Natural estrogens, including estrone (E1), 17β-estradiol (E2), and estriol (E3), are potentially carcinogenic pollutants commonly found in water and soil environments. Bacterial metabolic pathway of E2 has been studied; however, the catabolic products of E3 have not been discovered thus far. In this study, Novosphingobium sp. ES2-1 was used as the target strain to investigate its catabolic pathway of E3. The metabolites of E3 were identified by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) combined with stable 13C3-labeling. Strain ES2-1 could almost completely degrade 20 mg∙L-1 of E3 within 72 h under the optimal conditions of 30°C and pH 7.0. When inoculated with strain ES2-1, E3 was initially converted to E1 and then to 4-hydroxyestrone (4-OH-E1), which was then cleaved to HIP (metabolite A6) via the 4, 5-seco pathway or cleaved to the B loop via the 9,10-seco pathway to produce metabolite with a long-chain ketone structure (metabolite B4). Although the ring-opening sequence of the above two metabolic pathways was different, the metabolism of E3 was achieved especially through continuous oxidation reactions. This study reveals that, E3 could be firstly converted to E1 and then to 4-OH-E1, and finally degraded into small molecule metabolites through two alternative pathways, thereby reducing E3 pollution in water and soil environments.

The platform chemical cis,cis-muconic acid (ccMA) provides facile access to a number of monomers used in the synthesis of commercial plastics. It is also a metabolic intermediate in the β-ketoadipic acid pathway of many bacteria and, therefore, a current target for microbial production from abundant renewable resources via metabolic engineering. This study investigates Novosphingobium aromaticivorans DSM12444 as a chassis for the production of ccMA from biomass aromatics. The N. aromaticivorans genome predicts that it encodes a previously uncharacterized protocatechuic acid (PCA) decarboxylase and a catechol 1,2-dioxygenase, which would be necessary for the conversion of aromatic metabolic intermediates to ccMA. This study confirmed the activity of these two enzymes in vitro and compared their activity to ones that have been previously characterized and used in ccMA production. From these results, we generated one strain that is completely derived from native genes and a second that contains genes previously used in microbial engineering synthesis of this compound. Both of these strains exhibited stoichiometric production of ccMA from PCA and produced greater than 100% yield of ccMA from the aromatic monomers that were identified in liquor derived from alkaline pretreated biomass. Our results show that a strain completely derived from native genes and one containing homologs from other hosts are both capable of stoichiometric production of ccMA from biomass aromatics. Overall, this work combines previously unknown aspects of aromatic metabolism in N. aromaticivorans and the genetic tractability of this organism to generate strains that produce ccMA from deconstructed biomass.IMPORTANCEThe production of commodity chemicals from renewable resources is an important goal toward increasing the environmental and economic sustainability of industrial processes. The aromatics in plant biomass are an underutilized and abundant renewable resource for the production of valuable chemicals. However, due to the chemical composition of plant biomass, many deconstruction methods generate a heterogeneous mixture of aromatics, thus making it difficult to extract valuable chemicals using current methods. Therefore, recent efforts have focused on harnessing the pathways of microorganisms to convert a diverse set of aromatics into a single product. Novosphingobium aromaticivorans DSM12444 has the native ability to metabolize a wide range of aromatics and, thus, is a potential chassis for conversion of these abundant compounds to commodity chemicals. This study reports on new features of N. aromaticivorans that can be used to produce the commodity chemical cis,cis-muconic acid from renewable and abundant biomass aromatics.

In microgravity, bacteria undergo intriguing physiological adaptations. There have been few attempts to assess global bacterial physiological responses to microgravity, with most studies only focusing on a handful of individual systems. This study assessed the fitness of each gene in the genome of the aromatic compound-degrading Alphaproteobacterium Novosphingobium aromaticavorans during growth in spaceflight. This was accomplished using Comparative TnSeq, which involves culturing the same saturating transposon mutagenized library under two different conditions. To assess gene fitness, a novel comparative TnSeq analytical tool was developed, named TnDivA, that is particularly useful in leveraging biological replicates. In this approach, transposon diversity is represented numerically using a modified Shannon diversity index, which was then converted into effective transposon density. This transformation accounts for variability in read distribution between samples, such as cases where reads were dominated by only a few transposon inserts. Effective density values were analyzed using multiple statistical methods, including log2-fold change, least-squares regression analysis, and Welch\'s t-test. The results obtained across applied statistical methods show a difference in the number of significant genes identified. However, the functional categories of genes important to growth in microgravity showed similar patterns. Lipid metabolism and transport, energy production, transcription, translation, and secondary metabolite biosynthesis and transport were shown to have high fitness during spaceflight. This suggests that core metabolic processes, including lipid and secondary metabolism, play an important role adapting to stress and promoting growth in microgravity.

