Knockout of pde gene in Arthrobacter sp. CGMCC 3584 and transcriptomic analysis of its effects on cAMP production
Knockout of pde gene in Arthrobacter sp. CGMCC 3584 and transcriptomic analysis of its effects on cAMP production
复制标题
节杆菌 pde 基因的敲除。
DOI：
10.1007/s00449-019-02280-w
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发表时间：
2020-05-01
影响因子：
3.8
通讯作者：
Liu, Dong
中科院分区：
文献类型：
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作者：
Niu, Huanqing;Sun, Xinzeng;Liu, Dong
节杆菌属。 CGMCC 3584用于工业生产环磷酸腺苷（cAMP）。然而，由于缺乏用于对节杆菌属进行基因操作的基因工程工具，仅构建和研究了少数代谢工程节杆菌。在本研究中，我们首次通过基于PCR靶向的同源重组方法构建了不带有任何抗生素抗性标记的节杆菌arpde敲除突变体。我们的结果表明，arpde 的缺失对生物量产量影响不大，并且 cAMP 产量提高了 31.1%。此外，我们还比较了arpde敲除菌株和野生菌株的转录组，旨在从分子水平了解arpde失活导致的cAMP产生能力。比较转录组分析显示，arpde失活对代谢有两个主要影响：抑制糖酵解、PP途径和氨基酸代谢（苯丙氨酸、色氨酸、支链氨基酸和谷氨酸代谢）；促进嘌呤代谢和前体5'-磷酸核糖基1-焦磷酸的碳通量，有利于cAMP的产生。
Arthrobacter sp. CGMCC 3584 is used for the industrial production of cyclic adenosine monophosphate (cAMP). However, because of the paucity of genetic engineering tools for genetic manipulation on Arthrobacter species, only a few metabolically engineered Arthrobacter have been constructed and investigated. In this study, for the first time, we constructed an arpde knockout mutant of Arthrobacter without any antibiotic resistance marker by a PCR-targeting-based homologous recombination method. Our results revealed that the deletion of arpde had little effect on biomass production and improved cAMP production by 31.1%. Furthermore, we compared the transcriptomes of the arpde knockout strain and the wild strain, aiming to understand the capacities of cAMP production due to arpde inactivation at the molecular level. Comparative transcriptomic analysis revealed that arpde inactivation had two major effects on metabolism: inhibition of glycolysis, PP pathway, and amino acid metabolism (phenylalanine, tryptophan, branched-chain amino acids, and glutamate metabolism); promotion of the purine metabolism and carbon flux from the precursor 5 '-phosphoribosyl 1-pyrophosphate, which benefited cAMP production.