Dissecting and engineering of the TetR family regulator SACE_7301 for enhanced erythromycin production in Saccharopolyspora erythraea.
Dissecting and engineering of the TetR family regulator SACE_7301 for enhanced erythromycin production in Saccharopolyspora erythraea.
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TetR 家族调节因子 SACE_7301 的解剖和工程设计，用于增强红霉素糖多孢菌的产量。
DOI：
10.1186/s12934-014-0158-4
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发表时间：
2014-11-13
影响因子：
6.4
通讯作者：
Zhang B
中科院分区：
文献类型：
--
作者：
Wu H;Chen M;Mao Y;Li W;Liu J;Huang X;Zhou Y;Ye BC;Zhang L;Weaver DT;Zhang B
红糖多孢菌被广泛用于工业规模生产红霉素 A (Er-A)，红霉素 A 是一种常用于人类医学的大环内酯类抗生素。然而，S. erythraea 缺乏红霉素生物合成基因 (ery) 簇中的调节基因，从而阻碍了通过调节基因工程来增强 Er-A 产量的努力。通过基于线性DNA片段同源重组的染色体基因失活技术，我们灭活了多个候选TetR家族转录调节因子（TFR），并鉴定了一个TFR（SACE_7301）在S. erythraea A226中正控制红霉素生物合成。 qRT-PCR和EMSA分析表明，SACE_7301通过与红霉素生物合成基因eryAI和抗性基因ermE的启动子区域以低亲和力相互作用，激活它们的转录，类似于先前鉴定的调节红霉素生物合成和形态分化的BldD (SACE_2077)。因此，我们设计了在A226中在PermE*控制下以1至3个额外拷贝过度表达SACE_7301的策略。在 SACE_7301、eryAI 和 ermE 转录表达上调后，SACE_7301 过表达菌株均比 A226 增加了 Er-A 产量，且与拷贝数成正比。同样，当SACE_7301在工业红斑狼疮WB菌株中过表达时，突变体WB/7301、WB/2×7301和WB/3×7301的Er-A产量相对于WB分别增加了17%、29%和42%。在 5 L 发酵罐中，产量最高的菌株 WB/3×7301 的 Er-A 积累量增加至 4,230 mg/L，比 WB（3,322 mg/L）产量提高约 27%。我们在红葡萄球菌中鉴定并鉴定了一个 TFR SACE_7301，它可以正向调节红霉素生物合成，并且在野生型和工业红葡萄球菌菌株中过度表达 SACE_7301 可提高 Er-A 产量。这项研究显着提高了我们对红霉素生物合成异常调节机制的理解，并通过改造红霉素转录调节因子，提供了一种解决 Er-A 过量产生的新策略。本文的在线版本 (doi:10.1186/s12934-014-0158-4) 包含补充材料，可供授权用户使用。
Saccharopolyspora erythraea was extensively utilized for the industrial-scale production of erythromycin A (Er-A), a macrolide antibiotic commonly used in human medicine. Yet, S. erythraea lacks regulatory genes in the erythromycin biosynthetic gene (ery) cluster, hampering efforts to enhance Er-A production via the engineering of regulatory genes. By the chromosome gene inactivation technique based on homologous recombination with linearized DNA fragments, we have inactivated a number of candidate TetR family transcriptional regulators (TFRs) and identified one TFR (SACE_7301) positively controlling erythromycin biosynthesis in S. erythraea A226. qRT-PCR and EMSA analyses demonstrated that SACE_7301 activated the transcription of erythromycin biosynthetic gene eryAI and the resistance gene ermE by interacting with their promoter regions with low affinities, similar to BldD (SACE_2077) previously identified to regulate erythromycin biosynthesis and morphological differentiation. Therefore, we designed a strategy for overexpressing SACE_7301 with 1 to 3 extra copies under the control of PermE* in A226. Following up-regulated transcriptional expression of SACE_7301, eryAI and ermE, the SACE_7301-overexpressed strains all increased Er-A production over A226 proportional to the number of copies. Likewise, when SACE_7301 was overexpressed in an industrial S. erythraea WB strain, Er-A yields of the mutants WB/7301, WB/2×7301 and WB/3×7301 were respectively increased by 17%, 29% and 42% relative to that of WB. In a 5 L fermentor, Er-A accumulation increased to 4,230 mg/L with the highest-yield strain WB/3×7301, an approximately 27% production improvement over WB (3,322 mg/L). We have identified and characterized a TFR, SACE_7301, in S. erythraea that positively regulated erythromycin biosynthesis, and overexpression of SACE_7301 in wild-type and industrial S. erythraea strains enhanced Er-A yields. This study markedly improves our understanding of the unusual regulatory mechanism of erythromycin biosynthesis, and provides a novel strategy towards Er-A overproduction by engineering transcriptional regulators of S. erythraea. The online version of this article (doi:10.1186/s12934-014-0158-4) contains supplementary material, which is available to authorized users.
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3.2
作者：
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影响因子：
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DOI：
10.1073/pnas.0803622105
发表时间：
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影响因子：
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