Microbial synthesis of functional odd-chain fatty acids: a review.

Odd-chain fatty acids (OCFAs) naturally occur in bacteria, higher animals, and in plants. During recent years, they have received increasing attention due to their unique pharmacological properties and usefulness for agricultural and industrial applications. Recently, OCFAs have been identified and quantified in a few organisms, and new pharmacological functions of OCFAs have been reported. Some of the publications are related to the optimization of OCFA production through fermentation and genetic engineering. The present review aims to provide a summary on the recent progress in the field of microbial-derived OCFAs. More specifically, we outline the publications of OCFAs related to (i) different sources of OCFAs; (ii) endogenous synthesis of OCFAs; (iii) production of OCFAs through fermentation; (iv) genetic engineering related to OCFA; and (v) role of OCFAs in human health and disease. Finally, some areas that require further research are discussed.

Metabolic engineering of a robust Escherichia coli strain with a dual protection system.

Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino)propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 107 plaque-forming units/ml and produce GFP with a yield of up to 30 µg/109 colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.

Using 1-propanol to significantly enhance the production of valuable odd-chain fatty acids by Rhodococcus opacus PD630.

Odd-chain fatty acids (OCFAs) have been reported to possess pharmacological activity and have been used in the manufacture of agricultural and industrial chemicals. We here provided a new method to increase the OCFAs content in oil produced by Rhodococcus opacus PD630 through addition of 1-propanol to the fermentation media. The OCFAs in oil of R. opacus PD630 are primarily pentadecanoic acid (C15:0), heptadecanoic acid (C17:0) and heptadecenoic acid (C17:1). After adding 0.5-1.5% (v/v) 1-propanol, the production of oil increased from 1.27 g/L to 1.31-1.61 g/L, and the OCFAs content in oil increased by 46.7-55.1%. Metabolic intermediates determination and transcriptome analysis revealed that R. opacus assimilated 1-propanol through methylmalonyl-CoA pathway. When the nitrogen source was limited, propionyl-CoA was converted to propionyl-acyl carrier protein (ACP) which could be used as primer during the elongation of fatty acid synthesis. Then OCFAs were produced when odd number of propionyl-ACP was incorporated in the cycles of fatty acid synthesis.

[Thoughts on improving the design quality of acupuncture clinical randomized controlled trials].

The British Medical Journal published the Acupuncture research album which carried viewpoints, expert consensus, research methods and other genres of papers, providing an authoritative reference for standardizing the design of clinical randomized controlled trial (RCT) of acupuncture and improving the quality of clinical research. Based on the experts' consensus of "acupuncture album" on RCT, combining clinical practice and the related literature reports, and many existing problems existing in the design, we, in the present paper, analyzed ways to improve the design quality of clinical RCT of acupuncture, starting from improving the methodological quality and repeatability of clinica RCT, so as to provide methodological guidance for the design of clinical trials and to promote the standardization of clinical trials of acupuncture.

[Governor vessel moxibustion combined with western medication for mild to moderate depression with kidney-yang deficiency: a randomized controlled trial].

