An aldolase-dependent phloroglucinol degradation pathway in Collinsella sp. zg1085

Abstract

Phloroglucinol (1,3,5-trihydroxybenzene) is a key intermediate in the degradation of polyphenols such as flavonoids and hydrolysable tannins and can be used by certain bacteria as a carbon and energy source for growth. The identification of enzymes that participate in the fermentation of phloroglucinol to acetate and butyrate in Clostridia was recently reported. In this study, we present the discovery and characterization of a novel metabolic pathway for phloroglucinol degradation in the bacterium Collinsella sp. zg1085, from marmot respiratory tract. In both the Clostridial and Collinsella pathways, phloroglucinol is first reduced to dihydrophoroglucinol by the NADPH-dependent phloroglucinol reductase (PGR), followed by ring opening to form (S)-3-hydroxy-5-oxohexanoate by a Mn²⁺-dependent dihydrophloroglucinol cyclohydrolase (DPGC). In the Collinsella pathway, (S)-3-hydroxy-5-oxohexanoate is then cleaved to form malonate semialdehyde and acetone by a newly identified aldolase (HOHA). Finally, a NADP⁺-dependent malonate-semialdehyde dehydrogenase converts malonate semialdehyde to CO₂ and acetyl-CoA, an intermediate in carbon and energy metabolism. Recombinant expression of the Collinsella PGR, DPGC, and HOHA in E. coli enabled the conversion of phloroglucinol into acetone, providing support for the proposed pathway. Experiments with Olsenella profusa, another bacterium containing the gene cluster of interest, show that the PGR, DPGC, HOHA, and MSDH are induced by phloroglucinol. Our findings add to the variety of metabolic pathways for the degradation of phloroglucinol, a widely distributed phenolic compound, in the anaerobic microbiome.IMPORTANCEPhloroglucinol is an important intermediate in the bacterial degradation of polyphenols, a highly abundant class of plant natural products. Recent research has identified key enzymes of the phloroglucinol degradation pathway in butyrate-producing anaerobic bacteria, which involves cleavage of a linear triketide intermediate by a beta ketoacid cleavage enzyme, requiring acetyl-CoA as a co-substrate. This paper reports a variant of the pathway in the lactic acid bacterium Collinsella sp. zg1085, which involves cleavage of the triketide intermediate by a homolog of deoxyribose-5-phosphate aldolase, highlighting the variety of mechanisms for phloroglucinol degradation by different anaerobic bacterial taxa.

Keywords: (S)-3-hydroxy-5-oxohexanoate aldolase; acetyl-CoA; anaerobic bacteria; malonate-semialdehyde dehydrogenase; phloroglucinol degradation.

Fig 1

A new pathway for phloroglucinol degradation in Collinsella sp. zg1085. (A) Anaerobic phloroglucinol degradation gene clusters in Clostridium scatologenes (24) and Collinsella sp. zg1085. The percentages correspond to the sequence identities between the PGR and DPGC homologs present in the two bacterial strains. (B) Phloroglucinol degradation pathway in Clostridium scatologenes (24) and proposed pathway in Collinsella sp. zg1085. The new enzymes and/or reactions reported in this paper are highlighted in gray.

Fig 2

CzPGR enzyme activities assay. (A) CzPGR-catalyzed reaction. (B) Assays monitoring the consumption of NADPH. (C) Assays monitoring the formation of the dihydrophloroglucinol. Michaelis-Menten plot with (D) NADPH or (E) phloroglucinol as the variable substrate.

Fig 3

CzDPGC enzyme activities assay. (A) CzDPGC-catalyzed reaction. (B) UV-Vis spectra of the coupled reaction of CzPGR and CzDPGC. (C) Time-dependent UV-Vis spectra of the CzDPGC assay. (D) Specificity of CzDPGC for different divalent metal cofactors. (E) Michaelis-Menten plot with dihydrophloroglucinol as the variable substrate.

