公开(公告)号：US20150031085A1

公开(公告)日：2015-01-29

申请号：US14251579

申请日：2014-04-12

申请人： Jian Chen ,  Guocheng Du ,  Zhen Kang ,  Peng Jin

发明人： Jian Chen ,  Guocheng Du ,  Zhen Kang ,  Peng Jin

IPC分类号： C12N9/24 ,  C12P19/26

CPC分类号： C12N9/2402 ,  C12P19/26 ,  C12P21/02 ,  C12Y302/01036

摘要： The present invention provides a novel leech HAase and a method of producing low-molecular-weight HA oligosaccharides using the leech HAase. This invention successfully cloned the first leech HAase gene and provides a method for high-level expression of the leech HAase gene. By controlling the incubation condition, different HA oligosaccharides, particularly HA4, HA6, HA8 and HA10, can be selectively generated using the leech HAase. The large-scale expression of the leech HAase and the enzymatic production of specific HA oligosaccharides are not only useful for the cosmetic, healthcare and the medical industries but also can be a great help to polysaccharides chemical synthesis and cancer research.

摘要翻译： 本发明提供了一种新的水蛭HAase和使用该水蛭HAase生产低分子量HA寡糖的方法。 本发明成功克隆了第一个水蛭HAase基因，提供了一种高水平表达水蛭HAase基因的方法。 通过控制孵育条件，可以使用水蛭HAase选择性地产生不同的HA寡糖，特别是HA4，HA6，HA8和HA10。 水蛭HAase的大规模表达和特异性HA寡糖的酶促生产不仅可用于化妆品，保健和医疗行业，而且可以有助于多糖化学合成和癌症研究。

公开(公告)号：US20160319272A1

公开(公告)日：2016-11-03

申请号：US15140408

申请日：2016-04-27

申请人： Zhen Kang ,  Jian Chen ,  Guocheng Du ,  Peng Jin

发明人： Zhen Kang ,  Jian Chen ,  Guocheng Du ,  Peng Jin

IPC分类号： C12N15/10

CPC分类号： C12N15/1058 ,  C12N15/1031 ,  C12Q2521/101 ,  C12Q2521/501 ,  C12Q2531/113 ,  C12Q2533/101 ,  C12Q2563/179

摘要： The present invention provides a method of rapid directed DNA evolution based on single-stranded combinatorial DNA mutant library. The single- and double-stranded mutant library are constructed either separately or simultaneously using editing primers that contain mutated nucleotides and are targeted to different regions of the parent DNA sequence. The mutant library is then inserted into expression vectors and mutants with desired property are obtained by high throughput screening. Evolution of promoters, enzymes and metabolic pathways were successfully achieved using this method and mutants with excellent properties were obtained. The method of the present invention is simple, rapid, and efficient. It can be used for directed evolution of regulatory sequence such as promoters and ribosome binding sites, and is especially suitable for introducing diverse mutations into protein encoding genes, leading to rapid directed evolution of gene of interest.

摘要翻译： 本发明提供了基于单链组合DNA突变体文库的快速定向DNA进化的方法。 使用含有突变核苷酸的编辑引物单独或同时构建单链和双链突变体文库，并且靶向母DNA序列的不同区域。 然后将突变型文库插入表达载体中，并通过高通量筛选获得具有所需性质的突变体。 使用该方法成功实现了启动子，酶和代谢途径的进化，获得了具有优异性能的突变体。 本发明的方法简单，快速，高效。 它可以用于调节序列的定向进化，如启动子和核糖体结合位点，特别适用于将多种突变引入到蛋白质编码基因中，导致感兴趣的基因的快速定向进化。

