APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2026
·Vol. 92
·期 2
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IF 3.7
Yating Zou,Jinghui Zhou,Yongyi Zeng,Bishuang Chen,Lan Liu,Gang Xu
The development of sustainable biocatalytic processes for pharmaceutical synthesis represents a major goal in green chemistry. Ene-reductases (ERs) are attractive biocatalysts for asymmetric hydrogenation of activated alkenes, yet their industrial application is often constrained by limited substrate scope and stability. In this study, we explored the deep-sea sediment metagenome of the South China Sea and identified 41 putative ER genes, with 22 successfully solubly expressed in Escherichia coli. Biochemical characterization revealed broad substrate specificity, achieving up to 90% conversion for diverse α,β-unsaturated compounds. Notably, three enzymes (S2gene2614772, S2gene1139, and S2gene22028) exhibited exceptional adaptability, maintaining high activity over a wide pH range (5.5–8.5) and at low temperatures (15°C). However, none of the wild-type ERs showed significant activity toward the prochiral substrate 2-oxo-4-phenyl-3-butenoic acid, a key intermediate for angiotensin-converting enzyme inhibitors (ACEIs). Through directed evolution, we obtained a mutant (S2gene22028-G102S) with 30-fold enhanced activity, reaching 90% conversion at 10 mM substrate. Scale-up synthesis (5 mmol substrate) afforded 2-oxo-4-phenylbutyric acid (OPBA) at 11 mg/mL, demonstrating industrial potential. This study highlights marine metagenomes as valuable sources of novel ERs and provides an efficient biocatalytic route to ACEI precursors.
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2026
·
IF 4.2
Mengmeng Ji,Hong Sun,Guifang Shi,Wei E. Huang,Yun Wang
Conventional whole-cell biosensors function as “one analyte, one sensor” systems to ensure high specificity relying on transcriptional regulatory protein cascades to induce reporter gene expression, leading to the development of distinct sensor systems to monitor multiple target substances. To address this constraint and broaden analyte detection capabilities, we developed a modular biosensing platform that couples a key metabolite-responsive biosensor with various substrate-specific enzymes to detect a range of target molecules. This study demonstrated that the integration of a general hydrogen peroxide (H2O2)-responsive biosensor with glucose oxidase, uricase, and cholesterol oxidase enabled the detection of three clinically relevant biomarkers: glucose, uric acid, and cholesterol. Upon substrate-specific enzyme conversion, these three biomarkers generate H2O2, which triggers the expression of the luxCDABE reporter gene cluster and produces bioluminescence in the H2O2-responsive biosensor Pseudomonas putida KT2440[pPahpc]. The system has demonstrated sensitive detection of glucose (10–200 µM), uric acid (5–125 µM), and cholesterol (1.25–100 µM). Validation with 17 clinical urine specimens confirmed the reliability of our system for quantifying glucose and uric acid, thereby establishing its potential diagnostic utility.
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·
IF 3.9
Chao-Fan Yin,Yong Nie,Tao Li,Ning-Yi Zhou
Many Acinetobacter species can grow on n-alkanes of varying lengths (≤C40). AlmA, a unique flavoprotein in these Acinetobacter strains, is the only enzyme proven to be required for the degradation of long-chain (LC) n-alkanes, including C32 and C36 alkanes. Although it is commonly presumed to be a terminal hydroxylase, its role in n-alkane degradation remains elusive. In this study, we conducted physiological, biochemical, and bioinformatics analyses of AlmA to determine its role in n-alkane degradation by Acinetobacter baylyi ADP1. Consistent with previous reports, gene deletion analysis showed that almA was vital for the degradation of LC n-alkanes (C26–C36). Additionally, enzymatic analysis revealed that AlmA catalyzed the conversion of aliphatic 2-ketones (C10–C16) to their corresponding esters, but it did not conduct n-alkane hydroxylation under the same conditions, thus suggesting that AlmA in strain ADP1 possesses Baeyer–Villiger monooxygenase (BVMO) activity. These results were further confirmed by bioinformatics analysis, which revealed that AlmA was closer to functionally identified BVMOs than to hydroxylases. Altogether, the results of our study suggest that LC n-alkane degradation by strain ADP1 possibly follows a novel subterminal oxidation pathway that is distinct from the terminal oxidation pathway followed for short-chain n-alkane degradation. Furthermore, our findings suggest that AlmA catalyzes the third reaction in the LC n-alkane degradation pathway.
