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Plant roots assemble unique microbial communities in rhizosphere, which are critical for plant adapting to natural environment. Given the pivotal importance of plant-microbe interactions, this study was conducted to uncover the assembly of Artemisia annua on root-associated bacterial and fungal communities and their co-occurrence networks. Soil samples were collected from a field experiment with 7-year plantation of Artemisia annua, including unplanted, bulk and rhizosphere soil. The microbial communities were investigated by amplicon sequencing targeting bacteria and fungi. The soil microbiomes were highly diverse among the three treatments. Bacterial and fungal communities were significantly influenced by AP (available phosphorus), AK (available potassium), TOC (total organic carbon), TN (total nitrogen) and WSN (water soluble nitrogen). Two plant growth-promoting bacteria, Sphingomonas and Sphingobium, and the fungal ASVs defined as Saprotroph were dramatically enriched in rhizosphere. Network analysis revealed that Artemisia annua built the less complex root-associated microbial network, compared to unplanted and bulk soils. Specially, the percentage of inter-kingdom interactions between bacteria and fungi increased in rhizosphere network, and showed the highest proportion of negative relationship. These results indicate that A. annua could assemble the specific root-associated microbial communities with increased abundance of plant growth promoting microorganisms and build inter-kingdom co-occurrence networks, which may be beneficial for the fitness of plants to natural environment.

The polyethylene terephthalate hydrolase (PETase) has been proved to have a high activity to degrade polyethylene terephthalate (PET), but few studies have been carried on its secretion in Bacillus subtilis. In this study, the coding gene of PETase, which was isolated from the Ideonella sakaiensis, was synthesized and expressed in B. subtilis. Then, we evaluated the ability of five Bacillus signal peptides to enhance PETase secretion by B. subtilis. The results indicated that the SPamy -induced secretion of PETase was the highest, and its activity against p-Nitrophenyl palmitate was about fourfold that of the natural signal peptide SPPETase . The weak promoter P43 provided sufficient time for translation and folding of PETase, resulting in increased extracellular expression. Use of P43 and SPamy in combination yielded the greatest bis-(2-hydroxyethyl) terephthalate degradation and PET-film etching activity due to maximized secretion of PETase by B. subtilis. Our findings will facilitate biodegradation of PET plastic. SIGNIFICANCE AND IMPACT OF THE STUDY: High-level expression of polyethylene terephthalate hydrolase (PETase) facilitates biodegradation of PET. In this study, the expression elements, signal peptide and promoter, in the secretory expression system, were optimizing for maximizing secreted expression of PETase in Bacillus subtilis. The constructed strains yielded the greatest bis-(2-hydroxyethyl) terephthalate degradation and PET-film etching activities.

Although the plant-growth promotion by algae have been studied comprehensively, their impacts on indigenous soil microbiome remain largely unexplored. Herein we conducted a greenhouse experiment to investigate the changes in soil properties and corresponding microbial communities (bacterial, fungal and protists) after 2-year application of algae and their dynamic variation within 60 days immediately after algae addition. In comparison with Control treatment, the impact of algae on soil properties and microbial communities was huge, especially the content of nitrate was decreased however soluble organic nitrogen (SON) was increased. The increased copies of nifH gene suggested the improved potential of nitrogen fixation in algae treated soil. By constructing multitrophic ecological network, soil microorganisms were divided into several modules, and two key-stone microbial taxa (module 1 and 2) showed strong associations with the content of nitrate and SON. With addition of algae, the abundance of most microbial taxa was decreased and increased in module 1 and module 2, respectively. Particularly, module 1 and module 2 were proved to be taxonomically and functionally comprised of different microbes. Moreover, random forest analysis and structural equation model indicated that the key-stone microbial taxa were more important factors affecting the content of nitrate and SON than algae, bacterial, fungal and protistan communities and the influence of algae on soil nitrogen cycling mostly depended on their indirect effects via module 1 and 2.

