Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation.

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived ß-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

Engineering Novosphingobium aromaticivorans to produce cis,cis-muconic acid from biomass aromatics.

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 ß-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.

Conversion of estriol to estrone: A bacterial strategy for the catabolism of estriol.

Natural estrogens, including estrone (E1), 17ß-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.

Whole genome-scale assessment of gene fitness of Novosphingobium aromaticavorans during spaceflight.

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.

Production of carotenoids from aromatics and pretreated lignocellulosic biomass by Novosphingobium aromaticivorans.

IMPORTANCE: 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.

Novosphingobium mangrovi sp. nov., isolated from mangrove sediment.

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).

Novosphingobium album sp. nov., Novosphingobium organovorum sp. nov. and Novosphingobium mangrovi sp. nov. with the organophosphorus pesticides degrading ability isolated from mangrove sediments.

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).

Novosphingobium cyanobacteriorum sp. nov., isolated from a eutrophic reservoir during the Microcystis bloom period.

A novel Gram-stain-negative, aerobic and rod-shaped bacterial strain, HBC54T, was isolated from periphyton during a Microcystis bloom. Based on the results of the 16S rRNA gene sequence analysis, strain HBC54T was closely related to Novosphingobium aerophilum 4Y4T (98.36 %), Novosphingobium aromaticivorans DSM 12444T (98.08 %), Novosphingobium huizhouense c7T (97.94 %), Novosphingobium percolationis c1T (97.65 %), Novosphingobium subterraneum DSM 12447T (97.58 %), Novosphingobium olei TW-4T (97.58 %) and Novosphingobium flavum UCT-28T (97.37 %). The average nucleotide identity and digital DNA-DNA hybridization values between HBC54T and its related type stains were below 78.97 and 23.7 %, which are lower than the threshold values for species delineation. The major fatty acids (>10.0 %) were identified as C14 : 0 2-OH, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and the respiratory quinone was ubiquinone Q-10. The main polar lipids detected in the strain were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol and three unidentified phospholipids. The genomic DNA G+C content was 64.8 mol%. Strain HBC54T is considered to represent a novel species within the genus Novosphingobium, for which the name Novosphingobium cyanobacteriorum sp. nov. is proposed. The type strain is HBC54T (=KCTC 92033T=LMG 32427T).

Novosphingobium kaempferiae sp. nov., a phosphate-solubilizing bacterium isolated from stem of Kaempferia marginata Carey.

A Gram-stain-negative, aerobic, rod-shaped, non-spore-forming and yellow-pigment-producing bacterium, designated as Sx8-5T, was isolated from stem tissue of Kaempferia marginata Carey in Kanchanaburi Province, Thailand. The strain exhibited tricalcium phosphate solubilizing activity. Its taxonomic position was investigated using a polyphasic approach. Sx8-5T grew at 25-37 °C (optimum 30 °C), pH 6-9 (optimum 7) and with 0 and 1% NaCl (optimum 0 %). According to the 16S rRNA gene phylogeny, Sx8-5T represents a member of genus Novosphingobium and shared the highest sequence similarities to Novosphingobium barchaimii LL02T of 99.4 % and shared sequence similarities with other species of the genus Novosphingobium of less than 99.4 %. The whole-genome size was 5.7 Mb, comprised of one contig, with a DNA G+C content of 66 %. The average nucleotide identity using BLASTn (ANIb) or MUMMER (ANIm) values for whole genome comparisons between Sx8-5T and Novosphingobium barchaimii LL02T and six closely related type strains were 72.33-82.14 % and 83.82-87.38 %, respectively, and the digital DNA-DNA hybridization (dDDH) values ranged from 21.0 to 28.6% when compared with the type strains of the members of the genus Novosphingobium. Major fatty acids were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), respectively. Polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, unidentified phospholipids and unidentified polar lipids. The major isoprenoid quinone was Q-10. According to results obtained using a polyphasic approach, Sx8-5T represents a novel species of the genus Novosphingobium, the name Novosphingobium kaempferiae sp. nov. is proposed. The type strain is Sx8-5T (=JCM 35076T =TBRC 15600T).

Novosphingobium beihaiensis sp. nov., a novel pesticide-tolerant bacterium isolated from mangrove sediments.

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.

