还原酶抑制剂聚酮 FR901512 的丝状真菌 No.14919 的野生型菌株和随机诱变菌株进行了基于全基因组测序的点突变分析。在所发现的突变基因中,固醇调节元件结合蛋白(SREBP)切割激活蛋白(SCAP)的突变对增加 FR901512 生产力有积极影响。通过在 SCAP 突变株中互补 SCAP 基因,生产力降低到 SCAP 完整株的水平。相反,当 SREBP 或 SCAP 基因缺失时,生产力显著增加。通过基因组转录分析,发现 SCAP 突变导致甾醇生物合成途径中的三种酶的表达水平降低。这些发现导致了这样的假设,即由于 SREBP 系统的敲除导致甾醇生物合成受到抑制,聚酮,如乙酰辅酶 A 和丙二酰辅酶 A 的原料变得更有利于 FR901512 生物合成。这一机制在结构上类似于 FR901512 的多烯 lovastatin 的产生菌 Aspergillus terreus 中得到了证实。因此,敲除 SREBP 系统应该被认为是通过丝状真菌增加 HMG-CoA 还原酶抑制剂等聚酮类化合物的生产力的重要手段。