OBJECTIVE: To compare the therapeutic efficacy of governor vessel moxibustion combined with fluoxetine hydrochloride capsule, simple fluoxetine hydrochloride capsule and placebo moxibustion combined with fluoxetine hydrochloride capsule for mild to moderate depression with kidney-yang deficiency. METHODS: A total of 126 patients with mild to moderate depression with kidney-yang deficiency were randomized into a governor vessel moxibustion group (42 cases, 2 cases dropped off), a western medication group (42 cases, 1 case dropped off) and a placebo moxibustion group (42 cases, 1 case dropped off). The western medication group was given fluoxetine hydrochloride capsule orally, 20 mg a time, once a day. On the basis of the treatment in the western medication group, governor vessel moxibustion was applied from Dazhui (GV 14) to Yaoshu (GV 2) in the governor vessel moxibustion group, once a week; placebo moxibustion was applied in the placebo moxibustion group, once a week. Treatment of 8 weeks was required in the 3 groups. Before and after treatment, the scores of Hamilton depression scale-17 (HAMD-17), Asberg's rating scale for side effects (SERS) and TCM clinical symptom were compared, and the clinical efficacy was evaluated. RESULTS: After treatment, the scores of HAMD-17, SERS and TCM clinical symptom were decreased compared before treatment in the 3 groups (P<0.05), the decrease ranges of above scores in the governor vessel moxibustion group were larger than those in the western medication group and the placebo moxibustion group (P<0.05). The total effective rate was 92.5% (37/40) in the governor vessel moxibustion group, which was higher than 75.6% (31/41) in the western medication group and 80.5% (33/41) in the placebo moxibustion group (P<0.05). CONCLUSION: Governor vessel moxibustion combined with fluoxetine hydrochloride capsule can improve the degree of depression and relieve the clinical symptoms in mild to moderate depression patients with kidney-yang deficiency, the efficacy is superior to simple fluoxetine hydrochloride capsule, and can reduce the fluoxetine hydrochloride capsule-induced adverse effect to a certain extent.

Du-Moxibustion in a Mouse Model of Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a progressively worsening and disabling form of arthritis that primarily affects the axial skeleton. This disease mainly involves the spine and the sacroiliac joint. Fusion of the spine and the sacroiliac joint may occur in the later stage of the disease, resulting in spinal stiffness and kyphosis, as well as difficulty in walking, which seriously affects the quality of work and daily living activities and imposes a heavy burden on the patient, the family, and society. Increasing attention has been paid to non-pharmacotherapy as an alternative therapy for AS. Moxibustion is an ancient therapeutic technique used in Traditional Chinese Medicine (TCM). Du-moxibustion therapy, a unique and innovative external treatment developed on the basis of ordinary moxibustion, has a definite therapeutic effect on AS. Du-moxibustion skillfully combines the compatible techniques of TCM to integrate meridians, acupoints, Chinese herbal medicine, and moxibustion. This paper describes the operation procedures and precautions to be taken during Du-moxibustion in experimental mice in detail to provide an experimental basis for the study of the mechanism of Du-moxibustion in the treatment of AS.

[Professor YANG Ji-guo's experience in treatment of functional gastrointestinal diseases with acupuncture combined with umbilicus moxibustion].

Professor YANG Ji-guo's clinical experience in treatment of functional gastrointestinal diseases was summerized. Professor YANG Ji-guo believes that this disease is caused by the deficiency of six fu organs. Dysfunction of six fu organs in descending transportation is the basic pathogenesis. The principle of acupoint selection includes benefiting gastrointestinal functions, unblocking and purging six fu, soothing liver qi and calming down the mind. In treatment, acupuncture is combined with umbilicus moxibustion. In acupuncture, the deqi promoting technique by rotating and trembling needle is adopted. Focusing on the deficiency of six fu organs, umbilicus moxibustion is adopted to benefit the spleen and stomach and harmonize the functions of six fu organs for both biao (symptoms) and ben (root cause).

[Evaluation of clinical efficacy and mechanisms of Governor Vessel moxibustion therapy on carotid atherosclerosis].

