Metabolic Engineering Strategies Based on Secondary Messengers (p)ppGpp and C-di-GMP To Increase Erythromycin Yield in Saccharopolyspora erythraea
Metabolic Engineering Strategies Based on Secondary Messengers (p)ppGpp and C-di-GMP To Increase Erythromycin Yield in Saccharopolyspora erythraea
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基于二级信使 (p)ppGpp 和 C-di-GMP 的代谢工程策略提高红糖多孢菌中红霉素的产量
DOI：
10.1021/acssynbio.8b00372
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发表时间：
2019
影响因子：
4.7
通讯作者：
Ye Bang-Ce
中科院分区：
文献类型：
--
作者：
Xu Zhen;You Di;Tang Li-Ya;Zhou Ying;Ye Bang-Ce
次级信使（如(p)ppGpp和c-di-GMP）在放线菌的抗生素生物合成中起着重要作用。在本研究中，我们发现红霉素生物合成（ery）基因的转录水平在营养限制下上调，这取决于saccharopolyspora erythraea的(p)ppGpp。进一步的研究表明，在培养过程中，血红蛋白的表达和细胞内(p)ppGpp的浓度呈同步变化。通过引入链霉菌（streptomyces colicolorintos） c端截断的(p)ppGpp合成酶RelA (n端1.43 kb)，提高细胞内(p)ppGpp的浓度，红霉素产量显著提高（约200%）。红霉菌株NRRL2338（命名为WT/ pIB-PBAD-relA1-489）。由于工业红霉素高产菌株E3的细胞内(p)ppGpp浓度比WT菌株高得多（约10- 100倍），上述策略在E3菌株中不适用。进一步研究发现，低浓度的2-氧葡萄糖酸盐在E3菌株中产生“富氮”假信号，导致氮代谢基因下调，限制了氮源的利用，从而导致细胞内(p)ppGpp浓度高。此外，二级信使c-di-GMP被证明能够通过增强BldD与血红基因启动子的结合来激活血红基因的转录。过表达二胍酸环化酶CdgB。coelicolorinS。红红素增加胞内c-二gmp浓度，促进红霉素的产生。这些发现表明，增加细胞内次级信使的浓度可以激活基因的转录，并为设计基于次级信使的代谢工程来提高放线菌的抗生素产量提供了新的策略。
Secondary messengers (such as (p)ppGpp and c-di-GMP) were proved to play important roles in antibiotic biosynthesis in actinobacteria. In this study, we found that transcription levels of erythromycin-biosynthetic (ery) genes were upregulated in nutrient limitation, which depended on (p)ppGpp inSaccharopolyspora erythraea. Further study demonstrated that the expression oferygenes and intracellular concentrations of (p)ppGpp showed synchronization during culture process. The erythromycin yield was significantly improved (about 200%) by increasing intracellular concentration of (p)ppGpp through introduction of C-terminally truncated (p)ppGpp synthetase RelA (1.43 kb of the N-terminal segment) fromStreptomyces coelicolorintoS. erythraeastrain NRRL2338 (named as WT/pIB-PBAD-relA1–489). As the intracellular concentration of (p)ppGpp in an industrial erythromycin-high-producing strain E3 was greatly higher (about 10- to 100-fold) than WT strain, the applications of the above-described strategy did not work in E3 strain. Further research revealed that low concentration of 2-oxoglutarate in E3 strain exerted a “nitrogen-rich” pseudosignal, leading to the downregulation of nitrogen metabolism genes, which limited the use of nitrogen sources and thus the high intracellular (p)ppGpp concentration. Furthermore, the secondary messenger, c-di-GMP, was proved to be able to activateerygenes transcription by enhancing binding of BldD to promoters oferygenes. Overexpressing the diguanylate cyclase CdgB fromS. coelicolorinS. erythraeaincreased the intracellular c-di-GMP concentration, and improved erythromycin production. These findings demonstrated that increasing the concentration of intracellular secondary messengers can activateerygenes transcription, and provided new strategies for designing metabolic engineering based on secondary messengers to improve antibiotics yield in actinobacteria.