Environmental impact assessment of battery boxes based on lightweight material substitution.

Power battery is one of the core components of electric vehicles (EVs) and a major contributor to the environmental impact of EVs, and reducing their environmental emissions can help enhance the sustainability of electric vehicles. Based on the principle of stiffness equivalence, the steel case of the power cell is replaced with lightweight materials, a life cycle model is established with the help of GaBi software, and its environmental impact is evaluated using the CML2001 method. The results can be summarized as follows: (1) Based on the four environmental impact categories of GWP, AP, ADP (f), and HTP, which are the global warming potential (GWP), acidification potential (AP), abiotic depletion potential (ADP (f)) and human toxicity potential (HTP), the environmental impact of lightweight materials is lower than that of the steel box. Among them, the aluminum alloy box has the largest reduction, and the Carbon Fiber Sheet Molding Compound (CF-SMC) box is the second. (2) In the sensitivity analysis of electric structure, an aluminum alloy box is still the most preferable choice for environmental impact. (3) In the sensitivity analysis of driving mileage, the aluminum alloy box body is also the best choice for vehicle life. (4) Quantitative assessment using substitution factors measures the decrease in greenhouse gas emissions following the substitution of steel battery box with lightweight materials. The adoption of aluminum alloy battery box can lead to a reduction of 1.55 tons of greenhouse gas emissions, with a substitution factor of 1.55 tC sb-1. In the case that composite materials have not been recycled commercially on a large scale, aluminum alloy is still one of the best materials for the integrated environmental impact of the whole life cycle of the battery boxes.

Effects of different strains fermentation on nutritional functional components and flavor compounds of sweet potato slurry.

In this paper, we study the effect of microbial fermentation on the nutrient composition and flavor of sweet potato slurry, different strains of Aspergillus niger, Saccharomyces cerevisiae, Lactobacillus plantarum, Bacillus coagulans, Bacillus subtilis, Lactobacillus acidophilus, and Bifidobacterium brevis were employed to ferment sweet potato slurry. After 48 h of fermentation with different strains (10% inoculation amount), we compared the effects of several strains on the nutritional and functional constituents (protein, soluble dietary fiber, organic acid, soluble sugar, total polyphenol, free amino acid, and sensory characteristics). The results demonstrated that the total sugar level of sweet potato slurry fell significantly after fermentation by various strains, indicating that these strains can utilize the nutritious components of sweet potato slurry for fermentation. The slurry's total protein and phenol concentrations increased significantly, and many strains demonstrated excellent fermentation performance. The pH of the slurry dropped from 6.78 to 3.28 to 5.95 after fermentation. The fermentation broth contained 17 free amino acids, and the change in free amino acid content is closely correlated with the flavor of the sweet potato fermentation slurry. The gas chromatography-mass spectrometry results reveal that microbial fermentation can effectively increase the kinds and concentration of flavor components in sweet potato slurry, enhancing its flavor and flavor profile. The results demonstrated that Aspergillus niger fermentation of sweet potato slurry might greatly enhance protein and total phenolic content, which is crucial in enhancing nutrition. However, Bacillus coagulans fermentation can enhance the concentration of free amino acids in sweet potato slurry by 64.83%, with a significant rise in fresh and sweet amino acids. After fermentation by Bacillus coagulans, the concentration of lactic acid and volatile flavor substances also achieved its highest level, which can considerably enhance its flavor. The above results showed that Aspergillus niger and Bacillus coagulans could be the ideal strains for sweet potato slurry fermentation.

Allergenicity of wheat protein in diet: Mechanisms, modifications and challenges.

Wheat is widely available in people's daily diets. However, some people are currently experiencing IgE-mediated allergic reactions to wheat-based foods, which seriously impact their quality of life. Thus, it is imperative to provide comprehensive knowledge and effective methods to reduce the risk of wheat allergy (WA) in food. In the present review, recent advances in WA symptoms, the major allergens, detection methods, opportunities and challenges in establishing animal models of WA are summarized and discussed. Furthermore, an updated overview of the different modification methods that are currently being applied to wheat-based foods is provided. This study concludes that future approaches to food allergen detection will focus on combining multiple tools to rapidly and accurately quantify individual allergens in complex food matrices. Besides, biological modification has many advantages over physical or chemical modification methods in the development of hypoallergenic wheat products, such as enzymatic hydrolysis and fermentation. It is worth noting that using biotechnology to edit wheat allergen genes to produce allergen-free food may be a promising method in the future which could improve the safety of wheat foods and the health of allergy sufferers.

