Cofactor-independent C-C bond cleavage reactions catalyzed by the AlpJ family of oxygenases in atypical angucycline biosynthesis.

Biosynthesis of atypical angucyclines involves unique oxidative B-ring cleavage and rearrangement reactions, which are catalyzed by AlpJ-family oxygenases, including AlpJ, JadG, and GilOII. Prior investigations established the essential requirement for FADH2/FMNH2 as cofactors when utilizing the quinone intermediate dehydrorabelomycin as a substrate. In this study, we unveil a previously unrecognized facet of these enzymes as cofactor-independent oxygenases when employing the hydroquinone intermediate CR1 as a substrate. The enzymes autonomously drive oxidative ring cleavage and rearrangement reactions of CR1, yielding products identical to those observed in cofactor-dependent reactions of AlpJ-family oxygenases. Furthermore, the AlpJ- and JadG-catalyzed reactions of CR1 could be quenched by superoxide dismutase, supporting a catalytic mechanism wherein the substrate CR1 reductively activates molecular oxygen, generating a substrate radical and the superoxide anion O2 •-. Our findings illuminate a substrate-controlled catalytic mechanism of AlpJ-family oxygenases, expanding the realm of cofactor-independent oxygenases. Notably, AlpJ-family oxygenases stand as a pioneering example of enzymes capable of catalyzing oxidative reactions in either an FADH2/FMNH2-dependent or cofactor-independent manner.

Environmental Adaptability and Organic Pollutant Degradation Capacity of a Novel Rhodococcus Species Derived from Soil in the Uninhabited Area of the Qinghai-Tibet Plateau.

The Qinghai-Tibet Plateau (QTP) is known for extreme natural environments and, surprisingly, has been reported to contain widespread organic pollutants. Rhodococcus can survive a variety of extreme environments and degrade many organic contaminants. Here, we isolated a Rhodococcus strain (FXJ9.536 = CGMCC 4.7853) from a soil sample collected in the QTP. Phylogenomic analysis indicated that the strain represents a novel Rhodococcus species, for which the name Rhodococcus tibetensis sp. nov. is proposed. Interestingly, R. tibetensis FXJ9.536 maintained a fast growth rate and degraded 6.2% of p-nitrophenol (4-NP) and 50.0% of malathion even at 10 °C. It could degrade 53.6% of 4-NP and 99.9% of malathion at a moderate temperature. The genome of R. tibetensis FXJ9.536 contains 4-hydroxyphenylacetate 3-monoxygenase and carboxylesterase genes, which are likely associated with the degradation of 4-NP and malathion, respectively. Further genomic analysis revealed that the strain might employ multiple strategies to adapt to the harsh QTP environment. These include synthesizing cold shock proteins, compatible solutes, secondary metabolites, and storage compounds, utilizing inorganic compounds as energy and nutrition sources, as well as degrading a range of organic pollutants. Overall, our study reveals the potential of a QTP-derived new actinobacterial species for environmental adaptation and remediation in cold regions.

Isolation and Identification of Efficient Malathion-Degrading Bacteria from Deep-Sea Hydrothermal Sediment.

The genetic and metabolic diversity of deep-sea microorganisms play important roles in phosphorus and sulfur cycles in the ocean, distinguishing them from terrestrial counterparts. Malathion is a representative organophosphorus component in herbicides, pesticides, and insecticides and is analogues of neurotoxic agent. Malathion has been one of the best-selling generic organophosphate insecticides from 1980 to 2012. Most of the sprayed malathion has migrated by surface runoff to ocean sinks, and it is highly toxic to aquatic organisms. Hitherto, there is no report on bacterial cultures capable of degrading malathion isolated from deep-sea sediment. In this study, eight bacterial strains, isolated from sediments from deep-sea hydrothermal regions, were identified as malathion degradators. Two of the tested strains, Pseudidiomarina homiensis strain FG2 and Pseudidiomarina sp. strain CB1, can completely degrade an initial concentration of 500 mg/L malathion within 36 h. Since the two strains have abundant carboxylesterases (CEs) genes, malathion monocarboxylic acid (MMC α and MMC ß) and dibasic carboxylic acid were detected as key intermediate metabolites of malathion degradation, and the pathway of malathion degradation between the two strains was identified as a passage from malathion monocarboxylic acid to malathion dicarboxylic acid.

