Rational Design of a Flavoenzyme for Aerobic Nicotine Catabolism.

Enzymatic therapy with nicotine-degrading enzyme is a new strategy in treating nicotine addiction, which can reduce nicotine concentrations and weaken withdrawal in the rat model. However, when O2 is used as the electron acceptor, no satisfactory performance has been achieved with one of the most commonly studied and efficient nicotine-catabolizing enzymes, NicA2. To obtain more efficient nicotine-degrading enzyme, we rationally designed and engineered a flavoenzyme Pnao, which shares high structural similarity with NicA2 (RMSD = 1.143 Å) and efficiently catalyze pseudooxynicotine into 3-succinoyl-semialdehyde pyridine using O2. Through amino acid alterations with NicA2, five Pnao mutants were generated, which can degrade nicotine in Tris-HCl buffer and retained catabolic activity on its natural substrate. Nicotine-1'-N-oxide was identified as one of the reaction products. Four of the derivative mutants showed activity in rat serum and Trp220 and Asn224 were found critical for enzyme specificity. Our findings offer a novel avenue for research into aerobic nicotine catabolism and provides a promising method of generating additional nicotine-catalytic enzymes.

Design of a water-soluble transmembrane receptor kinase with intact molecular function by QTY code.

Membrane proteins are critical to biological processes and central to life sciences and modern medicine. However, membrane proteins are notoriously challenging to study, mainly owing to difficulties dictated by their highly hydrophobic nature. Previously, we reported QTY code, which is a simple method for designing water-soluble membrane proteins. Here, we apply QTY code to a transmembrane receptor, histidine kinase CpxA, to render it completely water-soluble. The designed CpxAQTY exhibits expected biophysical properties and highly preserved native molecular function, including the activities of (i) autokinase, (ii) phosphotransferase, (iii) phosphatase, and (iv) signaling receptor, involving a water-solubilized transmembrane domain. We probe the principles underlying the balance of structural stability and activity in the water-solubilized transmembrane domain. Computational approaches suggest that an extensive and dynamic hydrogen-bond network introduced by QTY code and its flexibility may play an important role. Our successful functional preservation further substantiates the robustness and comprehensiveness of QTY code.

Meta-omics assisted microbial gene and strain resources mining in contaminant environment.

Human activities have led to the release of various environmental pollutants, triggering ecological challenges. In situ, microbial communities in these contaminated environments are usually assumed to possess the potential capacity of pollutant degradation. However, the majority of genes and microorganisms in these environments remain uncharacterized and uncultured. The advent of meta-omics provided culture-independent solutions for exploring the functional genes and microorganisms within complex microbial communities. In this review, we highlight the applications and methodologies of meta-omics in uncovering of genes and microbes from contaminated environments. These findings may assist in future bioremediation research.

A meta-analysis of antibiotic residues in the Beibu Gulf.

Antibiotic residue stands as a significant ongoing environmental issue, with aquaculture being a major source of annual antibiotic discharge into the ocean. Nevertheless, there is still an incomplete evaluation of antibiotic residues in the Beibu Gulf, an area encompassed by two prominent aquaculture nations, China and Vietnam. The present systematic review and meta-analysis was conducted to examine the presence antibiotic residues in the Beibu Gulf based on published studies. Data were obtained through eight databases up to December 19th, 2023, and were updated on April 15th, 2024. The pooled concentration of antibiotic residues in seawater was 5.90 (ng/L), ranging from 5.73 to 6.06 (ng/L), and was 8.03 (ng/g), ranging from 7.77 to 8.28 (ng/g) in sediments. Fluoroquinolones, tetracyclines, and macrolides were identified as the main antibiotics found in both seawater and sediment samples. The Beibu Gulf showed higher antibiotic levels in its western and northeastern areas. Additionally, the nearshore mangrove areas displayed the highest prevalence of antibiotic residues. It is strongly advised to conduct regular long-term monitoring of antibiotic residues in the Beibu Gulf. Collaborative surveys covering the entire Beibu Gulf involving China and Vietnam are recommended.

Fine-tuning an aromatic ring-hydroxylating oxygenase to degrade high molecular weight polycyclic aromatic hydrocarbon.

Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) play pivotal roles in determining the substrate preferences of polycyclic aromatic hydrocarbon (PAH) degraders. However, their potential to degrade high molecular weight PAHs (HMW-PAHs) has been relatively unexplored. NarA2B2 is an RHO derived from a thermophilic Hydrogenibacillus sp. strain N12. In this study, we have identified four "hotspot" residues (V236, Y300, W316, and L375) that may hinder the catalytic capacity of NarA2B2 when it comes to HMW-PAHs. By employing structure-guided rational enzyme engineering, we successfully modified NarA2B2, resulting in NarA2B2 variants capable of catalyzing the degradation of six different types of HMW-PAHs, including pyrene, fluoranthene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene. Three representative variants, NarA2B2W316I, NarA2B2Y300F-W316I, and NarA2B2V236A-W316I-L375F, not only maintain their abilities to degrade low-molecular-weight PAHs (LMW-PAHs) but also exhibited 2 to 4 times higher degradation efficiency for HMW-PAHs in comparison to another isozyme, NarAaAb. Computational analysis of the NarA2B2 variants predicts that these modifications alter the size and hydrophobicity of the active site pocket making it more suitable for HMW-PAHs. These findings provide a comprehensive understanding of the relationship between three-dimensional structure and functionality, thereby opening up possibilities for designing improved RHOs that can be more effectively used in the bioremediation of PAHs.

Synergistic analysis of lignin degrading bacterial consortium and its application in rice straw fiber film.

Fiber film have received widespread attention due to its green friendliness. We can use microorganisms to degrade lignin in straw to obtain cellulose and make fiber films. Herein, a group of high-temperature (50 °C) lignin degrading bacterial consortium (LDH) was enriched and culture conditions for lignin degradation were optimized. Combined with high-throughput sequencing technology, the synergistic effect of LDH-composited bacteria was analyzed. Then LDH was used to treat rice straw for the bio-pulping experiment. The results showed that the lignin of rice straw was degraded 32.4 % by LDH at 50 °C for 10 d, and after the optimization of culture conditions, lignin degradation rate increased by 9.05 % (P < 0.001). The bacteria that compose in LDH can synergistically degrade lignin. Paenibacillus can encode all lignin-degrading enzymes present in the LDH. Preliminary tests of LDH in the pulping industry have been completed. This study is the first to use high temperature lignin degrading bacteria to fabricate fiber film.

Synergistic effect of two bacterial strains promoting anaerobic digestion of rice straw to produce methane.

A large amount of agricultural waste causes global environmental pollution. Biogas production by microbial pretreatment is an important way to utilize agricultural waste resources. In this study, Sporocytophaga CG-1 (A, cellulolytic strain) was co-cultured with Bacillus clausii HP-1 (B, non-cellulolytic strain) to analyze the effect of pretreatment of rice straw on methanogenic capacity of anaerobic digestion (AD). The results showed that weight loss rate of filter paper of co-culture combination is 53.38%, which is 29.37% higher than that of A. The synergistic effect of B on A can promote its degradation of cellulose. The cumulative methane production rate of the co-culture combination was the highest (93.04 mL/g VS substrate), which was significantly higher than that of A, B and the control group (82.38, 67.28 and 67.70 mL/g VS substrate). Auxiliary bacteria can improve cellulose degradation rate by promoting secondary product metabolism. These results provide data support for the application of co-culture strategies in the field of anaerobic digestion practices.

A multifunctional flavoprotein monooxygenase HspB for hydroxylation and C-C cleavage of 6-hydroxy-3-succinoyl-pyridine.

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCEPseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.

Production of acetic acid from wheat bran by catalysis of an acetoxylan esterase.

In this study, a gene encoding for acetylxylan esterase was cloned and expressed in E. coli. A single uniform band with molecular weight of 31.2 kDa was observed in SDS-PAGE electrophoresis. Served as the substrate, p-nitrophenol butyrate was employed to detect the recombinant enzyme activity. It exhibited activity at a wide temperature range (30-100 °C) and pH (5.0-9.0) with the optimal temperature of 70 °C and pH 8.0. Acetylxylan esterase showed two substrates' specificities with the highest Vmax of 177.2 U/mg and Km of 20.98 mM against p-nitrophenol butyrate. Meanwhile, the Vmax of p-nitrophenol acetate was 137.0 U/mg and Km 12.16 mM. The acetic acid yield of 0.39 g/g was obtained (70 °C and pH 8.0) from wheat bran pretreated using amylase and papain. This study showed the highest yield up to date and developed a promising strategy for acetic acid production using wheat bran.