公开(公告)号：US10793888B2

公开(公告)日：2020-10-06

申请号：US15347750

申请日：2016-11-09

申请人： Zhen Kang ,  Jian Chen ,  Peng Jin ,  Guocheng Du ,  Wenwen Ding

发明人： Zhen Kang ,  Jian Chen ,  Peng Jin ,  Guocheng Du ,  Wenwen Ding

IPC分类号： C12P19/34 ,  C12Q1/6806 ,  C12N15/10

摘要： The present invention provides a method for scarless in vitro DNA assembly using thermostable exonucleases and ligase, which relates to the field of genetic engineering. The present invention provides a fast method for assembling DNA subfragments with homologous ends, which employs thermostable polymerases and ligase in a thermal cycle of denaturation, annealing, digestion and ligation. After denaturation, DNA subfragments are assembled together via annealing of the homologous end sequences, the unpaired single-stranded overhangs are digested by polymerases, and the resulting nicked gaps are sealed by a ligase. Using this method, 2-6 DNA subfragments were successfully assembled within two hours. This method can be used in conventional DNA recombination and be adapted to high throughput assembly operations. In addition, combinatorial mutations can be easily introduced into the assembled sequence by use of primers with mutated bases. It is particularly suitable for making enzyme and synthetic pathways mutation libraries with high diversity, which can be used in directed evolution to screen for enzymes and synthetic pathways with desirable properties.

公开(公告)号：US09771607B2

公开(公告)日：2017-09-26

申请号：US15011509

申请日：2016-01-30

申请人： Jian Chen ,  Zhen Kang ,  Guocheng Du ,  Peng Jin

发明人： Jian Chen ,  Zhen Kang ,  Guocheng Du ,  Peng Jin

IPC分类号： C07K14/00 ,  C12P19/26 ,  C12N9/26 ,  C12N9/04 ,  C12N9/12 ,  C12N9/90 ,  C12N9/10

CPC分类号： C12P19/26 ,  C12N9/0006 ,  C12N9/1096 ,  C12N9/1241 ,  C12N9/2474 ,  C12N9/90 ,  C12Y101/01022 ,  C12Y206/01016 ,  C12Y207/07009 ,  C12Y207/07023 ,  C12Y302/01035 ,  C12Y504/0201

摘要： The present invention relates to the field of biotechnology engineering. It provides a method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids. By integranted expression of hasA from Streptococcus zooepidemicus and overexpression of genes of HA synthetic pathway, tuaD, glmU and glmS, high yield HA production was achieved in the recombinant strain. Additionally, introduction and functional expression of the leech hyaluronidase in the recombinant strain substantially increased the yield of HA to 19.38 g·L−1. Moreover, HAs with a broad range of molecular weights (103 Da to 106 MDa) were efficiently produced by controlling the expression level of hyaluronidase using RBS mutants with different translational strengths. The method of the present invention can be used to produce low molecular weight HAs at large scale in industrial applications.

公开(公告)号：US20170175156A1

公开(公告)日：2017-06-22

申请号：US15347750

申请日：2016-11-09

申请人： Zhen Kang ,  Jian Chen ,  Peng Jin ,  Guocheng Du ,  Wenwen Ding

发明人： Zhen Kang ,  Jian Chen ,  Peng Jin ,  Guocheng Du ,  Wenwen Ding

IPC分类号： C12P19/34

CPC分类号： C12P19/34 ,  C12N15/1031 ,  C12Q1/6806 ,  C12Q2521/319 ,  C12Q2521/501 ,  C12Q2531/113

摘要： The present invention provides a method for scarless in vitro DNA assembly using thermostable exonucleases and ligase, which relates to the field of genetic engineering. The present invention provides a fast method for assembling DNA subfragments with homologous ends, which employs thermostable polymerases and ligase in a thermal cycle of denaturation, annealing, digestion and ligation. After denaturation, DNA subfragments are assembled together via annealing of the homologous end sequences, the unpaired single-stranded overhangs are digested by polymerases, and the resulting nicked gaps are sealed by a ligase. Using this method, 2-6 DNA subfragments were successfully assembled within two hours. This method can be used in conventional DNA recombination and be adapted to high throughput assembly operations. In addition, combinatorial mutations can be easily introduced into the assembled sequence by use of primers with mutated bases. It is particularly suitable for making enzyme and synthetic pathways mutation libraries with high diversity, which can be used in directed evolution to screen for enzymes and synthetic pathways with desirable properties.

