Construction of Recombinant Corynebacterium glutamicum for L-threonine Production

Yangyong Lv,Zhanhong Wu,Shuangyan Han,Ying Lin,Suiping Zheng 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.1

L-threonine is an essential amino acid which is widely used in feed and pharmaceutical industries. We recently engineered Corynebacterium glutamicum R102(AHVr) for improved production of L-threonine. Inactivation of genes metX and dapA encoding dihydrodipicolinate synthase and homoserine O-acetyltransferase, respectively,was firstly conducted by homologous recombination, which differed from the common random mutagenesis method. Then operon gene hom-thrB (O) and export gene thrE (E)from R102 were over-expressed alone or together to obtain a series of recombinant strains. qPCR was employed to evaluate the transcript quantification of the target genes. In flask fermentation, the newly constructed strain R102ΔmetXΔdapA (pEC-Box) was able to accumulate 3.35 g threonine/L compared with 1.80 g threonine/L of strain R102 (AHVr). L-threonine is an essential amino acid which is widely used in feed and pharmaceutical industries. We recently engineered Corynebacterium glutamicum R102(AHVr) for improved production of L-threonine. Inactivation of genes metX and dapA encoding dihydrodipicolinate synthase and homoserine O-acetyltransferase, respectively,was firstly conducted by homologous recombination, which differed from the common random mutagenesis method. Then operon gene hom-thrB (O) and export gene thrE (E)from R102 were over-expressed alone or together to obtain a series of recombinant strains. qPCR was employed to evaluate the transcript quantification of the target genes. In flask fermentation, the newly constructed strain R102ΔmetXΔdapA (pEC-Box) was able to accumulate 3.35 g threonine/L compared with 1.80 g threonine/L of strain R102 (AHVr).

Effect and stability of miniscrew-assisted rapid palatal expansion: A systematic review and meta-analysis

Xinyi Huang,Yu Han,Shuangyan Yang 대한치과교정학회 2022 대한치과교정학회지 Vol.52 No.5

Objective: This study aimed to systematically analyze the effect and stability of miniscrew-assisted rapid palatal expansion (MARPE) to provide a reference for the clinical treatment of patients with maxillary transverse deficiency (MTD). Methods: We searched PubMed, Science Direct, Web of Science, Embase, Cochrane Library, CNKI, and Wanfang Database for relevant studies published before February 18, 2021 and selected them according to the eligibility criteria. The Cochrane Handbook for Systematic Reviews (version 5.1.0) criteria were used for the quality assessment of randomized controlled trials, while the scoring protocol of the methodological index for non-randomized studies was used for non-randomized controlled trials. Statistical analysis was performed using the RevMan5.3 software. Results: All the included studies showed a relatively high success rate of expansion. The changes in both the intermolar and alveolar widths after MARPE were statistically significant. MARPE exhibited greater skeletal expansion effects than did conventional RPE. The midpalatal suture was opened in parallel after MARPE. A small amount of relapse was observed 1 year after expansion. MARPE caused tooth inclination and a decrease in alveolar height, but it was less significant than in conventional RPE. Conclusions: MARPE may be an effective treatment modality for patients with MTD. It causes great transverse skeletal expansion in late adolescence. In comparison to conventional RPE, MARPE has lower detrimental periodontal effects and has certain clinical advantages.

Enhanced electrokinetic remediation of fluorine-contaminated soil by applying an ammonia continuous circulation system

Shufa Zhu,Ming Zhou,Shuangyan Zhang 한국화학공학회 2016 Korean Journal of Chemical Engineering Vol.33 No.2

The objective of this research was to investigate the effects of ammonia continuous circulation enhanced electrokinetic remediation of fluorine contaminated soil and to analyze its influence on soil pH after remediation. An experimental study was carried out in self-made electrokinetic apparatus. The voltage gradient was set at 1.0V/cm and ammonia water with different concentrations was used as electrolyte which circulated in series. Comparative studies were made by using deionized water as electrolyte which circulated separately in one experiment and continuously in another. According to the experiment the continuous circulation of ammonia water increased the current value during the remediation process and maintained current through the soil cell stabler, which not only increased fluorine migration but also reduced energy consumption. Among the given ammonia concentrations (0, 0.01, 0.1 and 0.2mol/L) the removal rate increased with ammonia concentration. 0.2mol/L had the highest current (26.8mA), and the removal rate amounted up to 57.3%. By using ammonia circulation enhanced electrokinetic technology, the difference between pH values of cathode soil and anode soil became smaller. Ammonia continuous circulation enhanced electrokinetics can effectively remediate fluorine contaminated soil and the residual ammonia in the soil can also improve soil fertility.