The role of wheat embryo globulin nutrients in improving cognitive dysfunction in AD rats.

Neuroinflammation and intestinal microbiota cause pathological progression of Alzheimer's disease (AD), leading to neurodegeneration and cognitive decline. This study investigates the effects of wheat embryo globulin nutrient (WEGN) on depression, neuroinflammation, and intestinal microbial disorder caused by AD and its protective mechanism on cognitive impairment. Results demonstrated that rats in the WEGN group have lower feed intake but higher body weight than those in the control group. Notably, rats in the WEGN group have a higher number of cross grids and uprights and a smaller amount of fecal particles than those in the control group. Biochemical examinations revealed that rats in the WEGN group had lower expression of interleukin-1ß, interleukin-6, and tumor necrosis factor α in hippocampus tissue and the expression of genes and proteins related to the TLR4/MyD88/NF-κB signaling pathway in AD rats was down-regulated compared to those in the control group. The 16S rRNA gene sequencing results demonstrated that WEGN treatment inhibits the increase of Erysipelotrichaceae, Erysipelatoclostridium, Erysipelotrichaceae, Corynebacterium, and Frisingicoccus, and the reduction of Lactobacillus in AD rats. WEGN has potential value as a practical food in alleviating neuroinflammation-related diseases such as AD.

Avilamycin production enhancement by mutagenesis and fermentation optimization in Streptomyces viridochromogenes.

Avilamycin, an excellent growth-promoting feed additive, produced by Streptomyces viridochromogenes, was widely used to promote the growth of poultry by inhibiting Gram-positive bacteria. In this work, the methods of combinational mutagenesis of UV (Ultraviolet) and ARTP (Atmospheric and room temperature plasma), and rational screening by high concentrations of CaCl2 were utilized to promote the production of avilamycin. The avilamycin high-yielding mutant strains of Z-6 (29.31 mg/L), A-9 (36.84 mg/L) and F-23 (45.73 mg/L) were screened out, with yields of avilamycin improved by 57.92%, 98.49% and 146.39%, respectively, compared with the wild strain (WT). The performance comparison showed that Z-6, A-9 and F-23 mutant strains had stronger abilities of substrate consumption, cell growth and antibiotic synthesis than WT. Furthermore, the composition of fermentation medium, inoculation parameters, supplementation strategies of oxygen vectors, glucose and precursors (L-valine, D-xylose and sodium acetate) had been optimized and the avilamycin yield of the mutant strain F-23 was significantly enhanced by 41.87% by fermentation optimization. In summary, the strategy of increasing the production of avilamycin in S. viridochromogenes in this work might provide an alternative method to enhance the synthesis of secondary metabolites in other Streptomyces.

Wheat embryo globulin nutrients ameliorate d-galactose and aluminum chloride-induced cognitive impairment in rats.

Wheat embryo globulin nutrient (WEGN), with wheat embryo globulin (WEG) as the main functional component, is a nutritional combination that specifically targets memory impairment. In this study, we explored the protective role of WEGN on Alzheimer's disease (AD)-triggered cognitive impairment, neuronal injury, oxidative stress, and acetylcholine system disorder. Specifically, we established an AD model via administration of d-galactose (d-gal) and Aluminum chloride (AlCl3) for 70 days, then on the 36th day, administered animals in the donepezil and WEGN (300, 600, and 900 mg/kg) groups with drugs by gavage for 35 days. Learning and memory ability of the treated rats was tested using the Morris water maze (MWM) and novel object recognition (NOR) test, while pathological changes and neuronal death in their hippocampus CA1 were detected via HE staining and Nissl staining. Moreover, we determined antioxidant enzymes by measuring levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) in serum, cortex, and hippocampus, whereas changes in the acetylcholine system were determined by evaluating choline acetyltransferase (ChAT), and acetylcholinesterase (AChE) activities, as well as choline acetylcholine (Ach) content. Results revealed that rats in the WEGN group exhibited significantly lower escape latency, as well as a significantly higher number of targeted crossings and longer residence times in the target quadrant, relative to those in the model group. Notably, rats in the WEGN group spent more time exploring new objects and exhibited lower damage to their hippocampus neuron, had improved learning and memory activity, as well as reversed histological alterations, relative to those in the model group. Meanwhile, biochemical examinations revealed that rats in the WEGN group had significantly lower MDA levels and AChE activities, but significantly higher GSH, SOD, and ChAT activities, as well as Ach content, relative to those in the model group. Overall, these findings indicate that WEGN exerts protective effects on cognitive impairment, neuronal damage, oxidative stress, and choline function in AD rats treated by d-gal/AlCl3.

