Aureobasidium pullulans dicarboxylic acid transport protein and recombinant vector and application thereof

A technology of Aureobasidium dicarboxylic acid and Aureobasidium pullulans, which is applied in the biological field to achieve the effect of improving fermentation yield

Active Publication Date: 2015-09-23
SOUTHWEST UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the A. pullulans transporter

Method used

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  • Aureobasidium pullulans dicarboxylic acid transport protein and recombinant vector and application thereof
  • Aureobasidium pullulans dicarboxylic acid transport protein and recombinant vector and application thereof
  • Aureobasidium pullulans dicarboxylic acid transport protein and recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, clone dicarboxylate transporter gene

[0021] According to the coding sequence of the dicarboxylate transporter gene, primers for clones g1688, g4644, g6666, g5215 and g6113 were designed. In order to facilitate the construction of recombinant vectors, an EcoRV restriction site was designed at the 5' end of the upstream primer, and an EcoRV restriction site was designed at the 5' end of the downstream primer. The XhoI restriction site was designed at the 'end, and the specific primers are shown in Table 1.

[0022] Table 1. Primers for cloning the dicarboxylate transporter gene

[0023] Primer name

[0024] 然后分别以g1688_up_EcoRV与g1688_down_XhoI,g4644_up_EcoRV与g4644_down_XhoI,g6666_up_EcoRV与g6666_down_XhoI,g5215_up_EcoRV与g5215_down_XhoI,g6113_up_EcoRI与g6113_down_XhoI为引物对,出芽短梗霉基因组DNA为模板进行PCR扩增,PCR扩增的退火温度为50-65℃ , the extension time is 1min 45s. The amplified product was subjected to agarose gel electrophoresis, and the results showed that bands wit...

Embodiment 2

[0025] Example 2, Aureobasidium pullulans overexpressing g1688 increases the yield of polymalic acid

[0026] 1. Plasmid construction

[0027] The g1688 gene cloned in Example 1 was cleaved with EcoR V and Xho I, and the g1688 gene cleavage fragment was reclaimed, while the pBARGPE1 plasmid was digested with EcoR V and Xho I ( figure 1 ), recovering the vector backbone. Then, the recovered g1688 gene fragment was ligated with the vector backbone of the pBARGPE1 plasmid, and the ligated product was transformed into DH5α competent cells, and the recombinant expression vector pBARGPE1-g1644 (abbreviated as OE::g1688) that overexpressed the g1688 gene was obtained by screening.

[0028] 2. Transformation of Aureobasidium pullulans with OE::g1688

[0029] Protoplasts were made from OE::g1688, and then transformed into Aureobasidium pullulans. After transformation, OE::g1688 transformants were obtained by double screening with 8 mg / mL glufosinate as the screening pressure. In the...

Embodiment 3

[0033] Example 3, Aureobasidium pullulans overexpressing g6666 increases the yield of polymalic acid

[0034] 1. Plasmid construction

[0035] The g6666 gene cloned in Example 1 was digested with EcoR V and Xho I, and the g6666 gene digested fragment was reclaimed, while the pBARGPE1 plasmid was digested with EcoR V and Xho I ( figure 1 ), recovering the vector backbone. Then, the recovered g6666 gene fragment was ligated with the vector backbone of the pBARGPE1 plasmid, and the ligated product was transformed into DH5α competent cells, and the recombinant expression vector pBARGPE1-g6666 (abbreviated as OE::g6666) that overexpressed the g6666 gene was obtained by screening.

[0036] 2. Transformation of Aureobasidium pullulans with OE::g6666

[0037] Protoplasts were made from OE::g6666, and then transformed into Aureobasidium pullulans. After transformation, OE::g6666 transformants were obtained by double screening with 8 mg / mL ammonium ammonium as the screening pressure. I...

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Abstract

The invention discloses aureobasidium pullulans dicarboxylic acid transport protein and a recombinant vector and application thereof. Amino acid sequence of the aureobasidium pullulans dicarboxylic acid transport protein is shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10 while nucleotide sequence of the same is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5. Overexpression of dicarboxylic acid transport protein in aureobasidium pullulans can increase yield of polymalic acid, and no free malic acid is detected in fermentation liquor, so that dicarboxylic acid transport protein gene is related to synthetic transport of the polymalic acid, and a foundation is laid for increasing yield of the polymalic acid.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically relates to the Aureobasidium pullulans dicarboxylic acid transporter, and also relates to a recombinant vector containing the Aureobasidium pullulans dicarboxylic acid transporter gene and the application of the Aureobasidium pullulans dicarboxylic acid transporter. Background technique [0002] Polymalic acid (PMA) is a new type of fully biodegradable polyester polymer. Due to its good water solubility, biodegradability and biocompatibility, it can be used as a drug carrier and microcapsule material. , biomedical materials, water-absorbing materials, cosmetics, food packaging and other materials, have a wide range of application prospects. In addition, the monomer of polymalic acid is L-malic acid, which is an excellent food sour agent and C4 platform compound, with an annual market demand of more than 100,000 tons. [0003] Polymalic acid is mainly synthesized by Aureobasidium pullula...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/80C12N1/15C12P7/62C12R1/645
CPCC07K14/37C12P7/62
Inventor 邹祥冯骏李正华李云政
Owner SOUTHWEST UNIVERSITY
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