Jiangnan University

Junling Guo

Li Zhou

Wenjing Cui

Zhongmei Liu

Zhemin Zhou

Chao Wang

Ting Li

Laichuang Han

Yu Yan

Wenqi Ye

Yufeng Liu

Yao Lan

Dezhi Chen

Sailan Zhang

Long Yu

Zhongyi Cheng

The present disclosure discloses a method for constructing an efficientpromoter, and belongs to the technical field of gene engineering. According to the present disclosure, natural promoters identified by different sigma subunits are connected in series to obtain some double-series and triple-series promoters, the lengths of intervening sequences between core areas of the promoters are optimized on the basis of series connection of the promoters to further improve the activity of the promoters, finally, different RBS designs are performed on the promoters, and it is verified that this strategy can not only improve the compatibility between the promoters and other gene expression regulating and controlling elements, but also controllably regulate the expression of exogenous genes. Through the method provided by the present disclosure, people can obtain the promoters with higher activity and stronger designability and compatibility through simple and convenient promoter design and modification methods. The method is simple and easy to implement and has wide application prospects in the construction of an exogenous protein efficient expression system and synthetic biology research.

Method for constructing efficientpromoter

The disclosure discloses a mutant of nitrile hydratase derived from, and belongs to the technical field of enzyme engineering. The nitrile hydratase mutantNhase-A20V provided by the disclosure has a half-life of about 10 min at 70° C., which does not change much compared with the thermal stability of the wild enzyme. The specific enzyme activity of the mutantNhase-A20V is 128% of that of the wild enzyme. At the same time, the mutant also has better tolerance to a substrate and a product, and the final yield of nicotinamide produced by whole-cell catalysis reaches 598 g/L. Therefore, the nitrile hydratase mutantNhase-A20V provided by the disclosure has good enzymatic properties and is beneficial to future industrial production.

Mutant of nitrile hydratase derived from

Disclosed is a double enzyme tandem preparation method of L-2-aminobutyric acid, and belongs to the field of bioengineering. In the disclosure, recombinantexpressing L-glutamate mutase and recombinantexpressing L-aspartate-β-decarboxylase are separately cultured to obtain L-glutamate mutase and L-aspartate-β-decarboxylase. The two enzymes are added to a reaction system at a certain mass ratio, and L-glutamate is used as a substrate to carry out an enzyme reaction to prepare the L-2-aminobutyric acid. When the dosage of the L-aspartate-β-decarboxylase is 2 mg/mL, and the reaction time is 24 h, 8.5 mmol/L L-2-aminobutyric acid is produced by conversion, with a molar conversion rate of 85.00%. Compared with a chemical production method, the method disclosed by the disclosure has a safe production process and no environmental pollution. Compared with a multi-enzyme synthesis system with threonine as a substrate, the substrate is cheaper and the process is simpler.

Double enzyme tandem preparation method of l-2-aminobutyric acid

The disclosure discloses an L-aspartate &#x3b1;-decarboxylase mutant and application thereof, and belongs to the technical field of enzyme engineering. In the disclosure, lysine at position 221 of L-aspartate &#x3b1;-decarboxylase is mutated to arginine, glycine at position 369 is mutated to alanine, and the obtained new mutant enzymes have better temperature tolerance and are beneficial to industrial production. The K221R and G369A recombinant strains are subjected to high-density fermentation, and with sodium L-aspartate as a substrate, a whole cell catalytic reaction is carried out to prepare &#x3b2;-alanine. Compared with a chemical production method, the method has the advantages that the production process is safe and clean, and has no environmental pollution. Compared with a pure enzyme catalysis method, the method has the advantages that the operation is simple and convenient. The yield of the final product &#x3b2;-alanine reaches 91% and 90% respectively, and the concentration reaches 162.15 g/L and 160.42 g/L respectively.

L-aspartate alpha-decarboxylase mutant and application thereof

The disclosure discloses a nitrile hydratase mutant, a genetically engineered bacterium containing the mutant and applications thereof, and belongs to the technical field of enzyme engineering. In the disclosure, glycine at position 47 of a nitrile hydratase mutant &#x3b1;L6T/A19V/F126Y-&#x3b2;M46K/E108R/S212Y (disclosed in the patent of disclosure CN102216455A) is mutated to asparagine. The obtained new mutant enzyme has better temperature tolerance and tolerance to a product, and is conducive to future industrial production. The recombinant strain containing the nitrile hydratase mutant is fermented at high density, and 3-cyanopyridine is used as a substrate to carry out a whole-cell catalytic reaction to prepare nicotinamide. Compared with a chemical production method, the method has a safe and clean production process and no environmental pollution. Compared with an enzymatic method, the substrate price is cheap and the catalytic efficiency is high. The yield of the final product nicotinamide is over 95%, the concentration reaches 680 g/L, and the separation and purification steps of the product are simplified.

Nitrile hydratase mutant, genetically engineered bacterium containing mutant and applications thereof

The invention relates to the technical field of bioengineering, and discloses a method for synthesizing L-aspartic acid with maleic acid by whole-cell biocatalysis. In the invention, a recombinant strain co-expressing maleate cis-trans isomerase and L-aspartate lyase is constructed, and engineered and optimized to produce L-aspartic acid from maleic acid with a high conversion rate by whole-cell catalyzing. Relatively inexpensive maleic acid is utilized by the recombinant strain to produce L-aspartic acid, where maleic acid is reacted completely in 40-120 min, there is almost no buildup of the intermediate fumaric acid, and the conversion rate is up to 98% or more.

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Wuxi, China

Junling Guo