Wheat embryo globulin protects against acute alcohol-induced liver injury in mice.

Wheat Embryo Globulin (WEG) is a high-quality plant-derived protein with anti-inflammatory, antioxidant, and immunity enhancement effects. WEG was prepared and characterized using free amino acid analysis, circular dichroism (CD), and scanning electron microscope (SEM). The liver protection effect of WEG on mice after acute alcohol stimulation was also investigated. Male KM mice were randomly divided into four groups (n = 10). Animals were orally administrated with WEG (60 mg/kg), silymarin (50 mg/kg), and the same volume of saline solution daily for 30 days, before administering an alcohol-intragastric injection. Results displayed that the liver index, the levels of serum total cholesterol (TC), serum triglyceride (TG), liver malondialdehyde (MDA) and the mRNA expression of CYP2E1were significantly decreased in WEG-treated mice compared with the model group. Meanwhile, the levels of serum high-density lipoprotein-cholesterol (HDL-C), hepatic reduced glutathione (GSH), superoxide dismutase (SOD) and the mRNA expression of ADH2 and ALDH2 were remarkably increased. Effect of WEG on histopathology of liver tissue confirmed its protective function. Meanwhile, GSH level of ileal was significantly increased, MDA was remarkably decreased in WEG-treated mice, which also indicated that WEG possessed a positive effect on intestinal micro ecological environment health to some extent. In conclusion, WEG is a promising agent for the prevention of acute alcoholic liver injury.

Retroperitoneal laparoscopic partial resection of the renal pelvis for urothelial carcinoma: A case report.

BACKGROUND: The standard treatment of transitional cell carcinoma of the upper urinary tract consists of radical nephroureterectomy with bladder cuff removal, which can be performed either in open or laparoscopy or robot-assisted laparoscopy. Treatment of chronic renal insufficiency patients with upper urothelial tumor is in a dilemma. Urologists weigh and consider the balance between tumor control and effective renal function preservation. European Association of Urology guidelines recommend that select patients may benefit from endoscopic treatment, but laparoscopic treatment is rarely reported. CASE SUMMARY: In this case report, we describe a case of 79-year-old female diagnosed with urothelial carcinoma of the renal pelvis and adrenal adenoma with chronic renal insufficiency. The patient was treated with retroperitoneal laparoscopic partial resection of the renal pelvis and adrenal adenoma resection simultaneously. CONCLUSION: Retroperitoneal laparoscopic partial resection of the renal pelvis is an effective surgical procedure for the treatment of urothelial carcinoma of the renal pelvis.

Immunity improvement and gut microbiota remodeling of mice by wheat germ globulin.

