圈卷产色链霉菌san7324和san7324L基因阻断对形态分化和尼可霉素产生的影响

Effects of san7324 and san7324L disruption on morphological differentiation and nikkomycin production of Streptomyces ansochromogenes

摘要:

[目的]圈卷产色链霉菌全局性调控基因wblA阻断突变后，尼可霉素不再产生。RNA-seq和转录分析表明san7324基因在野生型菌株中可以正常转录，而在wblA阻断突变株（ΔwblA）中不能转录，为此本文旨在揭示san7324与尼可霉素产生的关系。[方法]利用同源双交换策略对san7324进行基因阻断，而后通过基因遗传回补及对尼可霉素生物合成相关基因的转录分析等方法研究san7324的功能。[结果]在相同培养条件下，阻断突变株Δsan7324与野生型菌株相比失去了合成尼可霉素的能力。我们通过同源比对发现圈卷产色链霉菌中还存在一个与san7324同源的基因san7324L，该基因的阻断导致尼可霉素产量降低。当san7324和san7324L两个基因同时被阻断后，得到的突变株Δsan7324-san7324L生长稀疏而且不能正常发育分化形成灰色表型的孢子或孢子链，只能形成白色表型的气生菌丝，同时也丧失了合成尼可霉素的能力。当这两个基因（san7324-san7324L）回补双突变株后，则恢复了野生型的表型（能形成孢子链并恢复尼可霉素的产生）。进一步的研究初步表明san7324和san7324L的阻断主要影响了尼可霉素生物合成基因簇中途径特异性调控基因sanG的转录水平，从而影响圈卷产色链霉菌的发育分化和尼可霉素的产生。[结论]该结果为链霉菌形态分化与生理代谢关系的研究提供了更多的证据，同时为多效调控基因wblA作用机制的阐明奠定了基础。

Abstract:

[Objective] wblA disruption mutant of Streptomyces ansochromogenes fails to produce nikkomycin.RNA-seq and transcriptional analysis revealed that the san7324 could be transcribed normally in S.ansochromogenes(wild-type),whereas not transcribed in the wblA(a global regulatory gene) disruption mutant (ΔwblA).This research aims to reveal the function of san7324 in nikkomycin production.[Methods] To investigate the role of san7324 in nikkomycin biosynthesis,a san7324 disruption mutant was constructed via homologous recombination,and was subsequently confirmed by genetic complementation.The transcriptional levels of selected genes in the nikkomycin biosynthetic cluster were analyzed.[Results] The mutant (Δsan7324) lost ability to produce nikkomycin under the same culture conditions compared with the wild-type strain.Disruption of san7324L(a san7324 homologous gene) caused the decrease of nikkomycin production.However,when san7324 and san7324L were disrupted,the double mutant (Δsan7324-san7324L) failed to form grey spores or spore chains but conferred a white phenotype of aerial hyphae,and no nikkomycin production was observed in comparison with wild-type strain.In addition,the nikkomycin production and morphological differentiation could be restored by the complementation of san7324-san7324L in Δsan7324-san7324L.Further studies preliminarily indicated that disruption of san7324 and san7324L mainly affected the transcriptional level of pathway-specific regulatory gene sanG in nik cluster,affected the morphological differentiation and nikkomycin biosynthesis in S.ansochromogenes.[Conclusion] These results provided more evidence for investigating the relationship between morphological differentiation and physiological metabolism in Streptomyces,and lay the foundation for elucidating the regulatory mechanism of the pleiotropic regulatory gene wblA.