Abstract:
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.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention is related to fields of genetic engineering and fermentation engineering, and in particular, to a genetically recombinant  Saccharomyces cerevisiae  useful for degrading and utilizing kitchen waste. 
       BACKGROUND 
       [0002]    Fuel ethanol for motor vehicles is substantially generated using grain and corns at present in China. Large-scale production of fuel ethanol will results in competitive usage of foods, leading to rising food prices and potential food shortage. A possible solution uses non-food renewable biomass to generate ethanol. Kitchen waste comprises a huge amount of renewable biomass. Over 60 million tons of kitchen waste is produced every year in China, and most are used to feed livestock, or delivered to landfill or incineration, resulting in severe environmental pollution. Only a small part is utilized to for example make compost or biogas, but with low economic interest. Therefore, it is promising to use kitchen waste to produce fuel ethanol, which both converts wastes to valuables and relieves food and energy shortage crisis. 
         [0003]    Kitchen waste is a nutrition-enriched renewable biomass containing over 95% organics such as starch, saccharides, proteins, lipids, vitamins and elements such as N, P, S, K, Ca, and Mg. This enables kitchen waste to be biologically reusable. However, high contents of water and nutrition in the kitchen waste make microorganisms propagate rapidly at room temperature, taking advantage of the organics and mineral salts, causing the waste to decay and stink which brings challenges to process. 
         [0004]      Saccharomyces cerevisiae  is industrially preferable strain for ethanol fermentation which is capable of efficiently convert glucose to ethanol. However, the strain can not naturally use the starch and proteins contained in kitchen waste as carbon and nitrogen sources to generate ethanol due to lack of enzymes for degrading starch to glucose and enzymes for degrading proteins to polypeptides and amino acids. 
         [0005]    It is therefore necessary to introduce genes encoding enzymes degrading starch and proteins to the  Saccharomyces cerevisiae  so that the strain will be able to take advantage of the self-expressing amylase and protease to convert the starch and proteins in kitchen waste to carbon and nitrogen sources, such that the waste can be converted to fuel ethanol for industrial purpose. 
       SUMMARY 
       [0006]    One aspect of the invention provides a genetically recombinant  Saccharomyces cerevisiae  useful for degrading and utilizing kitchen wastes. 
         [0007]    Another aspect of the invention provides a construction method for a genetically recombinant  Saccharomyces cerevisiae  useful for degrading and utilizing kitchen wastes. 
         [0008]    The genetically recombinant  Saccharomyces cerevisiae  useful for degrading and utilizing kitchen wastes is constructed by introducing α-amylase (AMY) gene, glucoamylase (GA) gene and acid protease (AP) gene into  Saccharomyces cerevisiae  through a  saccharomyces cerevisiae  expression vector and achieving correct expression and secretion. 
         [0009]    The present invention relies on the introduction and expression of AMY, GA and AP genes. A  saccharomyces cerevisiae  expression vector is used as a tool. Preferably, the  saccharomyces cerevisiae  expression vector is a  saccharomyces cerevisiae  multi-gene co-expression vector which enables co-transduction of AMY, GA and AP genes into  saccharomyces cerevisiae.  Preferably, the  saccharomyces cerevisiae  multi-gene co-expression vector is pScIKP, the preparation of which was disclosed in Chinese Patent No. ZL 200810029630.6. Other types of  saccharomyces cerevisiae multi-gene co-expression vector can also be used. 
         [0010]    A method for constructing a genetically recombinant  Saccharomyces cerevisiae  useful for degrading and utilizing kitchen wastes is provided, which comprising 
         [0011]    S1. obtaining gene sequences encoding α-amylase, glucoamylase and acid protease respectively by using PCR amplification; introducing an artificial mutation to mutate the residue C at position 1566 to T for the gene encoding glucoamylase, and the residue C at position 1155 to T for the gene encoding acid protease; 
         [0012]    S2. introducing the genes encoding α-amylase, glucoamylase and acid protease into a  saccharomyces cerevisiae  expression vector to obtain a multi-gene co-expression vector; 
         [0013]    S3. linearizing the multi-gene co-expression vector by a restriction endonuclease, and transform to a  saccharomyces cerevisiae  to obtain a recombinant  saccharomyces cerevisiae.    
         [0014]    In a preferable embodiment, step S2 comprises steps of 
         [0015]    S11. digesting the  saccharomyces cerevisiae  expression vector, the α-amylase gene, the glucoamylase gene, and the acid protease gene by restriction endonucleases; 
         [0016]    S12. ligating the α-amylase gene, the glucoamylase gene, and the acid protease gene into the  saccharomyces cerevisiae  expression vector to obtain three recombinant single-gene vectors; 
         [0017]    S13. 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. 
         [0018]    Preferably, the restriction endonuclease used in step S3 is ApaI. 
         [0019]    Preferably, the transform step in step S3 is performed by electrotransformation, freezing, or chemical reagents. 
         [0020]    Preferably, the restriction endonucleases used in step S11 are BamHI and SpeI; and the restriction endonucleases used in step S13 are isocaudarners NheI and XbaI. 
