A new high-performance heterologous fungal expression system based on regulatory elements from the Aspergillus terreus terrein gene cluster.
A new high-performance heterologous fungal expression system based on regulatory elements from the Aspergillus terreus terrein gene cluster.
复制标题
DOI：
10.3389/fmicb.2015.00184
复制
发表时间：
2015
影响因子：
5.2
通讯作者：
Brock M
中科院分区：
文献类型：
--
作者：
Gressler M;Hortschansky P;Geib E;Brock M
近年来，对地曲霉terrein基因簇进行了鉴定和筛选，建立了新的异源表达体系。该簇编码特定的转录因子TerR，这是terrin簇诱导不可或缺的。为了确定TerR结合位点，分析了不同重组版本的TerR dna结合域，以识别特定的基序。高亲和力的共识基序TCGGHHWYHCGGH从产泰瑞因所需的基因中被鉴定出来，结合位点突变证实了它们对泰瑞因基因表达的重要贡献。在异源寄主黑曲霉中对TerR及其目标启动子的组合进行了重组表达体系的试验。TerR介导的目标启动子激活直接依赖于其转录水平。因此，terR在可调节的淀粉酶启动子PamyB的控制下表达，并将terA靶启动子的激活水平与从强gpdA控制启动子直接表达的报告子获得的激活水平进行比较。在这里，耦合系统胜过了直接表达系统。当偶联系统用于外源聚酮合成酶表达时，产生高代谢产物。此外，细粒曲霉聚酮合成酶基因orsA的表达表明，聚酮合成酶的主要产物是lecanoric酸，而不是orsellinic酸。结构域交换实验将这种从orsellinic酸到OrsA硫酯酶结构域的深度形成。这些实验证实了该表达系统在异源宿主中产生高水平代谢物的适用性。
Recently, the Aspergillus terreus terrein gene cluster was identified and selected for development of a new heterologous expression system. The cluster encodes the specific transcription factor TerR that is indispensable for terrein cluster induction. To identify TerR binding sites, different recombinant versions of the TerR DNA-binding domain were analyzed for specific motif recognition. The high affinity consensus motif TCGGHHWYHCGGH was identified from genes required for terrein production and binding site mutations confirmed their essential contribution to gene expression in A. terreus. A combination of TerR with its terA target promoter was tested as recombinant expression system in the heterologous host Aspergillus niger. TerR mediated target promoter activation was directly dependent on its transcription level. Therefore, terR was expressed under control of the regulatable amylase promoter PamyB and the resulting activation of the terA target promoter was compared with activation levels obtained from direct expression of reporters from the strong gpdA control promoter. Here, the coupled system outcompeted the direct expression system. When the coupled system was used for heterologous polyketide synthase expression high metabolite levels were produced. Additionally, expression of the Aspergillus nidulans polyketide synthase gene orsA revealed lecanoric acid rather than orsellinic acid as major polyketide synthase product. Domain swapping experiments assigned this depside formation from orsellinic acid to the OrsA thioesterase domain. These experiments confirm the suitability of the expression system especially for high-level metabolite production in heterologous hosts.