The wheat germ protein (WG) and it's proteolytic peptide have a variety of biological activities. Our previous work showed that WG could improve immunity of the immunosuppressive mice established by cyclophosphamide. However, in the healthy condition and normal diet, as a supplementary food, the effects of immunity improvement and gut microbiota remodeling by the wheat germ globulin has not been studied yet. Here, we reported that WG could improve the immunity and remodel the gut microbiota of the mice, as a potentially safe functional supplementary food for the first time. The increase of interleukin-6 (IL-6) and the decrease of tumor necrosis factor α (TNF-α) and interleukin-10 (IL-10) indicated that WG could enhance the levels of activated T cells and monocytes and anti-inflammatory ability, meanwhile, the significant increase of immunoglobin G (lgG) and the notable decrease of the immunoglobin M (lgM) and immunoglobin A (lgA) illustrated that WG could improve immunity by promoting the differentiation and maturation process of B cells, compared with the NC group (normal control group). 16S rRNA sequencing showed WG could remodel the gut microbiota. At the phylum level, the Bacteroidetes were reduced and Firmicutes were increased in WG group, compared with NC group. At the genus level, the SCFA producing genera of unclassified_f_Lachnospiraceae, Blautia and especially the Roseburia (increased more than threefold) increased notably. Further, the level changes of cytokines and immunoglobulins were associated with the gut microbiota. This work showed that WG could improve immunity and has potential application value as an immune-enhancing functional food.

Digestive characteristics and peptide release from wheat embryo proteins in vitro.

Due to the scarcity of the data on digestion and metabolism of wheat embryo proteins WEP, a simulated gastrointestinal digestion (SGID) scheme in vitro was utilized to explain the protein hydrolysis and biological activity of WEP during the digestion process. WEP had a certain degree of resistance to gastric digestion, especially the protein with a molecular weight of 50 kDa. In all the samples, no visually intact protein band emerged in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) during the intestinal phase, which was consistent with a gradually increasing content of released free amino acids. Moreover, the resistant digestion peptides (the amino acid sequences were ISQFXX and GTVX) were identified at the end of the gastrointestinal digestion (GID) product by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Although the complete protein in the sample was degraded, the antioxidant activity was not negatively affected, rather it showed an increasing trend and maintained a higher level of activity. The amount of the ß-sheet gradually increased as that of the α-helix declined, the random coil decreased, whereas no obvious change was noticed in ß-turn content. The results provide a better understanding for optimal selection of peptide candidates for designing protein products in the food processing industry as well as for WEP digestion and metabolism in the human body.

Pleiotropic regulation of daptomycin synthesis by DptR1, a LuxR family transcriptional regulator.

Daptomycin, produced by Streptomyces roseosporus is a novel cyclic lipopeptide antibiotic for treatment of Gram-positive bacteria caused infections. While, the regulatory mechanism of daptomycin synthesis has not been fully understood. Here we reported that DptR1, a LuxR family transcriptional regulator, played a pleiotropic regulatory role on daptomycin synthesis, for the first time. Deletion or over-expressing of dptR1 decreases the daptomycin's production, increases the transcriptional levels of the core dpt genes of day 3 and decreases the transcriptional levels of the core dpt genes of day 4, sharply, which indicates the transcriptional regulation of DptR1 on daptomycin synthesis is complex and time-ordered. The transcriptional levels of dptR2 increase in dptR1 deletion mutant (DR1), but decrease in dptR1 over-expression mutant (OR1), dramatically, compared to the starting strain of Streptomyces roseosporus N3 (WT), on the 3rd day, which indicates that DptR1 represses the transcription of dptR2. While, the transcriptional levels of dptR3 both in DR1 and OR1 decrease obviously, compared to WT, on the 3rd and 4th day. Comparative analysis of promoters' activities, using xylE gene as the reporter, showed that DptR1 activated the transcription of its own gene of dptR1 and represses the transcription of the dptR3 by affecting the promoter activities. While DptR1 may affect the expression of dptR2 indirectly, not by affecting the promoter activity of dptR2. DptR1, a LuxR family transcriptional regulator, played a pleiotropic regulation role on daptomycin synthesis.

Pleiotropic regulation of daptomycin synthesis by DptR1, a LuxR family transcriptional regulator.

