游动放线菌8-22中treY基因敲除对于降低阿卡波糖C组分的作用

Effect of treY gene inactivation on reducing of acarbose component C in Actinoplanes 8-22

摘要:

【目的】在阿卡波糖发酵过程中，C组分的存在严重影响阿卡波糖产品的质量，研究拟通过基因改造降低阿卡波糖C组分。【方法】通过构建treY同框敲除质粒pUAmT-YUD，以接合转移方法将其转入阿卡波糖工业菌株8-22，经同源重组将treY基因内部编码182个氨基酸的序列敲除，从而得到treY基因失活的突变株Y810。【结果】发酵结果显示突变菌株中C组分较出发菌株下降了约10倍，而阿卡波糖本身的效价末受影响。【结论】敲除treY基因可大幅降低阿卡波糖C组分的含量。研究的实施将大大简化阿卡波糖的纯化步骤，提升产品品质，降低生产成本，从而提高工业化生产的市场竞争力。研究同时还对游动放线菌的接合转移条件进行了优化，大大提高了转化效率。

Abstract:

[Objective] In acarbose producing strain Actinoplanes sp., treY, an important gene in trehalose synthesis, encodes an enzyme that catalyzes acarbose to produce component C during the fermentation. However, component C impacts the quality of acarbose products terribly. In current study, we construct a genetically engineered strain with reduced component C. [Methods] An in-frame knock-out plasmid pUAmT-YUD is constructed and transformed into acarbose-producing strain Actinoplanes 8-22 to inactivate treY by homologous recombination. [Results] Fermentation tests showed that component C produced by the recombinant strain Y810 was reduced by about 10-fold comparing with the parent strain, while the titer of acarbose was not affected. [Conclusion] Knockout of treY leads to compound C decrease in acarbose fermentation. The application of this trial will simplify the purification steps, improve the acarbose quality and reduce the cost to some extent. In addition, conjugation conditions of Actinoplanes are optimized and the conjugation efficiency is improved notably.