Journal: Microbial Biotechnology

Article Title: Identification of a self‐sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2‐hydroxyphenylacetic acid catabolism, via homogentisate pathway

doi: 10.1111/1751-7915.13865

Figure Lengend Snippet: Putative 2‐hydroxyphenylacetate (2‐HPA) degradation pathway and gene clusters involved in 2‐HPA catabolism in Cupriavidus pinatubonensis JMP134. A. C. pinatubonensis JMP134 route channelling degradation of 2‐HPA, 3‐HPA and 4‐HPA through homogentisate producing acetoacetate and fumarate (adapted from Pérez‐Pantoja et al ., ). B. Genes putatively involved in 2‐HPA/homogentisate catabolism in strain JMP134. C. Protein domain structures of self‐sufficient cytochrome P450 enzyme encoded by ohpA gene. The two fused domains are shown in bold. Specific functions are indicated in thin‐lines boxes. The protein residue 388 Cysteine (haem‐iron proximal ligand) is underlined. C1: Main chromosome; C2: Secondary chromosome.

Article Snippet: Notably, the gene coding for the OhpA homologue (locus tag Bmul_5958, previously BMULJ_05568) harboured by the 2‐HPA‐degrading Burkholderia multivorans ATCC 17616 strain (Fig. ) is induced in soil environment as revealed by in vivo expression technology (Nishiyama et al ., , see Table in the reference), suggesting that the substrate of CYP116B1 homologues should be a natural carbon source in such habitat.

Techniques: Residue

Journal: Microbial Biotechnology

Article Title: Identification of a self‐sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2‐hydroxyphenylacetic acid catabolism, via homogentisate pathway

doi: 10.1111/1751-7915.13865

Figure Lengend Snippet: Transcript levels of putative 2‐hydroxyphenylacetate (2‐HPA) degradation genes from Cupriavidus pinatubonensis cells exposed to different phenylacetate compounds. Real‐time PCR analysis was performed for ohpR , ohpT , ohpA and hmgA genes expression in cells grown on phenylacetate (PA), 2‐HPA, 3‐HPA, 4‐HPA or fructose (control) as a sole carbon and energy sources. Transcript levels were normalized to the average value of transcript levels in fructose treatment. Additionally, 16S rRNA levels were used as a reference gene (internal control). All experiments were performed in three biological replicates. Error bars represent SEM. Different letters indicate statistically significant differences between treatments for each gene (one‐way analysis of variance, P < 0.05; Tukey’s test, P < 0.05), specifically in this graph the statistical group including 2‐HPA treatment (a) had a significantly much higher transcript levels as compared to any other groups (b, c, bc). The transcript levels of hmgA when cells were grown on 3‐HPA or 4‐HPA are also included in this statistical group (a).

Article Snippet: Notably, the gene coding for the OhpA homologue (locus tag Bmul_5958, previously BMULJ_05568) harboured by the 2‐HPA‐degrading Burkholderia multivorans ATCC 17616 strain (Fig. ) is induced in soil environment as revealed by in vivo expression technology (Nishiyama et al ., , see Table in the reference), suggesting that the substrate of CYP116B1 homologues should be a natural carbon source in such habitat.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Microbial Biotechnology

Article Title: Identification of a self‐sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2‐hydroxyphenylacetic acid catabolism, via homogentisate pathway

doi: 10.1111/1751-7915.13865

Figure Lengend Snippet: Growth on 2‐HPA of Cupriavidus pinatubonensis JMP134, Pseudomonas putida KT2440 and their derivatives. A. Growth of C. pinatubonensis wild type (JMP134), ohpA mutant (JMP134d ohpA ) and ohpA mutant expressing ohpA gene driven by a heterologous P BAD promoter (JMP134d ohpA pBS1‐ ohpA ), on 2‐HPA as a sole carbon and energy source, in the presence or absence of l ‐arabinose. B. Growth of P. putida KT2440 and its derivative expressing ohpA gene driven by the heterologous P BAD promoter (KT2440 pBS1‐ ohpA ), on 2‐HPA as a sole carbon and energy source, in the presence or absence of l ‐arabinose. Three biological replicates were performed for growth measurements. Error bars indicate the standard deviation.

Article Snippet: Notably, the gene coding for the OhpA homologue (locus tag Bmul_5958, previously BMULJ_05568) harboured by the 2‐HPA‐degrading Burkholderia multivorans ATCC 17616 strain (Fig. ) is induced in soil environment as revealed by in vivo expression technology (Nishiyama et al ., , see Table in the reference), suggesting that the substrate of CYP116B1 homologues should be a natural carbon source in such habitat.

