Candida tropicalis genetically engineered bacterium with high yield of xylitol, and preparation method and application thereof
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A technology of Candida tropicalis and genetically engineered bacteria, applied in the directions of microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve problems such as reduced xylitol production
Inactive Publication Date: 2014-03-05
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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Studies have found that the biosynthesis of xylitol is closely connected with the pentose phosphate pathway (PPP), which requires the participation of xylose reductase and reducing coenzyme NADPH (xylose + NADPH Xylitol+NADP+), this reduction reaction is a key step for the smooth and continuous synthesis of xylitol. NADPH is mainly produced through the PPP pathway. If the PPP pathway is destroyed, the xylitol production will be significantly reduced.
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[0012] The present invention is a kind of Candida tropicalis genetically engineered bacteria with high yield of xylitol and its preparation method and application, specific implementation mode (technical scheme):
[0013] (1) PCR technology amplifies the g6pd gene from the Candida tropicalis DNA template, connects it to the shuttle plasmid expression vector pYES-pgk, and then introduces the recombinant vector pYES-pgk-g6pd into C.tropicalis CT 16 , the g6pd gene was overexpressed, positive transformants were screened, and the C.tropicalis engineering bacterium SYG5 with high xylitol production was constructed.
[0014] (2) Analysis of g6pd gene transcription level, G6PDH enzyme expression level and metabolic flux distribution of engineering bacteria SYG5.
[0015] (3) Using xylose as the main raw material, optimizing the fermentation medium and fermentation conditions of engineering bacteria, and using a fermenter (bioreactor) to ferment and produce xylitol.
[0016] technica...
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Abstract
The invention relates to a Candida tropicalis genetically engineered bacterium with high yield of xylitol, and a preparation method and application thereof, and belongs to the technical field of food sweetener biological manufacturing. The prepared xylitol can be widely used in the industries of food, medicine and daily chemicals. Taking Candida tropicalis CT16 as a starting strain, the method designs and constructs an engineering bacterium with glucose-6-phosphate dehydrogenase gene g6pd overexpression in xylitol synthetic and metabolic pathways, so as to enhance copy and expression of g6pd gene in the synthetic pathway; and the bred engineering bacterium (transformant) C.tropicalis SYG5 after several passages shows genetic stability. Therefore, the invention optimizes metabolic network and metabolic pathways in xylitol biosynthesis, obviously increases metabolic flux and yield of xylitol, and provides excellent strains and technical support for biological preparation of xylitol.
Description
technical field [0001] The invention relates to a Candida tropicalis genetically engineered bacterium with high xylitol production and its preparation method and application, belonging to the field of edible sweetener biomanufacturing, and the prepared xylitol can be widely used in food, medicine, daily chemical and other industries . Background technique [0002] Xylitol (Xylitol), also known as pentapentyl alcohol, is a five-carbon polyhydric sugar alcohol with a chemical formula of C 5 h 12 o 5 , the shape is crystalline powder or white crystal, easily soluble in water, sweet, comparable to sucrose, slightly soluble in methanol and ethanol, good thermal stability. Xylitol can be metabolized in the human body without the participation of insulin, and will not cause rapid changes in human blood sugar. Therefore, it can be used as a sweetener for diabetic foods, and has been widely used in chewing gum, chewing gum, toffee, etc. Sugar, fudge, chocolate, jelly, cold drink,...
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