Method for synthesis of D-psicose by aldolase whole cell
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A phosphate aldolase and fructose technology, which is applied in the biological field to achieve the effects of easy separation and purification, wide application prospects, and efficient synthesis method
Active Publication Date: 2015-05-13
天津怡和生物科技有限责任公司
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[0032] Corynebacterium glutamicum ( Corynebacterium glutamicum ) The construction of recombinant bacterial strain SY12 comprises the following steps:
[0033] 1. Construct the vector pXFTY of L-fucose-1-phosphate aldolase gene and fructose-1-phosphorylase gene
[0034] 1.1 According to the L-fucose-1-phosphate aldolase gene (Genbank number: 947282, 648bp, sequence 1 in the sequence listing) and fructose-1-phosphorylase ( YqaB) gene (Genbank number: 945776, 567bp, sequence 2 in the sequence listing), primer 1 and primer 2, primer 5 and primer 6 were designed. According to the sequence of the tac promoter (307bp, sequence 3 in the sequence table, which is used to add the promoter before the gene YqaB) in the vector pXMJ19 in Genbank, primer 3 and primer 4 were designed, and primer 1 and primer 5 contained the RBS site sequence (AAGGAGATATAG ), Primer 1 and P...
Embodiment 2
[0052] Embodiment 2, Corynebacterium glutamicum ( Corynebacterium glutamicum ) Application of recombinant strain SY12 in the production of D-psicose
[0053] 1. Cultivation and induction of the recombinant strain SY12 of Corynebacterium glutamicum
[0054] Select CGXII medium (recipe: (NH 4 ) 2 SO 4 (5g / L), urea (5g / L), KH 2 PO 4 (1g / L), K 2 HPO 4 (1g / L), MgSO 4 ?7H 2 O (0.25g / L), CaCl 2 (10mg / L), FeSO 4 ?7H 2 O (10mg / L), MnSO 4 ?H 2 O (0.1mg / L), ZnSO 4 ?7H 2 O (1mg / L), CuSO 4 ?5H 2 O (0.2mg / L), NiCl 2 ?6H 2 O (20μg / L), biotin (0.4mg / L), MOPS (42g / L) (pH 7.4)), glucose (10g / L) and chloramphenicol (12.5mg / L) were added to the medium. Cultivate the recombinant strain SY12 of Corynebacterium glutamicum at 30°C and 200rmp, when OD 600 When it reaches 0.6-0.8, add IPTG, the final concentration is 1mM, reduce the shaker speed to 120rmp, and induce for about 12h.
[0055] 2. Collection and concentration of the recombinant strain SY12 of Corynebacterium glu...
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Abstract
The invention discloses a method for synthesis of D-psicose by microbiological fermentation, specifically a method for synthesis of D-psicose by aldolase whole cell. The method includes: firstly constructing recombinant corynebacterium glutamicum SY12 carrying L-fucose-1-phosphate aldolase gene and fructose-1-phosphorylase gene (i.e. with aldehyde condensation approach), then adding glucose and D-glyceraldehyde into a basic salt medium, synthesizing dihydroxyacetone phosphate from glucose by means of intracellular glycolysis, synthesizing a single product D-psicose from D-glyceraldehyde and dihydroxyacetone phosphate (DHAP) under the action of the L-fucose-1-phosphate aldolase and fructose-1-phosphorylase carried by the corynebacterium glutamicum recombinant strain SY12, with the conversion rate of D-glyceraldehyde being 53%. Therefore, compared with the existing method for in vitro synthesis of D-psicose by ketose3-epimerase, the biosynthesis method of D-psicose provided by the invention has the advantages of high conversion rate, single product, easy separation and the like, and lays certain foundation for mass production of D-psicose.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for synthesizing D-psicose in whole cells using aldolase. That is, the recombinant strain of Corynebacterium glutamicum is obtained by using genetic engineering technology and microbiological methods, and the recombinant strain is used to efficiently synthesize D-psicose. Background technique [0002] Rare sugar is a type of monosaccharide and its derivatives that exist in nature but are very low in content (defined by ISRS in 2002). It has the advantages of low calorie, high stability, no cariogenicity, and high tolerance, and has attracted widespread attention in the fields of diet, health care, and medicine. They can be used as additives to improve the physical and chemical properties of food, improve the physiological function and health care of food Function. In recent years, the rare sugars that scientists have devoted to research mainly include D-Allose (D-Allose), ...
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