High-throughput screening of Saccharomyces cerevisiae efficiently producing tyrosine / 生物工程学报

Tyrosine is an important aromatic amino acid. Besides its nutritional value, tyrosine is also an important precursor for the synthesis of coumarins and flavonoids. Previously, our laboratory constructed a Saccharomyces cerevisiae strain LTH0 (ARO4K229L, ARO7G141S, Δaro10, Δzwf1, Δura3) where tyrosine feedback inhibition was released. In the present study, heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B. vulgaris) in strain LTH0 enabled production of yellow fluorescence. The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis, followed by flow cytometry screening. Among the mutants screened, the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD, which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1. Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability. The highest extracellular tyrosine titer reached 26.8 mg/L, which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1. Heterologous expression of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L, which was 1.02 times higher than that of the original strain LTH0-FjTAL.

Construction of a highly efficient synthetic lycopene engineered Saccharomyces cerevisiae / 生物工程学报

Lycopene, as a high value-added terpene compound, has been widely concerned by researchers at home and abroad. Firstly, the ability of lycopene synthesis of Saccharomyces cerevisiae model strains S288c and YPH499 was analyzed and compared. The results showed that YPH499 was more suitable for lycopene synthesis as yeast chassis. Subsequently, the effects of constitutive promoters GPDpr, TEF1pr and inducible promoters GAL1pr, GAL10pr on Lycopene synthesis were compared. The results showed that when GPDpr and TEF1pr were used as promoters of crtE, crtB and crtI in lycopene synthesis pathway, the production of lycopene was 15.31 mg/L after 60 h fermentation in shaking flask. When GAL1pr and GAL10pr were used as promoters, the production was 123.89 mg/L, which was 8.09 times higher. In addition, the methylvaleric acid (MVA) pathway was further modified to overexpress the key enzyme gene of N-terminal truncation, tHMG1 (3-hydroxy-3-methylglutaryl coenzyme A reductase). The lycopene production was 265.68 mg/L, and the yield per cell was 72.79 mg/g. The Saccharomyces cerevisiae strain designed and constructed in this study can express lycopene in high yield per cell, thus could be used in the industrial production of lycopene after further construction and optimization.

Diagnosis and treatment of recurrent clostridium difficile infection in children / 国际儿科学杂志

Clostridium difficile (CD),a gram-positive,spore-forming,obligate anaerobic bacillus,is a leading cause of antibiotic-associated diarrhea (AAD) worldwide.With the widespread use of broad-spectrum antibiotics,the incidence of clostridium difficile infection in children is rising,while recurrent clostridium difficile infection (RCDI) requires prolonged treatment and higher medical costs.Malignancy,recent surgery and antibiotic exposures have been identified as the risk factors in children.While the toxigenic strains culture and the cell cytotoxicity neutralization assay are gold standard for the diagnosis,new diagnostic approaches such as nucleic acid amplification method have become available.The use of antibiotics,fecal microbiota transplantation (FMT) or monoclonal antibodies are included in the current treatments for RCDI.This review will cover published studies to discuss the risk factors,diagnosis and treatment of RCDI in children.

Development and verification of an FLP/FRT system for gene editing in Bacillus licheniformis / 生物工程学报

Few tools of gene editing have been developed in Bacillus licheniformis at present. In order to enrich the tools, an FLP/FRT gene editing system that can repeatedly use a single selectable marker was constructed in Bacillus licheniformis, and the system was verified by knocking out an alpha amylase gene (amyL), an protease gene (aprE) and knocking in an exogenous Vitreoscilla hemoglobin gene (vgb). First, knock-out plasmids pNZTT-AFKF of amyL and pNZTT-EFKF of aprE were constructed using thermosensitive plasmid pNZT1 as a carrier. The two knock-out plasmids contained respective homology arms, resistance genes and FRT sites. Then the knock-out plasmids were transformed into Bacillus licheniformis and the target genes were replaced by respective deletion cassette via twice homologous exchange. Finally, an expression plasmid containing FLP recombinase reading frane was introduced and mediated the excision of resistance marker. In order to expand the practicability of the system, knock-in plasmid pNZTK-PFTF-vgb was constructed, with which knock-in of vgb at pflB site was carried out successfully. The results showed that amyL and aprE were successfully knocked out and the marker kanamycin cassette exactly excised. The activities of amylase and protease of deletion mutants were reduced by 95.3% and 80.4% respectively. vgb was successfully knocked in at pflB site and the marker tetracycline cassette excised. The expression of integrated vgb was verified via real-time PCR. It is the first time to construct an FLP/FRT system for gene editing in Bacillus licheniformis, which could provide an effective technical means for genetic modification.

