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Breeding method of erythritol producing strain

A technology of erythritol and erythrose, which is applied in the field of microorganisms, can solve the problems of poor strain viability, low breeding efficiency, and complicated breeding procedures, so as to reduce screening and testing work, improve breeding efficiency, and avoid blindness sexual effect

Inactive Publication Date: 2013-02-06
FUTASTE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a kind of breeding method of erythritol producing bacteria, to solve the complex breeding procedure of prior art erythritol producing bacteria, low breeding efficiency, poor strain vigor, specific strain Poor performance, low yield, low conversion rate, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] The gene sequences of erythrose 4-phosphate erythrose kinase and erythrose reductase amplified by the PCR instrument were respectively connected into the pBR327 plasmid, and then transformed into Yarrowia lipolytica. Conditions for PCR amplification: Denaturation at 90°C for 30 S, retreat at 53°C for 20 S, extension at 72°C for 30 S. The successfully transformed Yarrowia lipolytica was further enriched and cultured, made into a test tube slant and stored in a 4°C refrigerator until use;

[0013] Preparation medium: glucose monohydrate 32%, ammonium citrate 1%, magnesium sulfate heptahydrate 0.04%, copper chloride 0.0003%. Then sterilize at 110°C for 20 minutes for later use;

[0014] The selected culture temperature is 30°C, and the ventilation rate per minute is 30% of the volume of the feed liquid. The finally obtained high-yielding bacteria made the molar conversion rate of glucose to erythritol 79%.

Embodiment 2

[0016] The gene sequences of 4-phosphate erythrose kinase and erythrose reductase amplified by PCR were respectively connected into pBR327 plasmid, and then transformed into Yarrowia lipolytica. Conditions for PCR amplification: Denaturation at 90°C for 30 S, retreat at 53°C for 20 S, extension at 72°C for 30 S. The successfully transformed Yarrowia lipolytica was further enriched and cultured, made into a test tube slant and stored in a 4°C refrigerator until use;

[0017] Preparation medium: glucose monohydrate 30%, ammonium citrate 1.5%, magnesium sulfate heptahydrate 0.05%, copper chloride 0.0002%. Then sterilize at 110°C for 20 minutes for later use;

[0018] The selected culture temperature is 29°C, and the ventilation rate per minute is 25% of the volume of the feed liquid. The finally obtained high-yielding bacteria made the molar conversion rate of glucose to erythritol 84%.

Embodiment 3

[0020] The gene sequences of 4-phosphate erythrose kinase and erythrose reductase amplified by PCR were respectively connected into pBR327 plasmid, and then transformed into Yarrowia lipolytica. Conditions for PCR amplification: Denaturation at 90°C for 30 S, retreat at 53°C for 20 S, extension at 72°C for 30 S. The successfully transformed Yarrowia lipolytica was further enriched and cultured, made into a test tube slant and stored in a 4°C refrigerator until use;

[0021] Preparation medium: glucose monohydrate 31%, ammonium citrate 1.5%, magnesium sulfate heptahydrate 0.06%, copper chloride 0.0005%. Then sterilize at 110°C for 20 minutes for later use;

[0022] The selected culture temperature is 30°C, and the ventilation rate per minute is 35% of the volume of the feed liquid. The finally obtained high-yielding bacteria made the molar conversion rate of glucose to erythritol 82%.

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Abstract

The invention discloses a breeding method of an erythritol producing strain, belonging to the technical filed of microorganisms. The erythritol producing strain is reconstructed by adopting a genetic engineering means to increase gene dosage of 4-erythrose kinase phosphate and erythrose reductase; and then optimizing a component and a proportion of a culture medium, adding copper chloride in a raw material, optimizing a culture condition, and breeding Yarrowialipolytica strain with lower yield of erythritol so as to obtain a Yarrowialipolytica erythritol high-yield strain. The breeding method specifically comprises the following steps: 1, reconstructing the genetic engineering; 2, preparing a culture medium; and 3, optimizing the culture condition to obtain the high-yield erythritol producing strain. According to the invention, through the reconstruction of the genetic engineering, the blindness of the traditional process is avoided, a large quantity of subsequent screening and detecting works are reduced, the synthetic amount of the 4-erythrose kinase phosphate and erythrose reductase in the Yarrowialipolytica erythritol high-yield strain is greatly increased, and the production of erythritol is facilitated. The breeding efficiency, the strain activity and the yield of the erythritol producing strain are greatly increased.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a breeding method for erythritol-producing bacteria. Background technique [0002] Yarrowia lipolytica, the Chinese name is translated as Yarrowia lipolytica, Yarrowia lipolytica, Yarrowia lipolytica. The industrial production of erythritol has only emerged in recent years, and the actual global production is about 60,000 tons per year. Because erythritol is a zero-calorie natural sweetener, it is more and more popular in the market with the enhancement of people's health concept. Then scientific research institutes, universities and other scientific research workers are doing more and more research on erythritol production technology, especially strains. [0003] At present, the breeding methods of erythritol producing bacteria mainly adopt the methods of chemical mutagenesis and physical mutagenesis. Due to the difficulty in grasping the direction of mutage...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12R1/645
Inventor 王星云邱学良李雨王成福秦海青李毅
Owner FUTASTE PHARM CO LTD
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