Aldehyde dehydrogenases for oligo-isoprene aldehyde oxidation in Rhodococcus sp. RDE2

The microbial degradation of natural rubber (NR), which is mainly composed of cis-1,4-polyisoprene, is a promising sustainable strategy for rubber waste treatment. In rubber-degrading actinomycetes, the polymer is cleaved by the latex-clearing protein (Lcp) into oligo-isoprene aldehydes (OIA), which are then oxidized and channeled into the β-oxidation pathway. Some strains employ heterodimeric molybdenum hydroxylases (OxiAB) for this oxidation, whereas others, including Gordonia and Nocardia strains, lack oxiA/oxiB adjacent to lcp and instead use NAD(P)⁺-dependent aldehyde dehydrogenases (ALDHs); however, the ALDH repertoire differs among strains. Here, we identified the ALDHs responsible for OIA oxidation in Rhodococcus sp. RDE2. Cell-free extracts oxidized OIA preferentially with NAD⁺, although substantial NADP⁺-dependent activity (~37% of the NAD⁺-dependent activity) was also detected. Neither activity was induced during NR growth. Six candidate ALDHs were selected by combining homology searches, qRT-PCR, and heterologous expression analyses in Escherichia coli. Among the purified candidates, only LPH33_RS08240 and LPH33_RS17900 exhibited substantial NAD⁺-dependent OIA-oxidizing activity, which was 2.0- and 1.3-fold higher than that of the reference VH2 ALDH (GPOL_c02580), respectively. Deletion of either gene reduced both NR and OIA oxidation activities, identifying these two enzymes as major contributors to NAD⁺-dependent OIA oxidation in RDE2 and, to our knowledge, as the first OIA-oxidizing ALDHs identified in the genus Rhodococcus. Double knockouts did not abolish these activities, indicating contributions from additional ALDHs, consistent with the NADP⁺-dependent activities detected for the three other candidates. These findings extend the OIA-oxidizing ALDH repertoire of rubber-degrading actinomycetes and provide a foundation for engineering rubber-derived bioconversion. KEY POINTS: LPH33_RS08240 and RS17900 are the first OIA-oxidizing ALDHs found in Rhodococcus. Deleting both genes lowers rubber growth and OIA activity without abolishing either. NADP⁺-linked ALDHs may couple rubber degradation to reductive biosynthesis in RDE2.