Macrocycle molecule-based cryoprotectants for ice recrystallization inhibition and cell cryopreservation.

Cyclodextrin-based cryoprotectants were developed. α-TMCD, which can be easily put into large-scale production, showed enhanced cell viabilities of 19.97 ± 0.78%, 13.93 ± 4.46% and 19.10 ± 0.95% against GES-1, hucMSCs and A549 cells. Moreover, the viable cells observed by light microscope imaging showed that the enhanced hucMSC cell number percentage of α-TMCD was 103.2%. An α-TMCD-DMSO-based CPA exhibited an enhanced cryoprotective effect by a mechanism of DMSO-enhanced cell penetrating effect and α-TMCD-DMSO synergistically enhanced IMA ability. α-TMCD exhibited potential for the discovery of macrocycle-molecule-based cryoprotectants.

Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.

Dirammox Is Widely Distributed and Dependently Evolved in Alcaligenes and Is Important to Nitrogen Cycle.

Nitrogen cycle is an essential process for environmental health. Dirammox (direct ammonia oxidation), encoded by the dnfT1RT2ABCD cluster, was a novel pathway for microbial N2 production defined in Alcaligenes ammonioxydans HO-1. Here, a copy of the cluster dnfT1RT2ABCD as a whole was proved to have existed and very conserved in all Alcaligenes genomes. Phylogenetic analyses based on 16S rRNA gene sequences and amino acid sequences of DnfAs, together with G + C content data, revealed that dnf cluster was evolved associated with the members of the genus Alcaligenes. Under 20% O2 conditions, 14 of 16 Alcaligenes strains showed Dirammox activity, which seemed likely taxon-related. However, the in vitro activities of DnfAs catalyzing the direct oxidation of hydroxylamine to N2 were not taxon-related but depended on the contents of Fe and Mn ions. The results indicated that DnfA is necessary but not sufficient for Dirammox activity. The fact that members of the genus Alcaligenes are widely distributed in various environments, including soil, water bodies (both freshwater and seawater), sediments, activated sludge, and animal-plant-associated environments, strongly suggests that Dirammox is important to the nitrogen cycle. In addition, Alcaligenes species are also commonly found in wastewater treatment plants, suggesting that they might be valuable resources for wastewater treatment.

Identification of the Biosynthetic Pathway of Glycine Betaine That Is Responsible for Salinity Tolerance in Halophilic Thioalkalivibrio versutus D301.

Thioalkalivibrio versutus D301 has been widely used in the biodesulfurization process, as it is capable of oxidizing hydrogen sulfide to elemental sulfur under strongly halo-alkaline conditions. Glycine betaine contributes to the increased tolerance to extreme environments in some of Thioalkalivibrio species. However, the biosynthetic pathway of glycine betaine in Thioalkalivibrio remained unknown. Here, we found that genes associated with nitrogen metabolism of T. versutus D301 were significantly upregulated under high-salt conditions, causing the enhanced production of glycine betaine that functions as a main compatible solute in response to the salinity stress. Glycine betaine was synthesized by glycine methylation pathway in T. versutus D301, with glycine N-methyltransferase (GMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) as key enzymes in this pathway. Moreover, substrate specificities of GMT and SDMT were quite different from the well characterized enzymes for glycine methylation in halophilic Halorhodospira halochloris. Our results illustrate the glycine betaine biosynthetic pathway in the genus of Thioalkalivibrio for the first time, providing us with a better understanding of the biosynthesis of glycine betaine in haloalkaliphilic Thioalkalivibrio.

Transcriptome-guided identification of a four-component system, SbrH1-R, that modulates milbemycin biosynthesis by influencing gene cluster expression, precursor supply, and antibiotic efflux.