Acetogenic bacteria are a diverse group of anaerobes that use the reductive acetyl coenzyme A (acetyl-CoA) (Wood-Ljungdahl) pathway for CO2 fixation and energy conservation. The conversion of 2 mol CO2 into acetyl-CoA by using the Wood-Ljungdahl pathway as the terminal electron accepting process is the most prominent metabolic feature for these microorganisms. However, here, we describe that the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. Genome analysis of Clostridium bovifaecis revealed that it contains almost the complete genes of the Wood-Ljungdahl pathway but lacks the gene encoding formate dehydrogenase, which catalyzes the reduction of CO2 to formate as the first step of the methyl branch of the Wood-Ljungdahl pathway. The lack of a gene encoding formate dehydrogenase was verified by PCR, reverse transcription-PCR analysis, enzyme activity assay, and its formate-dependent acetogenic utilization of glucose on DNA, RNA, protein, and phenotype level, respectively. The lack of a formate dehydrogenase gene may be associated with the adaption to a formate-rich intestinal environment, considering the isolating source of strain BXX. The formate-dependent acetogenic growth of Clostridium bovifaecis provides insight into a unique metabolic feature of fecal acetogens.IMPORTANCE The acetyl-CoA pathway is an ancient pathway of CO2 fixation, which converts 2 mol of CO2 into acetyl-CoA. Autotrophic growth with H2 and CO2 via the acetyl-CoA pathway as the terminal electron accepting process is the most unique feature of acetogenic bacteria. However, the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. The formate-dependent acetogenic growth of Clostridium bovifaecis was associated with its lack of a gene encoding formate dehydrogenase, which may result from adaption to a formate-rich intestinal environment. This study gave insight into a unique metabolic feature of fecal acetogens. Because of the requirement of formate for the acetogenic growth of certain acetogens, the ecological impact of acetogens could be more complex and important in the formate-rich environment due to their trophic interactions with other microbes.

Acetogenic bacteria are a diverse group of anaerobes that use the reductive acetyl coenzyme A (acetyl-CoA) (Wood-Ljungdahl) pathway for CO(2) fixation and energy conservation. The conversion of 2 mol CO(2) into acetyl-CoA by using the Wood-Ljungdahl pathway as the terminal electron accepting process is the most prominent metabolic feature for these microorganisms. However, here, we describe that the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. Genome analysis of Clostridium bovifaecis revealed that it contains almost the complete genes of the Wood-Ljungdahl pathway but lacks the gene encoding formate dehydrogenase, which catalyzes the reduction of CO(2) to formate as the first step of the methyl branch of the Wood-Ljungdahl pathway. The lack of a gene encoding formate dehydrogenase was verified by PCR, reverse transcription-PCR analysis, enzyme activity assay, and its formate-dependent acetogenic utilization of glucose on DNA, RNA, protein, and phenotype level, respectively. The lack of a formate dehydrogenase gene may be associated with the adaption to a formate-rich intestinal environment, considering the isolating source of strain BXX. The formate-dependent acetogenic growth of Clostridium bovifaecis provides insight into a unique metabolic feature of fecal acetogens. IMPORTANCE The acetyl-CoA pathway is an ancient pathway of CO(2) fixation, which converts 2 mol of CO(2) into acetyl-CoA. Autotrophic growth with H(2) and CO(2) via the acetyl-CoA pathway as the terminal electron accepting process is the most unique feature of acetogenic bacteria. However, the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. The formate-dependent acetogenic growth of Clostridium bovifaecis was associated with its lack of a gene encoding formate dehydrogenase, which may result from adaption to a formate-rich intestinal environment. This study gave insight into a unique metabolic feature of fecal acetogens. Because of the requirement of formate for the acetogenic growth of certain acetogens, the ecological impact of acetogens could be more complex and important in the formate-rich environment due to their trophic interactions with other microbes.

Pseudomonas putida JYR-1 was isolated from soil contaminated with industrial oil because of its ability to utilize trans-anethole as a carbon and energy source. The complete genome is 5.41 Mb with 4,834 protein-coding genes. Study of this isolate will provide insight into biotransformation pathways for phenylpropanoids.

In this paper, the construction of 3D model structure of xylose dehydrogenase (XDH) by using homology modeling to guide the rational design of the enzyme for improving thermostability was reported. Three XDH mutants of NA-1 (+249L), NA-2 (G149P) and NA-3 (+249L/G149P) were designed and displayed on the surface of bacteria. Among them, bacteria displaying NA-1 (NA-1-bacteria) exhibited superior thermostability without compromising its activity and substrate specificity in comparison with its wild-type counterpart. NA-1-bacteria retained its original activity after incubation at room temperature for one month with the half-life of 9.8 days at 40 degrees C. Finally, the NA-1-bacteria were applied to construct xylose/O-2 based biofuel cell with good performance including enhanced operational stability. Thus, the approach described here could be explored for engineering of other enzymes for improving certain characters without three-dimensional structure identified by experimental methods. (C) 2015 Elsevier Inc. All rights reserved.