Characterization of a novel aromatic ring-hydroxylating oxygenase, NarA2B2, from thermophilic Hydrogenibacillus sp. strain N12.

Polycyclic aromatic hydrocarbons (PAHs) are harmful to human health due to their carcinogenic, teratogenic, and mutagenic effects. A thermophilic Hydrogenibacillus sp. strain N12 capable of degrading a variety of PAHs and derivatives was previously isolated. In this study, an aromatic ring-hydroxylating oxygenase, NarA2B2, was identified from strain N12, with substrate specificity including naphthalene, phenanthrene, dibenzothiophene, fluorene, acenaphthene, carbazole, biphenyl, and pyrene. NarA2B2 was proposed to add one or two atoms of molecular oxygen to the substrate and catalyze biphenyl at C-2, 2 or C-3, 4 positions with different characteristics than before. The key catalytic amino acids, H222, H227, and D379, were identified as playing a pivotal role in the formation of the 2-his-1-carboxylate facial triad. Furthermore, we conducted molecular docking and molecular dynamics simulations, notably, D219 enhanced the stability of the iron center by forming two stable hydrogen bonds with H222, while the mutation of F216, T223, and H302 modulated the catalytic activity by altering the pocket's size and shape. Compared to the wild-type (WT) enzyme, the degradation ratios of acenaphthene by F216A, T223A, and H302A had an improvement of 23.08%, 26.87%, and 29.52%, the degradation ratios of naphthalene by T223A and H302A had an improvement of 51.30% and 65.17%, while the degradation ratio of biphenyl by V236A had an improvement of 77.94%. The purified NarA2B2 was oxygen-sensitive when it was incubated with L-ascorbic acid in an anaerobic environment, and its catalytic activity was restored in vitro. These results contribute to a better understanding of the molecular mechanism responsible for PAHs' degradation in thermophilic microorganisms.IMPORTANCE(i) A novel aromatic ring-hydroxylating oxygenase named NarA2B2, capable of degrading multiple polycyclic aromatic hydrocarbons and derivatives, was identified from the thermophilic microorganism Hydrogenibacillus sp. N12. (ii) The degradation characteristics of NarA2B2 were characterized by adding one or two atoms of molecular oxygen to the substrate. Unlike the previous study, NarA2B2 catalyzed biphenyl at C-2, 2 or C-3, 4 positions. (iii) Catalytic sites of NarA2B2 were conserved, and key amino acids F216, D219, H222, T223, H227, V236, F243, Y300, H302, W316, F369, and D379 played pivotal roles in catalysis, as confirmed by protein structure prediction, molecular docking, molecular dynamics simulations, and point mutation.

Terrestrial inputs and physical processes control the distributions of potentially toxic elements (PTEs) in the seawater of the large-range Beibu Gulf, the northern South China Sea.

The potentially toxic elements (PTEs), Cu, Pb, Zn, Cd, Cr, Hg and As in the water from the Beibu Gulf, were investigated to reveal the contaminant characteristics and assess the risks to human health. The results showed that the concentration of PTEs in the Beibu Gulf varies significantly both seasonally and spatially, with higher concentrations in summer and in the northern and southern gulf. Terrestrial inputs and local anthropogenic discharge are responsible for the higher level in the northern gulf, and the transportation of water masses is also an important factor for the higher concentrations in the southern gulf. Ecological risk assessment suggested that Hg is the main ecological risk factor. The health risk assessment revealed that dermal exposure to PTEs in the gulf presents potentially carcinogenic health effects for humans. This study provides new insight into the transport of PTEs over a large area of the Beibu Gulf.

An Intelligent Synthetic Bacterium for Chronological Toxicant Detection, Biodegradation, and Its Subsequent Suicide.

