Transcriptome analysis and cytochrome P450 monooxygenase reveal the molecular mechanism of Bisphenol A degradation by Pseudomonas putida strain YC-AE1

Abstract

Background: Bisphenol A (BPA) is a rapid spreading organic pollutant that widely used in many industries especially as a plasticizer in polycarbonate plastic and epoxy resins. BPA reported as a prominent endocrine disruptor compound that possesses estrogenic activity and fulminant toxicity. Pseudomonas putida YC-AE1 was isolated in our previous study and exerted a strong degradation capacity toward BPA at high concentrations; however, the molecular degradation mechanism is still enigmatic.

Results: We employed RNA sequencing to analyze the differentially expressed genes (DEGs) in the YC-AE1 strain upon BPA induction. Out of 1229 differentially expressed genes, 725 genes were positively regulated, and 504 genes were down-regulated. The pathways of microbial metabolism in diverse environments were significantly enriched among DEGs based on KEGG enrichment analysis. qRT-PCR confirm the involvement of BPA degradation relevant genes in accordance with RNA Seq data. The degradation pathway of BPA in YC-AE1 was proposed with specific enzymes and encoded genes. The role of cytochrome P450 (CYP450) in BPA degradation was further verified. Sever decrease in BPA degradation was recorded by YC-AE1 in the presence of CYP450 inhibitor. Subsequently, CYP450bisdB deficient YC-AE1 strain △ bisdB lost its ability toward BPA transformation comparing with the wild type. Furthermore, Transformation of E. coli with pET-32a-bisdAB empowers it to degrade 66 mg l^-1 of BPA after 24 h. Altogether, the results showed the role of CYP450 in biodegradation of BPA by YC-AE1.

Conclusion: In this study we propose the molecular basis and the potential role of YC-AE1cytochrome P450 monooxygenase in BPA catabolism.

Keywords: Bisphenol A; Cytochrome P450; Degradation pathway; Pseudomonas putida YC-AE1; RNA sequencing.

Fig. 1

Expression and analysis of DEGs (a), volcano map of DEGs during BPA degradation showing the expression fold change (X axis) and significant value, representing the up-regulated (red), down-regulated (blue), and non-regulated genes (black), (b) heat map of significantly DEGs during BPA degradation with cluster analysis. The horizontal axis represents the samples for cluster analysis, and the vertical axis represents significant DEGs. The color represents the expression level after logarithmic conversion; the color difference implies the variation in gene expression

Fig. 2

(a) Classification of GO functions of significant DEGs. The abscissa represents the number of significant DEGs, and the ordinate represents GO terms. There are three categories of GO terms, which are marked with different colors, (b) Classification diagram of KEGG Pathway of significant DEGs [44]. The abscissa is the number of significantly DEGs, and the ordinate represents the pathway classification, which is marked with different colors according to the annotations

Fig. 3

Pathway classification map of significantly enriched DEGs. The abscissa is the pathway for the enrichment of significantly DEGs (arranged by the degree of enrichment significance, and the significance of enrichment decreases from left to right), and the ordinate indicates the number of significantly up-regulated DEGs in the corresponding pathway

Fig. 4

BPA biodegradation pathways by Pseudomonas putida YC-AE1 and recommended enzymes involved in the mineralization. Intermediates marked by orange indicate the degradation products detected by HPLC/MS in our previous study [21]. Abbreviations, BPA, bisphenol A; 1-BP, 1,2-bis(4-hydroxyphenyl)-2-propanol; 2-BP, 2,2-bis(4-hydroxyphenyl)-1-propanol; 4-DM, 4,4′-dihydroxyl-α-methylstilbene; 4-HPAH, 4-hydroxyphenacyl alcohol; 4-HBD, 4-hydroxybenzaldehyde; 4-HBZ, 4-hydroxybenzoate; 4-HAP, 4-hydroxy-acetophenone; 4-HPAT, 4′-hydroxyphenyl acetate; HQN, hydroquinone; 3,4- DHB, 3,4-dihydroxybenzoate; HMS, 4-hydroxymuconic semialdehyde; MLL, maleylacetate; β-CM, β-carboxy-muconate; γ-CL, gamma-carboxymucono-lactone; 3- OEL, 3-oxoadipate-enol-lactone; 3-ODP, 3-oxoadipate; 3-OAC, 3-oxoadipyl-CoA; SCA, succinyl-CoA. Gene names are summarized in Table S4 in Supplementary Material

Fig. 5

Validation of RNA-seq data by qRT-PCR. Ten DEGs involved in the BPA degradation by YC-AE1 were selected for verification. The data were expressed as the mean fold change relative to the control samples

Fig. 6

a Effect of CYP₄₅₀ inhibitor ABT on BPA degradation by Pseudomonas putida YC-AE1 after 24 h of incubation, (b) BPA degradation by bisdB- and bisdAB- recombinant E. coli cells after 24 h incubation in LB medium. E.coli cells with empty pET-32a were used as control, (c) BPA degradation by strain YC-AE1 and Knocked strain YC-AE1ΔbisdB in TEM medium

Fig. 7

Phylogenetic analysis of P450_bisdB from strain YC-AE1 and other bacterial CYP₄₅₀ proteins. The phylogenetic tree was constructed by the neighbor-joining method using MEGA 5.2 software. P450_bisdB from Pseudomonas putida YC-AE1 was marked using a dark triangle. The accession numbers of the protein sequences were in brackets