Guiding stars to the field of dreams: Metabolically engineered pathways and microbial platforms for a sustainable lignin-based industry.

Lignin is an important structural component of terrestrial plants and is readily generated during biomass fractionation in lignocellulose processing facilities. Due to lacking alternatives the majority of technical lignins is industrially simply burned into heat and energy. However, considering its vast abundance and a chemically interesting richness in aromatics, lignin is presently regarded both as the most under-utilized and promising feedstock for value-added applications. Notably, microbes have evolved powerful enzymes and pathways that break down lignin and metabolize its various aromatic components. This natural pathway atlas meanwhile serves as a guiding star for metabolic engineers to breed designed cell factories and efficiently upgrade this global waste stream. The metabolism of aromatic compounds, in combination with success stories from systems metabolic engineering, as reviewed here, promises a sustainable product portfolio from lignin, comprising bulk and specialty chemicals, biomaterials, and fuels.

Novosphingobium percolationis sp. nov. and Novosphingobium huizhouense sp. nov., isolated from landfill leachate of a domestic waste treatment plant.

Two strains designated as c1T and c7T, were isolated from the landfill leachate of a domestic waste treatment plant in Huizhou City, Guangdong Province, PR China. The cells of both strains were aerobic, rod-shaped, non-motile and formed yellow colonies on Reasoner's 2A agar plates. Strain c1T grew at 10-42 °C (optimum, 30 °C), pH 4.5-10.5 (optimum, pH 7.0) and 0-2.0 % (w/v) NaCl (optimum, 0-0.5 %). Strain c7T grew at 10-42 °C (optimum, 30 °C), pH 4.5-10.5 (optimum, pH 6.0) and 0-2.0 % (w/v) NaCl (optimum, 0-0.5 %). Phylogenetic analyses revealed that strains c1T and c7T belong to the genus Novosphingobium. The 16S rRNA gene sequence similarities of strains c1T and c7T to the type strains of Novosphingobium species were 94.5-98.2 % and 94.3-99.1 %, respectively. The calculated pairwise average nucleotide identity values among strains c1T, c7T and the reference strains were in the range of 75.2-85.9 % and the calculated pairwise average amino acid identity values among strains c1T, c7T and reference strains were in the range of 72.0-88.3 %. Their major respiratory quinone was Q-10, and the major cellular fatty acids were C18 : 1 ω7c, C18 : 0, C16 : 1 ω7c, C16 : 0 and C14 : 0 2OH. The major polar lipids of strains c1T and c7T were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, sphingoglycolipid, unidentified lipids and unidentified phospholipid. Based on phenotypic, chemotaxonomic, phylogenetic and genomic results from this study, strains c1T and c7T should represent two independent novel species of Novosphingobium, for which the names Novosphingobium percolationis sp. nov. (type strain c1T=GDMCC 1.2555T=KCTC 82826T) and Novosphingobium huizhouense sp. nov. (type strain c7T=GDMCC 1.2556T=KCTC 82827T) are proposed. The gene function annotation results of strains c1T and c7T suggest that they could play an important role in the degradation of organic pollutants.

Identifying the Active Phenanthrene Degraders and Characterizing Their Metabolic Activities at the Single-Cell Level by the Combination of Magnetic-Nanoparticle-Mediated Isolation, Stable-Isotope Probing, and Raman-Activated Cell Sorting (MMI-SIP-RACS).

Magnetic-nanoparticle-mediated isolation coupled with stable-isotope probing (MMI-SIP) is a cultivation-independent higher-resolution approach for isolating active degraders in their natural habitats. However, it addresses the community level and cannot directly link the microbial identities, phenotypes, and in situ functions of the active degraders at the single-cell level within complex microbial communities. Here, we used 13C-labeled phenanthrene as the target and developed a new method coupling MMI-SIP and Raman-activated cell sorting (RACS), namely, MMI-SIP-RACS, to identify the active phenanthrene-degrading bacterial cells from polycyclic aromatic hydrocarbon (PAH)-contaminated wastewater. MMI-SIP-RACS significantly enriched the active phenanthrene degraders and successfully isolated the representative single cells. Amplicon sequencing analysis by SIP, 13C shift of the single cell in Raman spectra, and the 16S rRNA gene from single cell sequencing via RACS confirmed that Novosphingobium was the active phenanthrene degrader. Additionally, MMI-SIP-RACS reconstructed the phenanthrene metabolic pathway and genes of Novosphingobium, including two novel genes encoding phenanthrene dioxygenase and naphthalene dioxygenase. Our findings suggested that MMI-SIP-RACS is a powerful method to efficiently and precisely isolate active PAH degraders from complex microbial communities and directly link their identities to functions at the single-cell level.

