Genetically recombinant
Disclosed is a genetically recombinant Saccharomyces cerevisiae useful for degrading and utilizing kitchen wastes. Genes encoding α-amylase (AMY), glucoamylase (GA) and acid protease (AP) were introduced into the genetically recombinant Saccharomyces cerevisiae using a Saccharomyces cerevisiae multi-gene co-expression vector and successfully expressed and secreted. The Saccharomyces cerevisiae so obtained are capable of secreting amylases and protease to degrade the starch and proteins in kitchen wastes to produce carbon and nitrogen sources such as glucose, polypeptides and amino acids, allowing fermentation into ethanol.
1. A genetically recombinant Saccharomyces cerevisiae useful for degrading and utilizing kitchen wastes, comprising genes encoding α-amylase (AMY), glucoamylase (GA) and acid protease (AP), wherein the genetically recombinant Saccharomyces cerevisiae is configured to be able to convert kitchen wastes to ethanol with a conversion rate up to 1 g ethanol per 4 g dry kitchen wastes.
2. The genetically recombinant Saccharomyces cerevisiae useful for degrading and utilizing kitchen wastes of claim 1 , wherein the genes are introduced into Saccharomyces cerevisiae through a Saccharomyces cerevisiae multi-gene co-expression vector, wherein the Saccharomyces cerevisiae multi-gene co-expression vector is vector pScIKP.
3. A method for constructing the genetically recombinant Saccharomyces cerevisiae useful for degrading and utilizing kitchen wastes of claim 1 , wherein the method comprises steps of
A. obtaining gene sequences encoding α-amylase, glucoamylase and acid protease respectively by using PCR amplification; introducing an artificial mutation to mutate nucleotide residue C at position 1566 to T for the gene encoding glucoamylase, and nucleotide residue C at position 1155 to T for the gene encoding acid protease;
B. introducing the genes encoding α-amylase, glucoamylase and acid protease into a Saccharomyces cerevisiae expression vector to obtain a multi-gene co-expression vector;
C. linearizing the multi-gene co-expression vector by a restriction endonuclease, and transform the linearized vector to a Saccharomyces cerevisiae to obtain the genetically recombinant Saccharomyces cerevisiae.
4. The method of claim 3 , wherein step B comprises steps of
D. digesting the Saccharomyces cerevisiae expression vector, the α-amylase gene, the glucoamylase gene, and the acid protease gene, by restriction endonucleases;
E. ligating the genes encoding α-amylase, glucoamylase, and acid protease respectively into the Saccharomyces cerevisiae expression vector to obtain three recombinant single-gene expression vectors;
F. cutting from the three recombinant single-gene vectors to obtain a complete α-amylase gene expression cassette, a glucoamylase gene expression cassette, and an acid protease gene expression cassette, respectively, by restriction endonucleases, with each gene expression cassette containing its own promoter and terminator fragments, and introducing the gene expression cassettes into one Saccharomyces cerevisiae expression vector in series in the form of cassettes amy-ga-ap.
5. The method of claim 3 , wherein the restriction endonuclease used in step C is ApaI.
6. The method of claim 3 , wherein the transform step in step C is performed by electrotransformation, freezing, or chemical reagents.
7. The method of claim 4 , wherein the restriction endonucleases used in step D are BamHI and SpeI; and the restriction endonucleases used in step F are isocaudomers NheI and XbaI.
8. The method of claim 3 , wherein the gene encoding α-amylase is the α-amylase gene originated from Aspergillus oryzae ; the gene encoding glucoamylase gene is the glucoamylase gene originated from Aspergillus niger ; and the gene encoding acid protease gene is the acid protease gene originated from Aspergillus niger.
9. The method of claim 4 , wherein the gene encoding α-amylase is the α-amylase gene originated from Aspergillus oryzae ; the gene encoding glucoamylase gene is the glucoamylase gene originated from Aspergillus niger ; and the gene encoding acid protease gene is the acid protease gene originated from Aspergillus niger.
10. The method of claim 7 , wherein the gene encoding α-amylase is the α-amylase gene originated from Aspergillus oryzae ; the gene encoding glucoamylase gene is the glucoamylase gene originated from Aspergillus niger ; and the gene encoding acid protease gene is the acid protease gene originated from Aspergillus niger.