OBJECTIVE: To explore the clinical efficacy and possible efficacy mechanisms of Governor Vessel moxibustion therapy in patients with carotid atherosclerosis (CAS). METHODS: A total of 100 patients with CAS were randomly divided into Governor Vessel moxibustion therapy group (n=51) and control group (n=49). The patients in the Governor Vessel moxibustion therapy group were treated by herbal cake-partitioned moxibustion ï¼»applied on Governor Vessel from Dazhui (GV14) to Yaoshu (GV2)ï¼½, once a week, for 8 weeks. Patients in the control group received oral administration of atorvastatin calcium tablets (10 mg) once daily for 8 weeks. Before and after treatment, the patients' constitution quantized score, body mass index (BMI), carotid intima-media thickness (IMT) and laboratory indicators ï¼»including total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), homocysteine (Hcy), fibrinogen (Fbg), and hypersensitive C-reactive protein (hs-CRP)ï¼½ were observed, and their clinical efficacy was evaluated. RESULTS: After the treatment, the constitution score and BMI were significantly reduced, and the IMT, TC, TG, LDL, Fbg and hs-CRP decreased significantly in the patients of the two groups as compared with those before treatment (P<0.05); and the constitution score and BMI were obviously lower in the Governor Vessel moxibustion therapy group than in the control group (P<0.05). In the Governor Vessel moxibustion therapy group, the effective rate of constitution score was 88.2% (45/51), the effective rate of weight loss was 54.9% (28/51), while the control group was 61.2% (30/49) and 34.7% (17/49) respectively, those in the Governor Vessel moxibustion therapy group were significantly higher than in the control group (P<0.05). Regarding to the CAS clinical effect, the effective rate was 80.4% (41/51) in the Governor Vessel moxibustion therapy group, and it was 73.5% (36/49) in the control group. There was no significant difference between the two groups in the CAS effective rate (P>0.05). CONCLUSION: The Governor Vessel moxibustion therapy can effectively treat CAS, and its mechanism may be related to the improvement of risk factors such as high level of TC, TG, LDL, Fbg, and hs-CRP.

Facile and Green Production of Human Milk Fat Substitute through Rhodococcus opacus Fermentation.

Human milk fat substitute (HMFS) is a class of structured lipids widely used in infant formulas. Herein, HMFS was prepared by Rhodococcus opacus fermentation. The substrate oils suitable for HMFS production were coconut oil (66.1-57.5%), soybean oil (17.5-26.5%), high oleic acid sunflower oil (5.4-4.5%), Antarctic krill oil (9-9.5%), and fungal oil (2%). Six HMFSs were prepared, among which HMFS V and VI were similar to human milk fat from Chinese in terms of fatty acid composition and triacylglycerol species. The sn-2 position of HMFS was occupied by palmitic acid (49.31 and 43.48% in HMFS V and VI, respectively). The major triacylglycerols were OPL, OPO, and LPL, accounting for 15.90, 9.49, and 6.84 and 17.52, 8.44, and 8.55% in HMFS V and VI, respectively. This study is the first to prepare structured lipids intended for infant formula through fermentation, providing a novel strategy for the edible oil industry.

Bioprospecting of a novel endophytic Bacillus velezensis FZ06 from leaves of Camellia assamica: Production of three groups of lipopeptides and the inhibition against food spoilage microorganisms.

Food contamination caused by microorganisms has become a threat to consumers' health. Exploring antagonistic endophytes from plants of food raw-material and applying bioactive metabolites to inhibit the contamination has been an alternative and safer solution. In this study, we isolated and screened potential antagonistic endophytes from fresh Camellia assamica leaves, which were widely used in tea beverage production. We focused on a strain that showed visible inhibitory activity to Gram-positive bacteria, Gram-negative bacteria, and fungi. It was identified as a member of Bacillus velezensis and named FZ06. The results of genome analysis showed the strain FZ06 had 167 single-copy specific genes, much higher than those of most related strains. Also, 11 potential gene clusters of antimicrobial metabolites were found. Three groups of lipopeptides (surfactin, iturin, and fengycin) were identified by UPLC-MS/MS in purified antimicrobial methanol fraction of strain FZ06. The results of minimum inhibitory concentration (MIC) test proved the lipopeptide extract showed significant inhibitory effect on food spoilage bacteria (MIC 512-2048 µg/mL) and toxigenic fungi (MIC 128-256 µg/mL). In conclusion, this study suggests that the endophytic B. velezensis FZ06 and its lipopeptide extract hold great potential applications in the inhibition of food spoilage bacteria and toxic fungi in food industry.

Using a novel polysaccharide BM2 produced by Bacillus megaterium strain PL8 as an efficient bioflocculant for wastewater treatment.