The genus Methylocystis belongs to the class Alphaproteobacteria, the family Methylocystaceae, and encompasses aerobic methanotrophic bacteria with the serine pathway of carbon assimilation. All Methylocystis species are able to fix dinitrogen and several members of this genus are also capable of using acetate or ethanol in the absence of methane, which explains their wide distribution in various habitats. One additional trait that enables their survival in the environment is possession of two methane-oxidizing isozymes, the conventional particulate methane monooxygenase (pMMO) with low-affinity to substrate (pMMO1) and the high-affinity enzyme (pMMO2). Here, we report the finished genome sequence of Methylocystis bryophila S285, a pMMO2-possessing methanotroph from a Sphagnum-dominated wetland, and compare it to the genome of Methylocystis sp. strain SC2, which is the first methanotroph with confirmed high-affinity methane oxidation potential. The complete genome of Methylocystis bryophila S285 consists of a 4.53 Mb chromosome and one plasmid, 175 kb in size. The genome encodes two types of particulate MMO (pMMO1 and pMMO2), soluble MMO and, in addition, contains a pxmABC-like gene cluster similar to that present in some gammaproteobacterial methanotrophs. The full set of genes related to the serine pathway, the tricarboxylic acid cycle as well as the ethylmalonyl-CoA pathway is present. In contrast to most described methanotrophs including Methylocystis sp. strain SC2, two different types of nitrogenases, that is, molybdenum–iron and vanadium–iron types, are encoded in the genome of strain S285. This unique combination of genome-based traits makes Methylocystis bryophila well adapted to the fluctuation of carbon and nitrogen sources in wetlands.

The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4- vinylcatechol, and 4-vinylguaiacol, respectively. Among var- ious ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic ac- tivity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/ V151I) showed approximately 34-fold higher catalytic activ- ity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to for- mation of compact active site compared with that of the wild- typeFADase.Consideringtheamountofphenoliccompounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

In this paper, glutamate dehydrogenase (Gldh) is reported to efficiently display on Escherichia coli cell surface by using N-terminal region of ice the nucleation protein as an anchoring motif. The presence of Gldh was confirmed by SDS-PAGE and enzyme activity assay. Gldh was detected mainly in the outer membrane fraction, suggesting that the Gldh was displayed on the bacterial cell surface. The optimal temperature and pH for the bacteria cell-surface displayed Gldh (bacteria-Gldh) were 70°C and 9.0, respectively. Additionally, the fusion protein retained almost 100% of its initial enzymatic activity after 1 month incubation at 4°C. Transition metal ions could inhibit the enzyme activity to different extents, while common anions had little adverse effect on enzyme activity. Importantly, the displayed Gldh is most specific to l-glutamate reported so far. The bacterial Gldh was enabled to catalyze oxidization of l-glutamate with NADP(+) as cofactor, and the resultant NADPH can be detected spectrometrically at 340nm. The bacterial-Gldh based l-glutamate assay was established, where the absorbance at 340nm increased linearly with the increasing l-glutamate concentration within the range of 10-400μM. Further, the proposed approach was successfully applied to measure l-glutamate in real samples.

Soil microorganisms have to rapidly respond to salt-induced osmotic stress. Type II methanotrophs of the genus Methylocystis are widely distributed in upland soils but are known to have a low salt tolerance. Here, we tested the ability of Methylocystis sp. strain SC2 to adapt to increased salinity. When exposed to 0.75% NaCl, methane oxidation was completely inhibited for 2.25 h and fully recovered within 6 h. Growth was inhibited for 23.5 h and then fully recovered. Its transcriptome was profiled after 0 min (control), 45 min (early response), and 14 h (late response) of stress exposure. Physiological and transcriptomic stress responses corresponded well. Salt stress induced the differential expression of 301 genes, with sigma factor σ 32 being a major controller of the transcriptional stress response. The transcript levels of nearly all the genes involved in oxidizing CH 4 to CO 2 remained unaffected, while gene expression involved in energy-yielding reactions ( nuoA-N ) recovered concomitantly with methane oxidation from salt stress shock. Glutamate acted as an osmoprotectant. Its accumulation in late stress response corresponded to increased production of glutamate dehydrogenase 1. Chromosomal genes whose products (stress-induced protein, DNA-binding protein from starved cells, and CsbD family protein) are known to confer stress tolerance showed increased expression. On plasmid pBSC2-1, genes encoding type IV secretion system and single-strand DNA-binding protein were upregulated in late response, suggesting stress-induced activation of the plasmid-borne conjugation machinery. Collectively, our results show that Methylocystis sp. strain SC2 is able to adapt to salt stress, but only within a narrow range of salinities. IMPORTANCE Besides the oxic interface of methanogenic environments, Methylocystis spp. are widely distributed in upland soils, where they may contribute to the oxidation of atmospheric methane. However, little is known about their ability to cope with changes in soil salinity. Growth and methane oxidation of Methylocystis sp. strain SC2 were not affected by the presence of 0.5% NaCl, while 1% NaCl completely inhibited its activity. This places strain SC2 into the low-salt-tolerance range reported for other Methylocystis species. Our results show that, albeit in a narrow range, strain SC2 is able to respond and adapt to salinity changes. It possesses various stress response mechanisms, which allow resumption of growth within 24 h when exposed to 0.75% NaCl. Presumably, these mechanisms allow Methylocystis spp., such as strain SC2, to thrive in upland soils and to adapt to certain fluctuations in soil salinity.