Daptomycin, produced by Streptomyces roseosporus is a novel cyclic lipopeptide antibiotic for treatment of Gram-positive bacteria caused infections. While, the regulatory mechanism of daptomycin synthesis has not been fully understood. Here we reported that DptR1, a LuxR family transcriptional regulator, played a pleiotropic regulatory role on daptomycin synthesis, for the first time. Deletion or over-expressing of dptR1 decreases the daptomycin's production, increases the transcriptional levels of the core dpt genes of day 3 and decreases the transcriptional levels of the core dpt genes of day 4, sharply, which indicates the transcriptional regulation of DptR1 on daptomycin synthesis is complex and time-ordered. The transcriptional levels of dptR2 increase in dptR1 deletion mutant (DR1), but decrease in dptR1 over-expression mutant (OR1), dramatically, compared to the starting strain of Streptomyces roseosporus N3 (WT), on the 3rd day, which indicates that DptR1 represses the transcription of dptR2. While, the transcriptional levels of dptR3 both in DR1 and OR1 decrease obviously, compared to WT, on the 3rd and 4th day. Comparative analysis of promoters' activities, using xylE gene as the reporter, shows that DptR1 activated the transcription of its own gene of dptR1 and represses the transcription of the dptR3 by affecting the promoter activities. While DptR1 may affect the expression of dptR2 indirectly, not by affecting the promoter activity of dptR2. DptR1, a LuxR family transcriptional regulator, played a pleiotropic regulation role on daptomycin synthesis.

BZW2 gene knockdown induces cell growth inhibition, G1 arrest and apoptosis in muscle-invasive bladder cancers: A microarray pathway analysis.

Bladder cancer is among the most common cancers all over the world. The function of basic leucine zipper and W2 domains 2 (BZW2) in tumour progression has been reported. However, the biological function of BZW2 in muscle-invasive bladder cancers (MIBCs) remains to be determined. The aim of the present study was to reveal the expression and roles of BZW2 in human MIBCs and to explore the molecular mechanisms underlying these functions. Clinically, BZW2 expression was higher in MIBC tissues than the adjacent non-tumour tissues. Knocking down BZW2 using shRNA inhibited cell proliferation and G1/S cell cycle progression in vitro, and induced apoptosis in both 5637 and T24 cells. Moreover, in vivo studies with mice xenograft models confirmed the anti-proliferative effects of BZW2-knockdown, providing a future therapeutic target. We also performed biochemical microarray analysis to identify the potential signalling pathways, disease states and functions which could be affected by suppressing BZW2 in MIBC cells. Collectively, our findings suggest BZW2 has an oncogenic role in MIBCs and serves as a promising target for molecular diagnosis and gene therapy.

[Interval of ≤2 weeks between 12-core prostate biopsy and laparoscopic radical prostatectomy does not affect perioperative parameters or surgical outcomes].

OBJECTIVE: To determine whether a short interval (≤2 weeks) between 12-core prostate biopsy and laparoscopic radical prostatectomy (LRP) affects perioperative parameters and the outcome of surgery. METHODS: This retrospective study included 102 cases of prostate cancer treated by LRP after 12-core prostate biopsy from January 2012 to December 2016. Based on the interval between prostate biopsy and LRP, we divided the patients into three groups: ≤2 wk (n = 35), >2－6 wk (n = 21), and >6 wk (n = 46). The patients averaged 69.87 (59－84) years in age, 24.99 (15.62－33.14) kg/m2 in the body mass index (BMI), 24.41 (0.41－111.78) µg/L in the baseline PSA level, 56.05 (15.97－216.52) ml in the prostate volume, and 7.51 (6－9) in the Gleason score. We analyzed the clinical data, perioperative parameters and outcomes of surgery, and compared them among the three groups of patients. RESULTS: Operations were completed successfully in all the 102 cases without transferring to open surgery. There were no statistically significant differences among the three groups of patients in age, BMI, baseline PSA level, prostate volume, Gleason score, or T stage, nor in the operation time, estimated intraoperative blood loss, blood transfusion rate, intestinal injury, positive incision margin rate, or urinary continence rate at 3 months after surgery. CONCLUSIONS: Laparoscopic radical prostatectomy at ≤2 weeks after 12-core prostate biopsy is safe and effective in the treatment of prostate cancer and does not affect the perioperative parameters and outcomes of surgery.

Optimization of the Biosynthesis Conditions of Daptomycin by the Biostatistical Methodology.