In this study, the purification and characterization of a novel polysaccharide-based bioflocculant BM2 produced by a bacterium Bacillus megaterium strain PL8 with self-flocculating property were investigated. The results showed that BM2 was an acidic polysaccharide composed of Gal, GalUA, Glc, GlcUA and Man at a molar ratio of 45.1: 33.8:9.3:9.2:2.4, respectively. The molecular weight of BM2 was 4.55 × 106 Da. BM2 had high flocculation efficiencies across a wide pH ranged from 4 to 11 and a wide temperature ranged from 20 to 100 °C towards kaolin clay. BM2 was a cation-independent bioflocculant which could achieve high flocculation activity without the addition of other cations. Adsorption bridging was the main mechanism in the flocculation process of BM2 towards kaolin clay. The BM2 also displayed a high removal efficiency in terms of Congo red (88.14%) and Pb2+ ions (82.64%). These results suggested that BM2 had a great potential as an efficient bioflocculant candidate in wastewater treatment.

Carbon source modify lipids composition of Rhodococcus opacus intended for infant formula.

Human milk fat substitutes (HMFSs) are the structured lipids intended for infant formula. It provides energy and essential fatty acid for infant. HMFSs are mainly prepared by enzymatic method. In this study, we aim to explore the potential for producing HMFSs by fermentation using R. opacus. The results indicated that different compounds with chain length from 12 to 18, used as carbon source, could be incorporated into triacylglycerols directly. Polyunsaturated fatty acids in term of ARA, EPA, DHA could enter the kennedy pathway directly and involved in the biosynthesis of triacylglycerols. GC, UPLC-MS and 13C-NMR analysis demonstrated that typical structured lipids ß-OPL (40.09%) was synthesized in R. opacus. Transcriptome analysis revealed that ß-oxidation, fatty acid elongation and kennedy pathways existed in R. opacus. It was concluded that fatty acid supplied as carbon source could enter the kennedy pathways directly or via the de novo fatty acid biosynthesis pathway depending on the chain length, thus, affect the triacylglycerol species formed in the Rhodococcus opacus.

Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design.

Chitinase is a promising biocatalyst for chitin biotransformation in the field of recalcitrant biomass degradation. Excellent catalytic performance is conducive to its commercial utilization. In this work, sequence- and structure-based semi-rational design was performed to evolve the thermostability and activity of a previously identified chitinase PpChi1 from Paenibacillus pasadenensis CS0611. After combinational mutagenesis, the mutant S244C-I319C/T259P with disulfide bond introduction and proline substitution exhibited higher specific activity at higher temperature, 26.3-fold in half-life value at 50 °C, and a 7.9 °C rise in half-inactivation temperature T1/215min compared to the wild-type enzyme. The optimal reaction temperature of the mutant was shifted from 45 °C to 52.5 °C. Molecular dynamic simulation and structure analysis confirmed that these improvements of the mutant were attributed to its stabilized folding form, possibly caused by the decreased entropy of unfolding. This work gives an initial insight into the effect of conserved proline residues in thermostable chitinases and proposes a feasible approach for improving chitinase thermostability to facilitate its application in chitin hydrolysis to valuable oligosaccharides.

Novel antibacterial polysaccharides produced by endophyte Fusarium solani DO7.

In this study, the primary antibacterial ingredients in Fusarium solani DO7 were confirmed as polysaccharides. After purification, two polysaccharides, DY1 and DY2, exhibited excellent antibacterial activity, especially to S. aureus and E. coli. Moreover, the glycosidic linkages of DY1 were composed of (1 → )-α-D-Glcp, (1 → 3)-ß-L-Rhaf, (1 → 4)-ß-D-Xylp, (1 → 6)-α-D-Glcp, (1 → 2,6)-α-D-Glcp and (1 → 2)-ß-D-Galp; while the glycosidic linkages of DY2 contained (1 → )-ß-D-Glcp, (1 → 2)-α-L-Rhaf, (1 → 3)-α-L-Araf, (1 → 4)-ß-D-Glcp, (1 → 4,6)-ß-D-Glcp and (1 → 3)-α-D-Galp. Additionally, DY1 and DY2 possessed nontoxicity to RAW 264.7 cells. Both polysaccharides could significantly promote the levels of TNF-α, IL-6 and NO by activating TNF-α, IL-6 and iNOs mRNAs expression. These results indicated that DY1 and DY2 had great potential as a food preservative and immunomodulatory agent.

