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Construction method and application of yarrowia lipolytica with reduced fermentation foam production capacity

A technology of Yarrowia lipolytica and Yarrowia lipolytica, which is applied in the field of food additive biosynthesis, can solve the problems of reduced foam-producing ability and large amount of foam, and achieves the effects of easy separation, cost reduction, and improvement of effective utilization.

Pending Publication Date: 2021-12-17
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to overcome the shortcomings of the existing Yarrowia lipolytica fermenting and synthesizing erythritol, that is, the deficiency of producing a large amount of foam during the fermentation process, and provide Yarrowia lipolytica with reduced ability to produce foam. Saccharomyces; also provides a method for constructing a recombinant lipolytic yeast engineering strain that reduces foam or produces no foam and synthesizes erythritol, and uses the engineering strain to directly ferment the method for synthesizing and purifying erythritol

Method used

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  • Construction method and application of yarrowia lipolytica with reduced fermentation foam production capacity
  • Construction method and application of yarrowia lipolytica with reduced fermentation foam production capacity
  • Construction method and application of yarrowia lipolytica with reduced fermentation foam production capacity

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Experimental program
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Effect test

Embodiment 1

[0038] Example 1. The method of site-directed insertion of exogenous DNA into Yarrowia lipolytica genome to obtain mutants

[0039] In this example, a section of artificial DNA sequence was constructed. The DNA sequence is shown as SEQ ID No.1. It was synthesized by the method of whole gene synthesis and directly used to transform the competent Yarrowia lipolytica. The transformation method used lithium acetate heat shock For the method, please refer to the method described by Cheng et al. (Cheng et al., 2018, Microb. Cell Fact., 17:133), and convert the in vitro synthesized DNA sequence SEQ ID No.2 into lipolytic synthesis of erythritol Yarrowia CGMCC NO.7326. 5 micrograms of this in vitro synthesized DNA sequence are contained in 100 microliters of transformation reaction solution. After transformation, spread on the nutrient plate containing screening (medium composition: glucose 20g / L, yeast powder 2g / L, peptone 2g / L, yeast nitrogen base 5g / L, ammonium sulfate 5g / L, hygro...

Embodiment 2

[0040] Embodiment 2, mutant strain primary screening method

[0041] Select the single colony grown on the screening plate and transfer it to a 2mL centrifuge tube containing 0.5ml liquid medium (200g / L glucose, 10g / L yeast powder, 3.5g / L ammonium citrate, 3g / L Peptone, initial pH 6.0), placed on a tube rack at 30°C, 800r / min shaking and shaking culture (MBR-420FL, Japan TAITEC company). The foam production performance of the mutant transformants was initially screened. The test results showed that the selected mutant transformants did not produce bubbles, while the comparison strain (Yarrowialipolytica CGMCC NO.7326, patent application number 201310282059.X) produced bubbles, and the bubbles were 2-3 mm above the liquid surface. figure 2 Show the bubble production performance of selected mutant strains and control strains in 2 ml centrifuge tubes. It can be seen from the figure that the selected mutant strain No. 1 has basically no foam production. After leaving standstil...

Embodiment 3

[0043] Embodiment 3, the shaking flask foaming test of mutant strain

[0044] The non-foaming mutants obtained through preliminary screening were inoculated into 250 mL shake flasks containing 30 mL liquid medium and 2000 mL shake flasks containing 150 mL liquid medium, and the composition of the medium was the same as that described in Example 2. At the same time, the unmutated wild strain was used for comparative experiments. Cultivate in a shaker at 30°C (230r / min) for 2-3 days, and observe the foam production. The foam production performance of the mutant strain and the control strain were compared in 250mL and 2000mL shake flasks respectively. The result is as image 3 shown.

[0045] Depend on image 3 It can be seen that No. 1 mutant strain did not produce foam after 40 h of cultivation in the 250 mL shake flask, while the control strain produced more foam. In a 2000mL shake flask, the No. 1 mutant strain produced only a small amount of foam when cultured in a reci...

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Abstract

The invention discloses a construction method of yarrowia lipolytica with reduced fermentation foam production capacity and a method for synthesizing erythritol by using the yarrowia lipolytica. According to the construction, yarrowia lipolytica for synthesizing erythritol is adopted as a base microorganism, and a yeast genome is mutated through a metabolic engineering improvement means to obtain a mutant strain. The obtained mutant strain remarkably reduces the foam production capacity in the fermentation process. In the fermentation process, the dosage of a defoaming agent is obviously reduced compared with that of a control strain, and even the defoaming agent is not added any more. Glucose with the initial fermentation concentration of 50-350 g / L and the like are used as a carbon source, a nitrogen source with the initial fermentation concentration of 5-30 g / L and inorganic salt are used as raw materials, the obtained yarrowia lipolytica mutant strain is inoculated after sterilization and cooling, and fermentation is conducted under the aerobic condition. And after the fermentation is finished, the erythritol is purified from the fermentation liquid.

Description

technical field [0001] The invention belongs to the technical field of biosynthesis of food additives, and relates to a method for constructing Yarrowia lipolytica with reduced ability to produce foam by fermentation and a method for synthesizing erythritol, in particular to a significantly reduced or no foam produced by fermentation A method for constructing bubbled Yarrowia lipolytica and a method for synthesizing erythritol; the constructed mutant strains ferment glucose and other carbon sources to synthesize erythritol. Background technique [0002] Foam production by fermentation is a common problem encountered in the field of fermentation. The solution to this problem is of great significance for improving fermentation performance and reducing fermentation cost. Yarrowia lipolytica is a commonly used synthetic biology chassis strain and a natural strain for the synthesis of functional sugar alcohols such as erythritol (Cheng et al., 2018, Microbial Cell Factories, 17:1...

Claims

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Application Information

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IPC IPC(8): C12N15/87C12N1/19C12P7/18C12R1/645
CPCC12N15/87C12P7/18C12N1/16
Inventor 程海荣黄艺园
Owner SHANGHAI JIAO TONG UNIV
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