Catabolism of Alkylphenols in Rhodococcus via a Meta-Cleavage Pathway Associated With Genomic Islands

The bacterial catabolism of aromatic compounds has considerable promise to convert lignin depolymerization products to commercial chemicals. Alkylphenols are a key class of depolymerization products whose catabolism is not well-elucidated. We isolated Rhodococcus rhodochrous EP4 on 4-ethylphenol and applied genomic and transcriptomic approaches to elucidate alkylphenol catabolism in EP4 and Rhodococcus jostii RHA1. RNA-Seq and RT-qPCR revealed a pathway encoded by the aphABCDEFGHIQRS genes that degrades 4-ethylphenol via the meta-cleavage of 4-ethylcatechol. This process was initiated by a two-component alkylphenol hydroxylase, encoded by the aphAB genes, which were upregulated ~3,000-fold. Purified AphAB from EP4 had highest specific activity for 4-ethylphenol and 4-propylphenol (~2,000 U/mg) but did not detectably transform phenol. Nevertheless, a ΔaphA mutant in RHA1 grew on 4-ethylphenol by compensatory upregulation of phenol hydroxylase genes (pheA1-3). Deletion of aphC, encoding an extradiol dioxygenase, prevented growth on 4-alkylphenols but not phenol. Disruption of pcaL in the β-ketoadipate pathway prevented growth on phenol but not 4-alkylphenols. Thus, 4-alkylphenols are catabolized exclusively via meta-cleavage in rhodococci while phenol is subject to ortho-cleavage. A putative genomic island encoding aph genes was identified in EP4 and several other rhodococci. Overall, this study identifies a 4-alkylphenol pathway in rhodococci, demonstrates key enzymes involved, and presents evidence that the pathway is encoded in a genomic island. These advances are of particular importance for wide-ranging industrial applications of rhodococci, including upgrading of lignocellulose biomass.


RT-qPCR
Gene expression was also assessed using RT-qPCR using primer sequences shown in Supplementary  Table 1. Reverse transcription to cDNA used SuperScript VILO (Life Technologies) and Turbo DNase (Thermo Fisher Scientific). Cycling conditions for all genes were: 95 o C for 5 min; 40 cycles of: 95 o C for 30s, 52 o C for 20s, 72 o C for 30s (read fluorescence) on the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Standard curves for all genes were made using 10x serial dilutions of pCR2.1-TOPO TA plasmids containing the target amplicon from 10 9 to 10 2 . RT-qPCR transcript abundances were compared statistically using two-tailed Student's t-tests with Bonferroni (bon) correction.

Protein purification
Full-length aphA and aphB genes were amplified from Rhodococcus EP4 DNA and aphC was amplified from RHA1 DNA using Phusion polymerase. Primers (Supplementary Table 4) were designed with N and C terminal NdeI/BamHI sites respectively. Amplicons were inserted into a pET15b plasmid and inserts sequenced. The pET15b backbone contained an N-terminal histidine tag with a TEV cut site inserted between the histidine tag and the coding sequence. Constructs were transformed into E. coli BL21 (DE3) cells using standard protocols. Individual colonies were pick and grown overnight in 5 mL of LB containing 100 mg/L ampicillin. These cells were then used to inoculate 2 x 1 L cultures in LB and grown until OD600 was ~0.5 and induced with 0.5 mM IPTG. Cultures containing the aphB construct contained 200 mg/L ammonium iron citrate to facilitate cofactor incorporation. Cells were grown overnight at 25°C and harvested by centrifugation the next day. Pellets were lysed using an EmulsiFlex-C5 homogenizer (Avestin, Ottawa, ON, Canada) and centrifuged a ~26,000 x g for 20 minutes to clarify the lysate. All enzymes were purified by Ni sepherose 6 fast flow resin according to the manufacturer's protocol. The histidine tag was removed by dialyzing overnight in 20 mM Tris pH 8.0 with TEV protease. TEV protease and any uncleaved enzyme were removed by flowing the lysate over the Ni resin. The AphAEP4 enzyme was further purified using a MonoQ 10/100 GL and an ÄKTA Purifier (GE Healthcare) using a linear gradient of 20 mM Tris pH 8.0 to 20 mM Tris 1 mM NaCl pH 8.0 over roughly 6 CV.

AphBEP4 reductase activity
The activity of AphBEP4 was measured by the reduction of cytochrome c spectrophotometrically using scanning kinetic mode on a Cary 60 spectrophotometer set to 550 nm as in (Guengerich et al., 2009). Roughly 7 nM AphCRHA1, 40 μM bovine cytochrome c, and 100 μM of either FAD, FMN or riboflavin were dissolved in 300 mM phosphate buffer pH 7.7 at 25°C. The reaction was initiated with the addition of 100 μM NADH and the increase in absorbance was measured. Specific activity was calculated using ε = 0.021 mM -1 cm -1 for cytochrome c.

Enzyme end point assays
For end point assays, 100 μM substrate (200 μM for 4-HPA and 4-NP) in 0.5 mL 20 mM MOPS, 90 mM NaCl, pH 7.2 containing 20 μM AphAEP4 and 2 μM AphBEP4, were incubated overnight at 30 °C. Samples were quenched with the addition of acetic acid to a final concentration of 1%. The samples were then centrifuged at 16,000 × g for 5 minutes and filtered through a 0.2-μm syringe filter. Samples were analyzed using a Waters 2695 HPLC (Waters, Milford, MA, USA) equipped with a Luna 5 µm C18(2) column 250 × 4.6 mm (Phenomenex, Torrance, CA, USA) and a UV detector. The column was operated at 0.7 ml min −1 and the sample was eluted using a 16.8 ml linear gradient of 1% formic acid in H2O to 100% methanol. Elution of compounds was monitored at 280 nm.

aphABEP4 transcriptional regulation
To understand how the transcriptional framework of the aphAB genes, we first aligned the assembled transcriptome to the EP4 genome, which provided experimental evidence of the transcription start site (TSS). Promotor prediction used BPROM (Solovyev and Salamov, 2011). PGAP gene prediction provided the coding start site (CS). Promotor sequences and AphR binding regions were predicted based on promotors in other Actinobacteria and alignment to sequences upstream of Rhodococcus pheA1 (Bashyam et al., 1996;Szőköl et al., 2014). The role of AphR transcriptional regulator proteins were predicted based on position in the EP4 genome and RAxML phylogenetic analysis against characterized AraC-family proteins following TCoffee sequence alignment.