Streptomyces can produce numerous antibiotics and many other bioactive compounds. Recently, the molecular mechanisms of transcriptional regulators in control of antibiotic production by influencing the expression of biosynthetic gene clusters (BGCs) have been extensively studied. However, for regulators that affect both antibiotic production and cell growth, the way to influence antibiotic production may be diverse, but related studies are limited. Here, based on time-course transcriptome analysis, a four-component system, SbrH1-R, consisting of the two-component system SbrKR (SBI_03479/3478) and two hypothetical proteins SbrH1 (SBI_03481) and SbrH2 (SBI_03480) potentially related with the biosynthesis of milbemycins was identified in Streptomyces bingchenggensis BC-101-4. Deletion of sbrH1-R resulted in weakened cell growth but a 110% increase of milbemycin production compared with that in BC-101-4. Comparative transcriptome analyses of the sbrH1-R mutant and BC-101-4 revealed that SbrH1-R not only indirectly represses milbemycin BGC expression, but also inhibits milbemycin production by modulating expression levels of genes related to precursor supply and antibiotic efflux. Further genetic experiments identified several new targets, including five precursor supply-associated reactions/pathways (e.g., the reaction from pyruvate to acetyl-CoA, the reaction from acetyl-CoA to citrate, the fatty acid ß-oxidation process, and the branched chain amino acid and phenylalanine acid degradation pathways) and a milbemycin exporter system (MilEX2) that can be engineered for milbemycin overproduction. These results shed new light on the understanding of regulation of milbemycin biosynthesis and provide useful targets for future metabolic engineering of the native host to improve milbemycin production.

TerC Is a Multifunctional and Promiscuous Flavoprotein Monooxygenase That Catalyzes Bimodal Oxidative Transformations.

The flavoprotein monooxygenase (FPMO) TerC is encoded by all known cyclopentene biosynthetic gene clusters. It can catalyze oxidative dearomatization toward a series of 6-HM analogues and further induces different skeletal distortions to form either benzoquinone or pyrone by bimodal reaction cascades, which is only governed by the C7 substitutions. Beyond our study demonstrated bimodal reaction cascades and advanced the biosynthetic knowledge of fungal cyclopentenes, this work also sets the stage for the bioengineering of 6-HM polyketides.

Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N2 with a previously unknown pathway.

Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen-rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO-1, oxidizing ammonia to hydroxylamine and ending in the production of N2 gas. Genome analysis revealed that strain HO-1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO-1 denitrified nitrite (not nitrate) to N2 and N2 O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N2 production. A gene cluster (dnfT1RT2ABCD) was cloned from strain HO-1 and enabled E. coli accumulated hydroxylamine. Sub-cloning showed that genetic cluster dnfAB or dnfABC already enabled E. coli cells to produce hydroxylamine and further to 15 N2 from (15 NH4 )2 SO4 . Transcriptome analysis revealed these three genes dnfA, dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO-1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of direct ammonia oxidation (Dirammox) to N2 .

Detection, Structural Elucidation, and Biological Effects of Diverse N-Acyl-homoserine Lactone Signaling Molecules in the Plant-Promoting Endophytic Bacterium Rhizobium oryzihabitans M15.

Quorum sensing (QS), usually performed by N-acyl-homoserine lactones (AHLs) in Gram-staining-negative bacteria, plays an important role in plant-bacteria interactions. Rhizobium oryzihabitans M15 is a plant-growth-promoting rhizobacterium (PGPR) isolated from rice roots. In this study, we found a QS system in the endogenous plasmid of R. oryzihabitans M15 and detected the activity of AHLs by a bioassay method. We identified five AHL analogues in R. oryzihabitans M15 using liquid chromatography-tandem mass spectrometry (LC-MS). The most dominant AHL analogue was N-(3R-hydroxy-7-cis-tetradecanoyl)-l-homoserine lactone according to nuclear magnetic resonance (NMR) and Mosher's reactions. Furthermore, the rosI mutant abolished AHL production and significantly decreased growth, exopolysaccharide (EPS) production, biofilm formation, and motility compared to the wild-type strain. These results lay the foundation for further investigating the QS regulation mechanism and signal pathway of R. oryzihabitans M15 and its interactions with the host plant.

Positional Isomeric Effects on the Optical Properties, Multivalent Glycosidase Inhibition Effect, and Hypoglycemic Effect of Perylene Bisimide-deoxynojirimycin Conjugates.

Although multivalent glycosidase inhibitors have shown enhanced glycosidase inhibition activities, further applications and research directions need to be developed in the future. In this paper, two positional isomeric perylene bisimide derivatives (PBI-4DNJ-1 and PBI-4DNJ-2) with 1-deoxynojirimycin conjugated were synthesized. Furthermore, PBI-4DNJ-1 and PBI-4DNJ-2 showed positional isomeric effects on the optical properties, self-assembly behaviors, glycosidase inhibition activities, and hypoglycemic effects. Importantly, PBI-4DNJ-1 exhibited potent hypoglycemic effects in mice with 41.33 ± 2.84 and 37.45 ± 3.94% decreases in blood glucose at 15 and 30 min, respectively. The molecular docking results showed that the active fragment of PBI-4DNJ-1 has the highest binding energy (9.649 kcal/mol) and the highest total hydrogen bond energy (62.83 kJ/mol), which were related to the positional isomeric effect on the hypoglycemic effect in mice. This work introduced a new means to develop antihyperglycemic agents in the field of multivalent glycomimetics.

