Haloferax mediterranei, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) producing haloarchaeon, possesses four PHA synthase encoding genes, phaC, phaC1, phaC2, and phaC3. In the wild-type strain, except phaC, the other three genes are cryptic and not transcribed under PHA-accumulating conditions. The PhaC protein together with PhaE subunit forms the active PHA synthase and catalyzes PHBV polymerization. Previously, it was observed that the deletion of a gene named pps-like significantly enhanced PHBV accumulation probably resulted from the upregulation of pha cluster genes (phaR-phaP-phaE-phaC). The present study demonstrated the influence of pps-like gene deletion on the cryptic phaC genes. As revealed by qRT-PCR, the expression level of the three cryptic genes was upregulated in the ΔEPSΔpps-like geneΔphaC mutant. Sequential knockout of the cryptic phaC genes and fermentation experiments showed that PhaC1 followed by PhaC3 had the ability to synthesize PHBV in ΔEPSΔpps-like geneΔphaC mutant. Both PhaC1 and PhaC3 could complex with PhaE to form functionally active PHA synthase. However, the expression of phaC2 did not lead to PHBV synthesis. Moreover, PhaC, PhaC1, and PhaC3 exhibited distinct substrate specificity as the 3HV content in PHBV copolymers was different. The EMSA result showed that PPS-like protein might be a negative regulator of phaC1 gene by binding to its promoter region. Taken together, PhaC1 had the most pronounced effect on PHBV synthesis in ΔEPSΔpps-like geneΔphaC mutant and deletion of pps-like gene released the negative effect from phaC1 expression and thereby restored PHBV accumulating ability in ΔphaC mutant. KEY POINTS: • Cryptic phaC genes were activated by pps-like gene deletion. • PPS-like protein probably regulated phaC1 expression by binding to its promoter. • Both PhaC1 and PhaC3 formed active PHA synthase with PhaE.

中文翻译：

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pps样基因的删除激活了嗜血哈拉发克斯中的phaC基因。

嗜盐哈拉法克斯（Haloferax mediterranei）是一种生产卤代古细菌的聚（3-羟基丁酸酯-co-3-羟基戊酸酯）（PHBV），具有四个PHA合酶编码基因phaC，phaC1，phaC2和phaC3。在野生型菌株中，除了phaC以外，其他三个基因是隐性的，在PHA积累条件下不会转录。PhaC蛋白与PhaE亚基一起形成活性PHA合酶并催化PHBV聚合。以前，已经观察到名为pps样的基因的缺失显着增强了PHBV积累，这可能是由于pha簇基因（phaR-phaP-phaE-phaC）的上调引起的。本研究证明了pps样基因缺失对phaC基因的影响。如qRT-PCR所揭示，在ΔEPSΔpps样基因ΔphaC突变体中，三个隐性基因的表达水平被上调。密码子phaC基因的顺序敲除和发酵实验表明，PhaC1随后是PhaC3具有在ΔEPSΔpps样基因ΔphaC突变体中合成PHBV的能力。PhaC1和PhaC3都可以与PhaE结合形成功能活跃的PHA合酶。但是，phaC2的表达并未导致PHBV合成。此外，由于PHBV共聚物中3HV的含量不同，PhaC，PhaC1和PhaC3表现出不同的底物特异性。EMSA结果表明，PPS样蛋白可能通过结合到其启动子区域而成为phaC1基因的负调控因子。两者合计，PhaC1对ΔEPSΔpps样基因ΔphaC突变体中的PHBV合成具有最显着的影响，而pps样基因的缺失释放了phaC1表达的负面影响，从而恢复了ΔphaC突变体中PHBV的积累能力。关键点：•隐匿性phaC基因被pps样基因缺失激活。•PPS样蛋白可能通过与其启动子结合来调节phaC1表达。•PhaC1和PhaC3均与PhaE形成了活性PHA合酶。