A review of BMP and Wnt signaling pathway in the pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by a progressive elevation in mean pulmonary arterial pressure. This occurs due to abnormal remodeling of small peripheral lung vasculature resulting in progressive occlusion of the artery lumen that eventually causes right heart failure and death. Current therapeutic options for PAH are limited and focused mainly on reversal of pulmonary vasoconstriction and proliferation of vascular cells. Although these treatments can relieve disease symptoms, PAH remains a progressive lethal disease.Bone morphogenetic proteins (BMPs) and their receptors were required for PAH-induced right ventricular hypertrophy. Emerging data suggest that restoration of BMP type II receptor (BMPR2) signaling in PAH is a promising alternative that could prevent and reverse pulmonary vascular remodeling. BMPR2 mutations have been identified in >70% of familial and roughly 15% of sporadic PAH cases. Wingless (Wnt) are a family of secreted glycoproteins with varying expression patterns and a range of functions, Wnt signaling pathway is divided into canonical signaling pathway and non-canonical signaling pathway. A recent study reports that interaction between BMP and Wnt closely associated with lung development, those cascade coordination regulation stem cell fate which determine lung branching morphogenes. The promoting effect of BMPR2 on proliferation, survival, and motility of endothelial cells was through recruiting Wnts signaling pathway, the interaction between BMP and Wnt closely associated with lung development.Therefore, in this review, we outline the latest advances of BMP and Wnt signaling pathway in the pathogenesis of PAH and disease progression.

Identification and characterization of a novel carboxylesterase EstQ7 from a soil metagenomic library.

A novel lipolytic gene, estq7, was identified from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42 kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174-Asp306-His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short-chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17 mM and 1910s-1, respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Structural modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/ß hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking revealed the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.

Enhancing the activity and thermal stability of a phthalate-degrading hydrolase by random mutagenesis.

Our previous work has reported that EstJ6 was a phthalate-degrading hydrolase. In the study, a random mutant library was constructed by two rounds of error-prone PCR, three mutants (ET1.1, ET2.1, and ET2.2) with enhanced hydrolytic activity against dibutyl phthalate (DBP) were obtained. The best mutant ET2.2, accumulated three amino acid substitutions (Thr91Met, Ala67Val, and Val249Ile) and exhibited 2.8-fold increase enzyme activity and 2.3-fold higher expression level. Meanwhile, compared with EstJ6, ET2.2 showed over 50% improvement in thermostability (at 50 °C for 1 h) and 1.2-fold increase in 50% methanol tolerance. Kinetic parameters analysis revealed that the Km value for ET2.2 decreased by 60% and the kcat/Km value increased by 166%. The molecular docking indicated that the shortening of hydrogen bond between Ser146-OH and DBP-CO, which may led to an increase in enzyme activity and catalytic efficiency, the enhancement of hydrophobicity of hydrophobic pocket was related to the improvement of organic solvents tolerance, and three hydrophobic amino acid substitutions Thr91Met, Ala67Val, and Val249Ile facilitated to improve the thermal stability and organic solvents tolerance. These results confirmed that random mutagenesis was an effective tool for improving enzyme properties and lay a foundation for practical applications of phthalate-degrading hydrolase in biotechnology and industrial fields.

Identification and characterization of a novel phthalate-degrading hydrolase from a soil metagenomic library.

Phthalate esters have raised public concerns owing to their effects on the environment and human health. We identified a novel phthalate-degrading hydrolase, EstJ6, from a metagenomic library using function-driven screening. Phylogenetic analysis indicated that EstJ6 is a member of family IV esterases. EstJ6 hydrolyzed various dialkyl and monoalkyl phthalate esters, and exhibited high hydrolytic activity (128 U/mg) toward dibutyl phthalate at 40 °C and pH 7.5. EstJ6 hydrolyzed not only common phthalate esters with simple side chains but also diethylhexyl phthalate and monoethylhexyl phthalate, which have complex and long side chains. Site-directed mutagenesis indicated that the catalytic triad residues of EstJ6 consists of Ser146, Glu240, and His270. EstJ6 is therefore a promising biodegradation enzyme, and our study illustrates the advantages of a metagenomic approach in identifying enzyme-coding genes for agricultural, food, and biotechnological applications.

Molecular cloning, expression and characterization of a novel feruloyl esterase from a soil metagenomic library with phthalate-degrading activity.

