[Construction of a novel carrimycin-producing strain by using CRISPR-Cas9 and ribosome engineering techniques].
[Construction of a novel carrimycin-producing strain by using CRISPR-Cas9 and ribosome engineering techniques].
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DOI：
10.13345/j.cjb.200763
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发表时间：
2021-06-25
期刊：
影响因子：
--
通讯作者：
He, Weiqing
中科院分区：
文献类型：
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作者：
Liu, Juanjuan;Zhang, Yan;He, Weiqing
可利霉素（CAM）是以异戊酰螺旋霉素（ISP）为主要成分的新型抗生素。它由整合了异源 4-O-异戊酰转移酶基因 (ist) 的螺旋链霉菌产生。然而，目前的CAM生产菌株携带两个抗性基因标记，这使得进一步的遗传操作变得困难。此外，螺旋霉素（SP）的异戊酰化效率可能较低，因为ist基因距离SP生物合成基因簇较远。本研究利用CRISPR-Cas9技术将ist及其正调控基因acyB2插入到螺旋霉菌1941中与SP生物合成基因簇相邻的orf54基因下游。通过同源重组和质粒脱落，获得了两个新的无标记CAM生产菌株54IA-1和54IA-2。有趣的是，菌株54IA-2中的ISP产量远高于菌株54IA-1。实时定量PCR检测表明，菌株54IA-2中ist、acyB2和一些与SP生物合成相关的基因表现出较高的表达水平。随后，对菌株54IA-2进行利福平（RFP）抗性选择，通过核糖体工程获得高产的CAM突变体。抗40 μg/mL RFP的突变体ISP产量显着增加，最高可达842.9 μg/mL，比菌株54IA-2高约6倍。对这7个突变体的rpoB基因序列进行分析发现，每个测序突变体中都存在576位丝氨酸突变为丙氨酸的情况。在携带其他错义突变的突变体中，携带谷氨酰胺(424)突变为亮氨酸的菌株RFP40-6-8显示出最高的ISP产量。总之，利用CRISPR-Cas9技术成功开发了两个无标记新型CAM生产菌株54IA-1和54IA-2。此外，通过核糖体工程获得了新的CAM高产菌株RFP40-6-8。因此，这项研究展示了一种提高 CAM 生产的有用组合方法。
Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 mug/mL RFP increased significantly, with the highest up to 842.9 mug/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.