Biology subjects

Philipp, B.

Publications and source records attributed to Philipp, B..

4 recordsLinked to original sources

bioRxiv2025

7β-hydroxysteroid dehydratase Hsh3 eliminates the 7-hydroxy group of the bile salt ursodeoxycholate during degradation by Sphingobium sp. strain Chol11 and other Sphingomonadaceae

Bile salts are steroids with distinctive hydroxylation patterns that are produced and excreted by vertebrates. In contrast to common human bile salts, ursodeoxycholate (UDCA) has a 7-hydroxy group in {beta}-configuration and is used in large amounts for treatment of diverse gastrointestinal diseases. We isolated the 7{beta}-hydroxysteroid dehydratase Hsh3 that is involved in UDCA degradation by Sphingobium sp. strain Chol11. Hsh3 eliminates the 7{beta}-hydroxy group as water, leading to a double bond in the B-ring. This is similar to 7-hydroxysteroid dehydratases in this and other strains, but Hsh3 is evolutionary different from these. Purified Hsh3 accepted steroids with and without side chain as substrates and had minor activity with 7-hydroxy groups. The deletion mutant strain Chol11 {Delta}hsh3 had impacted growth with UDCA and accumulated a novel compound. The compound was identified as 3,5-dihydroxy-H-methyl-hexahydro-5-indene-1-one-propanoate, consisting of steroid rings C and D with a C3-side chain carrying the former 7{beta}-hydroxy group, indicating that Hsh3 activity is important especially for the later stages of bile salt-degradation. Hsh3 homologs were found in other sphingomonads that were also able to degrade UDCA as well as in environmental metagenomes. Thus, Hsh3 adds to the bacterial enzyme repertoire for degrading a variety of differently hydroxylated bile-salts. ImportanceThe bacterial degradation of different bile salts is not only important for the removal of these steroidal compounds from the environment but also harbors interesting enzymes for steroid biotechnology. The 7{beta}-hydroxy bile salt UDCA naturally occurs in high concentration in the feces of black bears and has important pharmaceutical relevance for the treatment of different liver-related diseases, for which it is administered in high and increasing amounts. Additionally, it is present in the bile salt pool of humans in trace amounts. While ursodeoxycholate degradation is environmentally important, the enzyme Hsh3 modifies the hydroxy group that confers the medically relevant properties and thus might be interesting for microbiome analyses and biotechnological applications.

microbiology↗

bioRxiv2023

A patchwork pathway of apparently recent origin enables degradation of the synthetic buffer compound TRIS in bacteria

The widely used synthetic buffer compound TRIS was long considered to be biologically inert. Herein, we describe the discovery of a complete bacterial degradation pathway for TRIS. By serendipity, a Pseudomonas strain was isolated from sewage sludge that was able to grow with TRIS as only carbon and nitrogen source. Genome and transcriptome analyses revealed two adjacent gene clusters embedded in a mobile genetic element on a conjugative plasmid to be involved in TRIS degradation. Conjugational transfer of this plasmid into P. putida KT2440 enabled this strain to grow with TRIS, demonstrating that the complete TRIS degradation pathway can be transmitted by horizontal gene transfer. Heterologous gene expression revealed cluster I to encode a TRIS uptake protein, a TRIS alcohol dehydrogenase, and a TRIS aldehyde dehydrogenase, catalyzing the oxidation of TRIS into 2-hydroxymethylserine. Gene cluster II encodes a methylserine hydroxymethyltransferase and a D-serine dehydratase which plausibly catalyze the conversion of 2-hydroxymethylserine into pyruvate. Subsequent enrichments from wastewater purification systems led to the isolation of further TRIS-degrading bacteria from the Pseudomonas and Shinella genera carrying highly similar TRIS degradation gene clusters. Our data indicate that TRIS degradation evolved recently via gene recruitment and enzyme adaptation from multiple independent metabolic pathways and database searches suggest that the TRIS degradation pathway is now globally distributed. Our study illustrates how engineered environments can enhance the emergence of new microbial metabolic pathways in short evolutionary time scales. This knowledge is valuable for developing new water purification processes in times of increasing water scarcity.