研究领域:
微生物学
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酶学
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生物化学
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2021
·Vol. 87
·期 8
·
IF 4.792
Chao-Fan Yin,Ying Xu,Shi-Kai Deng,Wen-Long Yue,Ning-Yi Zhou
As a water-soluble polymer, the widely used polyvinyl alcohol (PVA) is produced from hydrolysis of polyvinyl acetate. Microbial PVA carbon backbone cleavage via a two-step reaction of dehydrogenation and hydrolysis has been well studied. The content of the acetyl group is a pivotal factor affecting performance of PVA derivatives in industrial applications, and deacetylation is a nonnegligible part of PVA degradation. However, the genetics and biochemistry of its deacetylation remain largely elusive. Here, Comamonas sp. strain NyZ500 was isolated for its capability of growing on acetylated PVA from activated sludge. The spontaneous PVA utilization-deficient mutant strain NyZ501 was obtained when strain NyZ500 was cultured in rich media. Comparative analysis between the genomes of these two strains revealed a fragment (containing a putative hydrolase gene, dacApva) deletion in strain NyZ501, and in the dacApva-complemented strain NyZ501 the ability to grow on PVA was restored. DacApva, which shares 21% identity with xylan esterase AxeA1 from Prevotella ruminicola 23, is a unique deacetylase catalyzing the conversion of acetylated PVA and its derivatives to deacetylated counterparts. This indicates that strain NyZ500 utilizes acetylated PVA via acetate as a carbon source to grow. DacApva also possesses the ability to deacetylate acetylated xylan and the antibiotic intermediate 7-aminocephalosporanic acid (7ACA), but the enzymes responsible for the conversion of those two compounds have no activity against PVA derivatives. This study enhanced our understanding of the diversity of microbial degradation of PVA, and DacApva characterized here is also a potential biocatalyst for the eco-friendly biotransformation of PVA derivatives and other acetylated compounds.IMPORTANCE Water-soluble PVA, which possesses a very robust ability to accumulate in the environment, has a very grave environmental impact due to its widespread use in industrial and household applications. On the other hand, chemical transformation of PVA derivatives is currently being carried out under high-energy-consumption and high-pollution conditions using hazardous chemicals (such as NaOH and methanol) under high temperatures. The DacApva reported here performs PVA deacetylation under mild conditions, so it has great potential to be developed into an eco-friendly biocatalyst for biotransformation of PVA derivatives. DacApva also has deacetylation activity for compounds other than PVA derivatives, which facilitates its development into a broad-spectrum deacetylation biocatalyst for production of certain desired compounds.
研究领域:
环境科学
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生物化学
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微生物学
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2016
·Vol. 82
·期 22
·
IF 3.823
Xiaodan Yan,Jianjun Wang,Yu Sun,Junge Zhu,Sheng Wu
Promiscuous enzymes are generally considered to be starting points in the evolution of offspring enzymes with more specific or even novel catalytic activities, which is the molecular basis of producing new biological functions. Mhg, a typical α/β fold hydrolase, was previously reported to have both γ-lactamase and perhydrolase activities. However, despite having high structural similarity to and sharing an identical catalytic triad with an extensively studied esterase from Pseudomonas fluorescens, this enzyme did not show any esterase activity. Molecular docking and sequence analysis suggested a possible role for the entry of the binding pocket in blocking the entrance tunnel, preventing the ester compounds from entering into the pocket. By engineering the entrance tunnel with only one or two amino acid substitutions, we successfully obtained five esterase variants of Mhg. The variants exhibited a very broad substrate acceptance, hydrolyzing not only the classical p-nitrophenol esters but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg was found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, the variant with the L233G mutation was a very specific esterase without any γ-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. In addition, we demonstrate that engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities.IMPORTANCE Promiscuous enzymes can act as starting points in the evolution of novel catalytic activities, thus providing a molecular basis for the production of new biological functions. In this study, we identified a critical amino acid residue (Leu233) at the entry of the substrate tunnel of a promiscuous enzyme, Mhg. We found that substitution of this residue with smaller amino acids such as Gly, Ala, Ser, or Pro endowed the enzyme with novel esterase activity. Different amino acids at this site can facilitate different catalytic activities. These findings exhibited universal significance in this subset of α/β fold hydrolases, including Mhg. Furthermore, we demonstrate that engineering the entrance tunnel is an efficient strategy to evolve new enzyme catalytic capabilities. Our study has important implications for the regulation of enzyme catalytic promiscuity and development of protein engineering methodologies.
