Zeng Xu, Zheng Junwei, Lu Feifei, Pan Li, Wang Bin
School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu Town, Guangzhou 510006, China.
Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, Guangzhou 510006, China.
J Fungi (Basel). 2022 Apr 16;8(4):407. doi: 10.3390/jof8040407.
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 , the MJ biosynthetic pathway genes (, , and ) were heterologously integrated into the genome of 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 . RT-PCR results proved that pathway synthesis-related genes could be heterologously expressed in . 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 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.
莫纳可林J（MJ）是洛伐他汀的关键前体，可通过水解洛伐他汀并添加不同侧链来合成重要的他汀类药物辛伐他汀。在本研究中，为减少天然菌株中洛伐他汀水解生成MJ的繁琐过程，通过酵母2μ同源重组将MJ生物合成途径基因（、、和）与强启动子和合适的整合位点异源整合到CBS513.88的基因组中，构建长链基因表达盒，并通过CRISPR/Cas9同源定向重组（CRISPR-HDR）将MJ基因整合到的基因组中。RT-PCR结果证明途径合成相关基因可在中异源表达。最后，我们构建了一株能够产生莫纳可林J的工程菌株，通过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，补料分批培养第4天MJ的最终效价为142.61 mg/L，与原始生长条件相比提高了53.5%。由于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中MJ的产量。