         [0021]    Preferably, the α-amylase gene is the α-amylase gene originated from  Aspergillus oryzae ; the glucoamylase gene is the glucoamylase gene originated from  Aspergillus niger ; and the acid protease gene is the acid protease gene originated from  Aspergillus niger.    
         [0022]    In a most preferable embodiment, the α-amylase gene is the α-amylase gene originated from  Aspergillus oryzae  CICC 40344 purchased from China Center of Industrial Culture Collection, CICC); the glucoamylase gene is the glucoamylase gene originated from  Aspergillus niger  CICC 40179 purchased from China Center of Industrial Culture Collection, CICC); and the acid protease gene is the acid protease gene originated from  Aspergillus niger  CICC 40179. 
         [0023]    The nucleic acid sequence of the α-amylase gene originated from  Aspergillus oryzae  CICC 40344 is shown in SEQ ID NO. 1, the nucleic acid sequence of the glucoamylase gene originated from  Aspergillus niger  CICC 40179 is shown in SEQ ID NO. 2 with the nucleotide residue C (cytimidine) at position 1566 artificially mutated to T (thymine), and the nucleic acid sequence of the acid protease gene originated from  Aspergillus niger  CICC 40179 is shown in SEQ ID NO. 3 with the nucleotide residue C (cytimidine) at position 1155 artificially mutated to T (thymine). 
         [0024]    The present invention is advantageous over conventional technologies. 
         [0025]      Saccharomyces cerevisiae  is not able to utilize the starch or proteins enriched in kitchen wastes as carbon or nitrogen sources to generate ethanol when it is not fermented. To enable the  Saccharomyces cerevisiae  to utilize kitchen wastes as raw materials to ferment ethanol, genes encoding enzymes for degrading starch and proteins are introduced into  Saccharomyces cerevisiae  and expressed, such that 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 for ethanol fermentation. 
         [0026]    The key point in the invention is to successfully introduce genes encoding α-amylase, glucoamylase and acid protease into a  Saccharomyces cerevisiae  through a  Saccharomyces cerevisiae  multi-gene co-expression vector and make the genes expressed. To this end, a recombinant vector carrying amy, ga, ap gene cassettes needs to be constructed firstly. It is required that the gene sequences can not harbor restriction sites for isocaudarners NheI and XbaI, because otherwise the gene sequences would be broken during the cassettes were cut from the single-expression vectors, resulting in incomplete introduction into the co-expression vector. For this purpose, the nucleotide residue C at position 1566 in the glucoamylase gene is mutated to T, causing the disruption of a restriction site of NheI, and the nucleotide residue C at position 1155 in the acid protease gene is mutated to T, causing the disruption of a restriction site of XbaI, without alternation to the amino acid sequences of the proteins encoded by these genes. A recombinant co-expression vector pScIKP-amy-ga-ap carrying three complete gene expression cassettes are obtained. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0027]      FIG. 1  is a flow chart showing construction of a recombinant  Saccharomyces cerevisiae  multi-gene expression vector pScIKP-amy-ga-ap. 
           [0028]      FIG. 2  shows the PCR results of positive transformants, (a) amplified fragments for α-amylase, (b) amplified fragments for glucoamylase and (c) amplified fragments for acid protease. 
           [0029]      FIG. 3 : Enzyme activity assay for α-amylase and glucoamylase of the recombinant yeast by iodine staining. 
           [0030]      FIG. 4 : Enzyme activity assay for acid protease of the recombinant yeast by casein developing process. 
           [0031]      FIG. 5 : HPLC results detecting ethanol levels in the fermentation broth by the present recombinant yeast. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0032]    The present invention will now be further illustrated with specific examples and accompany drawings which should not be constructed as limiting to the scope of the invention. 
         [0033]    The yeast  Saccharomyces cerevisiae  AS2.489 is purchased from Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences. The vector pScIKP is constructed and preserved by the Research Centre for Molecular Biology of Jinan University, the construction method of which can be found in Chinese Patent No. ZL 200810029630.6. 
       Example 1 
     Cloning of α-Amylase Gene amy, Glucoamylase Gene ga and Acid Protease Gene ap 
       [0034]    Primers were designed based on the sequences available from GenBank for the gene amy of  Aspergillus oryzae  [Accession number XM 001821384], the gene ga of  Aspergillus niger  [Accession number XM 001390493.1], and the gene ap of  Aspergillus niger  [Accession number XM 001401056.2]. Appropriate restriction sites were introduced into the primers: 
         [0035]    Primers for amy gene amplification: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0036]    Primers for ga gene amplification: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0037]    Primers for ap gene amplification: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0038]    The total RNA was extracted from  Aspergillus oryzae  CICC 40344 and the target gene was amplified by RT-PCR. The RT-PCR amplified products were ligated into a pGEM-T Easy vector (Promega) and verified by sequencing. 
         [0039]    PCR reaction conditions for the amy gene were set as follows. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 94° C. 
                 5 
                 min 
                   
