GENETICALLY ENGINEERED CANDIDA UTILIS CAPABLE OF DEGRADING AND UTILIZING KITCHEN WASTE AND CONSTRUCTION METHOD THEREFOR

• US 20190309315A1
• Filed: 06/24/2019
• Published: 10/10/2019
• Est. Priority Date: 12/19/2016
• Status: Active Grant

• Current Assignee: Guangdong Qizhi Biotechnology Co., Ltd.
• Original Assignee: Guangdong Qizhi Biotechnology Co., Ltd.
• Inventors: LIU, ZeHuan, LIN, JiangHai, LI, Shuai
• Granted Patent: US 10,577,615 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/815 for yeasts other than Saccharomyces
• CPC Class Codes: C12N 2800/102 for yeast
• CPC Class Codes: C12N 2800/22 Vectors comprising a coding region that has been codon optimised for expression in a respective host
• CPC Class Codes: C12N 9/2414 Alpha-amylase (3.2.1.1.)
• CPC Class Codes: C12N 9/242 Fungal source
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12N 9/62 from Aspergillus
• CPC Class Codes: C12P 7/08 produced as by-product or from waste or cellulosic material substrate
• CPC Class Codes: C12Y 302/01001 Alpha-amylase (3.2.1.1)
• CPC Class Codes: C12Y 302/01003 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12Y 304/23018 Aspergillopepsin I (3.4.23.18)

Abstract

Provided is a genetically engineered Candida utilis capable of degrading and utilizing kitchen waste. The genetically engineered Candida utilis is obtained by using a Candida utilis multigene co-expression vector to integrate alpha-amylase, glucoamylase and acid protease genes into the Candida utilis genome and to correctly express such three enzymes.

Claims

1. A method for constructing a genetically engineered Candida utilis capable of degrading and utilizing kitchen waste, wherein said genetically engineered Candida utilis is constructed and obtained by using a Candida utilis multigene co-expression vector to integrate alpha-amylase, glucoamylase and acid protease genes into the Candida utilis genome.

2. The method for constructing a genetically engineered Candida utilis capable of degrading and utilizing kitchen waste according to claim 1, wherein the Candida utilis multigene co-expression vector is obtained by using Saccharomyces cerevisiae multigene co-expression vector as the basis, removing the rDNA sequence from the Saccharomyces cerevisiae multigene co-expression vector, and replacing the phosphoglycerate kinase promoter of Saccharomyces cerevisiae in the Saccharomyces cerevisiae multigene co-expression vector with the glyceraldehyde-3-phosphate dehydrogenase promoter of Candida utilis.

3. The method for constructing a genetically engineered Candida utilis capable of degrading and utilizing kitchen waste according to claim 2, wherein the method for constructing the genetically engineered Candida utilis are specifically as follows:

S1;

performing the double digestion on the Candida utilis multigene co-expression vector, the Saccharomyces cerevisiae multigene co-expression vector, the alpha-amylase gene as shown in SEQ ID NO;

1, the glucoamylase gene as shown in SEQ ID NO;

2 and the acid protease gene as shown in SEQ ID NO;

3 by using restriction endonucleases, and purifying and recovering the desired fragments;

S2;

ligating the alpha-amylase gene and the glucoamylase gene into the Saccharomyces cerevisiae multigene co-expression vector respectively by using a DNA ligase, and ligating the acid protease gene into the Candida utilis multigene co-expression vector to form 3 recombinant single gene expression vectors;

S3;

cleaving the entire alpha-amylase gene expression cassette and glucoamylase gene expression cassette which contain the promoter fragment and the terminator fragment of the vector from the corresponding recombinant single gene expression vector by using a restriction endonuclease, then ligating same one by one into the restriction single gene expression vector which bears the acid protease gene in the form of tandem expression cassettes, thus constructing a three-gene co-expression vector of alpha-amylase, glucoamylase and acid protease; and

S4;

performing the single digestion on the three-gene co-expression vector constructed above by using a restriction endonuclease, then transforming the product into the Candida utilis after linearization and integrating same into the genome, thus obtaining the genetically engineered Candida utilis capable of degrading and utilizing kitchen waste.

4. The method for constructing a genetically engineered Candida utilis capable of degrading and utilizing kitchen waste according to claim 3, wherein in step S3, constructing the three-gene co-expression vector comprises the steps as follows:

S11;

performing double digestion on the recombinant single gene expression vectors of alpha-amylase and glucoamylase by using restriction endonucleases, recovering the entire expression cassettes of the alpha-amylase gene and the glucoamylase gene which bear the promoter and terminator of the vector respectively;

S12;

performing enzyme digestion on the recombinant single gene expression vector of acid protease by using a restriction endonuclease, then ligating the glucoamylase gene expression cassette into the recombinant single gene expression vector of acid protease, thus constructing a two-gene expression vector of glucoamylase and acid protease; and

S13;

performing enzyme digestion on the two-gene expression vector constructed in step S12 by using a restriction endonuclease, then ligating the alpha-amylase gene expression cassette into the two-gene expression vector, thus constructing a three-gene co-expression vector of alpha-amylase, glucoamylase and acid protease.

5. The method for constructing a genetically engineered Candida utilis capable of degrading and utilizing kitchen waste according to claim 3, wherein the restriction endonucleases in step S1 are BamHI and SpeI;

the restriction endonuclease in step S4 is SacI.

6. The method for constructing a genetically engineered Candida utilis capable of degrading and utilizing kitchen waste according to claim 4, wherein the restriction endonucleases in step S11 are NheI and XbaI;

the restriction endonuclease in step S12 is NheI;

the restriction endonuclease in step S13 is NheI.

7. A genetically engineered Candida utilis capable of degrading and utilizing kitchen waste, wherein the genetically engineered Candida utilis is prepared by the method of claim 1.