Recent progress in CRISPR-based bioengineering of microbial cell factories for important nutraceuticals synthesis.

Nutraceuticals are defined as food or food components with therapeutic capabilities that have few side effects and are regarded as a natural therapy for preventing the onset of several life-threatening illnesses. The use of microbial cell factories to produce nutraceuticals is considered to be sustainable and promising for meeting market demand. Among the diverse strategies for optimizing microbial cell factories, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system has emerged as a valuable tool for gene integration, deletion, activation, and downregulation. With the advent of multiplexed and precise CRISPR strategies, optimized microbial cell factories are revolutionizing the yield of nutraceuticals. This review focuses on the development of highly adaptable CRISPR strategies to optimize the production in microbial cell factories of some important nutraceuticals (belonging to the class of carotenoids, flavonoids, stilbenoids, polysaccharides, and nonprotein amino acids). Further, we highlighted current challenges related to the efficiency of CRISPR strategies and addressed potential future directions to fully harness CRISPR strategies to make nutraceutical synthesis in microbial cell factories an industrially favorable method.

Biocontrol Effect of Bacillus subtilis YPS-32 on Potato Common Scab and Its Complete Genome Sequence Analysis.

Potato common scab is caused by Streptomyces, which resides in soil and has become a serious disease in potato planting areas worldwide. In this study, we obtained a Bacillus subtilis YPS-32 strain by natural screening, and atmospheric and room-temperature plasma (ARTP) mutagenesis and field trial results showed that B. subtilis YPS-32 has a control efficacy of 83.70% against potato common scab. The complete genome of B. subtilis YPS-32 was sequenced, and multiple genes related to the synthesis of antibiotics and plant growth promoters were detected. Based on the genomic information for B. subtilis YPS-32, the sfp gene-inactivated (related to the synthesis of secondary metabolites) mutant strain B. subtilis YPS-32Δsfp was constructed. Analysis of crude extract metabolites using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) techniques revealed that strain YPS-32 encodes antagonists, such as surfactin and fengycin, which have antimicrobial effects. This study clarifies the mode of action by which B. subtilis YPS-32 antagonizes Streptomyces scabies and provides a reference for further research on antibacterial genes in the future.

Combination of Dichloroacetate and Atorvastatin Regulates Excessive Proliferation and Oxidative Stress in Pulmonary Arterial Hypertension Development via p38 Signaling.

Pulmonary arterial hypertension (PAH) is a lethal disease generally characterized by pulmonary artery remodeling. Mitochondrial metabolic disorders have been implicated as a critical regulator of excessively proliferative- and apoptosis-resistant phenotypes in pulmonary artery smooth muscle cells (PASMCs). Dichloroacetate (DCA) is an emerging drug that targets aerobic glycolysis in tumor cells. Atorvastatin (ATO) is widely used for hyperlipemia in various cardiovascular diseases. Considering that DCA and ATO regulate glucose and lipid metabolism, respectively, we hypothesized that the combination of DCA and ATO could be a potential treatment for PAH. A notable decrease in the right ventricular systolic pressure accompanied by reduced right heart hypertrophy was observed in the DCA/ATO combination treatment group compared with the monocrotaline treatment group. The DCA/ATO combination treatment alleviated vascular remodeling, thereby suppressing excessive PASMC proliferation and macrophage infiltration. In vitro, both DCA and ATO alone reduced PASMC viability by upregulating oxidative stress and lowering mitochondrial membrane potential. Surprisingly, when combined, DCA/ATO was able to decrease the levels of reactive oxygen species and cell apoptosis without compromising PASMC proliferation. Furthermore, suppression of the p38 pathway through the specific inhibitor SB203580 attenuated cell death and oxidative stress at a level consistent with that of DCA/ATO combination treatment. These observations suggested a complementary effect of DCA and ATO on rescuing PASMCs from a PAH phenotype through p38 activation via the regulation of mitochondrial-related cell death and oxidative stress. DCA in combination with ATO may represent a novel therapeutic strategy for PAH treatment.

The Application of Fermentation Technology in Traditional Chinese Medicine: A Review.

In Chinese medicine, fermentation is a highly important processing technology whereby medicinal herbs are fermented under appropriate temperature, humidity, and moisture conditions by means of the action of microorganisms to enhance their original characteristics and/or produce new effects. This expands the scope of such medicines and helps them to meet the stringent demands of clinical application. Since ancient times, Chinese medicine has been made into Yaoqu to reduce its toxicity and increase its efficiency. Modern fermentation technologies have been developed on the basis of traditional fermentation techniques and modern biological technology, and they can be divided into solid fermentation, liquid fermentation, and two-way fermentation technologies according to the fermentation form employed. This review serves as an introduction to traditional fermentation technology and its related products, modern fermentation technologies, and the application of fermentation technology in the field of Chinese medicine. Several problems and challenges facing the field are also briefly discussed.