microbiology↗

bioRxiv2021

Comparative analysis of bile-salt degradation in Sphingobium sp. strain Chol11 and P. stutzeri Chol1 reveals functional diversity of β-proteobacterial steroid degradation enzymes and suggests a novel reaction sequence for side-chain degradation involving a hydroxylation step

The reaction sequence for aerobic degradation of bile salts by environmental bacteria resembles degradation of other steroid compounds. Recent findings show that bacteria belonging to the Sphingomonadaceae use a pathway variant for bile-salt degradation. This study addresses this so-called {Delta}4,6 -variant by comparative analysis of unknown degradation steps in Sphingobium sp. strain Chol11 with known reactions found in Pseudomonas stutzeri Chol1. Investigations with strain Chol11 revealed an essential function of the acyl-CoA dehydrogenase Scd4AB for growth with bile salts. Growth of the scd4AB deletion mutant was restored with a metabolite containing a double bond within the side chain which was produced by the {Delta}22-acyl-CoA dehydrogenase Scd1AB from P. stutzeri Chol1. Expression of scd1AB in the scd4AB deletion mutant fully restored growth with bile salts while expression of scd4AB only enabled constricted growth in P. stutzeri Chol1 scd1A or scd1B deletion mutants. Strain Chol11 {Delta}scd4A accumulated hydroxylated steroid metabolites which were degraded and activated with coenzyme A by the wild type. Activities of five Rieske type monooxygenases of strain Chol11 were screened by heterologous expression and compared to the B-ring cleaving KshABChol1 from P. stutzeri Chol1. Three of the Chol11 enzymes catalyzed B-ring cleavage of only {Delta}4,6 -steroids while KshABChol1 was more versatile. Expression of a fourth KshA homolog, Nov2c228 led to production of metabolites with hydroxylations at an unknown position. These results indicate functional diversity of {beta}-proteobacterial enzymes for bile-salt degradation and suggest a novel side-chain degradation pathway involving an essential ACAD reaction and a steroid hydroxylation step. ImportanceThis study highlights the biochemical diversity of bacterial degradation of steroid compounds in different aspects. First, it further elucidates an unexplored variant in the degradation of bile-salt side chains by Sphingomonads, a group of environmental bacteria that is well-known for their broad metabolic capabilities. Moreover, it adds a so-far unknown hydroxylation of steroids to the reactions Rieske monooxygenases can catalyze with steroids. Additionally, it analyzes the first proteobacterial ketosteroid-9-hydroxylase and shows that this enzyme is able to catalyze side reactions with non-native substrates.

microbiology↗

bioRxiv2021

Proteome, bioinformatic and functional analyses reveal a distinct and conserved metabolic pathway for bile salt degradation in the Sphingomonadaceae

Bile salts are amphiphilic steroids with a C5 carboxylic side chain with digestive functions in vertebrates. Upon excretion, they are degraded by environmental bacteria. Degradation of the bile-salt steroid skeleton resembles the well-studied pathway for other steroids like testosterone, while specific differences occur during side-chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either {Delta}1,4- or {Delta}4,6-3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the {Delta}1,4- variant is well-known from many model organisms, the {Delta}4,6-variant involving a 7-hydroxysteroid dehydratase as key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic and functional analyses of the {Delta}4,6-variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to the {Delta}1,4-variant except for the elimination of the 7-OH as key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster for side-chain degradation encoding a CoA-ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase, but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the {Delta}4,6-variant to be widespread among the Sphingomonadaceae, which was verified for three type strains which also have the predicted side-chain degradation cluster. A second amidase in the side-chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile-salt hydrolases. This study signifies members of the Sphingomonadaceae remarkably well-adapted to the utilization of bile salts via a partially distinct metabolic pathway. ImportanceThis study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well-known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.

microbiology↗