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.