公开(公告)号：US20170073719A1

公开(公告)日：2017-03-16

申请号：US15011509

申请日：2016-01-30

申请人： Jian Chen ,  Zhen Kang ,  Guocheng Du ,  Peng Jin

发明人： Jian Chen ,  Zhen Kang ,  Guocheng Du ,  Peng Jin

IPC分类号： C12P19/26 ,  C12N9/10 ,  C12N9/12 ,  C12N9/90 ,  C12N9/26 ,  C12N9/04

CPC分类号： C12P19/26 ,  C12N9/0006 ,  C12N9/1096 ,  C12N9/1241 ,  C12N9/2474 ,  C12N9/90 ,  C12Y101/01022 ,  C12Y206/01016 ,  C12Y207/07009 ,  C12Y207/07023 ,  C12Y302/01035 ,  C12Y504/0201

摘要： The present invention relates to the field of biotechnology engineering. It provides a method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids. By integranted expression of hasA from Streptococcus zooepidemicus and overexpression of genes of HA synthetic pathway, tuaD, glmU and glmS, high yield HA production was achieved in the recombinant strain. Additionally, introduction and functional expression of the leech hyaluronidase in the recombinant strain substantially increased the yield of HA to 19.38 g·L−1. Moreover, HAs with a broad range of molecular weights (103 Da to 106 MDa) were efficiently produced by controlling the expression level of hyaluronidase using RBS mutants with different translational strengths. The method of the present invention can be used to produce low molecular weight HAs at large scale in industrial applications.

摘要翻译： 本发明涉及生物技术工程领域。 它提供了构建能产生特定分子量透明质酸的重组枯草芽孢杆菌的方法。 通过来自猪链球菌的hasA的整合表达和HA合成途径，tuaD，glmU和glmS的基因的过表达，在重组菌株中实现了高产量的HA产生。 此外，重组菌株中水蛭透明质酸酶的引入和功能表达显着提高了HA的产量至19.38g·L-1。 此外，通过使用具有不同平移强度的RBS突变体控制透明质酸酶的表达水平，有效地产生具有宽范围分子量（103Da至106Mpa）的HA。 本发明的方法可用于在工业应用中大规模生产低分子量的HAs。

公开(公告)号：US09279111B2

公开(公告)日：2016-03-08

申请号：US14251579

申请日：2014-04-12

申请人： Jian Chen ,  Guocheng Du ,  Zhen Kang ,  Peng Jin

发明人： Jian Chen ,  Guocheng Du ,  Zhen Kang ,  Peng Jin

IPC分类号： C12N9/24 ,  C12P19/26 ,  C12P21/02

CPC分类号： C12N9/2402 ,  C12P19/26 ,  C12P21/02 ,  C12Y302/01036

摘要： The present invention provides a novel leech HAase and a method of producing low-molecular-weight HA oligosaccharides using the leech HAase. This invention successfully cloned the first leech HAase gene and provides a method for high-level expression of the leech HAase gene. By controlling the incubation condition, different HA oligosaccharides, particularly HA4, HA6, HA8 and HA10, can be selectively generated using the leech HAase. The large-scale expression of the leech HAase and the enzymatic production of specific HA oligosaccharides are not only useful for the cosmetic, healthcare and the medical industries but also can be a great help to polysaccharides chemical synthesis and cancer research.

摘要翻译： 本发明提供了一种新的水蛭HAase和使用该水蛭HAase生产低分子量HA寡糖的方法。 本发明成功克隆了第一个水蛭HAase基因，提供了一种高水平表达水蛭HAase基因的方法。 通过控制孵育条件，可以使用水蛭HAase选择性地产生不同的HA寡糖，特别是HA4，HA6，HA8和HA10。 水蛭HAase的大规模表达和特异性HA寡糖的酶促生产不仅可用于化妆品，保健和医疗行业，而且可以有助于多糖化学合成和癌症研究。

