利用CRISPR-Cas9系统与核糖体工程获得新型可利霉素产生菌

Construction of a novel carrimycin-producing strain by using CRISPR-Cas9 and ribosome engineering techniques

摘要:

可利霉素 (Carrimycin，CAM) 是将异戊酰基转移酶基因 (Isovaleryltransferase gene，ist) 导入到螺旋链霉菌中产生的以异戊酰螺旋霉素 (Isovalerylspiramycin，ISP) 为主组分的抗生素。原工程菌中的ist基因与螺旋霉素 (Spiramycin，SP) 生物合成基因簇相距较远，且具有两种抗性基因，难以对其进行基因改造，因此需要构建新型CAM工程菌株。文中通过CRISPR-Cas9基因编辑系统靶向切割2个位点，将ist和其正调控基因acyB2通过同源重组插入到SP生物合成基因簇附近且不参与SP合成的orf54基因下游，获得2种无外源抗性基因插入的CAM产生菌54IA-1和54IA-2，经发酵产物检测发现54IA-2菌株中的ISP产量明显高于54IA-1菌株。通过实时定量PCR (Quantitative real-time PCR，qPCR) 检测证实54IA-2菌株中ist和acyB2基因以及部分SP生物合成基因的表达量均高于54IA-1菌株。为进一步获得高产菌株，以54IA-2为出发菌株，利用核糖体工程的方法筛选利福平 (Rifampicin，RFP) 抗性菌株，在RFP浓度为40 μg/mL的抗性菌株中，ISP的产量明显提高，最高可达842.9 μg/mL，比原始菌株提高约6倍。对其中7株菌的rpoB基因进行测序分析，每株菌的第576位丝氨酸都突变为丙氨酸，在其他错义突变中产量最高的菌株RFP40-6-8在第424位的谷氨酰胺突变为亮氨酸。综上所述，本研究应用CRISPR-Cas9系统成功构建了无任何抗性标记的新型CAM工程菌株54IA-1和54IA-2，并通过核糖体工程技术筛选获得了新型CAM高产菌株RFP40-6-8，为CAM工程菌株的进一步优化改造奠定了基础。

Abstract:

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 μg/mL RFP increased significantly, with the highest up to 842.9 μg/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.