Synthesisof Fructose Laurate Esters Catalyzed by a CALB-displaying Pichia pastoris Whole-cell Biocatalyst in a Non-aqueous System

Zi Jin,Shuli Liang,Xiuqin Zhang,Shuangyan Han,Changqiong Ren,Ying Lin,Suiping Zheng 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.2

Earlier studies on fructose laurate ester products have shown that recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) on the cell surface acts as an efficient whole-cell biocatalyst for sugar ester production from fructose and lauric acid in an organic solvent. The effects of various reaction factors, including solvent composition, substrate molar ratio, enzyme dose,temperature and water activity, on esterification catalyzed by the CALB-displaying P. pastoris whole-cell biocatalyst were examined in the present study. Under the preferred reaction conditions, specifically, 5 mL organic solvent mixture of 2-methyl-2-butanol/DMSO (20% v/v), 2 mmol fructose with a lauric acid to fructose molar ratio of 2:1, 0.3 g whole-cell biocatalyst (1,264 U/g dry cell) with an initial water activity of 0.11, 1.2 g 4Å molecular sieve, reaction temperature of 55oC and 200 rpm stirring speed, the fructose mono laurate ester yield was 78% (w/w). The CALBdisplaying P. pastoris whole-cell biocatalyst exhibited good operational stability, with an evident increase, rather than decrease, in relative activity after the continuous recover and reuse cycle. The relative activity of the biocatalyst remained 50% higher than that of the first batch, even following reuse for 15 batches. Our results collectively indicate that the CALB-displaying P. pastoris whole-cell biocatalyst may be potentially utilized in lieu of free or immobilized enzyme to effectively produce non-ionic surfactants such as fatty acid sugar esters, offering the significant advantages of cost-effectiveness, good operational stability and mild reaction conditions.

Production of Transgenic Goats by Sperm-mediated Exogenous DNA Transfer Method

Zhao, Yongju,Wei, Hong,Wang, Yong,Wang, Lingbin,Yu, Mingju,Fan, Jingsheng,Zheng, Shuangyan,Zhao, Cong Asian Australasian Association of Animal Productio 2010 Animal Bioscience Vol.23 No.1

In this study, the production of transgenic goats using sperm to integrate exogenous DNA and artificial insemination (AI) was carried out and the technical protocols for sperm-mediated gene transfer (SMGT) in the goat were optimized. The standard sperm parameters and the ability to bind foreign genes were assessed to select suitable sperm donor bucks. A total of 134 oestrous does were divided into 4 groups and inseminated using different methods and sperm numbers. The does of Groups I to III were inseminated with fresh semen ($1-2\times10^{7}$ and $10^{6}$ sperm) or frozen-thawed semen ($10^{6}$ sperm), respectively, through conventional intra-cervical AI, and the does of Group IV with frozen-thawed semen ($10^{6}$ sperm) through intrauterine AI. Total genomic DNAs were extracted from ear biopsies of the offspring. The presence of $pEGFP-N_{1}$ DNA was screened by PCR and then by Southern blotting analysis. A total of 76 live kids were produced and 8 kids were tested transgene positive on the basis of agarose gel electrophoresis of the PCR-amplified fragment. Southern blotting analysis of the samples showed 5 positive kids. A transgenic ratio of 10.53% was detected using PCR and 6.58% using Southern blotting. The positive kid rate assayed by PCR and Southern blotting of frozen-thawed goat semen was 3.61% and 9.27% higher than that of untreated semen. The results show that transgenic goats can be produced efficiently by the method of artificial insemination using sperm cells to integrate the exogenous DNA and intrauterine insemination allowed low numbers of DNA-transfected spermatozoa to be used, with satisfactory fertility.