研究领域:
生物化学
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蛋白质工程
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2025
·
IF 3.7
Yan Zhang,Huan Liu,Shan Yu,Ruocheng Pei,Haiyang Hu,Weiwei Wang,Ping Xu,Hongzhi Tang
Oil contamination poses significant risks to human health and ecosystems, emphasizing the importance of studying alkane biodegradation. In this study, we found that Rhodococcus erythropolis XP can utilize various alkanes, including C16–C36 n-alkanes and iso-alkane (pristane). The degradation capacity was significant, with over 95% of C20 degraded (500–2,500 mg/L) within 72 h. The bioremediation capacity in oily sludge was determined by a novel Low Pressure Gas Chromatography-Mass Spectrometry methodology especially for rapid analysis (within 12 min) of n-alkanes. Notable biodegradation of C14–C30 alkanes was observed in sludge treated with Rhodococcus erythropolis XP. In addition, metabolic intermediates of C16 and C20 were identified, indicating the presence of both terminal and subterminal pathways in Rhodococcus erythropolis XP. A new Baeyer-Villiger monooxygenase (BVMO_4041) was characterized, which catalyzes a key step in the subterminal pathway of alkane degradation. These results reflect the promise of Rhodococcus erythropolis XP in addressing the pressing need for efficient alkane degradation in contaminated environments.
研究领域:
环境微生物学
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生物修复
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石油烃降解
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应用微生物学
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分析化学
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2025
·
IF 3.7
Beibei Wang,Jianchao Zhang,Xiangyu Zhu,Yuebo Wang,H. Henry Teng
Minerals are fundamental yet underappreciated drivers of microbial ecology. Traditionally viewed as passive nutrient sources or inert scaffolds, their broader ecological roles remain poorly defined. This study investigates the evolutionary influence of substrates (minerals and rocks) on soil bacterial communities through serial passage evolution experiments. Soil-derived microbial consortia from three distinct locations were exposed to nutritive (olivine, granite, diorite) and non-nutritive (quartz, kaolinite, montmorillonite) substrates under nutrient-rich conditions to isolate substrate-specific effects. Results revealed systemic variations of community structure across all treatments, characterized by elevated Firmicutes/Bacteroidetes ratio and taxonomic changes predominantly driven by rare taxa. These discoveries indicate that, under the influence of substrates, the communities shifted toward ones that preferentially utilize more labile carbon. Crucially, the acute responsiveness of rare taxa to mineral-induced environmental selection suggests that, although abundant taxa appeared to maintain core community functions, the rare biosphere facilitated niche specialization and functional diversification. These findings position minerals as dynamic drivers of microbial ecology and evolution, highlighting the mineralosphere as a critical microhabitat where abiotic properties govern biodiversity, functional redundancy, and evolutionary innovation in soil ecosystems.
