Study on extraction and characterization of new antibiotics violacein from engineered Escherichia coli VioABCDE-SD.

To better understand the characteristic properties of violacein biosynthesized by engineered Escherichia coli VioABCDE-SD, a convenient and simplified method was designed to extract violacein and its stability, antimicrobial activity, and antioxidant capacity were analyzed. Different from the traditional extraction methods, our new method is easier and less time consuming and can directly obtain violacein dry powder product with a higher extraction rate. Low temperature, dark condition, neutral pH, reducing agents, Ba2+ , Mn2+ , Ni2+ , Co2+ , and some food additives of sucrose, xylose, and glucose were conducive to maintaining its stability. The violacein also exhibited surprisingly high bacteriostatic action against Gram-positive Bacillus subtilis, Deinococcus radiodurans R1, and Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa, but no effect on E. coli. The violacein of VioABCDE-SD exhibited strong antioxidant activity, with the scavenging rate of 1,1-diphenyl-2-picrylhydrazyl free radicals reaching 60.33%, the scavenging efficiency of hydroxyl radical scavenging reaching 56.34%, and the total antioxidant capacity reaching 0.63 U/mL. Violacein from VioABCDE-SD can be synthesized directionally with better stability, antibacterial, and antioxidant properties compared with that from the original strain Janthinobacterium sp. B9-8. Therefore, our study indicated that violacein from engineered E. coli VioABCDE-SD was a kind of new antibiotic with potential biological activities, which may have potential utility in multiple areas such as pharmacological, cosmetics, and healthy food industries.

Functional characterization of a novel violacein biosynthesis operon from Janthinobacterium sp. B9-8.

Violacein is a secondary metabolite mainly produced by Gram-negative bacteria that is formed from tryptophan by five enzymes encoded by a single operon. It is a broad-spectrum antibacterial pigment with various important biological activities such as anti-tumor, antiviral, and antioxidative effects. The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8. The key enzyme-encoding genes were cloned to construct the multigene coexpression plasmids pET-vioAB and pRSF-vioCDE. The violacein biosynthesis pathway was heterologously introduced into engineered Escherichia coli VioABCDE and VioABCDE-SD. The factors affecting violacein production, including temperature, pH, inoculum size, carbon and nitrogen source, precursor, and inducers were investigated. The violacein titer of VioABCDE-SD reached 107 mg/L in a two-stage fermentation process, representing a 454.4% increase over the original strain. The violacein operon from B9-8 provides a new microbial gene source for the analysis of the violacein synthesis mechanism, and the constructed engineering E. coli strains lay a foundation for the efficient and rapid synthesis of other natural products.Key points• The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8.• The violacein synthesis pathway was reconstructed in E. coli using two compatible plasmids.• A two-stage fermentation process was optimized for improved violacein accumulation.

Pathway engineering of Saccharomyces cerevisiae for efficient lycopene production.

To construct a Saccharomyces cerevisiae strain for efficient lycopene production, we used a pathway engineering strategy based on expression modules comprising fusion proteins and a strong constitutive promoter. The two recombinant plasmids pEBI encoding the fusion genes with an inducible promoter, as well as pIETB with a constitutive promoter and terminator were introduced into S. cerevisiae YPH499 and BY4741 to obtain the four recombinant strains ypEBI, ypIETB, byEBI and byIETB. The lycopene production and the transcription levels of key genes were higher in the BY4741 chassis than in YPH499. Accordingly, the content of total and unsaturated fatty acids was also higher in BY4741, which also exhibited a decrease of glucose, increase of trehalose, increase of metabolite in citrate cycle, and low levels of amino acids. These changes rerouted metabolic fluxes toward lycopene synthesis, indicating that the BY4741 chassis was more suitable for lycopene synthesis. The lycopene content of bpIETB in SG-Leu medium supplemented with 100 mg/L of linolenic acid reached 10.12 mg/g dry cell weight (DCW), which was 85.7% higher than without the addition of unsaturated fatty acids. The constitutive promoter expression strategy employed in this study achieved efficient lycopene synthesis in S. cerevisiae, and the strain bpIETB was obtained a suitable chassis host for lycopene production, which provides a basis for further optimization of lycopene production in artificial synthetic cells and a reference for the multi-enzyme synthesis of other similar complex terpenoids.

Design and tailoring of an artificial DNA scaffolding system for efficient lycopene synthesis using zinc-finger-guided assembly.