                   
               
               
                   
                 94° C. 
                 30 
                 s 
               
               
                   
                 53.3° C.   
                 30 
                 s 
                  {close oversize brace}  
                 30 cycles 
               
               
                   
                 72° C. 
                 100 
                 s 
               
               
                   
                 72° C. 
                 10 
                 min 
               
               
                   
                   
               
             
          
         
       
     
         [0040]    The total RNA was extracted from  Aspergillus niger  CICC 40179 and the target gene was amplified by RT-PCR. The RT-PCR amplified products of genes ga and ap were ligated into a pGEM-T Easy vector (Promega), respectively, and verified by sequencing. 
         [0041]    PCR reaction conditions for the ga gene were set as follows. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 94° C. 
                 5 
                 min 
                   
                   
               
               
                   
                 94° C. 
                 30 
                 s 
               
               
                   
                 57° C. 
                 30 
                 s 
                  {close oversize brace}  
                 30 cycles 
               
               
                   
                 72° C. 
                 100 
                 s 
               
               
                   
                 72° C. 
                 10 
                 min 
               
               
                   
                   
               
             
          
         
       
     
         [0042]    PCR reaction conditions for the ap gene were set as follows. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 94° C. 
                 5 
                 min 
                   
                   
               
               
                   
                 94° C. 
                 30 
                 s 
               
               
                   
                 58° C. 
                 30 
                 s 
                  {close oversize brace}  
                 30 cycles 
               
               
                   
                 72° C. 
                 100 
                 s 
               
               
                   
                 72° C. 
                 10 
                 min 
               
               
                   
                   
               
             
          
         
       