Publisher Correction: Harnessing the intracellular triacylglycerols for titer improvement of polyketides in Streptomyces.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Harnessing the intracellular triacylglycerols for titer improvement of polyketides in Streptomyces.

Pharmaceutically important polyketides such as avermectin are mainly produced as secondary metabolites during the stationary phase of growth of Streptomyces species in fermenters. The source of intracellular metabolites that are funneled into polyketide biosynthesis has proven elusive. We applied multi-omics to reveal that intracellular triacylglycerols (TAGs), which accumulates in primary metabolism, are degraded during stationary phase. This process could channel carbon flux from both intracellular TAGs and extracellular substrates into polyketide biosynthesis. We devised a strategy named 'dynamic degradation of TAG' (ddTAG) to mobilize the TAG pool and increase polyketide biosynthesis. Using ddTAG we increased the titers of actinorhodin, jadomycin B, oxytetracycline and avermectin B1a in Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces rimosus and Streptomyces avermitilis. Application of ddTAG increased the titer of avermectin B1a by 50% to 9.31 g l-1 in a 180-m3 industrial-scale fermentation, which is the highest titer ever reported. Our strategy could improve polyketide titers for pharmaceutical production.

Transcriptional Regulator AcrR Increases Ethanol Tolerance through Regulation of Fatty Acid Synthesis in Lactobacillus plantarum.

Lactobacillus plantarum is a versatile bacterium with significant adaptability to harsh habitats containing excessive ethanol concentrations. It was found that the L. plantarum NF92-TetR/AcrR family regulator, AcrR, significantly enhanced the growth rate of this lactic acid bacterium in the presence of ethanol. Through screening 172 ethanol-resistant related genes by electrophoretic mobility shift and quantitative reverse transcription-PCR (RT-qPCR) assays, six genes were identified to be regulated by AcrR under ethanol stress. Among these was a gene coding for a 3-hydroxyacyl-ACP dehydratase (fabZ1) regulated by AcrR under ethanol stress. AcrR regulated fabZ1 under ethanol stress by binding to its promoter, P fabZ1 DNase I footprinting analysis indicated that there were two specific AcrR binding sites on P fabZ1 RT-PCR results showed fabZ1 could cotranscribe with its downstream 12 genes and conform a fatty acid de novo biosynthesis (fab) gene cluster under the control of P fabZ1 Both RT-qPCR of the fab gene cluster in acrR knockout and overexpression strains and fatty acid methyl ester analysis of the acrR knockout strain showed that AcrR could promote fatty acid synthesis in L. plantarum NF92. Membrane fluorescence anisotropy analysis of acrR knockout and overexpression strains showed that AcrR could increase membrane fluidity under ethanol stress. Thus, AcrR could regulate fatty acid synthesis and membrane fluidity to promote the adaption of L. plantarum NF92 to a high ethanol concentration.IMPORTANCE Ethanol tolerance is essential for L. plantarum strains living in substances with more than 9% ethanol, such as wine and beer. The details regarding how L. plantarum adapts to ethanol are still lacking. This study demonstrates that AcrR regulates the de novo synthesis of fatty acids in L. plantarum adapting to toxic levels of ethanol. We also identified the ability of the TetR/AcrR family regulator to bind to the fatty acid biosynthesis gene promoter, P fabZ1 , in L. plantarum and defined the binding sites. This finding facilitates the induction of the adaptation of L. plantarum strains to ethanol for food fermentation applications.

A novel family of intrinsic chloramphenicol acetyltransferase CATC in Vibrio parahaemolyticus: Naturally occurring variants reveal diverse resistance levels against chloramphenicol.