A highly efficient lycopene production system was constructed by assembling enzymes fused to zinc-finger motifs on DNA scaffolds in vitro and in vivo. Three key enzymes of the lycopene synthesis pathway, geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, were fused with zinc-finger proteins, expressed and purified. Recombinant plasmids of the pS series containing DNA scaffolds that the zinc-finger proteins can specifically bind to were constructed. In the in vitro system, the production efficiency of lycopene was improved greatly after the addition of the scaffold plasmid pS231. Subsequently, the plasmid pET-AEBI was constructed and introduced into recombinant Escherichia coli BL21 (DE3) for expression, together with plasmids of the pS series. The lycopene production rate and content of the recombinant strain pp231 were higher than that of all strains carrying the DNA scaffold and the control. With the addition of cofactors and substrates in the lycopene biosynthesis pathway, the lycopene yield of pp231 reached 632.49 mg/L at 40 h, representing a 4.7-fold increase compared to the original recombinant strain pA1A3. This DNA scaffold system can be used as a platform for the construction and production of many biochemicals synthesized via multi-enzyme cascade reactions.

Cemental Tears: A Report of Four Cases and Literature Review.

PURPOSE: To present four different cases with a diagnosed cemental tear. The differences in aetiology, clinical diagnosis and treatment of cemental tears are described and discussed in order to provide guidance for clinical practice. CASES: Four patients of different ages presented with gingival swelling and other different complaints. Clinically, localised deep periodontal pockets and inflammation were noted on affected aspects in four cases. Radiographic examination revealed a prickly or flakey structure and bone loss on the affected side of the involved tooth. Different treatments, such as extraction, traditional periodontal initial therapy, periodontal flap surgery, or no clinical intervention were given based on different extents of tooth mobility and bone loss. In the first two cases, teeth with cemental tear were extracted due to poor prognosis. In case 3, deep pockets recovered to a normal condition, while cemental tears on the distal aspect of tooth 21 had no abnormal sensation or clinical symptoms. In case 4, a periapical radiograph showed distal bone loss was interrupted, and the tooth also recovered normal mobility. Histopathological evaluation of the specimens with H&E staining all resulted in a definitive diagnosis of cemental/cementodentinal tears. CONCLUSIONS: Cementodentinal or cemental tears are unique, localized, tooth-related factors associated with attachment loss. Aging, trauma and occlusal overload are the main aetiological factors. Early diagnosis and appropriate treatment will avoid unnecessary tooth extraction and result in a better prognosis. Detailed clinical and radiographic examinations as well as explorative surgery may help to make a diagnosis of cemental tears, but histopathological analysis is the only method for a definitive diagnosis.

A lipase with broad solvent stability from Burkholderia cepacia RQ3: isolation, characteristics and application for chiral resolution of 1-phenylethanol.

Using both polar and low polar organic solvents (DMSO and toluene) as screening stress, a solvent-stable bacterium Burkholderia cepacia RQ3 was newly isolated. An organic solvent-stable lipase from strain RQ3 was purified in a single step with 50.1% recovery by hydrophobic chromatography. The purified lipase was homogenous on SDS-PAGE and had an apparent molecular mass of 33 kDa. The gene of lipase RQ3 with an open reading frame of 1095 bp encoding 364-amino acid residues was cloned. The optimal pH and temperature for lipase activity were 9.0 and 40 °C. The lipase was stable in a wide pH range of 6.0-10.0 and at temperature below 50 °C. Strikingly, all the tested hydrophilic and hydrophobic organic solvents significantly extended the half-life of lipase RQ3 compared with that in a solvent-free system, which indicated that lipase RQ3 showed a broad solvent tolerance to various organic solvents. The lipase demonstrated excellent enantioselective transesterification toward the (S)-1-phenylethanol with a theoretical conversion yield of 50% and ee p of 99.9%, which made it an exploitable biocatalyst for organic synthesis and pharmaceutical industries.

Recent Advances on Key Enzymes of Microbial Origin in the Lycopene Biosynthesis Pathway.