公开(公告)号：US20160138057A1

公开(公告)日：2016-05-19

申请号：US14557468

申请日：2014-12-02

申请人： Jian Chen ,  Jingwen Zhou ,  Hongwei Guo ,  Weizhu Zeng ,  Guocheng Du

发明人： Jian Chen ,  Jingwen Zhou ,  Hongwei Guo ,  Weizhu Zeng ,  Guocheng Du

IPC分类号： C12P7/50 ,  C12N9/06

CPC分类号： C12P7/50 ,  C12N9/0016 ,  C12Y104/01002

摘要： The present invention provides methods for enhancing α-KG production in Yarrowia lipolytica, relates to the field of metabolic engineering. This invention successfully overexpresses the glutamate dehydrogenase in wild type strain Y. lipolytica WSH-Z06 to construct the recombinant Y. lipolytica WSH-Z06 which regulates the glutamate catabolism to synthesis α-KG. L-methionine imine is added into the fermentation medium during the process to strengthen the supply of intracellular glutamate and inhibite the intracellular glutamine synthesis from glutamate metabolism and then enhance the accumunation of α-KG. Therefor, the present invention provides an effective method for enhancing the accumunation of α-KG through regulation of intracellular amino acid metabolism.

摘要翻译： 本发明提供了在解脂耶氏酵母中增强α-KG生产的方法，涉及代谢工程领域。 本发明成功地过表达了野生型脂肪分解杆菌WSH-Z06中的谷氨酸脱氢酶，构建了重组解脂亚罗威阿酵母WSH-Z06，其调节合成α-KG的谷氨酸分解代谢。 在加工过程中将L-甲硫氨酸亚胺加入到发酵培养基中，以加强细胞内谷氨酸的供应，并从谷氨酸代谢抑制胞内谷氨酰胺合成，从而增强α-KG的积累。 因此，本发明提供了通过调节细胞内氨基酸代谢来增强α-KG的积累的有效方法。

公开(公告)号：US09187773B2

公开(公告)日：2015-11-17

申请号：US14047968

申请日：2013-10-07

申请人： Jian Chen ,  Guocheng Du ,  Long Liu ,  Jianghua Li ,  Ningzi Guan

发明人： Jian Chen ,  Guocheng Du ,  Long Liu ,  Jianghua Li ,  Ningzi Guan

IPC分类号： C12P7/52

CPC分类号： C12P7/52

摘要： The invention provides a simple and effective method for improving acid tolerance of P. acidipropionici by adding arginine and/or aspartic acid to the culture medium. The acid tolerance of P. acidipropionici was improved by 60% and 20% respectively through adding arginine or aspartic acid into the culture medium. Consequently, PA production was improved by 36% and 26%, respectively. The maximal PA production was obtained by adding both 20 mM arginine and 20 mM aspartic acid. This method can be applied to large scale production of PA.

摘要翻译： 本发明提供了一种通过向培养基中加入精氨酸和/或天冬氨酸来改善酸性丙酸酸的耐酸性的简单和有效的方法。 通过向培养基中加入精氨酸或天冬氨酸，分别将酸性丙酸的耐酸性提高了60％和20％。 因此，PA产量分别提高了36％和26％。 通过加入20mM精氨酸和20mM天冬氨酸获得最大的PA产生。 该方法可应用于大规模生产PA。

公开(公告)号：US09040274B2

公开(公告)日：2015-05-26

申请号：US13870509

申请日：2013-04-25

申请人： Jian Chen ,  Guocheng Du ,  Xinyao Lu ,  Song Liu ,  Juan Zhang

发明人： Jian Chen ,  Guocheng Du ,  Xinyao Lu ,  Song Liu ,  Juan Zhang

IPC分类号： C12N9/96 ,  C12N9/02 ,  C12N9/26 ,  C12N9/10

CPC分类号： C12N9/96 ,  C12N9/0069 ,  C12N9/1044 ,  C12N9/2411

摘要： The invention provides a simple and effective method for increasing thermal stability of a wide range of proteins, comprising fusing a self-assembling amphipathic peptide to the C- or N-terminal of target proteins. The fusion protein can have a half life up to 26 times longer than that of the wild type protein.

摘要翻译： 本发明提供了一种用于增加大范围蛋白质的热稳定性的简单且有效的方法，其包括将自组装两亲肽融合到靶蛋白的C-或N-末端。 融合蛋白的半衰期比野生型蛋白质长达26倍。