OBJECTIVES: To discover novel feruloyl esterases (FAEs) by the function-driven screening procedure from soil metagenome. RESULTS: A novel FAE gene bds4 was isolated from a soil metagenomic library and over-expressed in Escherichia coli. The recombinant enzyme BDS4 was purified to homogeneity with a predicted molecular weight of 38.8 kDa. BDS4 exhibited strong activity (57.05 U/mg) toward methyl ferulate under the optimum pH and temperature of 8.0 and 37°C. Based on its amino acid sequence and model substrates specificity, BDS4 was classified as a type-C FAE. The quantity of the releasing ferulic acid can be enhanced significantly in the presence of xylanase compared with BDS4 alone from de-starched wheat bran. In addition, BDS4 can also hydrolyze several phthalates such as diethyl phthalate, dimethyl phthalate and dibutyl phthalate. CONCLUSION: The current investigation discovered a novel FAE with phthalate-degrading activity and highlighted the usefulness of metagenomic approaches as a powerful tool for discovery of novel FAEs.

An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification.

An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H2O2. The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5-104 CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry.

Comparative genomic analysis of isoproturon-mineralizing sphingomonads reveals the isoproturon catabolic mechanism.

The worldwide use of the phenylurea herbicide, isoproturon (IPU), has resulted in considerable concern about its environmental fate. Although many microbial metabolites of IPU are known and IPU-mineralizing bacteria have been isolated, the molecular mechanism of IPU catabolism has not been elucidated yet. In this study, complete genes that encode the conserved IPU catabolic pathway were revealed, based on comparative analysis of the genomes of three IPU-mineralizing sphingomonads and subsequent experimental validation. The complete genes included a novel hydrolase gene ddhA, which is responsible for the cleavage of the urea side chain of the IPU demethylated products; a distinct aniline dioxygenase gene cluster adoQTA1A2BR, which has a broad substrate range; and an inducible catechol meta-cleavage pathway gene cluster adoXEGKLIJC. Furthermore, the initial mono-N-demethylation genes pdmAB were further confirmed to be involved in the successive N-demethylation of the IPU mono-N-demethylated product. These IPU-catabolic genes were organized into four transcription units and distributed on three plasmids. They were flanked by multiple mobile genetic elements and highly conserved among IPU-mineralizing sphingomonads. The elucidation of the molecular mechanism of IPU catabolism will enhance our understanding of the microbial mineralization of IPU and provide insights into the evolutionary scenario of the conserved IPU-catabolic pathway.

Tafuketide, a phylogeny-guided discovery of a new polyketide from Talaromyces funiculosus Salicorn 58.

A phylogeny-guided approach was applied to screen endophytic fungi containing type I polyketide synthase (PKS I) biosynthetic gene sequences and aimed to correlate genotype to chemotype for the discovery of novel bioactive polyketides. Salicorn 58, which was identified as Talaromyces funiculosus based on its internal transcribed spacer (ITS) and ribosomal large-subunit (LSU) DNA sequences, showed significant target bands. A chemical investigation of the culture of Salicorn 58 was allowed for the isolation of a new polyketide, Talafun (1), and a new natural product, N-(2'-hydroxy-3'-octadecenoyl)-9-methyl-4,8-sphingadienin (2), together with six known compounds, including chrodrimanin A (3), chrodrimanin B (4), N-(4-hydroxy-2-methoxyphenyl) acetamide (5), butyl ß-glucose (6), 3ß,15ß-dihydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7-dione (7), and (3ß,5a,8a,22E)-5,8-epidioxyergosta-6,22-dien-3-ol (8). Their chemical structures were elucidated by extensive spectroscopic analysis and electro circular dichroism (ECD) spectrum calculations. Antioxidant experiments revealed that compound 5 showed strong ABTS(+) radical scavenging activity with an IC50 value of 11.43 ± 1.61 µM and potent ferric reducing activity (FRAP assay) with FRAP value of 187.52 ± 2.97. Antimicrobial assays revealed that compounds 1 and 4 showed high levels of selectivity toward Escherichia coli with MIC values of 18 ± 0.40 and 43 ± 0.52 µM, respectively. Compounds 2 and 3 exhibited broad-spectrum antimicrobial activity against Staphylococcus aureus, Mycobacterium smegmatis, Micrococcus tetragenus, Mycobacterium phlei, and E. coli, respectively. The results from the current research highlight the advantage of phylogeny-guided pipeline for the screening of new polyketides from endophytic fungi containing PKS I genes.

