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Every year, several million tonnes of anaerobic digestate are produced worldwide as a by‐product of the biogas industry, most of which is applied as agricultural fertilizer. However, in the context of a circular bioeconomy, more sustainable uses of residual digestate biomass would be desirable. This study investigates the fate of the sterol lipids β‐sitosterol and cholesterol from the feedstocks to the final digestates of three agricultural and one biowaste biogas plants to assess if sterols are degraded during anaerobic digestion or if they remain in the digestate, which could provide a novel opportunity for digestate cascade valorization. Gas chromatographic analyses showed that feedstock sterols were not degraded during anaerobic digestion, resulting in their accumulation in the digestates to up to 0.15% of the dry weight. The highest concentrations of around 1440 mg β‐sitosterol and 185 mg cholesterol per kg dry weight were found in liquid digestate fractions, suggesting partial sterol solubilization. Methanogenic batch cultures spiked with β‐sitosterol, cholesterol, testosterone and β‐oestradiol confirmed that steroids persist during anaerobic digestion. Mycobacterium neoaurum was able to transform digestate sterols quantitatively into androstadienedione, a platform chemical for steroid hormones, without prior sterol extraction or purification. These results suggest that digestate from agricultural and municipal biowaste is an untapped resource for natural sterols for biotechnological applications, providing a new strategy for digestate cascade valorization beyond land application. [ABSTRACT FROM AUTHOR]

Bile acids are surface-active steroid compounds with toxic effects for bacteria. Recently, the isolation and characterization of a bacterium, Pseudomonas sp. strain Chol1, growing with bile acids as the carbon and energy source was reported. In this study, initial reactions of the aerobic degradation pathway for the bile acid cholate were investigated on the biochemical and genetic level in strain Chol1. These reactions comprised A-ring oxidation, activation with coenzyme A (CoA), and [beta]-oxidation of the acyl side chain with the [C.sub.19]-steroid dihydroxyandrostadienedione as the end product. A-ring oxidizing enzyme activities leading to [[DELTA].sub.1,4]-3-ketocholyl-CoA were detected in cell extracts and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cholate activation with CoA was demonstrated in cell extracts and confirmed with a chemically synthesized standard by LC-MS/MS. A transposon mutant with a block in oxidation of the acyl side chain accumulated a steroid compound in culture supernatants which was identified as 7[alpha],12[alpha]-dihydroxy-3-oxopregna-l,4-diene-20- carboxylate (DHOPDC) by nuclear magnetic resonance spectroscopy. The interrupted gene was identified as encoding a putative acyl-CoA-dehydrogenase (ACAD). DHOPDC activation with CoA in cell extracts of strain Choll was detected by LC-MS/MS. The growth defect of the transposon mutant could be complemented by the wild-type ACAD gene located on the plasmid pBBR1MCS-5. Based on these results, the initiating reactions of the cholate degradation pathway leading from cholate to dihydroxyandrostadienedione could be reconstructed. In addition, the first bacterial gene encoding an enzyme for a specific reaction step in side chain degradation of steroid compounds was identified, and it showed a high degree of similarity to genes in other steroid-degrading bacteria.

The bile salts cholate, deoxycholate, chenodeoxycholate and lithocholate are released from vertebrates into soil and water where environmental bacteria degrade these widespread steroid compounds. It was investigated whether different enzymes are required for the degradation of these tri-, di- and monohydroxylated bile salts in the model organism P seudomonas sp. strain Chol1. Experiments with available and novel mutants showed that the degradation of the C5-carboxylic side chain attached to the steroid skeleton is catalysed by the same set of enzymes. A difference was found for the degradation of partially degraded bile salts consisting of H-methylhexahydroindanone-propanoates ( HIPs). With deoxycholate and lithocholate, which lack a hydroxy group at C7 of the steroid skeleton, an additional acyl-coenzyme A ( CoA) dehydrogenase was required for β-oxidation of the C3-carboxylic side chain attached to the methylhexahydroindanone moiety. The β-oxidation of this side chain could be measured in vitro. With cholate and deoxycholate, a reductive dehydroxylation of the C12-hydroxy group of HIP was required. Deletion of candidate genes for this reaction step revealed that a so-far unknown steroid dehydratase and a steroid oxidoreductase were responsible for this CoA-dependent reaction. These results showed that all bile salts are channelled into a common pathway via bypass reactions with 3'-hydroxy- HIP- CoA as central intermediate. [ABSTRACT FROM AUTHOR]

P seudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation ( std) genes encoding a single catabolic pathway. The latter includes three predicted acyl- CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The Δ stdA1 and Δ stdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione ( AD). In contrast, a Δ stdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, Δ stdA1 accumulated Δ1/4-3-ketocholate and Δ1,4-3-ketocholate, whereas Δ stdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate ( DHOPDC). When incubated with testosterone or bile acids, Δ stdA3 accumulated 3aα- H-4α(3′propanoate)-7aβ-methylhexahydro-1,5-indanedione ( HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ1/4-3-ketocholate, and Δ1,4-3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC- CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl- CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between P roteobacteria and A ctinobacteria. [ABSTRACT FROM AUTHOR]

Bacterial degradation of steroids is widespread, but the metabolic pathways have rarely been explored. Previous studies with Pseudomonas sp. strain Choll and the C24 steroid cholate have shown that cholate degradation proceeds via oxidation of the A ring, followed by cleavage of the C5 acyl side chain attached to C-17, with 7α,12β-dihydroxy-androsta-l,4-diene-3,17-dione (12β-DHADD) as the product. In this study, the pathway for degradation of the acyl side chain of cholate was investigated in vitro with cell extracts of strain Choll. For this, intermediates of cholate degradation were produced with mutants of strain Choll and submitted to enzymatic assays containing coenzyme A (CoA), ATP, and NAD+ as cosubstrates. When the C24 steroid (22E)-7α, 12α-dihydroxy-3 -oxochola- 1,4,22-triene-24-oate (DHOCTO) was used as the substrate, it was completely transformed to 12α-DHADD and 7α-hydroxy-androsta-l,4-diene-3,12,17-trione (HADT) as end products, indicating complete removal of the acyl side chain. The same products were formed with the C22 steroid 7α,12α-dihydroxy-3-oxopregna- 1,4-diene-20-carboxylate (DHOPDC) as the substrate. The 12-keto compound HADT was transformed into 12β-DHADD in an NADPH-depende reaction. When NAD+ was omitted from assays with DHOCTO, a new product, identified as 7β,12β-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA), was formed. This aldehyde was transformed to DHOPDC and DHOPDC-CoA in the presence of NAD+, CoA, and ATP. These results revealed that degradation of the C5 acyl side chain of cholate does not proceed via classical β-oxidation but via a free aldehyde that is oxidized to the corresponding acid. The reaction leading to the aldehyde presumably catalyzed by an aldolase encoded by the gene skt, which was previously predicted to be a β-ketothiolase. [ABSTRACT FROM AUTHOR]

Bile salts such as cholate are steroid compounds with a C 5 carboxylic side chain and occur ubiquitously in vertebrates. Upon their excretion into soils and waters, bile salts can serve as growth substrates for diverse bacteria. Novosphingobium sp. strain Chol11 degrades 7-hydroxy bile salts via 3-keto-7-deoxy-Δ 4,6 metabolites by the dehydration of the 7-hydroxyl group catalyzed by the 7α-hydroxysteroid dehydratase Hsh2. This reaction has not been observed in the well-studied 9-10-seco degradation pathway used by other steroid-degrading bacteria indicating that strain Chol11 uses an alternative pathway. A reciprocal BLASTp analysis showed that known side chain degradation genes from other cholate-degrading bacteria ( Pseudomonas stutzeri Chol1, Comamonas testosteroni CNB-2, and Rhodococcus jostii RHA1) were not found in the genome of strain Chol11. The characterization of a transposon mutant of strain Chol11 showing altered growth with cholate identified a novel steroid-24-oyl-coenzyme A ligase named SclA. The unmarked deletion of sclA resulted in a strong growth rate decrease with cholate, while growth with steroids with C 3 side chains or without side chains was not affected. Intermediates with a 7-deoxy-3-keto-Δ 4,6 structure, such as 3,12-dioxo-4,6-choldienoic acid (DOCDA), were shown to be likely physiological substrates of SclA. Furthermore, a novel coenzyme A (CoA)-dependent DOCDA degradation metabolite with an additional double bond in the side chain was identified. These results support the hypothesis that Novosphingobium sp. strain Chol11 harbors an alternative pathway for cholate degradation, in which side chain degradation is initiated by the CoA ligase SclA and proceeds via reaction steps catalyzed by so-far-unknown enzymes different from those of other steroid-degrading bacteria. IMPORTANCE This study provides further evidence of the diversity of metabolic pathways for the degradation of steroid compounds in environmental bacteria. The knowledge about these pathways contributes to the understanding of the CO 2 -releasing part of the global C cycle. Furthermore, it is useful for investigating the fate of pharmaceutical steroids in the environment, some of which may act as endocrine disruptors., (Copyright © 2017 American Society for Microbiology.)