Manipulation of the Global Regulator mcrA Upregulates Secondary Metabolite Production in Aspergillus wentii Using CRISPR-Cas9 with In Vitro Assembled Ribonucleoproteins.
Manipulation of the Global Regulator mcrA Upregulates Secondary Metabolite Production in Aspergillus wentii Using CRISPR-Cas9 with In Vitro Assembled Ribonucleoproteins.
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DOI：
10.1021/acschembio.2c00456
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发表时间：
2022-10-21
影响因子：
4
通讯作者：
Wang, Clay C. C.
中科院分区：
文献类型：
--
作者：
Yuan, Bo;Keller, Nancy P.;Oakley, Berl R.;Stajich, Jason E.;Wang, Clay C. C.
丝状真菌的基因组测序表明，大多数次级代谢物生物合成基因簇（BGC）在标准实验室条件下是沉默的。在这项工作中，我们已经在文氏曲霉中建立了体外CRISPR-Cas9系统。为了激活沉默的BGC，我们删除了负转录调节因子mcrA。当该菌株在马铃薯葡萄糖培养基（PDA）上培养时，mcrA的缺失（mcrAΔ）导致总共17个SM的差异产生。这些SM中有9种得到了充分表征，包括大黄素（1）、大黄素甲醚（2）、磺罗胆素（3）、大黄素甲醚双蒽酮（4）、14-O-去甲基磺罗胆素（5）、（反式/顺式）-大黄素双蒽酮（6和7）和（反式/顺式）-大黄素大黄素甲醚双蒽酮（8和9）。发现这些化合物都是由相同的聚酮合酶（PKS）BGC产生的。然后，我们在mcrAΔ背景下针对该PKS簇进行了二次敲除。双敲除菌株的代谢产物谱显示了先前在mcrAΔ亲本菌株中未检测到的新代谢产物。从双敲除菌株中纯化了另外两种SM，并将其表征为曲霉酸B（16）和一种结构相关但先前未鉴定的化合物（17）。这项工作首次提出了一种能够在A.文蒂。这项工作还说明了执行双重敲除以消除主要代谢产物的实用性，从而能够发现额外的SM。
Genome sequencing of filamentous fungi has demonstrated that most secondary metabolite biosynthetic gene clusters (BGCs) are silent under standard laboratory conditions. In this work, we have established an in vitro CRISPR-Cas9 system in Aspergillus wentii. To activate otherwise silent BGCs, we deleted the negative transcriptional regulator mcrA. Deletion of mcrA (mcrAΔ) resulted in differential production of 17 SMs in total when the strain was cultivated on potato dextrose media (PDA). Nine out of fifteen of these SMs were fully characterized, including emodin (1), physcion (2), sulochrin (3), physcion bianthrone (4), 14-O-demethylsulochrin (5), (trans/cis)-emodin bianthrone (6 and 7), and (trans/cis)-emodin physcion bianthrone (8 and 9). These compounds were all found to be produced by the same polyketide synthase (PKS) BGC. We then performed a secondary knockout targeting this PKS cluster in the mcrAΔ background. The metabolite profile of the dual-knockout strain revealed new metabolites that were not previously detected in the mcrAΔ parent strain. Two additional SMs were purified from the dual-knockout strain and were characterized as aspergillus acid B (16) and a structurally related but previously unidentified compound (17). For the first time, this work presents a facile genetic system capable of targeted gene editing in A. wentii. This work also illustrates the utility of performing a dual knockout to eliminate major metabolic products, enabling additional SM discovery.
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影响因子：
8.4
作者：
de Mattos-Shipley KMJ;Spencer CE;Greco C;Heard DM;O'Flynn DE;Dao TT;Song Z;Mulholland NP;Vincent JL;Simpson TJ;Cox RJ;Bailey AM;Willis CL
通讯作者：
Willis CL
影响因子：
4.8
作者：
Grahl N;Demers EG;Crocker AW;Hogan DA
通讯作者：
Hogan DA
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
2.6
作者：
Easwar, S;Argade, NP
通讯作者：
Argade, NP
影响因子：
6.4
作者：
Kjaerbolling, Inge;Vesth, Tammi;Andersen, Mikael R.
通讯作者：
Andersen, Mikael R.