Lycopene is a common carotenoid found mainly in ripe red fruits and vegetables that is widely used in the food industry due to its characteristic color and health benefits. Microbial synthesis of lycopene is gradually replacing the traditional methods of plant extraction and chemical synthesis as a more economical and productive manufacturing strategy. The biosynthesis of lycopene is a typical multienzyme cascade reaction, and it is important to understand the characteristics of each key enzyme involved and how they are regulated. In this paper, the catalytic characteristics of the key enzymes involved in the lycopene biosynthesis pathway and related studies are first discussed in detail. Then, the strategies applied to the key enzymes of lycopene synthesis, including fusion proteins, enzyme screening, combinatorial engineering, CRISPR/Cas9-based gene editing, DNA assembly, and scaffolding technologies are purposefully illustrated and compared in terms of both traditional and emerging multienzyme regulatory strategies. Finally, future developments and regulatory options for multienzyme synthesis of lycopene and similar secondary metabolites are also discussed.

Sustained and Microenvironment-Accelerated Release of Minocycline from Alginate Injectable Hydrogel for Bacteria-Infected Wound Healing.

During wound healing, bacterial infection is one of the main limiting factors for the desired efficiency. Wound dressing-mediated antibiotics therapies could overcome this problem to a great extent due to sustained drug release and controllable dose. Here, we designed a kind of alginate injectable hydrogel loaded with minocycline (SA@MC) as a dressing for staphylococcus aureus-infected wound healing. SA@MC hydrogel possessed good injectability and can be injected by syringes. MC participated in the gel formation, causing the microstructure change based on the morphology characterization. The element mapping and FT-IR spectra further confirmed the successful loading of MC in SA hydrogel. Interestingly, MC was released more efficiently in a weakly alkaline condition (pH 7-8) than in a weakly acidic condition (pH 4-6) from SA@MC injectable hydrogel, which means that there is an accelerated release to respond to the weakly alkaline wound microenvironment. Meanwhile, SA@MC injectable hydrogel had high biocompatibility and excellent antibacterial activity due to the sustained release of MC. Further, in vivo experiment results demonstrated that SA@MC injectable hydrogel promoted staphylococcus aureus-infected wound healing efficiently. In summary, the injectable composite hydrogel can serve as an ideal dressing to prevent bacterial infection and promote wound healing.

Draft genome sequence of a new carotenoid-producing strain Brevibacterium sp. XU54, isolated from radioactive soil in Xinjiang, China.

Some species of the genus Brevibacterium are orange bacteria involved in cheese ripening, synthesis of odoriferous compounds, and carotenoids with aromatic end groups. Here, we report the genome sequence of Brevibacterium sp. XU54, isolated from radioactive soil in Xinjiang, China. The genome of XU54 consists of 4,899,099 base pairs with a GC content of 62.2%. The genome sequence was annotated with 4453 genes, encoding 4260 proteins, 13 rRNAs, and 49 tRNAs. 16S rRNA BLAST and comparative genomic analysis both indicated that XU54 may be a new species of Brevibacterium. In addition, compared to the type strains, some enzymes related to sulfur metabolism showed a low similarity of 66.85, 79.53 and 14.61%, respectively. The carotenoids biosynthesis gene cluster was identified and analyzed according to the genomic data, which revealed relatively low identity (5-85%) with existing strains. The optimum conditions for its growth and carotenoid production were then discussed. The whole-genome sequence of Brevibacterium sp. XU54 will be beneficial for utilizing these newly identified genes in carotenoid biosynthesis and regulation of sulfur metabolism pathway to promote the production of novel carotenoids and other structurally diverse compounds through combinatorial biosynthesis, which facilitates cheese ripening and coloration. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03366-1.

Analysis and expression of the carotenoid biosynthesis genes from Deinococcus wulumuqiensis R12 in engineered Escherichia coli.

Deinococcus wulumuqiensis R12 is a red-pigmented extremophilic microorganism with powerful antioxidant properties that was isolated from radiation-contaminated soil in Xinjiang Uyghur Autonomous Region of China. The key carotenoid biosynthesis genes, crtE, crtB and crtI, which are related to the cells' antioxidant defense, were identified in the sequenced genome of R12 and analyzed. In order to improve the carotenoid yield in engineered Escherichia coli, the origin of carotenoid biosynthesis genes was discussed, and a strain containing the R12 carotenoid biosynthesis genes was constructed to produce lycopene, an important intermediate in carotenoid metabolism. The gene order and fermentation conditions, including the culture medium, temperature, and light, were optimized to obtain a genetically engineered strain with a high lycopene production capacity. The highest lycopene content was 688 mg L-1 in strain IEB, which corresponds to a 2.2-fold improvement over the original recombinant strain EBI.

