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.

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.

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.

Unveiling degradation mechanism of PAHs by a Sphingobium strain from a microbial consortium.

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent pollutants with adverse biological effects and pose a serious threat to ecological environments and human health. The previously isolated phenanthrene-degrading bacterial consortium (PDMC) consists of the genera Sphingobium and Pseudomonas and can degrade a wide range of PAHs. To identify the degradation mechanism of PAHs in the consortium PDMC, metagenomic binning was conducted and a Sphingomonadales assembly genome with 100% completeness was obtained. Additionally, Sphingobium sp. SHPJ-2, an efficient degrader of PAHs, was successfully isolated from the consortium PDMC. Strain SHPJ-2 has powerful degrading abilities and various degradation pathways of high-molecular-weight PAHs, including fluoranthene, pyrene, benzo[a]anthracene, and chrysene. Two ring-hydroxylating dioxygenases, five cytochrome P450s, and a pair of electron transfer chains associated with PAH degradation in strain SHPJ-2, which share 83.0%-99.0% similarity with their corresponding homologous proteins, were identified by a combination of Sphingomonadales assembly genome annotation, reverse-transcription quantitative polymerase chain reaction and heterologous expression. Furthermore, when coexpressed in Escherichia coli BL21(DE3) with the appropriate electron transfer chain, PhnA1B1 could effectively degrade chrysene and benzo[a]anthracene, while PhnA2B2 degrade fluoranthene. Altogether, these results provide a comprehensive assessment of strain SHPJ-2 and contribute to a better understanding of the molecular mechanism responsible for the PAH degradation.

Guided by the principles of microbiome engineering: Accomplishments and perspectives for environmental use.

Although the accomplishments of microbiome engineering highlight its significance for the targeted manipulation of microbial communities, knowledge and technical gaps still limit the applications of microbiome engineering in biotechnology, especially for environmental use. Addressing the environmental challenges of refractory pollutants and fluctuating environmental conditions requires an adequate understanding of the theoretical achievements and practical applications of microbiome engineering. Here, we review recent cutting-edge studies on microbiome engineering strategies and their classical applications in bioremediation. Moreover, a framework is summarized for combining both top-down and bottom-up approaches in microbiome engineering toward improved applications. A strategy to engineer microbiomes for environmental use, which avoids the build-up of toxic intermediates that pose a risk to human health, is suggested. We anticipate that the highlighted framework and strategy will be beneficial for engineering microbiomes to address difficult environmental challenges such as degrading multiple refractory pollutants and sustain the performance of engineered microbiomes in situ with indigenous microorganisms under fluctuating conditions.