There are many unidentified microbes in polluted soil needing to be explored and nominated to benefit the study of microbial ecology. In this study, a taxonomic research was carried out on five bacterial strains which were isolated and cultivated from polycyclic aromatic hydrocarbons, and heavy metals polluted soil of an abandoned coking plant. Phylogenetical analysis showed that they belonged to the phyla Proteobacteria and Actinobacteria, and their 16S rRNA gene sequence identities were lower than 98.5% to any known and validly nominated bacterial species, suggesting that they were potentially representing new species. Using polyphasic taxonomic approaches, the five strains were classified as new species of the families Microbacteriaceae and Sphingomonadaceae. Genome sizes of the five strains ranged from 3.07 to 6.60 Mb, with overall DNA G+C contents of 63.57-71.22 mol%. The five strains had average nucleotide identity of 72.38-87.38% and digital DNA-DNA hybridization of 14.0-34.2% comparing with their closely related type strains, which were all below the thresholds for species delineation, supporting these five strains as novel species. Based on the phylogenetic, phylogenomic, and phenotypic characterizations, the five novel species are proposed as Agromyces chromiiresistens (type strain H3Y2-19aT = CGMCC 1.61332T), Salinibacterium metalliresistens (type strain H3M29-4T = CGMCC 1.61335T), Novosphingobium album (type strain H3SJ31-1T = CGMCC 1.61329T), Sphingomonas pollutisoli (type strain H39-1-10T = CGMCC 1.61325T), and Sphingobium arseniciresistens (type strain H39-3-25T = CGMCC 1.61326T). Comparative genome analysis revealed that the species of the family Sphingomonadaceae represented by H39-1-10T, H39-3-25T, and H3SJ31-1T possessed more functional protein-coding genes for the degradation of aromatic pollutants than the species of the family Microbacteriaceae represented by H3Y2-19aT and H3M29-4T. Furthermore, their capacities of resisting heavy metals and metabolizing aromatic compounds were investigated. The results indicated that strains H3Y2-19aT and H39-3-25T were robustly resistant to chromate (VI) and/or arsenite (III). Strains H39-1-10T and H39-3-25T grew on aromatic compounds, including naphthalene, as carbon sources even in the presence of chromate (VI) and arsenite (III). These features reflected their adaptation to the polluted soil environment.

There is economic and environmental interest in generating commodity chemicals from renewable resources, such as lignocellulosic biomass, that can substitute for chemicals derived from fossil fuels. The bacterium Novosphingobium aromaticivorans is a promising microbial platform for producing commodity chemicals from lignocellulosic biomass because it can produce these from compounds in pretreated lignocellulosic biomass, which many industrial microbial catalysts cannot metabolize. Here, we show that N. aromaticivorans can be engineered to produce several valuable carotenoids. We also show that engineered N. aromaticivorans strains can produce these lipophilic chemicals concurrently with the extracellular commodity chemical 2-pyrone-4,6-dicarboxylic acid when grown in a complex liquor obtained from alkaline pretreated lignocellulosic biomass. Concurrent microbial production of valuable intra- and extracellular products can increase the economic value generated from the conversion of lignocellulosic biomass-derived compounds into commodity chemicals and facilitate the separation of water- and membrane-soluble products.

Environmental 17β-estradiol (E2) can cause potential harm to ecological balance and human health. Novosphingobium sp. ES2-1 is an E2-degrading bacterium previously obtained, which converts E2 to estrone (E1) and then to 4-hydroxyestrone (4-OH-E1) followed by oxidation to form metabolites with long-chain structure during upstream degradation. Herein, we found that intracellular enzymes were the major contributors to E2 biodegradation by strain ES2-1. A total of 243 proteins were dys-expressed under E2 condition, 123 were up-regulated and 120 were down-regulated thereinto. The up-regulated members of ABC transport systems, aromatics degradation, and fatty acid degradation indicated a reinforced transfer and utilization of E2. Cytochrome P450 monooxygenase (EstP1), 2-keto-4-pentenoate hydratase, pyruvate dehydrogenase, acetyl-CoA acetyltransferase, TonB-dependent receptor were involved in E2 catabolism. During downstream degradation, the metabolites with long-chain structure were decomposed adopting β-oxidation pattern and ultimately entered the TCA cycle; 2-keto-4-pentenoic acid might be an emblematic product of such process. Furthermore, E2 converting to E1 was catalyzed by 17β-dehydrogenase probably encoded by IM701_16645 or IM701_16910; 4-OH-E1 meta-cleavage was catalyzed by a dioxygenase encoded by IM701_20340 or IM701_21000 or IM701_09625. Our study provided an in-depth insight into the adaptive responses and metabolic strategies of Novosphingobium to E2.

