Dicarboxylic acid synthesis-related enzyme, and method for producing dicarboxylic acid using same
A dicarboxylic acid, a technology involved in dicarboxylic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0045] [Example 1] Development of DAME-resistant strains using evolutionary engineering methods
[0046] In order to develop strains tolerant to DAME, which is a cytotoxic substrate, Candida tropicalis (C.tropicalis) MYA_3404 strain was cultured in YNB medium (10 g / L yeast extract) supplemented with DAME at a concentration of 10 g / L. and 20g / L peptone). In this case, it was confirmed that the concentration of DAME in the medium was maintained at about 0.45 g / L (maximum solubility) due to the low solubility of the DAME substrate (confirmed by the results of in-house experiments). The growth curve of the inoculated strain was determined by measuring the absorbance value at a wavelength of 600 nm.
[0047] The absorbance of the medium inoculated with the strain was observed in real time, and then the strain was subcultured in fresh medium until the growth of the strain reached the mid-exponential phase. The specific growth rate of the strain was calculated from the measured abs...
Embodiment 2
[0048] [Example 2] Transcriptome analysis of DAME resistant mutant strain (ES5)
[0049] To examine transcriptome changes in media with and without DAME, the transcriptomes of ES5 strains grown in DAME-supplemented media and ES5 strains grown in DAME-free media were analyzed.
[0050] The ES5 strain was cultured at 30° C. for 24 hours in YNB medium without DAME and in YNB medium supplemented with 10 g / LDAME. Cultured cells were collected and washed with water. Afterwards, the collected cells were used as samples for total RNA extraction. RNA extraction was performed using RNeasy Mini Kit (Qiagen, Hilden, Germany), and NanoDrop (Thermo Scientific, Wilmington, DE, USA) and Agilent Bioanalyzer 2100 (Santa Clara, Ca, USA) were used to measure the concentration and purity of the extracted RNA, respectively .
[0051] The transcriptome of the mutant ES5 strain was analyzed and compared with that of the parental strain. The results confirmed that a total of 453 genes were up-regu...
Embodiment 3
[0056] [Example 3] Utilize cloning technology to obtain SA biosynthetic pathway related genes
[0057] Based on the transcriptome analysis results in Example 2, five genes (LIP1, CYP52B1, NCP1, FAO1 and ALD1) expected to be associated with the sebacic acid biosynthetic pathway were selected, and LIP1 (Uniprot.ID: C5MD87), CYP52B1 were obtained by cloning (Uniprot.ID: C5MAM3), NCP1 (Uniprot.ID: C5M346), NADPH-cytochrome P450 reductase, FAO1 (Uniprot.ID: Q6QIR6) and ALD1 (Uniprot.ID: C5M346) genes to examine genes derived from 5 Enzyme (lipase, cytochrome P450 52B1, long-chain alcohol oxidase and aldehyde dehydrogenase) activities of genes. The CYP450 gene is known to have two subunits, CYP1 and NCP1.
[0058] Candida tropicalis MYA_3404 strain was cultured at 30°C for 48 hours in YPD medium (10g / L yeast extract, 20g / L peptone and 20g / L glucose), and then was purified using the Yeast DNA Isolation Kit (Epicentre, Madison, WI). , USA) to extract template DNA for cloning. The c...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More