Purification and characterization of cellulase produced by Novosphingobium sp. Cm1 and its waste hydrolysis efficiency and bio-stoning potential.

AIM: The aim of the study was to purify and characterize cellulase from a previously isolated Novosphingobium sp. strain Cm1 and to evaluate its waste hydrolysis and bio-stoning efficiency. MATERIALS AND METHODS: There is a growing demand for cellulase, a multipurpose enzyme widely used in industrial applications. Here, we purified cellulase from Novosphingobium sp. Cm1 by cellulose chromatography. SDS-PAGE revealed a molecular mass of 25 kDa. After 18-fold purification, the cellulase had an activity of 31.4 U/mg at pH of 5 and 40°C, and it retained activity at a wide range of pH and temperatures. The presence of Fe2+ and Co2+ boosted the enzyme activity by 57% and 25% respectively. The hydrolysing capacity of the strain towards cellulosic material was assessed for two paper types and the highest activity (2.6 ± 0.05 U/ml) was found with filter paper as the sole carbon source. Alterations in the structure of the papers as a result of bacterial hydrolysis were confirmed by scanning electron microscope and Fourier-transform infrared spectroscopy. The strain was also tested for its potential in various industrial applications and exhibited pectinolytic activity (6.78 ± 0.68 U/ml), xylanolytic activity (0.22 ± 0.14 U/ml) and bio-stoning ability. CONCLUSION: The highly active purified cellulase has a broad pH and temperature range. The strain possesses waste-hydrolysing ability, pectinolytic and xylanolytic ability along with bio-stoning capacity. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficacy and versatility of the enzyme from Novosphingobium sp. Cm1 make it an excellent candidate for diverse industrial applications.

Using fermentation waste of ethanol-producing yeast for bacterial riboflavin production and recycling of spent bacterial mass for enhancing the growth of oily plants.

AIM: This study aims to use fermentation waste of ethanol production (solid and liquid) for riboflavin and recycling of bacterial biomass as biofertilizers to enhance the growth of some oily crop plants. METHODS AND RESULTS: Out of 10 yeast isolates from fresh milk, Clavispora lusitaniae ASU 33 (MN583181) was able to ferment different concentrations of glucose (2.5%, 5%, 7.5%, 10%, 15% and 20%) into ethanol with high efficiency at 10%. Among seven non-Lactobacillus bacterial isolates recovered from cheese samples, two bacterial isolates Bacillus subtlis-SR2 (MT002768) and Novosphingobium panipatense-SR3 (MT002778) were selected for their high riboflavin production. Different media (control medium, fermentation waste medium and a mixture of the fermentation waste medium and control medium [1:1]) were used for riboflavin production. These media were inoculated by a single or mixture of B. subtlis-SR2, N. panipatense-SR3. The addition of the waste medium of ethanol production to the control medium (1:1) had a stimulatory effect on riboflavin production whether inoculated with either a single strain or a mixture of B. subtlis-SR2 and N. panipatense-SR3. A mixture of fermentation waste and control media inoculated with N. panipatense produced a high riboflavin yield in comparison with other media. Inoculation of Zea mays and Ocimum basilicum plants with either the bacterial biomass waste of riboflavin production (B. subtlis or N. panipatense) or a mixture of B. subtlis and N. panipatense) shows a stimulatory effect on the plant growth in comparison with control (uninoculated plants). CONCLUSIONS: These results demonstrate the possibility of minimizing the cost of riboflavin and biofertilizer manufacturing via interlinking ethanol and riboflavin with the biofertilizer production technology. SIGNIFICANCE AND IMPACT OF STUDY: This study outlines the methods of evaluating the strength of spent media by applying procedures developed in the vitamin production industries. Furthermore, bacterial biomass waste can act as an environmentally friendly alternative for agrochemicals.

Pathway discovery and engineering for cleavage of a ß-1 lignin-derived biaryl compound.

