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定点突变 2

丝孢堆黑粉菌 1

二聚体 1

分子动力学模拟 1

分子对接 1

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理性设计 1

脂肪酶 1

莫西沙星 1

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Combination of ARTP mutagenesis and color-mediated high-throughput screening to enhance 1-naphthol yield

Chenggang Qiu, Alei Zhang, Sha Tao, Kang Li, Kequan Chen, Pingkai Ouyang

《化学科学与工程前沿（英文）》 2020年 第14卷 第5期   页码 793-801 doi: 10.1007/s11705-019-1876-2

摘要： Strain QCG of the aerobic bacteria is capable of producing 1-naphthol from naphthalene, this strain was first isolated and characterized in this study. Strain QCG was mutagenized to enhance 1-naphthol production, using atmospheric and room temperature plasma (ARTP) technology. Then, a microbial clone screening system was used to accelerate the operation. Meanwhile, a novel color-mediated high-throughput screening using 4-aminoantipyrine was performed to screen mutants. The optimal mutant strain QCG4 produced 19.58±0.34 mg∙L 1-naphthol from naphthalene that was 47.32% higher than that of the original strain (13.29±0.28 mg∙L ). In addition, the optimal conditions for 1-naphthol production via whole-cell catalysis of strain QCG4 were determined to be an OD of 40, 150 mg∙L naphthalene, and 7.5% dimethyl formamide as a co-solvent at pH 7.5 and 26°C for 3 h, resulting in 41.18±0.12 mg∙L 1-naphthol, i.e., the mutant strain produces a 2.1-fold higher yield compared to the original strain.

关键词： Bacillus cereus QCG     naphthalene     1-naphthol     ARTP mutagenesis     high-throughput screening     4-aminoantipyrine    

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analysis of with enhanced poly--glutamic acid production through atmospheric and room temperature plasma mutagenesis

《化学科学与工程前沿（英文）》 2022年 第16卷 第12期   页码 1751-1760 doi: 10.1007/s11705-022-2211-x

摘要： Poly-γ-glutamic acid is an extracellular polymeric substance with various applications owing to its valuable properties of biodegradability, flocculating activity, water solubility, and nontoxicity. However, the ability of natural strains to produce poly-γ-glutamic acid is low. Atmospheric and room temperature plasma was applied in this study to conduct mutation breeding of Bacillus licheniformis CGMCC 2876, and a mutant strain M32 with an 11% increase in poly-γ-glutamic acid was obtained. Genome resequencing analysis identified 7 nonsynonymous mutations of ppsC encoding lipopeptide synthetase associated with poly-γ-glutamic acid metabolic pathways. From molecular docking, more binding sites and higher binding energy were speculated between the mutated plipastatin synthase subunit C and glutamate, which might contribute to the higher poly-γ-glutamic acid production. Moreover, the metabolic mechanism analysis revealed that the upregulated amino acids of M32 provided substrates for glutamate and promoted the conversion between L- and D-glutamate acids. In addition, the glycolytic pathway is enhanced, leading to a better capacity for using glucose. The maximum poly-γ-glutamic acid yield of 14.08 g·L–1 was finally reached with 30 g·L–1 glutamate.

关键词： ARTP mutagenesis     Bacillus licheniformis     poly-γ-glutamic acid     metabolomics    

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Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity

Chuanfeng Wu, Cynthia E. Dunbar

《医学前沿（英文）》 2011年 第5卷 第4期   页码 356-371 doi: 10.1007/s11684-011-0159-1

摘要： Virus-based vectors are widely used in hematopoietic stem cell (HSC) gene therapy, and have the ability to integrate permanently into genomic DNA, thus driving long-term expression of corrective genes in all hematopoietic lineages. To date, HSC gene therapy has been successfully employed in the clinic for improving clinical outcomes in small numbers of patients with X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy (ALD), thalassemia, chronic granulomatous disease (CGD), and Wiskott-Aldrich syndrome (WAS). However, adverse events were observed during some of these HSC gene therapy clinical trials, linked to insertional activation of proto-oncogenes by integrated proviral vectors leading to clonal expansion and eventual development of leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity, with the aim of developing safer vectors and lower-risk gene therapy protocols. This review will summarize current information on the mechanisms of insertional mutagenesis in hematopoietic stem and progenitor cells due to integrating gene transfer vectors, discuss the available assays for predicting genotoxicity and mapping vector integration sites, and introduce newly-developed approaches for minimizing genotoxicity as a way to further move HSC gene therapy forward into broader clinical application.

关键词： gene therapy     hematopoietic stem cells     insertional mutagenesis     genotoxicity     induced pluripotent stem cell    

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Site-directed mutagenesis of long QT syndrome KCNQ1 gene

LI Wei, WANG Bin, XU Qiumei, KE Qinmei, YANG Junguo, DU Rong, TIAN Li, WANG Qing

《医学前沿（英文）》 2008年 第2卷 第1期   页码 100-104 doi: 10.1007/s11684-008-0018-x

摘要： To construct a polymerase chain reaction (PCR) site-directed mutagenesis of the long QT syndrome KCNQ1 gene , two sets of primers were designed according to the sequence of KCNQ1 cDNA and a mismatch was introduced into primers. Mutagenesis was performed in a two-step PCR. The amplified fragments from the third PCR which contained the mutation site were sub-cloned into the T-vector pCR2.1. Then, the fragments containing the mutation site was obtained from pCR2.1 using restriction enzymes digestion and inserted into the same restriction site of pIRES-EGFP-KCNQ1. The sequencing analysis shows that the mutation site was correct. Mutation from A to G in site 983 of KCNQ1 cDNA was found. Using the Effectene transfection reagent, pIRES-EGFP-KCNQ1 (G983A) was transfected into HEK cells successfully. These results may shed light on further functional study of KCNQ1 gene.

关键词： restriction     digestion     syndrome     sequence     site-directed mutagenesis    

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Mutagenesis and selective breeding of a high producing

Tian WANG, Shiru JIA, Zhilei TAN, Yujie DAI, Shuai SONG, Guoliang WANG

《化学科学与工程前沿（英文）》 2012年 第6卷 第2期   页码 179-183 doi: 10.1007/s11705-012-1273-6

摘要： -Poly- -lysine ( -PL) is an -lysine linear homopolymer, which is produced by bacteria belonging to the family and by ergot fungi. However, the production of -PL by the wild bacteria strain is very low, which limits its utilization. In most bacteria including the genus, -lysine is a precursor of -PL and is biosynthesized by the -aspartate pathway. Aspartokinase (Ask) is the first key enzyme in this pathway and is subject to complex regulation such as the feedback inhibition by the end product amino acids. In addition, phosphoenolpyruvate carboxykinase is feedback-regulated by -aspartate. To reduce these feedback inhibitions and to improve -PL productivity, resistant mutants were produced using sulfaguanidine (SG) + glycine+ -lysine+ -3-hydroxynorvaline (AHV) as selective markers. Using the interaction between -PL and the charged dye in the solid culture medium, hundreds of colonies were simultaneously screened in a quick and effective manner. Finally, one -PL-producing strain, L9, was selected. The productivity of this strain during flask fermentation was 0.77 g/L, which was 15% higher than that of the original strain. Moreover, its fermentation performance and genetic characteristics were very stable.