A PKS I gene-based screening approach for the discovery of a new polyketide from Penicillium citrinum Salicorn 46.

Salicorn 46, an endophytic fungus isolated from Salicornia herbacea Torr., was identified as Penicillium citrinum based on its internal transcribed spacer and ribosomal large-subunit DNA sequences using a type I polyketide synthase (PKS I) gene screening approach. A new polyketide, penicitriketo (1), and seven known compounds, including ergone (2), (3ß,5α,8α,22E)-5,8-epidioxyergosta-6,9,22-trien-3-ol (3), (3ß,5α,8α,22E)-5,8-epidioxyergosta-6,22-dien-3-ol (4), stigmasta-7,22-diene-3ß,5α,6α-triol (5), 3ß,5α-dihydroxy-(22E,24R)-ergosta-7,22-dien-6ß-yl oleate (6), N b-acetyltryptamine (7), and 2-(1-oxo-2-hydroxyethyl) furan (8), were isolated from the culture of Salicorn 46, and their chemical structures were elucidated by spectroscopic analysis. Antioxidant experiments revealed that compound 1 possessed moderate DPPH radical scavenging activity with an IC50 value of 85.33 ± 1.61 µM. Antimicrobial assays revealed that compound 2 exhibited broad-spectrum antimicrobial activity against Candida albicans, Clostridium perfringens, Mycobacterium smegmatis, and Mycobacterium phlei with minimal inhibitory concentration (MIC) values of 25.5, 25.5, 18.5, and 51.0 µM, respectively. Compound 3 displayed potent antimicrobial activities against C. perfringens and Micrococcus tetragenus with a MIC value of 23.5 µM. Compounds 5 and 6 showed high levels of selectivity toward Bacillus subtilis and M. phlei with MIC values of 22.5 and 14.4 µM, respectively. The results of this study highlight the use of PCR-based techniques for the screening of new polyketides from endophytic fungi containing PKS I genes.

Non-invasive anticipation of infusion taste in fine-manipulated green teas through hyperspectral appearance analysis guided by ECG content.

The high catechin content in summer-to-autumn tea leaves often results in strong, unpleasant tastes, leading to significant resource waste and economic losses due to lignification of unpicked leaves. This study aims to improve the taste quality of summer-to-autumn green teas by combining fine manipulation techniques with hyperspectral observation. Fine manipulation notably enhanced infusion taste quality, particularly in astringency and its aftertaste (aftertasteA). Using Partial Least Squares Discriminant Analysis (PLSDA) on hyperspectral data, 100% prediction accuracy was achieved for dry tea appearance in the near-infrared spectrum. Astringency and aftertasteA correlated with hyperspectral data, allowing precise estimation with over 90% accuracy in both visible and near-infrared spectrums. Epicatechin gallate (ECG) emerged as a key taste compound, enabling non-invasive taste prediction. Practical applications in processing and quality control are demonstrated by the derived equations (Astringency = -0.88 × ECG + 45.401, AftertasteA = -0.353 × ECG + 18.609), highlighting ECG's role in shaping green tea taste profiles.

An Integrated Pipeline and Overexpression of a Novel Efflux Transporter, YoeA, Significantly Increases Plipastatin Production in Bacillus subtilis.

Plipastatin, an antimicrobial peptide produced by Bacillus subtilis, exhibits remarkable antimicrobial activity against a diverse range of pathogenic bacteria and fungi. However, the practical application of plipastatin has been significantly hampered by its low yield in wild Bacillus species. Here, the native promoters of both the plipastatin operon and the sfp gene in the mono-producing strain M-24 were replaced by the constitutive promoter P43, resulting in plipastatin titers being increased by 27% (607 mg/mL) and 50% (717 mg/mL), respectively. Overexpression of long chain fatty acid coenzyme A ligase (LCFA) increased the yield of plipastatin by 105% (980 mg/mL). A new efflux transporter, YoeA, was identified as a MATE (multidrug and toxic compound extrusion) family member, overexpression of yoeA enhanced plipastatin production to 1233 mg/mL, an increase of 157%, and knockout of yoeA decreased plipastatin production by 70%; in contrast, overexpression or knockout of yoeA in mono-producing surfactin and iturin engineered strains only slightly affected their production, demonstrating that YoeA acts as the major exporter for plipastatin. Co-overexpression of lcfA and yoeA improved plipastatin production to 1890 mg/mL, which was further elevated to 2060 mg/mL after abrB gene deletion. Lastly, the use of optimized culture medium achieved 2514 mg/mL plipastatin production, which was 5.26-fold higher than that of the initial strain. These results suggest that multiple strain engineering is an effective strategy for increasing lipopeptide production, and identification of the novel transport efflux protein YoeA provides new insights into the regulation and industrial application of plipastatin.