Lignin biosynthesis typically results in a polymer with several inter-monomer bond linkages, and the heterogeneity of linkages presents a challenge for depolymerization processes. While several enzyme classes have been shown to cleave common dimer linkages in lignin, the pathway of bacterial ß-1 spirodienone linkage cleavage has not been elucidated. Here, we identified a pathway for cleavage of 1,2-diguaiacylpropane-1,3-diol (DGPD), a ß-1 linked biaryl representative of a ring-opened spirodienone linkage, in Novosphingobium aromaticivorans DSM12444. In vitro assays using cell lysates demonstrated that RS14230 (LsdE) converts DGPD to a lignostilbene intermediate, which the carotenoid oxygenase, LsdA, then converts to vanillin. A Pseudomonas putida KT2440 strain engineered with lsdEA expression catabolizes erythro-DGPD, but not threo-DGPD. We further engineered P. putida to convert DGPD to a product, cis,cis-muconic acid. Overall, this work demonstrates the potential to identify new enzymatic reactions in N. aromaticivorans and expands the biological funnel of P. putida for microbial lignin valorization.

Aromatic Dimer Dehydrogenases from Novosphingobium aromaticivorans Reduce Monoaromatic Diketones.

Lignin is a potential source of valuable chemicals, but its chemical depolymerization results in a heterogeneous mixture of aromatics and other products. Microbes could valorize depolymerized lignin by converting multiple substrates into one or a small number of products. In this study, we describe the ability of Novosphingobium aromaticivorans to metabolize 1-(4-hydroxy-3-methoxyphenyl)propane-1,2-dione (G-diketone), an aromatic Hibbert diketone that is produced during formic acid-catalyzed lignin depolymerization. By assaying genome-wide transcript levels from N. aromaticivorans during growth on G-diketone and other chemically-related aromatics, we hypothesized that the Lig dehydrogenases, previously characterized as oxidizing ß-O-4 linkages in aromatic dimers, were involved in G-diketone metabolism by N. aromaticivorans. Using purified N. aromaticivorans Lig dehydrogenases, we found that LigL, LigN, and LigD each reduced the Cα ketone of G-diketone in vitro but with different substrate specificities and rates. Furthermore, LigL, but not LigN or LigD, also reduced the Cα ketone of 2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one (GP-1) in vitro, a derivative of G-diketone with the Cß ketone reduced, when GP-1 was provided as a substrate. The newly identified activity of these Lig dehydrogenases expands the potential range of substrates utilized by N. aromaticivorans beyond what has been previously recognized. This is beneficial both for metabolizing a wide range of natural and non-native depolymerized lignin substrates and for engineering microbes and enzymes that are active with a broader range of aromatic compounds. IMPORTANCE Lignin is a major plant polymer composed of aromatic units that have value as chemicals. However, the structure and composition of lignin have made it difficult to use this polymer as a renewable source of industrial chemicals. Bacteria like Novosphingobium aromaticivorans have the potential to make chemicals from lignin not only because of their natural ability to metabolize a variety of aromatics but also because there are established protocols to engineer N. aromaticivorans strains to funnel lignin-derived aromatics into valuable products. In this work, we report a newly discovered activity of previously characterized dehydrogenase enzymes with a chemically modified by-product of lignin depolymerization. We propose that the activity of N. aromaticivorans enzymes with both native lignin aromatics and those produced by chemical depolymerization will expand opportunities for producing industrial chemicals from the heterogenous components of this abundant plant polymer.

Novosphingobium decolorationis sp. nov., an aniline blue-decolourizing bacterium isolated from East Pacific sediment.

Aniline blue-decolourizing bacterial strain 502str22T, isolated from sediment collected in the East Pacific, was subjected to characterization by a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 502str22T belongs to the genus Novosphingobium, with closely related type strains 'Novosphingobium profundi' F72T (97.6%), N. mathurense SM117T (97.1%) and N. arvoryzae Jyi-02T (97.0%). Digital DNA-DNA hybridization and average nucleotide identity values between strain 502str22T and closely related type strains were 20.3-24.8% and 74.1-81.9%, respectively. The major cellular fatty acid (>10%) was C18:1 ω7c. The polar lipid profile consisted of a mixture of phosphatidylcholine, one sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The DNA G+C content of strain 502str22T was 65.5 mol%. The polyphasic taxonomic results indicated that strain 502str22T represents a novel species of the genus Novosphingobium, for which the name Novosphingobium decolorationis sp. nov is proposed. The type strain is 502str22T (=KCTC 82134T= MCCC 1K04799 T).