Techniques: Mutagenesis, Expressing, Standard Deviation

Journal: Microbial Biotechnology

Article Title: Identification of a self‐sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2‐hydroxyphenylacetic acid catabolism, via homogentisate pathway

doi: 10.1111/1751-7915.13865

Figure Lengend Snippet: Resting cell assays of Cupriavidus pinatubonensis JMP134, Pseudomonas putida KT2440 and their derivatives in presence of hydroxyphenylacetate isomers. A. Resting cells of C. pinatubonensis JMP134, and the ohpA mutant (JMP134d ohpA ) were grown on fructose 20 mM plus 2‐HPA 2.5 mM as inducer, washed and subsequently exposed to 1 mM 2‐HPA, 3‐HPA or 4‐HPA. B. Resting cells of P. putida KT2440, and the derivative expressing ohpA gene driven by the heterologous P BAD promoter (KT2440 pBS1‐ ohpA ) were grown on succinate 30 mM plus arabinose 2.5 mM as inducer, washed and subsequently exposed to 1 mM 2‐HPA, 3‐HPA or 4‐HPA. Two biological replicates were performed for growth measurements. Error bars indicate the standard deviation.

Article Snippet: Notably, the gene coding for the OhpA homologue (locus tag Bmul_5958, previously BMULJ_05568) harboured by the 2‐HPA‐degrading Burkholderia multivorans ATCC 17616 strain (Fig. ) is induced in soil environment as revealed by in vivo expression technology (Nishiyama et al ., , see Table in the reference), suggesting that the substrate of CYP116B1 homologues should be a natural carbon source in such habitat.

Techniques: Mutagenesis, Expressing, Standard Deviation

Journal: Microbial Biotechnology

Article Title: Identification of a self‐sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2‐hydroxyphenylacetic acid catabolism, via homogentisate pathway

doi: 10.1111/1751-7915.13865

Figure Lengend Snippet: Evolutionary relationships among several OhpA homologues. Maximum likelihood topology provided by IQ‐TREE (Nguyen et al ., ) based on sequence alignments calculated using MAFFT (Katoh et al ., ) is shown with SH‐like approximate likelihood ratio support values ( n = 1000) given at each node (values > 50% are shown). Sequences indicated in bold belong to strains tested by its ability to grow in 2‐HPA as a sole carbon and energy source. Blue, α‐proteobacteria; orange, β‐proteobacteria; green, γ‐proteobacteria; yellow, Actinobacteria; red, Hydrogenophilalia class.

Article Snippet: Notably, the gene coding for the OhpA homologue (locus tag Bmul_5958, previously BMULJ_05568) harboured by the 2‐HPA‐degrading Burkholderia multivorans ATCC 17616 strain (Fig. ) is induced in soil environment as revealed by in vivo expression technology (Nishiyama et al ., , see Table in the reference), suggesting that the substrate of CYP116B1 homologues should be a natural carbon source in such habitat.

Techniques: Sequencing

Journal: Microbial Biotechnology

Article Title: Identification of a self‐sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2‐hydroxyphenylacetic acid catabolism, via homogentisate pathway

doi: 10.1111/1751-7915.13865

Figure Lengend Snippet: Growth on 2‐hydroxyphenylacetate (2‐HPA) of several bacterial species included in different taxonomic groups. Strains belonging to Acinetobacter , Burkholderia , Cupriavidus , Paraburkholderia , Rhodococcus and Sphingomonas genera were grown in mineral salt medium with 2.5 mM 2‐HPA as a sole carbon and energy sources. Bacterial strains that carry the ohpA gene are indicated in bold. Shading indicates optical density (OD) at 600 nm on 24, 48 and 72 h (average of two biological replicates). Lower OD at stationary phase is the result of cell clumping in some strains. A dendrogram based in GyrB sequences is shown as reference of phylogenetic relationship among close related 2‐HPA + and 2‐HPA − strains. The phylogenetic tree was constructed using IQ‐TREE (Nguyen et al ., ) based on sequence alignments calculated utilizing MAFFT (Katoh et al ., ). Acinetobacter species were supplemented with 0.25 mM tyrosine to overcome amino acid auxotrophy. The S. wittichii RW1 strain needed a previous adaptation in 2‐HPA to obtain reproducible growth.

Article Snippet: Notably, the gene coding for the OhpA homologue (locus tag Bmul_5958, previously BMULJ_05568) harboured by the 2‐HPA‐degrading Burkholderia multivorans ATCC 17616 strain (Fig. ) is induced in soil environment as revealed by in vivo expression technology (Nishiyama et al ., , see Table in the reference), suggesting that the substrate of CYP116B1 homologues should be a natural carbon source in such habitat.

Techniques: Construct, Sequencing

Journal: Microbial Biotechnology

Article Title: Identification of a self‐sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2‐hydroxyphenylacetic acid catabolism, via homogentisate pathway

doi: 10.1111/1751-7915.13865

Figure Lengend Snippet: Bacterial strains and plasmids used in this study.

Article Snippet: Notably, the gene coding for the OhpA homologue (locus tag Bmul_5958, previously BMULJ_05568) harboured by the 2‐HPA‐degrading Burkholderia multivorans ATCC 17616 strain (Fig. ) is induced in soil environment as revealed by in vivo expression technology (Nishiyama et al ., , see Table in the reference), suggesting that the substrate of CYP116B1 homologues should be a natural carbon source in such habitat.

Techniques: Plasmid Preparation, Expressing, Control