Antiviral alkaloids produced by the mangrove-derived fungus Cladosporium sp. PJX-41.

Six new indole alkaloids including five new glyantrypine derivatives (1, 2a, 2b, 3, 4) and a new pyrazinoquinazoline derivative (5), together with eight known alkaloids (6-13), were isolated from the culture of the mangrove-derived fungus Cladosporium sp. PJX-41. Their structures were elucidated primarily by spectroscopic and physical data. The absolute configurations of compounds 1-9 were established on the basis of CD, NOESY data, and single-crystal X-ray diffraction analysis. Compounds 2b, 5, 7-9, and 11 exhibited significant activities against influenza virus A (H1N1), with IC50 values of 82-89 µM.

Identification and Characterization of a Novel Carboxylesterase Belonging to Family VIII with Promiscuous Acyltransferase Activity Toward Cyanidin-3-O-Glucoside from a Soil Metagenomic Library.

An alkaline esterase, designated as EstXT1, was identified through functional screening from a metagenomic library. Sequence analysis revealed that EstXT1 belonged to the family VIII carboxylesterases and contained a characteristic conserved S-x-x-K motif and a deduced catalytic triad Ser56-Lys59-Tyr165. EstXT1 exhibited the strongest activity toward methyl ferulate at pH 8.0 and temperature 55°C and retained over 80% of its original activity after incubation in the pH range of 7.0-10.6 buffers. Biochemical characterization of the recombinant enzyme showed that it was activated by Zn2+ and Co2+ metal ion, while inhibited by Cu2+ and CTAB. EstXT1 exhibited significant promiscuous acyltransferase activity preferred to the acylation of benzyl alcohol acceptor using short-chain pNP-esters (C2-C8) as acyl-donors. A structure-function analysis indicated that a WAG motif is essential to acyltransferase activity. This is the first report example that WAG motif plays a pivotal role in acyltransferase activity in family VIII carboxylesterases beside WGG motif. Further experiment indicated that EstXT1 successfully acylated cyanidin-3-O-glucoside in aqueous solution. The results from the current investigation provided new insights for the family VIII carboxylesterase and lay a foundation for the potential applications of EstXT1 in food and biotechnology fields.

Engineering Modular and Highly Sensitive Cell-Based Biosensors for Aromatic Contaminant Monitoring and High-Throughput Enzyme Screening.

Although a variety of whole-cell-based biosensors have been developed for different applications in recent years, most cannot meet practical requirements due to insufficient sensing performance. Here, we constructed two sets of modular genetic circuits by serial and parallel modes capable of significantly amplifying the input/output signal in Escherichia coli. The biosensors are engineered using σ54-dependent phenol-responsive regulator DmpR as a sensor and enhanced green fluorescent protein as a reporter. Cells harboring serial and parallel genetic circuits displayed nearly 9- and 16-fold higher sensitivity than the general circuit. The genetic circuits enabled rapid detection of six phenolic contaminants in 12 h and showed the low limit of detection of 2.5 and 2.2 ppb for benzopyrene (BaP) and tetracycline (Tet), with a broad detection range of 0.01-1 and 0.005-5 µM, respectively. Furthermore, the positive rate was as high as 73% when the biosensor was applied to screen intracellular enzymes with ester-hydrolysis activity from soil metagenomic libraries using phenyl acetate as a phenolic substrate. Several novel enzymes were isolated, identified, and biochemically characterized, including serine peptidases and alkaline phosphatase family protein/metalloenzyme. Consequently, this study provides a new signal amplification method for cell-based biosensors that can be widely applied to environmental contaminant assessment and screening of intracellular enzymes.

