Rhizobacter gummiphilus NS21 has two rubber oxygenases (RoxA and RoxB) acting synergistically in rubber utilisation

Biodegradation of poly(c is -1,4-isoprene) (rubber) by Gram-negative bacteria has been investigated on the enzymatic level only in Steroidobacter cummioxidans 35Y (previously Xanthomonas sp. 35Y). This species produces two kinds of rubber oxygenases, RoxA 35Y and RoxB 35Y , one of which (RoxB 35Y ) cleaves polyisoprene to a mixture of C 20 - and higher oligoisoprenoids while the other (RoxA 35Y ) cleaves polyisoprene and RoxB 35Y -derived oligoisoprenoids to the C 15 -oligoisoprenoid 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). ODTD can be taken up by S. cummioxidans and used as a carbon source. Gram-positive rubber-degrading bacteria employ another type of rubber oxygenase, latex clearing protein (Lcp), for the initial oxidative attack of the polyisoprene molecule. In this contribution, we examined which type of rubber oxygenase is present in the only other well-documented Gram-negative rubber-degrading species, Rhizobacter gummiphilus NS21. No homologue for an Lcp protein but homologues for a putative RoxA and a RoxB protein (the latter identical to a previously postulated LatA-denominated rubber cleaving enzyme) were identified in the genome of strain NS21. The roxA NS21 and roxB NS21 genes were separately expressed in a ∆roxA 35Y /∆roxB 35Y background of S. cummioxidans 35Y and restored the ability of the mutant to produce oligoisoprenoids. The RoxA NS21 and RoxB NS21 proteins were each purified and biochemically characterised. The results—in combination with in silico analysis of databases—indicate that Gram-negative rubber-degrading bacteria generally utilise two synergistically acting rubber oxygenases (RoxA/RoxB) for efficient cleavage of polyisoprene to ODTD.