关键词： ?-poly-L-lysine     plasma     AHV     Streptomyces diastatochromogenes     fermentation    

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Cofactor engineering in cyanobacteria to overcome imbalance between NADPH and NADH: A mini review

Jongmoon Park,Yunnam Choi

《化学科学与工程前沿（英文）》 2017年 第11卷 第1期   页码 66-71 doi: 10.1007/s11705-016-1591-1

摘要： Cyanobacteria can produce useful renewable fuels and high-value chemicals using sunlight and atmospheric carbon dioxide by photosynthesis. Genetic manipulation has increased the variety of chemicals that cyanobacteria can produce. However, their uniquely abundant NADPH-pool, in other words insufficient supply of NADH, tends to limit their production yields in case of utilizing NADH-dependent enzyme, which is quite common in heterotrophic microbes. To overcome this cofactor imbalance and enhance cyanobacterial fuel and chemical production, various approaches for cofactor engineering have been employed. In this review, we focus on three approaches: (1) utilization of NADPH-dependent enzymes, (2) increasing NADH production, and (3) changing cofactor specificity of NADH-dependent enzymes from NADH to NADPH.

关键词： NADH-dependent enzyme     NADPH-dependent enzyme     transhydrogenase     site-directed mutagenesis     enzyme engineering    

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Improved degradation of azo dyes by lignin peroxidase following mutagenesis at two sites near the catalytic

Karla Ilić Đurđić, Raluca Ostafe, Olivera Prodanović, Aleksandra Đurđević Đelmaš, Nikolina Popović, Rainer Fischer, Stefan Schillberg, Radivoje Prodanović

《环境科学与工程前沿（英文）》 2021年 第15卷 第2期 doi: 10.1007/s11783-020-1311-4

摘要： Abstract • Mutations in Lignin peroxidase Trp171 environment improved azo dyes degradation. • Expression on yeast cell surface and cell lysis allowed reusability of biocatalyst. • Aga2-LiP chimeric variants were characterized. The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation methods. Lignin peroxidase (LiP) from Phanerochaete chrysosporium is a heme-containing lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules, including industrial dyes. This enzyme is therefore ideal as a starting point for protein engineering. Accordingly, we subjected two positions (165 and 264) in the environment of the catalytic Trp171 residue to saturation mutagenesis, and the resulting library of 104 independent clones was expressed on the surface of yeast cells. This yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more efficiently. We identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes (Evans blue, amido black 10B and Guinea green) and up to 13-fold higher catalytic activity. Additionally, cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis, achieving significant increases in both enzyme activity and stability compared to a whole-cell biocatalyst. LiP-coated cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 h. The best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.

关键词： Bioremediation     Enzyme immobilization     Protein engineering     Yeast surface display.    

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一种新型葡激酶分子的设计 基因构建 表达纯化及性质研究

宋钢,于敏,莫炜,宋后燕

《中国工程科学》 2000年 第2卷 第11期   页码 68-72

摘要：

在葡激酶构效关系的研究过程中发现，葡激酶易形成二聚体，甚至多聚体，不利于葡激酶的临床应用。为了探究聚合体的形成机制以研制不易聚合的新型葡激酶分子，在葡激酶X射线晶体衍射结构模型的基础上，采用分子对接软件GRAMM V1.03，以高分辨率整体对接方式预测了葡激酶二聚体可能的结合区。结合区主要有两种可能的形成方式，通过强疏水相互作用及氢键结合。根据模型设计了旨在降低聚合能力的葡激酶突变体RGD-Sak,将Fill置换为D111，并且改变K109为R109，恰好使分子中形成RGD结构，使新型分子还可能具有抑制血小板聚集作用。利用定点突变及DNA重组技术，构建了RGD-Sak基因，并利用大肠杆菌原核表达系统进行了高效表达。RGD-Sak以包涵体形式存在，包涵体经洗涤，8 mol/L尿素溶解，稀释复性，离子交换色谱一步分离得到电泳纯的RGD-Sak,纯度达95%以上，分子量与理论值相符，比活性5×104 HU/mg。RGD-Sak与纤溶酶形成的复合物催化纤溶酶原的Km、Kcat值分别为12.40 μmol/L、0.81 s-1。RGD-Sak显示了很弱的聚合能力。此研究为研制防止二聚体形成的新型葡激酶分子打下了基础。