Response surface methodology (RSM) was employed to optimize medium components including oxygen vector of n-dodecane of a mutant strain GC-63 of Streptomyces roseosporus NRRL 11379. The two-level Plackett-Burman design (PB factorial design) with fourteen variables including oxygen vector was used to screen the most significant factors affecting antibiotic production. Then, the RSM based on center composite design was used to identify the optimum levels of the significant variables to generate optimal response. Glucose, soybean meal, asparagine and n-dodecane were screened to significantly influence the daptomycin production. The medium composition optimized with response surface methodology was (g/L): glucose, 9.46; soluble starch, 25; dextrin, 12.5; yeast extract, 12.5; soybean meal, 21.34; peptone, 25; casein, 5; asparagine, 2.68; K2SO4, 6; (NH4)2Fe(SO4)2, 2; MgSO4, 1; CaCO3, 5; MnCl2, 0.5; n-dodecane, 7.47 % (v/v). The maximum daptomycin concentration reached 979.36 mg/L which was nearly 2.2-fold higher compared to that in the basal medium, with predicted optimal concentrations in a 7.5-L fermentor.

[Laparoscopic radical prostatectomy for prostate cancer found in transurethral resection of the prostate: Report of 14 cases].

OBJECTIVE: To sum up the experience in the treatment of prostate cancer found in transurethral resection of the prostate (TURP) by laparoscopic radical prostatectomy (LRP). METHODS: Fourteen patients found with prostate cancer during TURP underwent LRP in our hospital between 2011 and 2016. We reviewed our experience in the treatment and analyzed the clinical and follow-up data. RESULTS: LRP was successfully performed in all the cases at 1－4 months after TURP, with a mean operation time of (113 ± 94) min (80－220 min), a mean blood loss of (188 ± 152) ml (100－500 ml), a mean catheterization time of (11.7 ± 3.7) d (7－16 d), and a median follow-up time of 28 (4－68) months. There were no rectal injuries, conversion to open surgery, or blood transfusion during the operation. Positive surgical margin was found in 1 case, in which the tumor involved the nerve and vessel, and lymphatic fistula occurred in another. Urinary continence was desirable in 13 cases at 12 months after surgery, and no incontinence was observed in the other, which had been followed up for less than 12 months. The patient with positive surgical margin received radiotherapy and endocrine therapy postoperatively and was still alive without pathologic progression. No biochemical or clinical recurrence was found in the other 13 cases. CONCLUSIONS: LRP at 1 month after TURP can provide a proper anatomical plane, make the operation easier, and achieve a satisfactory functional and oncological prognosis for patients with prostate cancer.

Molecular Design, Structural Analysis and Antifungal Activity of Derivatives of Peptide CGA-N46.

Chromogranin A (CGA)-N46, a derived peptide of human chromogranin A, has antifungal activity. To further research the active domain of CGA-N46, a series of derivatives were designed by successively deleting amino acid from both terminus of CGA-N46, and the amino acid sequence of each derivative was analyzed by bioinformatic software. Based on the predicted physicochemical properties of the peptides, including half-life time in mammalian reticulocytes (in vitro), yeast (in vivo) and E. coli (in vivo), instability index, aliphatic index and grand average of hydropathicity (GRAVY), the secondary structure, net charge, the distribution of hydrophobic residues and hydrophilic residues, the final derivatives CGA-N15, CGA-N16, CGA-N12 and CGA-N8 were synthesized by solid-phase peptide synthesis. The results of bioinformatic analysis showed that CGA-N46 and its derivatives were α-helix, neutral or weak positive charge, hydrophilic, and CGA-N12 and CGA-N8 were more stable than the other derivatives. The results of circular dichroism confirmed that CGA-N46 and its derived peptides displayed α-helical structure in an aqueous solution and 30 mM sodium dodecylsulfate, but α-helical contents decreased in hydrophobic lipid vesicles. CGA-N15, CGA-N16, CGA-N12 and CGA-N8 had higher antifungal activities than their mother peptide CGA-N46. Among of the derived peptides, CGA-N12 showed the least hemolytic activity. In conclusion, we have successfully identified the active domain of CGA-N46 with strong antifungal activity and weak hemolytic activity, which provides the possibility to develop a new class of antibiotics.

Optimization of the Expression Conditions of CGA-N46 in Bacillus subtilis DB1342(p-3N46) by Response Surface Methodology.