Phenylpropanoids are common aromatic compounds synthesized by plants that are often used as starting compounds for the production of various flavor and fragrance compounds. The use of bacterial metabolism as a means to produce valueadded compounds from natural resources has been given much attention as an alternative method to replace conventional chemical syntheses. This review describes bacterial metabolisms of the phenylpropanoid compounds trans-anethole, isoeugenol, and isosafrole to better understand efficient production of natural fragrance and other value-added compounds.

The cytochrome P450 enzymes (CYPs) CYP-sb21 from Sebekia benihana and CYP-pa1 from Pseudonocardia autotrophica are able to hydroxylate the immunosuppressant cyclosporin A (CsA) in a regioselective manner, giving rise to the production of two hair-stimulating agents (with dramatically attenuated immunosuppressant activity), γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH) and γ-hydroxy-N-methyl-l-Leu9-CsA (CsA-9-OH). Recently, the in vitro activity of CYP-sb21 was identified using several surrogate redox partner proteins. Herein, we reconstituted the in vitro activity of CYP-pa1 for the first time via a similar strategy. Moreover, the supporting activities of a set of ferredoxin (Fdx)/ferredoxin reductase (FdR) pairs from the cyanobacterium Synechococcus elongatus PCC 7942 were comparatively analyzed to identify the optimal redox systems for these two CsA hydroxylases. The results suggest the great value of cyanobacterial redox partner proteins for both academic research and industrial application of P450 biocatalysts.

α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1' residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1' site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.

For the highly nonlinear, strong coupling and underactuated, an effective controller for quadrotor unmanned aerial vehicle (UAV) is not easy to obtain, especially in conditions of the parameters variations and external disturbance. In this paper, an adaptive inverse model control method (AIMCM) is proposed for this plant. Two BP networks are used in control system to achieve the model identification and control. Both offline trained and online learned are used to ensure the fast learning and the robustness. The convergence of the learning algorithm is proved based on Lyapunov function. At last, a quadrotor UAV control simulation based on the full model shows the superiority and robustness of the control system.

This paper investigates the problem of the position and attitude control of quad-rotor UAV. A new simplified model which is more reasonable for some actual cases is derived. Based on the simplified model, a T-S fuzzy state feedback controller to stabilize the attitude of quad-rotor UAV is designed. Proposed control strategy can steer quad-rotor UAV flight with a desired acceleration. In addition, it can guarantee the assumption for simplifying quad-rotor model is reasonable during the flight. Finally, numerical simulations are provided to illustrate the effectiveness of the obtained theoretical results.

The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

A novel electrochemical microbial biosensor for the rapid monitoring of p-nitrophenyl-substituted organophosphates (OPs) compounds based on glass carbon electrode (GCE) modified with both ordered mesopore carbons (OMCs) and cell surface-expressed organophosphorus hydrolase (OPH) (OPH-bacteria/ OMCs/GCE) was described in this paper. The genetically engineered Escherichia coli strain surface displayed mutant OPH (55) with improved enzyme activity and favorable stability was constructed using a newly identified N-terminal of ice nucleation protein as an anchoring motif, which can be used directly without further time-consuming enzyme-extraction and purification, thereafter greatly improved the stability of the enzyme. Compared to OPH-bacteria modified GCE (OPH-bacteria/GCE), the OPH-bacteria/OMCs/GCE not only significantly enhanced the current response but also reduced the oxidation overpotential towards oxidizable p-nitrophenol (p-NP), which was the hydrolysate of p-nitrophenyl-substituted OPs. Under the optimized experimental conditions, at +0.84 V (vs. SCE), the current-time curve was performed with varying OPs concentration. The current response was linear with paraoxon concentration within 0.05-25 mu M. Similarly, linear range of 0.05-25 mu M was found for parathion, and 0.08-30 mu M for methyl parathion. The low limits of detection were evaluated to be 9.0 nM for paraoxon, 10 nM for parathion and 15 nM for methyl parathion (S/N=3). Thus, a highly specific, sensitive and rapid microbial biosensor was established, which holds great promise for on-site detection of trace p-nitrophenyl-substituted OPs. (C) 2014 Elsevier B.V. All rights reserved.

