Jiangnan University

Roquette Freres

Wanmeng Mu

Tao Zhang

Bo Jiang

Ming Miao

Hangyu Jiang

Yingying Zhu

Pierre Lanos

Liuming Zhou

Wenli Zhang

Min Jia

Shuhuai Yu

Guocong Luo

Zeyu Li

Disclosed are a construction method and application of a microorganism capable of realizing high production of lacto-N-neotetraose, belonging to the field of microbial genetic engineering. Coding genes of ß-1,3-acetyl glucosamine transferase, ß-1,4-galactosyl transferase and/or UDP-glucose 4 epimerase are over-expressed on the basis of a strain which is previously constructed by the team and is subjected to related-gene knockout, thus enabling the strain to have a synthesis capability of producing the lacto-N-neotetraose. The present disclosure accurately regulates the carbon flux of a metabolic pathway and relieves the metabolic stress by screening the high-efficiency ß-1,4-galactosyl transferase gene and regulating the expression of IgtA, Aa-β-1,4-GalT and galE in a lacto-N-neotetraose synthesis pathway in a combined manner. In a shake flask experiment, the lacto-N-neotetraose production capacity ofis 0.91 g/L. The lacto-N-neotetraose yield in a 3 L fermentation tank reaches 12.14 g/L. Therefore, the microorganism has an industrial application prospect.

Construction method and application of microorganism capable of realizing high production of lacto-N-neotetraose

Disclosed are a construction method and application of a microorganism capable of realizing high production of lacto-N-tetrose, belonging to the field of microbial genetic engineering. The present disclosure employs a strain which has been constructed in the early stage for efficiently producing a precursor lacto-N-triose II as an original strain to synthesize a key gene of the lacto-N-tetrose via over-expression, thus enabling the strain to have a synthesis capability of producing the lacto-N-tetrose. The present disclosure improves the synthesis of the lacto-N-tetrose by screening a high-efficiency β-1,3-galactosyl transferase gene, and reasonably designing the co-expression of the β-1,3-galactosyl transferase gene and a key UDP-glucose 4 epimerase gene (galE) for strengthening a UDP-galactose pathway on a vector pCDFDuet-1. In a shake flask experiment, the lacto-N-tetrose production capacity ofis 3.04 g/L. The lacto-N-tetrose yield in a 3 L fermentation tank reaches 25.49 g/L. Therefore, the microorganism has an industrial application prospect.

Construction method and application of microorganism capable of realizing high production of lacto-N-tetrose

A genetically engineered arginine deiminase reconstructed by site-directed mutagenesis belongs to the technical field of genetic engineering technology. Its amino acid sequence is shown as SEQ ID No. 1. In the amino acid sequence of the arginine deiminase reconstructed by site-directed mutagenesis, glycine at position 264 is mutated to proline, compared to an amino acid sequence of native arginine deiminase. Compared with wild type enzyme, the effective pH range effect of the mutated arginine deiminase according to the present invention is broadened to a certain extent, and especially a good enzyme activity is achieved at physiological pH 7.4. With the broadening of the effective pH effect range, the mutant enzyme still has higher stability under the condition of pH 5.5-7.5. Therefore, the problem that the arginine deiminase generally is low in enzymatic activity and short in half-life in vivo under physiological conditions in clinical application for tumor therapy is solved, and a good condition for using the enzyme and an encoding gene thereof for clinical treatment is created.

Genetically engineered arginine deiminase modified by site-directed mutagenesis

An arginine deiminase mutant with improved enzyme activity and temperature stability and application thereof were provided, belonging to the technical field of genetic engineering and enzyme engineering. The arginine deiminase mutant is proline, namely Gly292 Pro, mutated from glycine near an enzyme active center. A wild-type arginine deiminase arcA coding gene is molecularly modified by a site-directed mutation technique to obtain a mutant enzyme ADIG292P, which has glycine at position 292 of an amino acid sequence of the wild type arginine deiminase mutated to proline. The arginine deiminase, modified by site-directed mutation, of the present invention has 1.5 times of increase in enzyme activity and 5.43 times of increase in half-life period at 40&#xb0; C. compared with the wild-type enzyme, which solves the problems of low catalytic ability and temperature stability during the catalytic synthesis of citrulline using arginine deiminase, and lays a foundation for industrial production of efficient synthesis of citrulline and medication application.

Arginine deiminase mutant methods of using the same

The present invention relates to an improved variant of a D-psicose 3-epimerase and its uses.

Variant of D-psicose 3-epimerase and uses thereof

Arginine deiminase mutant and methods of using the same

The invention discloses a high-efficiency synthesis method of difructose anhydride III. The method comprises the following steps: firstly converting sucrose into inulin by using inulosucrase without separating polysaccharide, and then converting inulin by using inulin fructotransferase to synthesize the functional disaccharide difructose anhydride III. The method has the advantages of simple process and high efficiency, and a conversion rate of synthesizing inulin into difructose anhydride III can reach 40%-54%. In order to obtain purer difructose anhydride III, yeast is utilized to remove small molecule monosaccharides in a reaction solution, and the finally obtained purer difructose anhydride III can be easily separated and purified. Thus, the method has broad market application prospects.

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Wuxi, China

Wanmeng Mu