Heterologous Synthesis of Monacolin J by Reconstructing Its Biosynthetic Gene Cluster in Aspergillus niger.
Heterologous Synthesis of Monacolin J by Reconstructing Its Biosynthetic Gene Cluster in Aspergillus niger.
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通过在黑曲霉中重建Monacolin J生物合成基因簇异源合成Monacolin J
DOI：
10.3390/jof8040407
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发表时间：
2022-04-16
期刊：
影响因子：
--
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莫纳可林J（MJ）是洛伐他汀的关键前体，通过水解洛伐他汀并添加不同的侧链可以合成重要的他汀类药物辛伐他汀。在这项研究中，为了减少洛伐他汀在本地菌株土曲霉中产生 MJ 的繁琐水解，将 MJ 生物合成途径基因（lovB、lovC、lovG 和 lovA）异源整合到曲霉基因组中。黑曲霉CBS513.88具有强启动子和合适的整合位点，通过酵母2μ同源重组构建长基因表达盒，并通过CRISPR/Cas9同源定向重组（CRISPR-HDR）将MJ基因整合到黑曲霉基因组中。 RT-PCR结果证明途径合成相关基因可以在黑曲霉中异源表达。最后，我们构建了一个可以产生莫纳可林 J 的工程菌株，经 LC-HR-ESIMS 检测（MJ，339.22 [M-H]+）。培养7天后MJ产量达到92.90 mg/L。通过优化培养条件并添加前体，补料培养第4天MJ的最终效价为142.61 mg/L，较原生长条件提高了53.5%。由于黑曲霉在食品和医药工业发酵中的广泛应用，后续工作将致力于优化代谢网络以提高工程菌株的MJ产量。
Monacolin J (MJ), a key precursor of Lovastatin, could synthesize important statin drug simvastatin by hydrolyzing lovastatin and adding different side chains. In this study, to reduce the cumbersome hydrolysis of lovastatin to produce MJ in the native strain Aspergillus terreus, the MJ biosynthetic pathway genes (lovB, lovC, lovG, and lovA) were heterologously integrated into the genome of Aspergillus. niger CBS513.88 with strong promoters and suitable integration sites, via yeast 2μ homologous recombination to construct expression cassettes of long-length genes and CRISPR/Cas9 homology-directed recombination (CRISPR-HDR) to integrate MJ genes in the genome of A. niger. RT-PCR results proved that pathway synthesis-related genes could be heterologously expressed in A. niger. Finally, we constructed an engineered strain that could produce monacolin J, detected by LC-HR-ESIMS (MJ, 339.22 [M-H]+). The yield of MJ reached 92.90 mg/L after 7-day cultivation. By optimizing the cultivation conditions and adding precursor, the final titer of MJ was 142.61 mg/L on the fourth day of fed-batch cultivation, which was increased by 53.5% compared to the original growth conditions. Due to the wide application of A. niger in industrial fermentation for food and medicine, the following work will be dedicated to optimizing the metabolic network to improve the MJ production in the engineered strain.
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影响因子：
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作者：
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DOI：
10.3390/jof7050374
发表时间：
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期刊：
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影响因子：
--
作者：
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通讯作者：
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