解硫胺类芽孢杆菌SY20中多粘菌素A1合成与调控相关基因表达分析

Polymyxin A1 Synthesis-related Gene Expression and Regulation in Paenibacillus thiaminolyticus SY20

该研究以前期筛选的多粘菌素A1产生菌株Paenibacillus thiaminolyticus SY20为研究对象,首先探究菌株在不同生长阶段的抑菌活性变化规律;随后,利用实时荧光定量PCR,研究多粘菌素A1合成基因簇(pmx)五个基因pmxA、pmxB、pmxC、pmxD和pmxE在发酵过程中的表达情况。最后,探究多粘菌素调控基因sfp、ectB、spo0A和abrB表达水平。结果表明,在M63T培养基中,P.thiaminolyticus SY20抑菌物质在菌株生长的对数前期产生,至对数末期或平台前期(84 h)达到最大值;pmx基因簇各基因在菌生长对数前期(36 h)表达水平显著提高且基本呈现同步趋势;ectB与pmxA、pmxE基因的表达水平也基本同步;sfp和spo0A基因表达与抑菌活性呈正相关,而abrB基因与抑菌活性呈负相关。因此,在P.thiaminolyticus SY20发酵过程中,pmx基因簇表达水平先于抑菌活性达到最大值,ectB基因与多粘菌素A1合成密切相关,sfp和spo0A基因为正调控基因,而abrB基因为负调控基因,为多粘菌素合成与调控机制提供理论基础。

The Paenibacillus thiaminolyticus SY20 strain,which was previously screened and identified as polymyxin A1-producing strain,was evaluated for antimicrobial activity at different stages of growth.Subsequently,the expression levels of five genes,namely pmxA,pmxB,pmxC,pmxD,and pmxE in the polymyxin A1 synthetic gene cluster(pmx),were investigated during the fermentation process using real-time PCR.Finally,the expression levels of regulatory genes for polymyxin(sfp,ectB,spo0A,and abrB)were also explored.The results indicated that,in the M63T medium,the antimicrobial substances were produced from P.thiaminolyticus SY20 in the early logarithmic phase of bacterial growth,and reached a maximum value at the end of the logarithmic phase or early stationary phase(84 h).Pmx expression showed a synchronous trend of significant increase in the early logarithmic stage(36 h).The ectB expression was synchronized with the pmxA and pmxE genes.The sfp and spo0A expression was positively correlated with antimicrobial activity,while abrB expression indicated the opposite.In conclusion,during fermentation in P.thiaminolyticus SY20,the pmx cluster expression reached a maximum before exhibiting antimicrobial activity,and ectB was verified as closely related to polymyxin A1 synthesis.Both sfp and spo0A were identified as positive regulatory genes,while abrB was a negative one.This study provides a theoretical basis for understanding the synthesis and regulation mechanisms of polymyxin.