Biguanide-Derived Polymeric Nanoparticles Kill MRSA Biofilm and Suppress Infection In Vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of drug-resistant infections. Its propensity to develop biofilms makes it especially resistant to conventional antibiotics. We present a novel nanoparticle (NP) system made from biocompatible F-127 surfactant, tannic acid (TA), and biguanide-based polymetformin (PMET) (termed FTP NPs), which can kill MRSA biofilm bacteria effectively in vitro and in vivo and which has excellent biocompatibility. FTP NPs exhibit biofilm bactericidal activity-ability to kill bacteria both inside and outside biofilm-significantly better than many antimicrobial peptides or polymers. At low concentrations (8-32 µg/mL) in vitro, FTP NPs outperformed PMET with ∼100-fold (∼2 log10) greater reduction of MRSA USA300 biofilm bacterial cell counts, which we attribute to the antifouling property of the hydrophilic poly(ethylene glycol) contributed by F-127. Further, in an in vivo murine excisional wound model, FTP NPs achieved 1.8 log10 reduction of biofilm-associated MRSA USA300 bacteria, which significantly outperformed vancomycin (0.8 log10 reduction). Moreover, in vitro cytotoxicity tests showed that FTP NPs have less toxicity than PMET toward mammalian cells, and in vivo intravenous injection of FTP NPs at 10 mg/kg showed no acute toxicity to mice with negligible body weight loss and no significant perturbation of blood biomarkers. These biguanide-based FTP NPs are a promising approach to therapy of MRSA infections.

Child compound Endothelium corneum attenuates gastrointestinal dysmotility through regulating the homeostasis of brain-gut-microbiota axis in functional dyspepsia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Nowadays, there is no specific effective western medicine for functional dyspepsia (FD), especially in children. Clinically, child compound Endothelium corneum (CCEC) has shown to be effective for the therapy of FD, however, the underlying mechanism has not been elucidated yet. MATERIALS AND METHODS: FD was induced in rats by irregular diet plus dilute hydrochloric acid feeding. Gastric emptying and small intestinal transit were examined by intragastric gavage with Evans blue. Histopathology was assessed by H&E staining. Gastrointestinal hormones and brain gut peptides were measured by ELISA assay. mRNA expression level was quantified by real-time PCR. Protein expression level was detected by western blotting assay. Gut microbiota was analyzed by 16S rRNA miseq sequencing. RESULTS: CCEC significantly enhanced gastric emptying and small intestinal transit of FD rats, and prominently suppressed gastrointestinal microinflammation. At phylum level, CCEC prevented the decrease of Firmicutes and the increase of Bacteroidetes in gut of FD rats. In stomach of FD rats, MTL, CCK and VIP levels were significantly increased, which could be repressed by CCEC; however, the decreased GAS level could not be elevated by CCEC. In small intestine of FD rats, MTL and GAS levels were decreased, while VIP content was increased. These alterations could be effectively reversed by CCEC. NPY levels in serum, small intestine and hypothalamus of FD rats were significantly decreased, which could be rescued by CCEC. Moreover, the over-activated POMC/Stat3/Akt pathway in hypothalamus of FD rats could be suppressed by CCEC. CONCLUSION: CCEC enhanced gastrointestinal motility probably through rebalancing the homeostasis of brain-gut-microbiota axis in FD rats. The novel findings may provide insightful theoretical basis for its clinical employment.

Acupuncture as an add-on therapy to sublingual allergen-specific immunotherapy for patients with allergic rhinitis.

This study retrospectively analyzed the effectiveness of acupuncture as add-on therapy (AAOT) to sublingual allergen-specific immunotherapy (SASIT) for patients with allergic rhinitis (AR). A total of 120 eligible cases of adult patients with AR were included in this retrospective study. Of these, 60 patients received AAOT plus SASIT and were assigned to a treatment group, while the other 60 subjects underwent SASIT only, and were assigned to a control group. Primary outcome was AR symptoms. The secondary outcome was quality of life, as evaluated by the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ). In addition, adverse events were also recorded during the study period. All outcomes were assessed before and after 8 weeks treatment. After 8 weeks treatment, patients in the treatment group had much better effectiveness in symptoms relief (P <.05), and quality of life improvement (activity domain, P = .04; practical domain, P = .03), compared with patients in the control group. In addition, although patients in the treatment group reported more mild pain at local area after the treatment than that in the control group (P <.01), no patients stopped the treatment. The results of this study showed that AAOT plus SASIT achieved more benefits in patients with AR than SASIT alone.

Traditional Uses, Origins, Chemistry and Pharmacology of Bombyx batryticatus: A Review.