关键词： 葡激酶     二聚体     分子对接     分子设计     定点突变    

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理性设计大幅提高一种丝孢堆黑粉菌来源脂肪酶催化活性——用于合成莫西沙星手性中间体 Article

蔡雪, 沈江伟, 强煜, 华婧, 马樟奇, 柳志强, 郑裕国

《工程（英文）》 2022年 第19卷 第12期   页码 207-216 doi: 10.1016/j.eng.2022.03.020

摘要：

通过脂肪酶不对称拆分外消旋N-乙酰基-哌啶-2,3-二甲酸二甲酯[cis-(±)-1]，从而获得手性中间体 (S,S)-2,8-二氮杂双环壬烷，是合成氟喹诺酮类抗生素莫西沙星重要手性中间体的一条具有吸引力的工艺路线。在前期研究中，筛选得到了一株丝孢堆黑粉菌（SRL）来源的脂肪酶，该脂肪酶具有高热稳定性和pH值稳定性。但SRL来源的脂肪酶活性较低，无法满足工业化应用的需求。因此本研究依据前期南极假丝酵母来源脂肪酶B（CALB）改造策略，采用理性设计方法对SRL进行定点突变改造，获得一个双突变体SRLI194N/V195L。随后，确定了位于活性口袋外的环6 上两个关键氨基酸残基L145 和L154；获得四位点突变体SRL-I194N/V195L/L145V/L154G（V13），活性显著提高，达到87.8 U·mg−1，是野生型SRL的2195 倍（E > 200）。该突变体在50 ℃下的半衰期达到92.5 h。突变体V13（100 mg·L−1）能够高效拆分1 mol·L−1cis-(±)-1，2 h 内底物转化率达到49.9%，实现严格立体选择性（E > 200）。总体而言，研究发现了一株对cis-(±)-1 (S,S)-2,8-二氮杂双环壬烷具有高催化活性、严格立体选择性的脂肪酶，可应用于工业化生产，并为其他具有相似结构的脂肪酶和其他种类酶的活性提高提供了一种通用策略。

关键词： 脂肪酶     丝孢堆黑粉菌     定点突变     分子动力学模拟     理性设计     莫西沙星    

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标题 作者 时间 类型 操作

Combination of ARTP mutagenesis and color-mediated high-throughput screening to enhance 1-naphthol yield

Chenggang Qiu, Alei Zhang, Sha Tao, Kang Li, Kequan Chen, Pingkai Ouyang

期刊论文

analysis of with enhanced poly--glutamic acid production through atmospheric and room temperature plasma mutagenesis

期刊论文

Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity

Chuanfeng Wu, Cynthia E. Dunbar

期刊论文

Site-directed mutagenesis of long QT syndrome KCNQ1 gene

LI Wei, WANG Bin, XU Qiumei, KE Qinmei, YANG Junguo, DU Rong, TIAN Li, WANG Qing

期刊论文

Mutagenesis and selective breeding of a high producing

Tian WANG, Shiru JIA, Zhilei TAN, Yujie DAI, Shuai SONG, Guoliang WANG

期刊论文

Cofactor engineering in cyanobacteria to overcome imbalance between NADPH and NADH: A mini review

Jongmoon Park,Yunnam Choi

期刊论文

Improved degradation of azo dyes by lignin peroxidase following mutagenesis at two sites near the catalytic

Karla Ilić Đurđić, Raluca Ostafe, Olivera Prodanović, Aleksandra Đurđević Đelmaš, Nikolina Popović, Rainer Fischer, Stefan Schillberg, Radivoje Prodanović

期刊论文

一种新型葡激酶分子的设计 基因构建 表达纯化及性质研究

宋钢,于敏,莫炜,宋后燕

期刊论文

理性设计大幅提高一种丝孢堆黑粉菌来源脂肪酶催化活性——用于合成莫西沙星手性中间体

蔡雪, 沈江伟, 强煜, 华婧, 马樟奇, 柳志强, 郑裕国

期刊论文