阿卡波糖生物合成调控蛋白的钓取与功能解析

Mining and function studies of regulators for acarbose biosynthesis in Actinoplanes sp. SE50/110

摘要:

[目的] 发现游动放线菌Actinoplanes sp.SE50/110中阿卡波糖生物合成的调控因子，并提高其产量。[方法] 首先，利用DNA亲和层析技术，钓取与阿卡波糖生物合成基因簇2个双向启动子区域结合的调控蛋白。然后，在阿卡波糖产生菌QQ-2中强化表达或敲除这些调控蛋白编码基因，进行体内功能验证。同时，利用大肠杆菌BL21（DE3）异源表达获得可溶性蛋白，通过凝胶阻滞实验验证蛋白与启动子区域的结合能力。[结果] 经DNA亲和层析及蛋白质质谱分析，钓取出9个与双向启动子P_WV和P_AB结合的调控蛋白。在QQ-2中分别强化表达和缺失这9个调控基因后发现，基因ACPL_1889的强化表达使阿卡波糖产量提高25%，而该基因的缺失使产量降低22%；基因ACPL_5445、ACPL_3989的强化表达使阿卡波糖产量分别降低12%和39%，而这两个基因的缺失使产量分别提高15%和8%。对阿卡波糖生物合成基因转录水平的检测发现，强化表达基因ACPL_1889使acbA、acbB、acbW、acbV的转录水平升高，而缺失该基因使这4个基因的转录水平降低；敲除基因ACPL_5445使这4个基因转录水平均有提高；强化表达基因ACPL_3989使这4个基因的转录水平均下降，而其敲除使acbW和acbA的转录水平分别提高了约100倍和40倍。在凝胶阻滞实验中，ACPL_1889与ACPL_3989均能与acb基因簇的启动子区域结合。最后将正调控基因的强化表达和负调控基因的敲除进行组合，使阿卡波糖产量提升32%。[结论] 本研究发现了9个与阿卡波糖生物合成基因簇的启动子区域结合的调控蛋白，通过体内、体外实验证明ACPL_1889为阿卡波糖生物合成的正调控因子、ACPL_5445和ACPL_3989为负调控因子，不但为揭示阿卡波糖生物合成的转录调控机制奠定了基础，而且这些调控基因的改造显著提升了阿卡波糖的产量。

关键词:阿卡波糖;DNA亲和层析;转录调控;凝胶阻滞实验

Abstract:

[Objective] Identification of regulatory factors for acarbose biosynthesis and harnessing them for the improvement of acarbose yield in Actinoplanes sp. SE50/110. [Methods] Firstly, regulatory proteins binding to the two bi-directional promoters of acarbose biosynthetic gene cluster were obtained using DNA affinity chromatography. Secondly, to validate functions, these coding genes of regulatory proteins were deleted or overexpressed in Actinoplanes sp. QQ-2. Next, soluble proteins were obtained by heterologous expression in E. coli BL21(DE3), and electrophoretic mobility shift assays were performed to verify the interaction between these regulatory proteins and promoter regions. [Results] By analyzing the results of affinity chromatography and mass spectra, we identified nine regulatory proteins (ACPL_1889, ACPL_4236, ACPL_7303, ACPL_6479, ACPL_8104, ACPL_8270, ACPL_5445, ACPL_3989, ACPL_7617). Furthermore, we studied the potential function of all the nine regulatory proteins by deleting or overexpressing their coding genes in the strain QQ-2. The overexpression of ACPL_1889 resulted in 25% yield increase, whereas its deletion led to 22% yield decrease of acarbose. Respective overexpression of ACPL_5445 and ACPL_3989 resulted in 12% and 39% yield decrease, whereas their deletions let to 15% and 8% yield increase, respectively. Meanwhile, transcription level of acarbose biosynthetic genes acbA, acbB, acbW and acbV increased when ACPL_1889 was overexpressed and decreased when it was deleted; the transcription of these four genes increased to a certain extent in ACPL_5445 mutant; whereas the transcription of these four genes decreased in the ACPL_3989-overexpressed mutant, the transcription of acbW and acbA increased by 100 times and 40 times in the ACPL_3989 deleted mutant, respectively. Moreover, we found both ACPL_1889 and ACPL_3989 were able to bind to promoters of the acb gene cluster in EMSA experiments. Eventually, we increased the yield of acarbose by 32% applying a combinatory strategy of overexpressing positive regulatory genes and deleting negative regulatory genes. [Conclusion] This study identified nine regulatory proteins binding to the two bi-directional promoters of acarbose biosynthetic gene cluster, among which ACPL_1889 is a positive regulatory factor, while ACPL_5445 and ACPL_3989 are negative regulatory factors. This work not only laid a foundation for studying the regulatory mechanism of acarbose biosynthesis, but also substantially improved acarbose yield by manipulating these regulatory genes.

Key words:acarbose;DNA affinity chromatography;transcriptional regulation;electrophoretic mobility shift assays