Novosphingobium aureum sp. nov., a marine bacterium isolated from salt flat sediment.

A Gram-stain-negative, aerobic, pale yellow-coloured, rod-shaped marine bacterium designated strain YJ-S2-02T was isolated from salt flat sediment sampled in Yongyu-do, Republic of Korea. Strain YJ-S2-02T grew at pH 6.0-9.0 (optimum, pH 7.0), 10-40 °C (optimum, 30 °C) and with optimum 1 % (w/v) NaCl. The 16S rRNA gene sequence analysis indicated that strain YJ-S2-02T was closely related to Novosphingobium naphthalenivorans NBRC 102051T (97.8 %) followed by Novosphingobium mathurense SM117T (97.5 %), Novosphingobium indicum H25T (97.3 %), Novosphingobium pentaromativorans US6-1T (96.8 %), Novosphingobium fontis STM-14T (96.6 %), Novosphingobium endophyticum EGI60015T (96.5 %), Novosphingobium naphthae D39T (96.5 %) and Novosphingobium malaysiense MUSC 273T (95.9 %). The average nucleotide identity and estimated DNA-DNA hybridization values between YJ-S2-02T and related type strains were 77.0-77.9 % and 19.1-24.0 %. Strain YJ-S2-02T was characterized as having Q-10 as the predominant respiratory quinone and the principal fatty acids (>10 %) were summed feature 8 (C18 : 1 ω6c/ω7c, 20.7 %), C18 : 3 ω6c (16.3 %) and C17 : 1 ω6c (11.8 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sphingolipids and two unidentified lipids. The DNA G+C content of strain YJ-S2-02T was 65.6 mol%. On the basis of the polyphasic taxonomic evidence presented in this study, YJ-S2-02T should be classified as representing a novel species within the genus Novosphingobium, for which name Novosphingobium aureum is proposed, with the type strain YJ-S2-02T (=KACC 21677T =KCTC 72891T=JCM 33996T).

Novosphingobium olei sp. nov., with the ability to degrade diesel oil, isolated from oil-contaminated soil and proposal to reclassify Novosphingobium stygium as a later heterotypic synonym of Novosphingobium aromaticivorans.

Two yellow-pigmented, non-motile, Gram-stain-negative, and rod-shaped bacteria, designated TW-4T and TNP-2 were obtained from oil-contaminated soil. Both strains degrade diesel oil, hydrolyse aesculin, DNA, Tween 40 and Tween 60. A phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain TW-4T formed a lineage within the family Erythrobacteraceae and clustered as members of the genus Novosphingobium. The closest members of strain TW-4T were Novosphingobium subterraneum DSM 12447T (97.9 %, sequence similarity), Novosphingobium lubricantis KSS165-70T (97.8 %), Novosphingobium taihuense T3-B9T (97.8 %), Novosphingobium aromaticivorans DSM 12444T (97.7 %), Novosphingobium flavum UCT-28T (97.7 %), and Novosphingobium bradum STM-24T (97.6 %). The sequence similarity for other members was ≤97.6 %. The genome of strain TW-4T was 4 683 467 bp long with 44 scaffolds and 4280 protein-coding genes. The sole respiratory quinone was Q-10. The major cellular fatty acids were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0 and C14 : 0 2-OH. The major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), phosphatidyl-n-methylethanolamine (PME) and sphingoglycolipid (SGL). The DNA G+C content of the type strain was 65.0 %. The average nucleotide identity (ANIu) and in silico DNA-DNA hybridization (dDDH) relatedness values between strain TW-4T and closest members were below the threshold value for species delineation. Based on polyphasic taxonomic analyses, strain TW-4T represents novel species in the genus Novosphingobium, for which the name Novosphingobium olei sp. nov. is proposed. The type strain is TW-4T (=KACC 21628T=NBRC 114364T) and strain TNP-2 (=KACC 21629=NBRC 114365) represents an additional strain. Based on new data obtained in this study, it is also proposed to reclassify Novosphingobium stygium as a later heterotypic synonym of Novosphingobium aromaticivorans.