Intrinsic resistance of bacteria to antibiotics plays an increasingly significant role in antibiotic resistance. However, the breadth of intrinsic resistance has not been fully elucidated. Here we identified a novel class of chloramphenicol acetyltransferase (type C CAT or CATC) in Vibrio parahaemolyticus and its closely related species V. alginolyticus, V. antiquarius, and V. diabolicus. The catC genes encoding the CATC clade are distributed among the four Vibrio species and are consistently found in the same conserved genomic regions. Based on their prevalence, these genes are considered to be intrinsic in V. parahaemolyticus, V. alginolyticus, V. antiquarius, and V. diabolicus. We also demonstrated that naturally occurring variants of CATC can confer diverse resistance levels against chloramphenicol in Escherichia coli. Furthermore, the enzyme kinetics of CATC variant proteins supported the diversity of their resistance phenotypes. This work provides insights into the distribution and resistance phenotypes of a novel class of intrinsic resistance genes in bacteria.

Photobacterium sp. NNA4, an efficient hydroxylamine-transforming heterotrophic nitrifier/aerobic denitrifier.

An efficient heterotrophic nitrifying/aerobic denitrifying strain, Photobacterium sp. NNA4 was isolated from a recirculating aquaculture system (RAS). NNA4 was capable of utilizing ammonia, nitrate or nitrite as sole N-source with maximal removal rates of 12.5 mg/L/h for NH4+N, 16.4 mg/L/h for NO3--N, and 4.5 mg/L/h for NO2--N, respectively. Optimal nitrification conditions were: sodium succinate as C-source, 30-37°C, NaCl 1-4%, pH 7.0-8.0, dissolved oxygen 5.89 mg/L, C/N > 10. Gas chromatography/mass spectrometry and gas chromatography/isotope ratio mass spectrometry analyses showed that N2 and N2O were aerobic denitrification products of nitrite and nitrate. NNA4 could tolerate high concentration of hydroxylamine and displayed efficient hydroxylamine-transforming capability. Hydroxylamine oxidoreductase activity using potassium ferricyanide as electron acceptor was 0.042 U. Results revealed that strain NNA4 could oxidize NH2OH directly to N2O at aerobic conditions. In view of its high removal ability of inorganic nitrogen pollutants and broad salinity tolerance range, NNA4 has great potential in denitrification treatment of types of wastewater with either low salinity (e.g., municipal facilities) or high salinity (e.g., aquaculture, seafood processing).

Transcriptome responses of Lactobacillus acetotolerans F28 to a short and long term ethanol stress.

Lactobacillus acetotolerans is a major microbe contributing to the Chinese liquor fermentation with unknown function. It can be grown well in a high concentration of ethanol. RNA sequencing (RNA-seq) was performed on L. acetotolerans F28 growing in 12% ethanol to determine important genetic mechanisms for both a short and long term adaption to this environment. A genome-wide transcriptional analysis revealed that the most important genetic elements for L. acetotolerans F28 grown in ethanol are related to high levels of stress response and fatty acid biosynthesis, and a reduction of amino acid transport and metabolism after both a short and long time stress. The fatty acid methyl ester analyses showed that most fatty acids were increased in L. acetotolerans F28 after exposure to ethanol while the unsaturated fatty acid octadecenoic acid (C18:1) was significantly increased. The increasing unsaturated fatty acid biosynthesis in L. acetotolerans F28 might enhance cell membrane fluidity and protect the cells against high concentration of ethanol. Overall, the transcriptome and functional analysis indicated that the elevated stress response and fatty acid biosynthesis, and the decrease of amino acid transport and metabolism might play important roles for L. acetotolerans F28 to adapt to environmental ethanol.

Engineered jadomycin analogues with altered sugar moieties revealing JadS as a substrate flexible O-glycosyltransferase.

Glycosyltransferases (GTs)-mediated glycodiversification studies have drawn significant attention recently, with the goal of generating bioactive compounds with improved pharmacological properties by diversifying the appended sugars. The key to achieving glycodiversification is to identify natural and/or engineered flexible GTs capable of acting upon a broad range of substrates. Here, we report the use of a combinatorial biosynthetic approach to probe the substrate flexibility of JadS, the GT in jadomycin biosynthesis, towards different non-native NDP-sugar substrates, enabling us to identify six jadomycin B analogues with different sugar moieties. Further structural engineering by precursor-directed biosynthesis allowed us to obtain 11 new jadomycin analogues. Our results for the first time show that JadS is a flexible O-GT that can utilize both L- and D- sugars as donor substrates, and tolerate structural changes at the C2, C4 and C6 positions of the sugar moiety. JadS may be further exploited to generate novel glycosylated jadomycin molecules in future glycodiversification studies.