The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

A novel flavoprotein monooxygenase, trans-anethole oxygenase (TAO), from Pseudomonas putida JYR-1, which is capable of catalyzing the oxidation of trans-anethole to p-anisaldehyde, was heterologously expressed in E. coli and purified. Enzymatic kinetics of diverse substrates and cofactors revealed that TAO is likely to be a novel self-sufficient flavoprotein monooxygenase. Enzyme assays of GST-TAO demonstrated that TAO catalyzed a trans-anethole oxidation reaction without auxiliary component enzyme-like electron-transfer flavin reductases. The single component TAO had the ability to reduce flavin cofactors and simultaneously oxidize trans-anthole to p-anisaldehyde. In the processes of reduction of flavin and oxidation of trans-anethole, TAO accepted various flavin and NAD(P)H cofactors. TAO also catalyzed oxidation of isoeugenol, O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure with different catalytic efficiency. TAO had the greatest catalytic efficiency (k(cat)/K-m) with the original substrate, trans-anethole. Investigation about partially deleted mutants of TAO indicated that reductase active sites appeared to be located near the N terminal. Site directed mutagenesis studies also proved that the proposed flavin binding sites, Trp-38, Thr-43, Tyr-55, were critical for flavin reduction. However, disruption of any portion of TAO eliminated the oxygenase activity.

A gene encoding p-anisaldehyde dehydrogenase (PAADH), which catalyzes the oxidation of p-anisaldehyde to p-anisic acid, was identified to be clustered with the trans-anethole oxygenase (tao) gene in Pseudomonas putida JYR-1. Heterologously expressed PAADH in Escherichia coli catalyzed the oxidation of vanillin, veratraldehyde, and piperonal to the corresponding aromatic acids vanillic acid, veratric acid, and piperonylic acid, respectively. Coexpression of trans-anethole oxygenase (TAO) and PAADH in E. coli also resulted in the successful transformation of trans-anethole, isoeugenol, O-methyl isoeugenol, and isosafrole to p-anisic acid, vanillic acid, veratric acid, and piperonylic acid, respectively, which are compounds found in plants as secondary metabolites. Because of the relaxed substrate specificity and high transformation rates by coexpressed TAO and PAADH in E. coli , the engineered strain has potential to be applied in the fragrance industry.

A plasmid, pTA163, in Escherichia coli contained an approximately 34-kb gene fragment from Pseudomonas putida JYR-1 that included the genes responsible for the metabolism of trans -anethole to protocatechuic acid. Three Tn 5 -disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyze trans -anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter in E. coli catalyzed oxidative cleavage of a propenyl group of trans -anethole to an aldehyde group, resulting in the production of para -anisaldehyde, and this gene was designated tao ( t rans - a nethole o xygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein of Agrobacterium vitis S4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water into p -anisaldehyde using 18 O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase from Pseudomonas putida IE27 and Pseudomonas nitroreducens Jin1, TAO from P. putida JYR-1 catalyzed isoeugenol, O -methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts of E. coli (pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

Due to the non-bioactivity and poor conjunction performance of present cartilage prostheses, the main work here is to develop the bioactive glass-polyvinyl alcohol hydrogel articular cartilage/bone (BG-PVA/bone) composite implants. The essential criterion for a biomaterial to bond with living bone is well-matched mechanical properties as well as biocompatibility and bioactivity. In vitro studies on the formation of a surface layer of carbonate hydroxyl apatite (HCA) and the corresponding variation of the properties of biomaterials are imperative for their clinical application. In this paper, the mineralization behavior and variation of the interface properties of BG-PVA/bone composites were studied in vitro by using simulated body fluid (SBF). The mineralization and HCA layer formed on the interface between the BG-PVA hydrogel and bone in SBF could provide the composites with bioactivity and firmer combination. The compression property, shear strength and interface morphology of BG-PVA/bone composite implants varying with the immersion time in SBF were characterized. Also, the influence laws of the immersion time, content of BG in the composites and aperture of bones to the mineralization behavior and interface properties were investigated. The good mineralization behavior and enhanced conjunction performance of BG-PVA/bone composites demonstrated that this kind of composite implant might be more appropriate cartilage replacements.