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Construction method of yarrowia lipolytica genetically engineered bacterium for producing citric acid or itaconic acid by using acetic acid

A technology of Yarrowia lipolytica and genetically engineered bacteria, applied in the field of genetic engineering, can solve unseen problems

Pending Publication Date: 2021-10-01
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there have been no reports on citric acid production using Yarrowia lipolytica using acetic acid as a carbon source

Method used

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  • Construction method of yarrowia lipolytica genetically engineered bacterium for producing citric acid or itaconic acid by using acetic acid
  • Construction method of yarrowia lipolytica genetically engineered bacterium for producing citric acid or itaconic acid by using acetic acid
  • Construction method of yarrowia lipolytica genetically engineered bacterium for producing citric acid or itaconic acid by using acetic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 uses acetic acid as carbon source to produce the construction of Yarrowia lipolytica genetically engineered bacterium of citric acid

[0042] A. Construction of plasmid PYcam

[0043](1) The citric acid synthase gene CAS and the acetyl-CoA synthetase gene ACS are all derived from the genome of Yarrowia lipolytica. The corresponding citrate synthase gene CAS and acetyl-CoA synthetase gene ACS were amplified by PCR above, which were derived from the mitochondrial carboxylic acid transporter gene MTT of Aspergillus terreus, and connected to the vector PYLXP' in turn to obtain the recombinant plasmid PYcam, whose nucleoside The acid sequence is shown in SEQ ID NO.5.

[0044] Primer sequences used in Table 1

[0045]

[0046] B. Yarrowia lipolytica transformation of plasmid pYcam

[0047] The transformation method of the plasmid is the lithium acetate yeast transformation method commonly used in yeast transformation.

[0048] The specific way is:

[0049...

Embodiment 2

[0057] Example 2 Production of itaconic acid Yarrowia lipolytica genetically engineered bacteria using acetic acid as a carbon source

[0058] A, construction of plasmid PYcama

[0059] (1) The citric acid synthase gene CAS and the acetyl-CoA synthetase gene ACS are all derived from the genome of Yarrowia lipolytica. The corresponding citrate synthase gene CAS and acetyl-CoA synthetase gene ACS were amplified by PCR above, which were derived from the mitochondrial carboxylate transporter gene MTT and aconitic acid decarboxylase gene CADA of Aspergillus terreus, and were sequentially connected to the vector PYLXP' The obtained recombinant plasmid is PYcama, the nucleotide sequence of which is shown in SEQ ID NO.6, and the sequence used is shown in Table 1.

[0060] B. Yarrowia lipolytica transformation of plasmid pYcam

[0061] The transformation method of the plasmid is the lithium acetate yeast transformation method commonly used in yeast transformation.

[0062] The speci...

Embodiment 3

[0071] Example 3 Experiment of YLA01 strain producing citric acid by fermenting acetic acid as carbon source

[0072] 1. Experimental material: strain YLA01

[0073] 2. Experimental method:

[0074] Seed medium: acetic acid leucine deficient medium, YNB1.5 g / L, acetic acid 23 g / L, ammonium sulfate 4.6 g / L, CSM-leu0.8 g / L. Sterilize at 121°C for 25 minutes.

[0075] Fermentation medium: acetic acid leucine deficient medium, YNB1.7 g / L, acetic acid 41 g / L, ammonium sulfate 0.825 g / L, CSM-leu 0.69 g / L. Sterilize at 121°C for 25 minutes.

[0076] Inoculate the strain YLA01 in 5mL of seed medium, and cultivate it for 48 hours at 28°C and 220rpm, take 100μL of seed liquid, inoculate it in 50mL of fermentation medium, and cultivate it at 28°C and 220rpm After 144 hours, the concentration of citric acid and the remaining amount of acetic acid in the fermentation broth were measured.

[0077] 3. Product detection method

[0078] Citric acid is easily soluble in water, so the prod...

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Abstract

The invention relates to a construction method of yarrowia lipolytica genetically engineered bacterium for producing citric acid or itaconic acid by using acetic acid, and belongs to the field of gene engineering. The invention discloses a yarrowia lipolytica genetically engineered bacterium for producing citric acid or itaconic acid, which is prepared by the following steps: introducing a citric acid synthetase gene CAS, an acetyl-coenzyme A synthetase gene ACS, a mitochondrial carboxylic acid transporter gene MTT and a cis-aconitic acid decarboxylase gene CADA into leucine auxotrophic yarrowia lipolytica to construct and the yarrowia lipolytica genetically engineered bacterium YLA01 (containing genes CAS, ACS and MTT) and YLA02 (containing genes CAS, ACS, MTT and CADA) capable of metabolizing acetic acid. The yarrowia lipolytica genetically engineered bacterium constructed by the research can efficiently produce citric acid or itaconic acid by using acetic acid as a carbon source, and a good solution is provided for the waste acetic acid in the fermentation industry.

Description

technical field [0001] This research belongs to the field of genetic engineering, and specifically relates to a Yarrowia lipolytica genetically engineered bacterium capable of producing citric acid or itaconic acid from acetic acid and its application. Background technique [0002] Citric acid is a natural component of various citrus fruits, pineapples, pears, peaches and figs. It is the most important food acidifier today. In addition to improving the flavor of food, citric acid can also inhibit the growth of microorganisms and play a role in food preservation. Therefore, its use in the food industry has always been the highest among organic acids. In addition, citric acid can also be used as a preferred additive for chelating agents and detergents, animal feed, lubricants and plasticizers. Therefore, the market demand for citric acid is huge and shows a continuous growth trend. Citric acid is mainly produced through chemical synthesis or fermentation by Aspergillus niger...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/48C12P7/44C12R1/645
CPCC12N9/1025C12N9/88C12N9/93C12N15/815C07K14/38C12Y203/03008C12Y401/01006C12Y602/01003C12P7/48C12P7/44
Inventor 邓利萧琦刘欢刘军锋王芳
Owner BEIJING UNIV OF CHEM TECH
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