Modules, toolboxes, and synthetic biology systems may be designed to address environmental bioremediation. However, weak and decentralized functional modules require complex control. To address this issue, an integrated system for toxicant detection and biodegradation, and subsequent suicide in chronological order without exogenous inducers is constructed. Salicylic acid, a typical pollutant in industrial wastewater, is selected as an example to demonstrate this design. Biosensors are optimized by regulating the expression of receptors and reporters to get 2-fold sensitivity and 6-fold maximum output. Several stationary phase promoters are compared, and promoter Pfic is chosen to express the degradation enzyme. Two concepts for suicide circuits are developed, with the toxin/antitoxin circuit showing potent lethality. The three modules are coupled in a stepwise manner. Detection and biodegradation, and suicide are sequentially completed with partial attenuation compared to pre-integration, except for biodegradation, being improved by the replacements of ribosome binding site. Finally, a long-term stability test reveals that the engineered strain maintained its function for ten generations. The study provides a novel concept for integrating and controlling functional modules that can accelerate the transition of synthetic biology from conceptual to practical applications.

Complete genome of Rossellomorea sp. DA94, an agarolytic and orange-pigmented bacterium isolated from mangrove sediment of the South China Sea.

Rossellomorea sp. DA94, isolated from mangrove sediment in the South China Sea (Beihai, Guangxi province), is an agarolytic and orange-pigmented bacterium. Here, we present the complete genome sequence of strain DA94, which comprises 4.63 Mb sequences with 43.5% GC content. In total, 4589 CDSs, 33 rRNA genes and 110 tRNA genes were obtained. Genomic analysis of strain DA94 revealed that 108 CAZymes were organized in 4578 PULs involved in polysaccharides degradation, transport, and regulation. Further, we performed the diversity of CAZymes and PULs comparison among Rossellomorea strains. Less CAZymes were organized more PULs, indicating highly efficiently polysaccharides utilization in Rossellomorea. Meanwhile, PUL0459, PUL0460 and PUL0316 related to agar degradation, and exolytic beta-agarase GH50, endo-type beta-agarase GH86 and arylsulfatase were identified in the genome of strain DA94. We verified that strain DA94 can degrade agar to form a bright clear zone around the bacterial colonies in the laboratory. Moreover, the carotenoid biosynthetic pathways were proposed, which may be responsible for orange-pigment of Rossellomorea sp. DA94. This study represents a thorough genomic characterization of CAZymes repertoire and carotenoid biosynthetic pathways of Rossellomorea, provides insight into diversity of related enzymes and their potential biotechnological applications.

Microbial production of cis,cis-muconic acid from aromatic compounds in engineered Pseudomonas.

Industrial expansion has led to environmental pollution by xenobiotic compounds like polycyclic aromatic hydrocarbons and monoaromatic hydrocarbons. Pseudomonas spp. have broad metabolic potential for degrading aromatic compounds. The objective of this study was to develop a "biological funneling" strategy based on genetic modification to convert complex aromatic compounds into cis,cis-muconate (ccMA) using Pseudomonas putida B6-2 and P. brassicacearum MPDS as biocatalysts. The engineered strains B6-2 (B6-2ΔcatBΔsalC) and MPDS (MPDSΔsalC(pUCP18k-catA)) thrived with biphenyl or naphthalene as the sole carbon source and produced ccMA, attaining molar conversions of 95.3% (ccMA/biphenyl) and 100% (ccMA/naphthalene). Under mixed substrates, B6-2ΔcatBΔsalC grew on biphenyl as a carbon source and transformed ccMA from non-growth substrates benzoate or salicylate to obtain higher product concentration. Inserting exogenous clusters like bedDC1C2AB and xylCMAB allowed B6-2 recombinant strains to convert benzene and toluene to ccMA. In mixed substrates, constructed consortia of engineered strains B6-2 and MPDS specialized in catabolism of biphenyl and naphthalene; the highest molar conversion rate of ccMA from mixed substrates was 85.2% when B6-2ΔcatBΔsalC was added after 24 h of MPDSΔsalC(pUCP18k-catA) incubation with biphenyl and naphthalene. This study provides worthwhile insights into efficient production of ccMA from aromatic hydrocarbons by reusing complex pollutants.

Characterization on nicotine degradation and research on heavy metal resistance of a strain Pseudomonas sp. NBB.

