Genetically engineered Candida utilis capable of degrading and utilizing kitchen waste and construction method therefor

• US 10,577,615 B2
• Filed: 06/24/2019
• Issued: 03/03/2020
• Est. Priority Date: 12/19/2016
• Status: Active Grant

• Current Assignee: Guangdong Qizhi Biotechnology Co., Ltd.
• Original Assignee: Guangdong Recyclean Low-Carbon Technology
• Inventors: Liu, ZeHuan, Lin, JiangHai, Li, Shuai
• Primary Examiner(s): Baskar, Padmavathi
• Application Number:
  US16/449,858
• Publication Number:
  US 20190309315A1
• Time in Patent Office: 253 Days
• Field of Search: None
• 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 the genetically engineered Candida utilis expresses a Candida utilis multigene co-expression vector that has an alpha-amylase comprising SEQ ID NO:

1, a glucoamylase gene comprising SEQ ID NO;

2, and an acid protease gene comprising SEQ ID NO;

3 and has been integrated into the Candida utilis genome, the method comprising steps of;

(1) obtaining the Candida utilis multigene co-expression vector by (a) providing a Saccharomyces cerevisiae multigene co-expression vector comprising an rDNA sequence, a phosphoglycerate kinase promoter of Saccharomyces cerevisiae, and a terminator, (b) removing the rDNA sequence from the Saccharomyces cerevisiae multigene co-expression vector, and (c) replacing the phosphoglycerate kinase promoter in the Saccharomyces cerevisiae multigene co-expression vector with a glyceraldehyde-3-phosphate dehydrogenase promoter of Candida utilis, thereby obtaining the Candida utilis multigene co-expression vector;

(2) performing double digestions on the Candida utilis multigene co-expression vector, the Saccharomyces cerevisiae multigene co-expression vector, the alpha-amylase gene, the glucoamylase gene, and the acid protease gene by using restriction endonucleases to obtain a fragment of DNA comprising the Candida utilis multigene co-expression vector, a fragment of DNA comprising the Saccharomyces cerevisiae multigene co-expression vector, a fragment of DNA comprising the alpha-amylase gene, a fragment of DNA comprising the glucoamylase gene, and a fragment of DNA comprising the acid protease gene;

(3) purifying and recovering the fragments of DNA comprising the Candida utilis multigene co-expression vector, the Saccharomyces cerevisiae multigene co-expression vector, the alpha-amylase gene, the glucoamylase gene, and the acid protease gene, respectively;

(4) ligating the fragment of DNA comprising the alpha-amylase gene into the Saccharomyces cerevisiae multigene co-expression vector, ligating the fragment of DNA comprising the glucoamylase gene into the fragment of DNA comprising the Saccharomyces cerevisiae multigene co-expression vector, and ligating the fragment of DNA comprising the acid protease gene into the fragment of DNA comprising the Candida utilis multigene co-expression vector, by using a DNA ligase, to form (i) an alpha-amylase gene expression cassette comprising the phosphoglycerate kinase promoter, the alpha-amylase gene, and the terminator, (ii) a glucoamylase gene expression cassette comprising the phosphoglycerate kinase promoter, the glucoamylase gene, and the terminator, and (iii) an acid protease gene expression cassette comprising the glyceraldehyde-3-phosphate dehydrogenase promoter, the acid protease gene, and the terminator, respectively;

(5) cleaving the alpha-amylase gene expression cassette, the glucoamylase gene expression cassette, and the acid protease gene expression cassette by using one or more restriction endonucleases to form a fragment of DNA comprising the alpha-amylase gene expression cassette, a fragment of DNA comprising the glucoamylase gene expression cassette, and a fragment of DNA comprising the acid protease gene expression cassette, respectively, then ligating the fragment of DNA comprising the alpha-amylase gene expression cassette and the fragment of DNA comprising the glucoamylase gene expression cassette, sequentially into the the fragment of DNA comprising the acid protease gene expression cassette, thus constructing a three-gene co-expression vector of alpha-amylase, glucoamylase, and acid protease;

(6) performing a single digestion on the three-gene co-expression vector by using a restriction endonuclease, and

(7) transforming the product into Candida utilis thereby integrating the product into the genome, thus obtaining the genetically engineered Candida utilis capable of degrading and utilizing kitchen waste.

2. The method for constructing a genetically engineered Candida utilis capable of degrading and utilizing kitchen waste according to claim 1, where step (5) is carried out as follows:

the cleaving of the alpha-amylase gene expression cassette and the cleaving of the glucoamylase gene expression cassette are performed as double digestions by using restriction endonucleases;

the fragment of DNA comprising the alpha-amylase gene expression cassette the fragment of DNA comprising the glucoamylase gene expression cassette are recovered;

the cleaving of the fragment of DNA comprising the acid protease gene expression cassette is performed as a single digestion by using a restriction endonuclease;

the recovered fragment of DNA comprising the glucoamylase gene expression cassette is ligated into the fragment of DNA comprising the acid protease gene expression cassette, thus constructing a two-gene co-expression vector of glucoamylase and acid protease;

a single digestion is performed on the two-gene co-expression vector of glucoamylase and acid protease by using a restriction endonuclease to obtain a DNA fragment comprising the two-gene co-expression vector; and

the recovered fragment of DNA comprising the alpha-amylase gene expression cassette is ligated into the two-gene co-expression vector, thus constructing the three-gene co-expression vector.

3. The method for constructing a genetically engineered Candida utilis capable of degrading and utilizing kitchen waste according to claim 1, wherein the restriction endonucleases in step (2) are BamHI and SpeI;

and the restriction endonuclease in step 6 is SacI.

4. The method for constructing a genetically engineered Candida utilis capable of degrading and utilizing kitchen waste according to claim 2, wherein the restriction endonucleases used for the cleaving of the alpha-amylase gene expression cassette and the cleaving of the glucoamylase gene expression cassette are NheI and XbaI;

the restriction endonuclease used for the cleaving of the fragment of DNA comprising the acid protease gene expression cassette is NheI; and

the restriction endonuclease used for performing the single digestion on the two-gene co-expression vector is NheI.