CGA-N46 is a small antifungal-derived peptide and consists of the 31st-76th amino acids of the N-terminus of human chromogranin A. Polycistronic expression of recombinant CGA-N46 in Bacillus subtilis DB1342 was used to improve its production, but the yield of CGA-N46 was still low. In the present study, response surface methodology (RSM) was used to optimize culture medium composition and growth conditions of the engineered strain B. subtilis DB1342(p-3N46) for the further increase in CGA-N46 yield. The results of two-level factorial experiments indicated that dextrin and tryptone were significant factors affecting CGA-N46 expression. Central composite design (CCD) was used to determine the ideal conditions of each significant factors. From the results of CCD, the optimal medium composition was predicted to be dextrin 16.6 g/L, tryptone 19.2 g/L, KH2PO4·H2O 6 g/L, pH 6.5. And the optimal culture process indicated inoculation of B. subtilis DB1342(p-3N46) seed culture into fresh culture medium at 5 % (v/v), followed by expression of CGA-N46 for 56 hours at 30 °C induced by 2 % (v/v) sucrose after one hour of shaking culture. To test optimal CGA-N46 peptide expression, the yeast growth inhibition assay was employed and it was found that under optimal culture conditions, CGA-N46 inhibited the growth of Candida albican by 42.17, 30.86 % more than that in the pre-optimization conditions. In summary, RSM can be used to optimize expression conditions of CGA-N46 in engineered strains B. subtilis DB1342(p-3N46).

Optimization of the expression conditions of CGA-N46 in Bacillus subtilis DB1342(p-3N46) by response surface methodology.

CGA-N46 is a small antifungal derived peptide and consists of the 31st to 76th amino acids of the N-terminus of human chromogranin A. Polycistronic expression of recombinant CGA-N46 in Bacillus subtilis DB1342 was used to improve its production, but the yield of CGA-N46 was still low. In the present study, response surface methodology (RSM) was used to optimize culture medium composition and growth conditions of the engineered strain B. subtilis DB1342(p-3N46) for the further increase of CGA-N46 yield. The results of two-level factorial experiments indicated that dextrin and tryptone were significant factors affecting CGA-N46 expression. Central composite design (CCD) was used to determine the ideal conditions of each significant factors. From the results of CCD, the optimal medium composition was predicted to be dextrin 16.6 g/L, tryptone 19.2 g/L, KH2PO4·3H2O 6 g/L, pH 6.5. And the optimal culture process was indicated that B. subtilis DB1342(p-3N46) seed culture was inoculated into fresh culture medium at 5% (v/v), followed by expression of CGA-N46 for 56 hours at 30°C induced by 2% (v/v) sucrose after one hour of shaking culture. To test optimal CGA-N46 peptide expression, the yeast growth inhibition assay was employed and it was found that under optimal culture conditions, CGA-N46 inhibited the growth of C. albican by 42.17%, 30.86% more than that in the pre-optimization conditions. In summary, RSM can be used to optimize expression conditions of CGA-N46 in engineered strains B. subtilis DB1342(p-3N46).

Genome shuffling of Streptomyces roseosporus for improving daptomycin production.

In this work, enhancement of daptomycin production by genome shuffling in Streptomyces roseosporus was conducted. Ultraviolet and NTG were used as mutagenizing agents to improve the volumetric productivity of the wild-type strain. Eight strains with enhanced daptomycin production were screened out as the starting population for genome shuffling. Daptomycin's production increased steadily with each round of genome shuffling. After the fourth round of fusion, a high-production strain (582 mg/L), named F4, was selected as a potential industrial production strain and its heredity stability was stable. Moreover, comparative analysis of the non-ribosomal peptide synthetase (NRPS) genes at the transcript level between the wild and the mutant was studied by RT-PCR in order to explore mechanism of genome shuffling. The transcript levels of NRPS genes dptA, dptBC, and dptD in the mutant were approximately 6.5 to 7 times higher than those in the wild. In summary, it is suggested that this strategy for increasing the daptomycin production in S. roseosporus by genome shuffling may provide an alternative approach to enhance the metabolite production in other Streptomyces.