Optimizing extraction conditions and isolation of bound phenolic compounds from corn silk (Stigma maydis) and their antioxidant effects.

During the processing of maize, Stigma maydis, also known as corn silk, is normally discarded as waste. Phytochemical research was carried out on the S. maydis to use it as a valuable source of bioactive components. This research aimed to maximize the recovery of free and bound phenolic compounds from corn silk under optimal experimental conditions. Response surface design was operated to optimize the alkaline hydrolysis extraction of bound phytochemicals from corn silk based on total phenolic content and DPPH radical scavenging activity. The optimum conditions (i.e., NaOH concentration 2 M, digestion time 135 min, digestion temperature of 37.5°C, the solid-to-solvent ratio of 1:17.5, and acetone) were obtained. The optimum parameters were used to extract the corn silk. The structures of two compounds isolated from ethyl acetate extracts were then identified as friedelin (1) and (E)-4-(4-hydroxy-3-methoxyphenyl) but-3-en-2-one (2). The DPPH, H2 O2 , and ABTS % inhibition of the compounds is as follows: compound (1) 74.81%, 76.8%, 70.33% and compound (2) 70.37%, 56.70% and 57.46%, respectively. The current study has opened previously unexplored perspectives of the composition of bound compounds in corn silk and established the foundations for more effective processing and utilization of corn waste. PRACTICAL APPLICATION: Bound phenolic compounds from corn silk under optimal experimental conditions were obtained. Corn silk can be utilized as a type of medicinal herb as well as a source of inexpensive natural antioxidants.

Improving the activity and expression level of a phthalate-degrading enzyme by a combination of mutagenesis strategies and strong promoter replacement.

Phthalic acid esters (PAEs) are widely used plasticizers found in consumer products, which enter the environment and pose severe threats to human health. Here, a new PAE-degrading enzyme EstJ6 was modified by combining mutagenesis strategies and a strong promoter replacement to improve its catalytic activity and expression level. Four mutants with enhanced activity were obtained by random mutation, among which EstJ6M1.1 exhibited the highest catalytic activity with an increase in catalytic activity by 2.9-fold toward dibutyl phthalate (DBP) than that of the wild-type (WT) enzyme. With these mutants as a template, a variant EstJ6M2 with 3.1-fold higher catalytic activity and 4.61 times higher catalytic efficiency (Kcat/Km) was identified by staggered extension PCR. Targeting four mutation sites of EstJ6M2, a variant EstJ6M3.1 was gained by site-directed saturation mutagenesis and displayed 4.3-fold higher activity and 5.97 times higher Kcat/Km than WT. The expression level of three mutants EstJ6M1.1, EstJ6M2, and EstJ6M3.1, as well as the WT, increased nearly threefold after a strong promoter replacement. These results provide a proof-theoretical basis and practicable pipeline for applying PAE-degrading enzymes.

BON domain-containing protein-mediated co-selection of antibiotic and heavy metal resistance in bacteria.

The widespread antibiotic resistance of bacteria has become one of the most severe threats to public health. However, the mechanisms that allow microbial acquisition of resistance are still poorly understood. In the present study, a novel BON domain-containing protein was heterologously expressed in Escherichia coli. It functions as an efflux pump-like to confer resistance to various antibiotics, especially for ceftazidime, with a >32-fold increase in minimum inhibitory concentration (MIC). The fluorescence spectroscopy experiment indicated that BON protein could interact with several metal ions, such as copper and silver, which has been associated with the induced co-regulation of antibiotic and heavy metal resistance in bacteria. Furthermore, the BON protein was demonstrated to spontaneously self-assemble into a trimer and generate a central pore-like architecture for antibiotic transporting. A WXG motif as a molecular switch is essential for forming the transmembrane oligomeric pores and controls the interaction between BON protein and cell membrane. Based on these findings, a mechanism termed "one-in, one-out", was proposed for the first time. The present study provides new insights into the structure and function of BON protein and a previously unidentified antibiotic resistance mechanism, filling the knowledge gap in understanding BON protein-mediated intrinsic antibiotic resistance.

Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method.