Hydroxyapatite nanowires modified polylactic acid membrane plays barrier/osteoinduction dual roles and promotes bone regeneration in a rat mandible defect model.

Periodontitis is an inflammatory disease leading to tooth loss, alveolar bone absorption and disorder of masticatory function. Guided tissue regeneration (GTR) is one of the most common strategies for regeneration of lost periodontium. During surgical process, barrier membranes, and osteoinductive/osteoconductive materials should be placed, respectively, which may increase risks of infection, bleeding, and difficulty of operation. Here, we introduced a new kind of hydroxyapatite (HAp) nanowires modified polylactic acid (PLA) membrane to achieve barrier/osteoinduction dual functions. The physicochemical property measurements suggested the two sides of the composite membrane did not change after composition. Then a rat mandibular defect model was established to investigate barrier and osteoinductive effects of this composite membrane. After implantation, effects of functional cells engraftment and osteoinduction were detected by scanning electron microscope (SEM), histomorphometric measurement, immunohistochemical staining, and Micro-CT scanning. SEM images showed HAp side engrafted more cells than PLA side. The result of immunohistochemical staining suggested HAp/PLA promoted the expression of bone-related markers. Moreover, there were more newly formed bones with better quality in HAp/PLA group. Therefore, this composite membrane would be a promising biomaterial in tissue engineering for bone regeneration due to its barrier/osteoinduction dual functions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3099-3110, 2018.

Resveratrol Suppresses Rotenone-induced Neurotoxicity Through Activation of SIRT1/Akt1 Signaling Pathway.

Rotenone is a common pesticide and has been reported as one of the risk factors for Parkinson disease. Rotenone can cause neuronal death or apoptosis through inducing oxidative injury and inhibiting mitochondrial function. As a natural polyphenolic compound, resveratrol possesses the antioxidant capacity and neuroprotective effect. However, the mechanism underlying the neuroprotective effect of resveratrol against rotenone-induced neurotoxicity remains elusive. Here, we treated PC12 cells with rotenone to induce neurotoxicity, and the neurotoxic cells were subjected to resveratrol treatment. The CCK8 and LDH activity assays demonstrated that resveratrol could suppress neurotoxicity induced by rotenone (P < 0.01). The DCFH-DA assay indicated that resveratrol reduced the production of reactive oxygen species (ROS). JC-1 and Hoechst 33342/PI staining revealed that resveratrol attenuated mitochondrial dysfunction and cell apoptosis. Moreover, resveratrol reversed rotenone-induced decrease in SIRT1 expression and Akt1 phosphorylation (P < 0.05). Furthermore, when the SIRT1 and Akt1 activity was inhibited by niacinamide and LY294002, respectively, the neuroprotective effect of resveratrol was remarkably attenuated, which implied that SIRT1 and Akt1 could mediate this process and may be potential molecular targets for intervening rotenone-induced neurotoxicity. In summary, our study demonstrated that resveratrol reduced rotenone-induced oxidative damage, which was partly mediated through activation of the SIRT1/Akt1 signaling pathway. Our study launched a promising avenue for the potential application of resveratrol as a neuroprotective therapeutic agent in Parkinson disease. Anat Rec, 301:1115-1125, 2018. © 2018 Wiley Periodicals, Inc.

[Research progress in multi-enzyme regulation of genetically engineered bacteria producing lycopene].

Lycopene plays a crucial role in the biosynthesis pathway of 2-methyl-derythritol-4-phosphate (MEP) and mevalonic acid (MVA). It is a representative product of isoprenoid family, and a typical product of multi-enzyme catalytic reaction in organism. In this paper, we first introduced the general regulation methods in multi-enzyme synthesis reaction, including the construction of multi gene co-expression plasmid, gene order regulation, promoter and ribosome binding site regulation, gene knockout and replacement, aiming at the optimization strategies of multi-enzyme catalytic reaction in lycopene synthesis pathway. Meanwhile, we introduced several new regulation methods in multi-enzyme reaction, including multi-fragment assembly technology, artificial scaffold self-assembly methods and so on. At last, we summarized the application of these multi-enzyme regulation methods in lycopene synthesis. These methods provide a great inspiration and research foundation for the construction of lycopene-producing strains with high yield.