State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
College of Life Science, University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China.
Appl Microbiol Biotechnol. 2020 Nov;104(22):9759-9771. doi: 10.1007/s00253-020-10898-0. Epub 2020 Sep 12.
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.
地中海盐杆菌是一种能够生产聚（3-羟基丁酸酯-co-3-羟基戊酸酯）（PHBV）的嗜盐古菌，它拥有四个 PHA 合酶编码基因，分别是 phaC、phaC1、phaC2 和 phaC3。在野生型菌株中，除了 phaC 之外，其他三个基因都是隐匿的，在 PHBV 积累条件下不转录。PhaC 蛋白与 PhaE 亚基一起形成有活性的 PHA 合酶，并催化 PHBV 的聚合。先前的研究表明，删除一个名为 pps-like 的基因可显著提高 PHBV 的积累量，这可能是由于pha 簇基因（phaR-phaP-phaE-phaC）的上调所致。本研究证实了 pps-like 基因缺失对隐匿性 phaC 基因的影响。qRT-PCR 结果显示，在ΔEPSΔpps-like 基因ΔphaC 突变体中，三个隐匿基因的表达水平上调。对隐匿性 phaC 基因进行连续敲除和发酵实验表明，在ΔEPSΔpps-like 基因ΔphaC 突变体中，PhaC1 随后是 PhaC3 具有合成 PHBV 的能力。PhaC1 和 PhaC3 都可以与 PhaE 形成有功能活性的 PHA 合酶。然而，phaC2 的表达并没有导致 PHBV 的合成。此外，PhaC、PhaC1 和 PhaC3 表现出不同的底物特异性，因为 PHBV 共聚物中的 3HV 含量不同。EMSA 结果表明，PPS-like 蛋白可能通过结合其启动子区域来抑制 phaC1 基因的表达。综上所述，在ΔEPSΔpps-like 基因ΔphaC 突变体中，PhaC1 对 PHBV 合成的影响最为显著，而 pps-like 基因的缺失解除了 phaC1 表达的负调控作用，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-like 基因缺失激活。• PPS-like 蛋白可能通过结合其启动子来调节 phaC1 表达。• PhaC1 和 PhaC3 与 PhaE 形成有活性的 PHA 合酶。