The remediation of polluted sites containing multiple contaminants like nicotine and heavy metals poses significant challenges, due to detrimental effects like cell death. In this study, we isolated a new strain Pseudomonas sp. NBB capable of efficiently degrading nicotine even in high level of heavy metals. It degraded nicotine through pyrrolidine pathway and displayed minimum inhibitory concentrations of 2 mM for barium, copper, and lead, and 5 mM for manganese. In the presence of 2 mM Ba2+ or Pb2+, 3 g L-1 nicotine could be completely degraded within 24 h. Moreover, under 0.5 mM Cu2+ or 5 mM Mn2+ stress, 24.13% and 72.56% of nicotine degradation were achieved in 60 h, respectively. Strain NBB tolerances metal stress by various strategies, including morphological changes, up-regulation of macromolecule transporters, cellular response to DNA damage, and down-regulation of ABC transporters. Notably, among the 153 up-regulated genes, cds_821 was identified as manganese exporter (MneA) after gene disruption and recovery experiments. This study presents a novel strain capable of efficiently degrading nicotine and displaying remarkable resistance to heavy metals. The findings of this research provide valuable insights into the potential application of nicotine bioremediation in heavy metal-contaminated areas.

Bioluminescence Contributes to the Adaptation of Deep-Sea Bacterium Photobacterium phosphoreum ANT-2200 to High Hydrostatic Pressure.

Bioluminescence is a common phenomenon in nature, especially in the deep ocean. The physiological role of bacterial bioluminescence involves protection against oxidative and UV stresses. Yet, it remains unclear if bioluminescence contributes to deep-sea bacterial adaptation to high hydrostatic pressure (HHP). In this study, we constructed a non-luminescent mutant of ΔluxA and its complementary strain c-ΔluxA of Photobacterium phosphoreum ANT-2200, a deep-sea piezophilic bioluminescent bacterium. The wild-type strain, mutant and complementary strain were compared from aspects of pressure tolerance, intracellular reactive oxygen species (ROS) level and expression of ROS-scavenging enzymes. The results showed that, despite similar growth profiles, HHP induced the accumulation of intracellular ROS and up-regulated the expression of ROS-scavenging enzymes such as dyp, katE and katG, specifically in the non-luminescent mutant. Collectively, our results suggested that bioluminescence functions as the primary antioxidant system in strain ANT-2200, in addition to the well-known ROS-scavenging enzymes. Bioluminescence contributes to bacterial adaptation to the deep-sea environment by coping with oxidative stress generated from HHP. These results further expanded our understanding of the physiological significance of bioluminescence as well as a novel strategy for microbial adaptation to a deep-sea environment.

lA multiple PAHs-degrading Shinella sp. strain and its potential bioremediation in wastewater.

Polycyclic aromatic hydrocarbons (PAHs) and heterocyclic derivatives are organic pollutants which threaten ecosystems and human beings. In this study, a new strain, Shinella sp. FLN 14, was isolated and characterized. It can utilize fluorene as its sole carbon source and effectively co-metabolize multiple PAHs and heterocyclic derivatives, including phenanthrene, acenaphthene, and fluoranthene. Two possible metabolic pathways are proposed (i.e., salicylic acid pathway and phthalic acid pathway). Whole-genome sequencing revealed that strain FLN14 possesses a chromosome and four plasmids. However, when combined with ensemble genetic information, novel fluorene-degrading functional gene clusters were not located within the genome of FLN 14, except for some new dioxygenases and electron transport chains, which typically initiate the oxidation of aromatic compounds. In wastewater bioremediation, strain FLN14 removed nearly 95 % of PAHs within 5 days and maintained high degrading activity during the 18-day reaction compared to the control. Overall, our study provides a promising candidate to achieve bioremediation of PAHs-contaminated environments.

Anaerobic biodegradation of polycyclic aromatic hydrocarbons.

Although many anaerobic microorganisms that can degrade PAHs have been harnessed, there is still a large gap between laboratory achievements and practical applications. Here, we review the recent advances in the biodegradation of PAHs under anoxic conditions and highlight the mechanistic insights into the metabolic pathways and functional genes. Achievements of practical application and enhancing strategies of anaerobic PAHs bioremediation in soil were summarized. Based on the concerned issues during research, perspectives of further development were proposed including time-consuming enrichment, byproducts with unknown toxicity, and activity inhibition with low temperatures. In addition, meta-omics, synthetic biology and engineering microbiome of developing microbial inoculum for anaerobic bioremediation applications are discussed. We anticipate that integrating the theoretical research on PAHs anaerobic biodegradation and its successful application will advance the development of anaerobic bioremediation.