利用CRISPR-Cas9系统构建新型异戊酰螺旋霉素Ⅰ产生菌

Construction of a new isovalerylspiramycin I producing strain by CRISPR-Cas9 system

摘要:

异戊酰螺旋霉素 (Isovalerylspiramycin，ISP)Ⅰ是必特螺旋霉素 (Bitespiramycin，BT) 的一种主组分，其抗菌活性与BT相似，而且作为单一组分在质控和剂型上更具优势，目前正在进行临床前研究。原有的ISPⅠ工程菌株经过3次基因改造，已经具有2种抗性基因，很难再进行遗传操作。前期研究利用经典同源重组的方法无法构建无抗性的ISPⅠ产生菌，文中利用CRISPR-Cas9基因编辑系统在螺旋霉素 (Spiramycin，SP) 产生菌中成功将3位酰基化酶的基因bsm4替换为组成型强启动子ermEp*控制下的异戊酰基转移酶基因 (Isovaleryltansferase gene，ist)。删除bsm4后突变株只能产生SPⅠ组分，外源基因ist的表达产物催化SPⅠ在其4″位进行异戊酰化修饰形成ISPⅠ。经过HPLC和质谱鉴定，阳性菌株ΔEI的发酵产物中只有ISPⅠ一种ISP组分，证实新的ISPⅠ工程菌株构建成功。ΔEI菌株不带有抗性基因，可重复利用CRISPR-Cas9系统进行基因操作来获得新的改良菌株。

Abstract:

Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.