Fig 4

CzHOHA enzyme activities assay. (A) CzHOHA-catalyzed reaction, coupled to CzADH or PfHBDH for spectrophotometric readout. The substrate (S)-3-hydroxyl-5-oxohexanoate was generated in situ by CzDPGC. (B) UV-Vis spectra of the CzDPGC-CzHOHA reaction, coupled with CzADH-NADPH for acetone detection. (C) UV-Vis spectra of the CzDPGC-CzHOHA reaction, coupled with PfHBDH-NADH for malonate semialdehyde detection. (D) Elution profiles of the DNPH-derivatized products of the CzPGR-CzDPGC-CzHOHA catalyzed reaction, monitoring the absorbance at 360 nm. DNPH and DNPH-acetone standards are included. (E) The EIC profiles of malonate semialdehyde-DNPH and acetaldehyde-DNPH. The mass spectrum of the malonate semialdehyde peak suggested decarboxylation accompanying the ionization process. (F) The EIC profiles of acetone-DNPH. (G) CzHOHA-catalyzed aldol reaction of acetone and malonic semialdehyde, detected using (S)−3-hydroxy-5-oxohexanoate dehydrogenase (CsTfD) as a coupling enzyme. (H) LC-MS elution profile of CzDPGC reaction products and CzHOHA reaction products, DPG (dihydrophloroglucinol), MSA (malonic semialdehyde). (I) LC-MS elution profile of CzHOHA aldol reaction products with succinic semialdehyde as acceptor and acetone as donor.

Fig 5

In vitro reconstitution of the CzPGR-CzDPGC-CzHOHA pathway. LC-MS analysis of reaction mixtures containing (A) PG (phloroglucinol) and NADPH; (B) PG, NADPH, and CzPGR; (C) PG, NADPH, CzPGR, and CzDPGC; (D) PG, NADPH, CzPGR, CzDPGC, and CzHOHA.

Fig 6

CzMSDH enzyme activity assay. (A) Sequence alignment of CzMSDH with NAD^+-dependent CoA-acylating malonate-semialdehyde dehydrogenase from Bacillus subtilis (33), Bacillus megaterium (32), and Pseudomonas aeruginosa (34). The conserved residues involved in cofactor binding were colored and labeled. (B) The active site of the crystal structure of structure of BsMSDH in complex with NAD⁺ (light purple, PDB: 1T90) is superimposed on a homology model of CzMSDH (green). The key residue involved in the 2'-hydroxyl of adenosine interaction is displayed and labeled, and the hydrogen bond is indicated by a dashed line. (C) CzMSDH-catalyzed reaction, with the malonate semialdehyde substrate generated in situ by BmPydD2. (D) UV-Vis spectra of the coupled reaction of CzMSDH and BmPydD2, monitoring NADPH formation. (E) Assays monitoring NAD(P)H formation accompanying malonic semialdehyde oxidation.

Fig 7

Physiological validation of genes involved in aldolase-dependent phloroglucinol degradation. (A) SDS-PAGE analysis of E. coli BL21 (pET28a-CzPGR-CzDPGC-CzHOHA) grown aerobically in TB medium supplemented with either glycerol or phloroglucinol, without the addition of IPTG. Source data are provided as Supplementary Data 1. (B) Detection of acetone in phloroglucinol fermentation products by enzymatic assay with CzADH. (C) Presence of the aldolase-dependent phloroglucinol degradation pathway in various anaerobic bacteria. The percentages correspond to the sequence identities between the proteins and their characterized homologs: PGR, DPGC, HOHA, MSDH from Collinsella sp. zg1085. (D) qPCR analyses of the transcription levels of PGR, DPGC, HOHA, and MSDH in Olsenella profusa. The transcriptional levels of genes of interest were normalized by that of the 16S rRNA. The induction by phloroglucinol was displayed in comparison with the transcriptional from glucose-grown cells. Bars in different samples were as indicated. (E) UV-Vis spectra of the CzDPGC-OpHOHA reaction, coupled with CzADH-NADPH for acetone detection. (F) UV-Vis spectra of the CzDPGC-OpHOHA reaction, coupled with PfHBDH-NADH for malonate semialdehyde detection.