研究领域:
微生物生态学
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土壤科学
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进化生物学
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地球生物学
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环境微生物学
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2025
·
IF 3.7
Ronghui Liu,Jiadun Liu,Kuo Zhang,Shujun He,Qishun Feng,Jing Dai,Xinrong Guo,Yang Fu,Yi Li
In motor-assisted nanopore sensing, increasing the salt concentration improves the signal-to-noise ratio (SNR); however, helicases used as motor proteins generally fail to sustain efficient unwinding under high-salt conditions. In this study, we characterized the structure and function of AAA+ ATPase helicases from acidophiles. Bioinformatic analysis indicated that such helicases are broadly distributed among acidophilic bacteria. A representative helicase from Leptospirillum spp., designated LfDda, adopts a dimeric architecture at 3.5 Å resolution with a flexible tower domain while retaining conserved 1A and 2A domains, akin to well-studied motor (T4 Dda helicase). Biochemical assays demonstrated that the dimeric form of LfDda is functional, acting as a 5′–3′ directional helicase that can utilize various divalent metal ions as cofactors, with Mn2+ supporting the highest catalytic activity. Crosslinking of the flexible 1B and 2B domains yielded a monomeric variant capable of efficient DNA translocation at 600 mM KCl, representing a 6-fold enhancement in ionic tolerance. These findings suggest that helicases derived from extremophilic bacteria can be engineered to modulate DNA translocation behavior, providing a potential avenue for developing new motor proteins for nanopore and other DNA-unwinding applications.
研究领域:
分子生物学
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结构生物学
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生物物理学
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合成生物学
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纳米技术
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2025
·
IF 3.7
Weiyu Xie,Zhenkun Cai,Zehua Bao
Saccharomyces cerevisiae is an important organism for basic research and applied biotechnology. Genome editing techniques, particularly CRISPR/Cas9 from Streptococcus pyogenes, have greatly facilitated saturation genome editing in yeast to understand mutant functions on a large scale. However, Cas9 is restricted by its targeting preference for G-rich protospacer-adjacent motif (PAM) sequences. To broaden the targeting scope, we established an efficient homology-integrated CRISPR/Cas12a system to install genetic variants through homologous recombination by targeting T-rich PAMs. We benchmarked the PAM compatibility of PAM-relaxed Cas12a variants and identified the improved LbCas12a (impLbCas12a) as the most efficient and PAM-relaxed variant in S. cerevisiae, showing high editing purity and an editing window centering the double-strand break. We show that our system can be used to perform targeted saturation mutagenesis to reveal functional variants not captured previously. By using a homology-integrated CRISPR RNA array, we utilized the multiplexing capability of CRISPR/Cas12a to realize multiplex genetic variant installation. Our system enriches the yeast genetic variant engineering toolbox, complementing the commonly used CRISPR/Cas9 system.
研究领域:
遗传学
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分子生物学
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合成生物学
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基因组工程
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生物技术
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2025
·
IF 3.7
Yu Li,Ke Luo,Chaofeng Jiang,Yihao Zhang,Yong Yang,Yitong Yao,Huai Li,Fei Gan,Xiao-Feng Tang,Bing Tang
Phage-encoded endolysins are getting increasing attention because of their potential to serve as alternative antimicrobials to combat antibiotic-resistant bacteria. Here, we report a novel endolysin LysBT1, which is encoded by a prophage of thermophilic Brevibacillus thermoruber WF146 and comprises an N-acetylmuramoyl-L-alanine amidase domain and an S-layer homology (SLH) domain not found in known endolysins. LysBT1 is not only extremely thermostable, retaining more than 60% activity after 1 h incubation at 95°C, but also highly stable over a wide pH range of 4.0–11.0. Moreover, the thermostability of LysBT1 could be enhanced by EDTA or reducing agents. Although none of the seven cysteine residues of LysBT1 participate in disulfide bond formation, six of them, including the catalytic Zn2+-coordinating Cys156, are involved in stabilizing the enzyme at elevated temperatures. The SLH domain contributes to the thermostability of LysBT1 and mediates cell surface binding of the enzyme to facilitate enzymatic lysis of strain WF146 cells via increasing local enzyme concentration around the substrate. LysBT1 is capable of trimerization, where the SLH domains are predicted to form a three-prong spindle-like trimer similar to that in S-layer proteins. The SLH domain of LysBT1 could bind to cell surfaces of both Gram-positive and Gram-negative bacteria. LysBT1 can lyse not only Gram-positive strain WF146, Geobacillus stearothermophilus, and Bacillus subtilis but also Gram-negative Escherichia coli and Acinetobacter baumannii with the aid of EDTA or citric acid. EDTA also facilitates LysBT1 to lyse Bacillus cereus, probably because EDTA-induced disorganization of the S-layer allows LysBT1 to access and hydrolyze the peptidoglycan.