A novel Novosphingobium species, designated strain B2638T, was isolated from mangrove sediments which was collected from Beibu Gulf, Beihai, P. R. China. The isolate could grow in the presence of chlorpyrifos. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the isolate belonged to the genus Novosphingobium, showing 99.9% sequence similarity with N. decloroationis 502str22T and less than 98% similarity with other type strain of species of this genus. Molecular typing by BOX-PCR divided strain B2638T and N. declorationis 502str22T into two clusters and indicated that they were not identical. Genomic comparison referenced by values of the DNA-DNA hybridization (dDDH) and the average nucleotide identity (ANI) between strain B2638T and its close phylogenetic neighbors were 20.0-29.5% and 75.3-85.3%, respectively, that were lower than proposed thresholds for bacterial species delineation. The major fatty acids (> 10%) were identified as C18:1 ω7c, C17:1 iso ω9c and C16:0. The main polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, phosphatidyl glycerol, unidentified lipid and unidentified aminolipid. Results from phenotypic, chemotaxonomic and genotypic analyses proposed that strain B2638T (= MCCC 1K07406T = KCTC 72968 T) is represented a novel species in the genus Novosphingobium, for which the names Novosphingobium beihaiensis sp. nov. is proposed.

Bacteria degrading large portion of saturated hydrocarbons are important for crude oil bioremediation. This study investigates Novosphingobium sp. S1, Gordonia amicalis S2 and Gordonia terrae S5 capability of degrading wide range of saturated hydrocarbons from Congo Bilondo crude oil and discusses the degradation pathway. A parallel analytical approach combining GC-MS and LC-HRMS enabled characterization of saturated hydrocarbons and comprehensive determination of carboxylic acid metabolites produced during biodegradation, respectively. Results showed that the three strains could efficiently degrade the n-alkanes (C10-C28) as well as methyl-substituted alkanes (C11-C26). The series of mono-, hydroxy- and dicarboxylic acids identified in this study confirmed the active biodegradation of the saturate fraction and suggest their degradation was via the bi-terminal oxidation pathway. This is the first study linking these bacterial species to bi-terminal oxidation of the saturated hydrocarbons. The study highlights the potential application of the bacterial strains in the bioremediation of crude oil contaminated sites. Additionally, while carboxylic acids is indicated as a suitable and valuable metabolic biomarker, its application is considered feasible and cost effective for rapid monitoring and evaluation of hydrocarbon biodegradation.

A novel Gram-stain-negative, aerobic and rod-shaped bacterial strain, designated as HK4-1T, was isolated from mangrove sediments in Hong Kong, PR China. Based on 16S rRNA gene sequence data, strain HK4-1T was found to belong to the genus Novosphingobium, family Erythrobacteraceae, and showed high similarity to Novosphingobium chloroacetimidivorans BUT-14T (96.88 %) and Novosphingobium indicum H25T (96.88 %). The G+C content of the whole genome of strain HK4-1T was 64.05 mol%. The major fatty acids were C16 : 0, C18 : 1  ω7c and summed feature 3 (C16 : 1  ω7c and/or C16 : 1  ω6c). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, sphingoglycolipid and two unknown lipids. The predominant respiratory quinone was Q-10. Based on genomic, phylogenetic, phenotypic, physiological and chemotaxonomic data, strain HK4-1T should be classified as representing a novel species of the genus Novosphingobium, for which the name Novosphingobium mangrovi sp. nov. is proposed. The type strain of Novosphingobium mangrovi sp. nov. is HK4-1T (=MCCC 1K08252T=JCM 35764T).

Members of the genus Novosphingobium were frequently isolated from polluted environments and possess great bioremediation potential. Here, three species, designated B2637T, B2580T and B1949T, were isolated from mangrove sediments and might represent novel species in the genus Novosphingobium based on a polyphasic taxonomy study. Phylogenomic analysis revealed that strains B2580T, B1949T and B2637T clustered with Novosphingobium naphthalenivorans NBRC 102051T, \'N. profundi\' F72 and N. decolorationis 502str22T, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between isolates and their closely related species were less than 94 and 54 %, respectively, all below the threshold of species discrimination. The sizes of the genomes of isolates B2580T, B2637T and B1949T ranged from 4.4 to 4.6 Mb, containing 63.3-66.4 % G+C content. Analysis of their genomic sequences identified genes related to pesticide degradation, heavy-metal resistance, nitrogen fixation, antibiotic resistance and sulphur metabolism, revealing the biotechnology potential of these isolates. Except for B2637T, B1949T and B2580T were able to grow in the presence of quinalphos. Results from these polyphasic taxonomic analyses support the affiliation of these strains to three novel species within the genus Novosphingobium, for which we propose the name Novosphingobium album sp. nov. B2580T (=KCTC 72967T=MCCC 1K04555T), Novosphingobium organovorum sp. nov. B1949T (=KCTC 92158T=MCCC 1K03763T) and Novosphingobium mangrovi sp. nov. B2637T (KCTC 72969T=MCCC 1K04460T).