Bombyx batryticatus (B. batryticatus), a well-known traditional animal Chinese medicine, has been commonly used in China for thousands of years. The present paper reviewed advances in traditional uses, origin, chemical constituents, pharmacology and toxicity studies of B. batryticatus. The aim of the paper is to provide more comprehensive references for modern B. batryticatus study and application. In Traditional Chinese Medicine (TCM) culture, drugs containing B. batryticatus have been used to treat convulsions, headaches, skin prurigo, scrofula, tonsillitis and fever. Many studies indicate B. batryticatus contains various compounds, including protein and peptides, fatty acids, flavonoids, nucleosides, steroids, coumarin, polysaccharide and others. Numerous investigations also have shown that extracts and compounds from B. batryticatus exert a wide spectrum of pharmacological effects both in vivo and in vitro, including effects on the nervous system, anticoagulant effects, antitumor effects, antibacterial and antifungal effects, antioxidant effects, hypoglycemic effects, as well as other effects. However, further studies should be undertaken to investigate bioactive compounds (especially proteins and peptides), toxic constituents, using forms and the quality evaluation and control of B. batryticatus. Furthermore, it will be interesting to study the mechanism of biological activities and structure-function relationships of bioactive constituents in B. batryticatus.

Development of GRAS strains for nutraceutical production using systems and synthetic biology approaches: advances and prospects.

Nutraceuticals are food substances with medical and health benefits for humans. Limited by complicated procedures, high cost, low yield, insufficient raw materials, resource waste, and environment pollution, chemical synthesis and extraction are being replaced by microbial synthesis of nutraceuticals. Many microbial strains that are generally regarded as safe (GRAS) have been identified and developed for the synthesis of nutraceuticals, and significant nutraceutical production by these strains has been achieved. In this review, we systematically summarize recent advances in nutraceutical research in terms of physiological effects on health, potential applications, drawbacks of traditional production processes, characteristics of production strains, and progress in microbial fermentation. Recent advances in systems and synthetic biology techniques have enabled comprehensive understanding of GRAS strains and its wider applications. Thus, these microbial strains are promising cell factories for the commercial production of nutraceuticals.

Engineering propionibacteria as versatile cell factories for the production of industrially important chemicals: advances, challenges, and prospects.

Propionibacteria are actinobacteria consisting of two principal groups: cutaneous and dairy. Cutaneous propionibacteria are considered primary pathogens to humans, whereas dairy propionibacteria are widely used in the food and pharmaceutical industries. Increasing attention has been focused on improving the performance of dairy propionibacteria for the production of industrially important chemicals, and significant advances have been made through strain engineering and process optimization in the production of flavor compounds, nutraceuticals, and antimicrobial compounds. In addition, genome sequencing of several propionibacteria species has been completed, deepening understanding of the metabolic and physiological features of these organisms. However, the metabolic engineering of propionibacteria still faces several challenges owing to the lack of efficient genome manipulation tools and the existence of various types of strong restriction-modification systems. The emergence of systems and synthetic biology provides new opportunities to overcome these bottlenecks. In this review, we first introduce the major species of propionibacteria and their properties and provide an overview of their functions and applications. We then discuss advances in the genome sequencing and metabolic engineering of these bacteria. Finally, we discuss systems and synthetic biology approaches for engineering propionibacteria as efficient and robust cell factories for the production of industrially important chemicals.

Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach.

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04-02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (µset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12 ml h(-1) at post-induction phase. The total PGL yield reached 371.86 U mL(-1), which is the highest PGL production by recombinant E. coli expression system.

A newly isolated Paecilomyces sp. WSH-L07 for laccase production: isolation, identification, and production enhancement by complex inducement.

Laccase can catalyze the oxidation of a wide range of organic and inorganic substrates. In this study, an easily detectable method was employed for screening laccase-producing microorganisms by using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as laccase-secretion indicator. A novel laccase-producing strain was isolated and identified as Paecilomyces sp. WSH-L07 according to the morphological characteristics and the comparison of internal transcribed spacer (ITS) ribosomal DNA (rDNA) gene sequences. In further investigation, the production of laccase by Paecilomyces sp. WSH-L07 was greatly enhanced by the nontoxic inducers of copper sulphate and methylene blue. Under the induction of 50 microM copper sulphate and 20 microM methylene blue, the maximum laccase production was obtained. When these inducers were added into cultivation medium at 24 h and 12 h, respectively, an increment of about 100 times of laccase activity compared with that of in inducer-free medium and about two times of that of in single copper-supplemented medium was observed. Compared with other Paecilomyces species, Paecilomyces sp. WSH-L07 exhibit the better laccase-producing characteristics with an activity of 1,650 U/l on the eighth day, suggesting its potential ability for industrial application.