Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
Technische Universität Berlin, Faculty III Process Sciences, Institute of Biotechnology, Chair of Applied and Molecular Microbiology, Berlin, Germany.
Sci Rep. 2020 May 6;10(1):7630. doi: 10.1038/s41598-020-64893-6.
We present a Penicillium rubens strain with an industrial background in which the four highly expressed biosynthetic gene clusters (BGC) required to produce penicillin, roquefortine, chrysogine and fungisporin were removed. This resulted in a minimal secondary metabolite background. Amino acid pools under steady-state growth conditions showed reduced levels of methionine and increased intracellular aromatic amino acids. Expression profiling of remaining BGC core genes and untargeted mass spectrometry did not identify products from uncharacterized BGCs. This platform strain was repurposed for expression of the recently identified polyketide calbistrin gene cluster and achieved high yields of decumbenone A, B and C. The penicillin BGC could be restored through in vivo assembly with eight DNA segments with short overlaps. Our study paves the way for fast combinatorial assembly and expression of biosynthetic pathways in a fungal strain with low endogenous secondary metabolite burden.
我们展示了一株具有工业背景的红色青霉菌株，该菌株中用于生产青霉素、罗奎福特菌素、金雀花碱和真菌孢子素的四个高度表达的生物合成基因簇 (BGC) 已被去除。这导致最低限度的次生代谢物背景。在稳态生长条件下的氨基酸池显示出蛋氨酸水平降低和细胞内芳香族氨基酸增加。剩余 BGC 核心基因的表达谱和非靶向质谱分析未鉴定出未表征 BGC 的产物。该平台菌株被重新用于最近鉴定的聚酮类 calbistrin 基因簇的表达，并实现了 decumbenone A、B 和 C 的高产。青霉素 BGC 可以通过体内组装带有 8 个短重叠 DNA 片段来恢复。我们的研究为在低内源性次生代谢物负担的真菌菌株中快速组合组装和表达生物合成途径铺平了道路。