Structure and immunomodulatory activity of polysaccharides from Fusarium solani DO7 by solid-state fermentation.

Two polysaccharides, DGS1 and DGS2, were obtained by solid-state fermentation (SSF) of Fusarium solani DO7, an endophytic fungus isolated from the orchid Dendrobium officinale. Structural characterizations revealed that DGS1 consisted of arabinose, glucose, mannose and galactose with a molar ratio of 2.9:13.4:3.0:1, respectively, and contained (1 → 5)-Araf, (1 → 4)-Glcp, (1 → 6)-Glcp, (1 → 3)-Manp, (1 → 2,6)-Manp and (1 → 6)-Galp glycosidic linkages, while DGS2 was composed of arabinose, glucose, mannose and galactose in a molar ratio of 3.5:8.1:2.1:1, respectively, and contained (1 → 5)-Araf, (1 → 4)-Glcp, (1 → 6)-Glcp, (1 → 3)-Manp and (1 → 6)-Galp glycosidic linkages. Neither polysaccharide was toxic to human embryonic kidney cells or mouse RAW 264.7 macrophage cells. An immunomodulatory activity assay indicated that both polysaccharides could significantly enhance the levels of TNF-α, IL-6 and NO by activating TNF-α, IL-6 and iNOs gene expression, respectively, especially DGS2. Interestingly, DGS2 also possessed relatively high antioxidant activity. These results illustrate that, due to its cost-effectiveness and environmentally friendly features, SSF has significant potential as a commercially competitive source of natural products, including fungal polysaccharides, with immunomodulatory activity.

Fungal polysaccharide similar with host Dendrobium officinale polysaccharide: Preparation, structure characteristics and biological activities.

Based on the concept of endophytic fungus, if endophytic fungus can produce the same or similar product as the host plant, which will get rid of the restrictions of farmland, seasonal and pest, the active product could be sustainably obtained. In this study, an endophytic fungus polysaccharide FP showing the similar structure with the host Dendrobium officinale polysaccharide (DOP) was sustainably and cost-effectively obtained under preferred reaction conditions with different C/N ratio. The FP with high yield up to 2.77 ±â€¯0.51 g/L showed same monosaccharide composition with DOP as well as some host-plant-associated polysaccharides in published literatures. The main chain of FP was composed by →3,6)-ß-L-Man-(1→, α-D-Glc-(1→, →4)-α-D-Glc-(1→, →3,6)-ß-D-Gal-(1→, and →6)-ß-D-Gal-(1→, while the side chain was α-D-Glc-(1→. Meanwhile, FP was confirmed as a safe polysaccharide with good antioxidant, antiglycation and immunomodulatory activities. Furthermore, TLR2 and TLR4 were confirmed as the membrane receptors of FP on RAW264.7 cells.

Structure-activity relationship of flavonoids active against lard oil oxidation based on quantum chemical analysis.

In this study, the antioxidant activities of 15 flavonoids against lard oil oxidation were determined by using the Rancimat test. Quercetin, dihydromyricetin, luteolin and kaempferol showed the strongest antioxidant activity, with protection factor values (PF) of 11.50, 11.29, 4.24 and 2.49, respectively. The role of conjugated hydroxyl groups of the B and C ring is discussed. By using the following descriptors: DeltaH(f) (the difference in heat of formation between flavonoids and their free radicals resulted after hydrogen atom donation) and H(BC) (the number of conjugated hydroxyl groups of the B and C ring), the result obtained in the antioxidant Rancimat test could be successfully explained.

4-(4-Nitro-benzene-sulfonamido)pyridinium nitrate.

A short C-N distance [1.394 (2) Å] in the title compound, C(11)H(10)N(3)O(4)S(+)·NO(3) (-), is indicative of some conjugation of the sulfonamide π electrons with those of the pyridinium ring. The crystal structure is stabilized by N-H⋯O hydrogen bonds.