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2025
·
IF 3.7
Zhili Shi,Ya Li,Weibo Shi,Zhixin Mu,Qingxi Han,Weiwei Zhang
Probiotics are prior agents for treating bacterial infection with advantages of inhibiting pathogenic bacteria and improving immune responses of hosts, thus increasing the survival rate of cultured animals. In this study, one Vibrio parahaemolyticus YDE17 pathogenic to shrimp and its antagonist Glutamicibacter sp. ZY1 were screened, and ZY1 showed stable inhibitory effects on diverse Vibrio spp., especially V. parahaemolyticus. ZY1 secreted inhibitory substances into supernatant, and the activity of inhibitory substances did not change after being treated under different temperatures, proteinase K, and pH (6-10), which indicated that the inhibitory substances might be small molecules, which led us to trace the siderophore production. The siderophore production of YDE17 co-incubated with the cell-free supernatant of ZY1 was greater than that of YDE17 alone, which indicated that the cell-free supernatant of ZY1 created iron-limiting conditions for YDE17. This finding was confirmed by iron supplementation assays, in which the inhibitory activity of the cell-free supernatant of ZY1 on YDE17 as well as the siderophore production of YDE17 decreased in the presence of FeCl3. The effect of iron on inhibition was further confirmed by in vivo infection. The relative percent survival of ZY1 to shrimp challenged by YDE17 was 83.3%, but the survival rates of shrimp challenged with YDE17/ZY1/FeCl3 were similar to that of YDE17, both of which were significantly lower than the 70% survival rate of shrimps simultaneously challenged by ZY1/YDE17. Our study offers a new probiotic resource to control vibriosis, which works through iron competition with the opportunistic pathogens of Vibrio spp.
研究领域:
微生物学
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水产养殖
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病原生物学
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高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2025
·
IF 3.7
Qian Zhu,Kangning Wei,Kaihua Pan,Gang Hu,Weihao Zhu,Yanni Huang,Changchang Wang,Qian Li,Mingliang Zhang,Jiguo Qiu,Qing Hong
Isoprocarb (IPC), a widely used carbamate insecticide, causes environmental contamination and poses risks to humans and ecosystems. Rhodococcus sp. D-6, capable of utilizing IPC as the sole growth substrate, was isolated by our lab. Strain D-6 initiates IPC degradation through ester bond hydrolysis by the hydrolase IpcH, yielding 2-isopropylphenol (IPP). In this study, the catabolic pathway of IPP in strain D-6 was elucidated, and the ipp gene cluster responsible for its degradation was predicted by transcriptomic analysis. Key genes, including a novel two-component IPP monooxygenase gene ippA1A2 and a 2-isopropylhydroquinone dioxygenase gene ippB, are responsible for the successive conversion of IPP, resulting in the ring cleavage of IPP. IppA1, a group D flavin-dependent monooxygenase, along with its reductase component IppA2, hydroxylates IPP to 2-isopropylhydroquinone using reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) as cofactors. The optimal molar ratio of IppA1 to IppA2 was 3:1. The apparent Km and kcat values of IppA1 for IPP were 60.52 ± 1.27 µM and 70.07 ± 0.85 s−1, respectively. IppB, an extradiol dioxygenase, catalyzes 2-isopropylhydroquinone ring cleavage to generate 2-isopropyl-4-hydroxymuconic semialdehyde. The apparent Km and kcat values of IppB for 2-isopropylhydroquinone were 29.07 ± 1.54 μM and 147.65 ± 1.92 s−1, respectively. This study provides critical insights into the molecular mechanisms of IPP degradation, contributing to a comprehensive understanding of IPC degradation.
研究领域:
环境微生物学
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生物降解
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分子生物学
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生物化学
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基因组学
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农药代谢
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高影响因子
危险品化学品经营许可证（带存储）