     
         [0043]    The nucleic acid sequence of the α-amylase gene amy originated from  Aspergillus oryzae  CICC 40344 is shown in SEQ ID NO. 1, the nucleic acid sequence of the glucoamylase gene ga originated from  Aspergillus niger  CICC 40179 is shown in SEQ ID NO. 2 with the nucleotide residue C (cytimidine) at position 1566 artificially mutated to T (thymine), and the nucleic acid sequence of the acid protease gene ap originated from  Aspergillus niger  CICC 40179 is shown in SEQ ID NO. 3 with the nucleotide residue C (cytimidine) at position 1155 artificially mutated to T (thymine). 
       Example 2 
     CONSTRUCTION of the Co-Expression Vector Carrying Genes Encoding the Three Enzymes 
       [0044]    The process of construction is shown in  FIG. 1 . 
         [0045]    The amy, ga and ap coding sequences obtained from Example 1 were double digested from pGEM-T Easy vectors using BamHI and SpeI, and then ligated into the vector pScIKP previously digested by the same restriction endonucleases, to obtain recombinant plasmidspScIKP-amy, pScIKP-ga and pScIKP-ap. 
         [0046]    pScIKP-ga was double digested by NheI and XbaI to obtain the ga gene expression cassette containing the PGK promoter and the PGK terminator from  S. cerevisiae.  NheI single digestion of pScIKP-amy results in linearization. T4 DNA ligase was used to ligate the ga cassette and the linearized pScIKP-amy, taking advantage of the fact that NheI and XbaI are isocaudarners, to give a recombinant plasmid pScIKP-amy-ga. Similarly, pScIKP-ap was double digested by NheI and Xba I to obtain the ap gene expression cassette containing the PGK promoter and the PGK terminator from  S. cerevisiae , which is then ligated with the linearized pScIKP-amy-ga digested by NheI to give a recombinant plasmidpScIKP-amy-ga-ap. 
       Example 3 
     Screening and Validation of Recombinant Yeast Transformants 
       [0047]    Resistance tolerance was performed for  Saccharomyces cerevisiae  AS2.489 over resistance selection markers G418 prior to electrotransformation. It was found that the yeast can not grow on an YPD plate containing G418 of 150 μg/ml, so concentrations above 150 μg/ml G418 can be used for recombinant screening. 
         [0048]    The recombinant plasmid pScIKP-amy-ga-ap obtained from Example 2 was linearized by ApaI and introduced into  Saccharomyces cerevisiae  AS2.489 by electrotransformation. The yeast was cultured on an YPD agar plate for 3-4 days in the presence of G418 at 200 μg/ml. The colony normally grown was selected for screening for the positive transformant by PCR using specific primers for each of the genes. The positive PCR results ( FIG. 2 ), demonstrated that the genes were incorporated into the genome of the  Saccharomyces cerevisiae.    
       Example 4 
     Enzyme Activity Assay for the Amylases and Protease Secreted by the Recombinant  Saccharomyces cerevisiae    
       [0049]    The positive transformant obtained from Example 3 was inoculated on an YNBS plate (YNB 6.7 g/l, soluble starch 10 g/l, and agar powder 15 g/l) containing 1% soluble starch, and cultured in an incubator at 30° C. for 72 h. The plate was stained by iodine vapor. The results were shown in  FIG. 3 , where obvious transparent zones formed because of the hydrolysis of starch were observed around the colony, indicating that the transformant can utilize the starch in the plate as carbon source to grow. 
         [0050]    The positive transformant obtained from Example 3 was inoculated in a solid YPD medium (0.5 g yeast extract, 2 g peptone, agar 1.5 g, added to 100 ml using 1% casein solution) containing 1% casein and incubated at 30° C. for 3-4 days. As shown in  FIG. 4 , the casein was degraded by protease and thus obvious transparent zones formed by casein hydrolysis were observed around the colony. 
       Example 5 
     Ethanol Production by Fermentation of Kitchen Waste Using the Recombinant Yeast 
       [0051]    (1) Medium Composition 
         [0052]    The seeding medium: YPD medium (yeast extract 10 g/l, tryptone 20 g/l, glucose 20 g/l), subject to autoclaved sterilization. 
         [0053]    Fermentation medium: kitchen wastes collected from food residues from a canteen in a university in Guangzhou. Non-food residues were removed and the kitchen wastes were crushed by a crushing processor dedicated for garbage treatment. The wastes were mixed thoroughly and loaded to 1 L conical flasks and sterilized at 121° C. for 20 min for fermentation by the recombinant yeast. The composition of the kitchen waste mixture was determined as the following: water 73.8%, dry matter 26.2% (including starch 9.7%, protein 1.0%, soluble saccharides 4.4%, others 11.1%), pH 6.1. 
         [0054]    (2) Fermentation 
         [0055]    The recombinant  Saccharomyces cerevisiae  was activated before inoculated to a 25 ml YPD seeding yeast medium at 2% inoculation. The yeast was cultured in an air shaker incubator at 30° C., 200 rpm for 24 h and then inoculated to a 200 ml YPD medium at 10% inoculation for an enlarged culture at 30° C., 250 rpm until the logarithmic phase. When cell reaches about 0.8˜1.2×10 8 /mL, about 20% budding rate and no more than 1% mortality, it indicated that seeding yeast was mature. 
         [0056]    The culture was transferred to the sterilized conical flasks containing the kitchen wastes at a volume of 10% of the fermentation medium and started fermentation. The fermentation conditions were set as follows: 30° C., 250 rpm, natural pH, aerated fermentation for 4 h; then 30° C., 150 rpm, natural pH, and anaerobic fermentation for 60 h. Sampling was performed every 12 h during fermentation, and the production of ethanol was detected by HPLC ( FIG. 5 ). It was shown that the maximum ethanol production was achieved at 52 h, reaching a concentration of 66 g/L. The conversion rate of kitchen waste-ethanol reached up to 1 g ethanol per 4 g kitchen wastes (dry weight). 
         [0057]    The results showed that the recombinant  Saccharomyces cerevisiae  as constructed by the present invention was able to degrade and utilize kitchen wastes and converted them to ethanol. The recombinant yeast was therefore named by the inventors as “Waste-swallow Yeast 1”.