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. 2020 Apr;22(4):1356-1369.
doi: 10.1111/1462-2920.14947. Epub 2020 Feb 27.

Beyond Oil Degradation: Enzymatic Potential of Alcanivorax to Degrade Natural and Synthetic Polyesters

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Free PMC article

Beyond Oil Degradation: Enzymatic Potential of Alcanivorax to Degrade Natural and Synthetic Polyesters

Vinko Zadjelovic et al. Environ Microbiol. .
Free PMC article

Abstract

Pristine marine environments are highly oligotrophic ecosystems populated by well-established specialized microbial communities. Nevertheless, during oil spills, low-abundant hydrocarbonoclastic bacteria bloom and rapidly prevail over the marine microbiota. The genus Alcanivorax is one of the most abundant and well-studied organisms for oil degradation. While highly successful under polluted conditions due to its specialized oil-degrading metabolism, it is unknown how they persist in these environments during pristine conditions. Here, we show that part of the Alcanivorax genus, as well as oils, has an enormous potential for biodegrading aliphatic polyesters thanks to a unique and abundantly secreted alpha/beta hydrolase. The heterologous overexpression of this esterase proved a remarkable ability to hydrolyse both natural and synthetic polyesters. Our findings contribute to (i) better understand the ecology of Alcanivorax in its natural environment, where natural polyesters such as polyhydroxyalkanoates (PHA) are produced by a large fraction of the community and, hence, an accessible source of carbon and energy used by the organism in order to persist, (ii) highlight the potential of Alcanivorax to clear marine environments from polyester materials of anthropogenic origin as well as oils, and (iii) the discovery of a new versatile esterase with a high biotechnological potential.

Figures

Figure 1
Figure 1
Determination of polyester degradation by Alcanivorax sp. 24.A. Clear zone hydrolysis test of Alcanivorax sp. 24 on the five aliphatic polyesters PHB, PHBV, PES, PBS and PCL. Polyesters were added at 0.3% w/v to BH mineral media containing 1% agarose (w/v). Arrows highlight the hydrolysis halos surrounding the 5 mm‐diameter wells made for Alcanivorax sp. 24 inoculation.B. Growth curves of Alcanivorax sp. 24 when incubated in the presence of three different polyesters, succinate (labile substrate control) and BH mineral media (MM; negative control). Increase in biological biomass was assessed by protein quantification. Error bars indicate the standard deviation of three biological replicates.C. Degradation of the PET intermediate BHET by Alcanivorax sp. 24. The substrate BHET and its hydrolysed product TPA were monitored by LC–MS. ‘No inoculum’ represents the replicate control condition where Alcanivorax sp. 24 was not inoculated. [Color figure can be viewed at http://wileyonlinelibrary.com]
Figure 2
Figure 2
Proteomic analysis and identification of the esterase (i.e. ALC24_4107) involved in the aliphatic polyester degradation of Alcanivorax sp. 24.A. PCA of the exoproteomes produced by Alcanivorax sp. 24 when grown in the presence of different substrates including the three aliphatic polyesters PES, PHB and PHBV as well as BHET and succinate.B. Relative abundance of the esterase ALC24_4107 in each one of the exoproteomes of Alcanivorax sp. 24 when grown in the presence of different substrates. Error bars indicate the standard deviation of three biological replicates.C. Protein domains and genomic context of ALC24_4107.D. Hydrolytic activity of the heterologously overexpressed ALC24_4107 in E. coli BL21 assessed by a clear zone hydrolysis test on five different aliphatic polyesters. Halos surround 5 mm‐diameter wells. [Color figure can be viewed at http://wileyonlinelibrary.com]
Figure 3
Figure 3
Similarities and hydrolytic activity of ALC24_4107 homologous esterases.A. Phylogenetic tree of ALC24_4107 and its closest homologues in other bacteria retrieved from an NCBI BLASTp search. The tree was generated using Neighbour‐Joining and Jukes‐Cantor as the generic distance, with bootstrap set to 1000 replicates represented at the base of the nodes, and using the PHB‐depolymerase from Streptomyces hygrogroscopicus as the outgroup. Arrows indicate the strains that were purchased and for which hydrolytic activity was assessed on each one of the five aliphatic polyesters. The tests that gave a positive clearing halo (information available in Supplementary Fig. S4) are indicated by red circles.B. Multiple alignment of ALC24_4107 with 10 relevant homologues, including the esterases encoded by the six strains tested for their hydrolytic activity. Only the catalytic and substrate‐binding domains are shown. [Color figure can be viewed at http://wileyonlinelibrary.com]
Figure 4
Figure 4
Ecological strategy of Alcanivorax spp. in the environment.The biodegradation and assimilation of hydrocarbons (left) and polyesters (right) of both natural (bottom) and anthropogenic origin (top) is depicted. [Color figure can be viewed at http://wileyonlinelibrary.com]

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