Rapeutic evaluation of 30 cases of perianal fistulising Crohn's disease who were removed setons at fixed time / 国际外科学杂志

Objective To analyze the time of removing setons by observing 30 cases of the infliximab (IFX)combined drainage setons therapy in the treatment of perianal fistulising Crohn's disease.Methods Thirty patients of perianal fistulising Crohn's disease of Department of Colorectal Surgery,Jiangsu Prorince Hospital of TCM in Aug.2015 to Jul.2017 were treated with drainage setons followed by intravenous IFX injections according to the standard,and setons were removed after the third injection of IFX.The method of repeated measurement were adopted to record.Clinical symptoms,perianal manifestations,laboratory indexes,healing rate and general linear model and nonparametric test of SPSS 23.0 software were used for statistical analysis.Results The differences of WBC,CRP,ESR,HB,PLT,BMI,CDAI and PDAI between post treatment and pretreatment were statistically significant (P ＜ 0.05).WBC,CRP and ESR will rerise at some time after removing the rubber band (sixth week).The closure rate of fistular at the 14th week was 53.3％,and after the follow-up in the ward at 22th week and 30th week,the clinical efficacy of fistular was not statistically significant (P ＞0.05) at the 14th week,22th week and 30th week.Conclusion IFX combined drainage setons which was removed when the induction therapy finished can induce Crohn's disease remission,improve laboratory index and promote fistula closure.the clinical efficacy in short time was stable.

Infliximab combined with sphincter-sparing surgery for the treatment of Perianal Crohn's disease / 国际外科学杂志

Objective To evaluate the efficacy of infliximab combined with sphincter-sparing surgery in treatment of perianal Crohn's disease.Methods Clinical data of 49 patients with perianal Crohn's disease (3 cases with nonfistulizing perianal Crohn's disease and 46 cases with fistulizing perianal Crohn's disease) receiving infliximab combined with sphincter-sparing surgery in Jiangsu Province Hospital of TCM from May 2010 to June 2014 were analyzed retrospectively.The situation of fistula closure was assessed.C-reactive protein and perianal disease activity index were estimated by Wilcoxon method.Results No patient was failed to respond to primary treatment.patients (26/46) had a complete response to treatment,28.3％ patients (13/46) had a partial response and 15.2％ patients (7/46) had lost response to treatment;2 cases of anal ulcer healing;No other perianal lesion occurred in 1 case of verrucous skin.At final follow-up,CRP was decreased to [4.5 (1.5,15.9)] mg/Lfrom [16.9(6.6,35.6)] mg/L(Z=-3.994,P=0.00);PDAI was decreased to[1(0,4.5)]from [7(4.5,10.5)] (Z=-6.100,P=0.00).Conclusion Infliximab combined with sphincter-sparing surgery is effective for perianal Crohn's disease.

Infliximab combined with sphincter-sparing surgery for the treatment of Perianal Crohn's disease / 国际外科学杂志

Objective To evaluate the efficacy of infliximab combined with sphincter-sparing surgery in treatment of perianal Crohn's disease.Methods Clinical data of 49 patients with perianal Crohn's disease (3 cases with nonfistulizing perianal Crohn's disease and 46 cases with fistulizing perianal Crohn's disease) receiving infliximab combined with sphincter-sparing surgery in Jiangsu Province Hospital of TCM from May 2010 to June 2014 were analyzed retrospectively.The situation of fistula closure was assessed.C-reactive protein and perianal disease activity index were estimated by Wilcoxon method.Results No patient was failed to respond to primary treatment.patients (26/46) had a complete response to treatment,28.3％ patients (13/46) had a partial response and 15.2％ patients (7/46) had lost response to treatment;2 cases of anal ulcer healing;No other perianal lesion occurred in 1 case of verrucous skin.At final follow-up,CRP was decreased to [4.5 (1.5,15.9)] mg/Lfrom [16.9(6.6,35.6)] mg/L(Z=-3.994,P=0.00);PDAI was decreased to[1(0,4.5)]from [7(4.5,10.5)] (Z=-6.100,P=0.00).Conclusion Infliximab combined with sphincter-sparing surgery is effective for perianal Crohn's disease.