In the present study, a feruloyl esterase DLFae4 identified in our previous research was modified by error-prone PCR and site-directed saturation mutation to enhance the catalytic efficiency and acyltransferase activity further. Five mutants with 6.9-118.9% enhanced catalytic activity toward methyl ferulate (MFA) were characterized under the optimum conditions. Double variant DLFae4-m5 exhibited the highest hydrolytic activity (270.97 U/mg), the Km value decreased by 83.91%, and the Kcat/Km value increased by 6.08-fold toward MFA. Molecular docking indicated that a complex hydrogen bond network in DLFae4-m5 was formed, with four of five bond lengths being shortened compared with DLFae4, which might account for the increase in catalytic activity. Acyl transfer activity assay revealed that the activity of DLFae4 was as high as 1550.796 U/mg and enhanced by 375.49% (5823.172 U/mg) toward 4-nitrophenyl acetate when residue Ala-341 was mutated to glycine (A341G), and the corresponding acyl transfer efficiency was increased by 7.7 times, representing the highest acyltransferase activity to date, and demonstrating that the WGG motif was pivotal for the acyltransferase activity in family VIII carboxylesterases. Further experiments indicated that DLFae4 and variant DLFae4 (A341G) could acylate cyanidin-3-O-glucoside effectively in aqueous solution. Taken together, our study suggested the effectiveness of error-prone PCR and site-directed saturation mutation to increase the specific activity of enzymes and may facilitate the practical application of this critical feruloyl esterase.

Research on the Regulation of Plipastatin Production by the Quorum-Sensing ComQXPA System of Bacillus amyloliquefaciens.

Plipastatin is a cyclic lipopeptide synthesized by non-ribosomal peptide synthetases (NRPS), which has a diverse range of applications in postharvest preservation of fruits and vegetables, biological control, and feed processing. Whereas the yield of plipastatin in wild Bacillus sp. is low, its chemical structure is complex and challenging to synthesize, significantly limiting its production and application. ComQXPA-PsrfA, a quorum-sensing (QS) circuit from Bacillus amyloliquefaciens, was constructed in this study. Two QS promoters MuPsrfA and MtPsrfA, with 35 and 100% increased activity, respectively, were obtained by mutating the original promoter PsrfA. Thus, the natural promoter of plipastatin was replaced by a QS promoter to achieve the dynamic regulation of plipastatin, which increased the yield of plipastatin by 3.5 times. Integrating ComQXPA into plipastatin mono-producing M-24:MtPsrfA increased the yield of plipastatin to 3850 mg/L, representing the highest yield reported to date. Four new plipastatins were identified via UPLC-ESI-MS/MS and GC-MS analysis of fermentation products of mono-producing engineered strains. Among them, three plipastatins contained two double bonds in the fatty acid side chain, representing the first example of a new type of plipastatin. Our results indicate that the QS system ComQXPA-PsrfA of Bacillus can dynamically regulate plipastatin production, and the pipeline could be extended to the other strains to regulate target products dynamically.

Improving Degradation of Polycyclic Aromatic Hydrocarbons by Bacillus atrophaeus Laccase Fused with Vitreoscilla Hemoglobin and a Novel Strong Promoter Replacement.

Laccases catalyze a variety of electron-rich substrates by reducing O2 to H2O, with O2 playing a vital role as the final electron acceptor in the reaction process. In the present study, a laccase gene, lach5, was identified from Bacillus atrophaeus through sequence-based screening. LacH5 was engineered for modification by fusion expression and promoter replacement. Results showed that the purified enzyme LacH5 exhibited strong oxidative activity towards 2,2'-azinobis(3-ehtylbenzothiazolin-6-sulfnic acid) ammonium salt (ABTS) under optimum pH and temperature conditions (pH 5.0, 60 °C) and displayed remarkable thermostability. The activity of the two fusion enzymes was enhanced significantly from 14.2 U/mg (LacH5) to 22.5 U/mg (LacH5-vgb) and 18.6 U/mg (Vgb-lacH5) toward ABTS after LacH5 fusing with Vitreoscilla hemoglobin (VHb). Three of six tested polycyclic aromatic hydrocarbons (PAHs) were significantly oxidized by two fusion laccases as compared with LacH5. More importantly, the expression level of LacH5 and fusion protein LacH5-vgb was augmented by 3.7-fold and 7.0-fold, respectively, by using a novel strong promoter replacement. The results from the current investigation provide new insights and strategies for improving the activity and expression level of bacterial laccases, and these strategies can be extended to other laccases and multicopper oxidases.