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.
Keywords: Cryptic phaC genes; Haloferax mediterranei; Poly(3-hydroxybutyrate-co-3-hydroxyvalerate); pps-like gene.