WO2015100856A1 - 一种能降解利用餐厨废弃物的基因重组酿酒酵母 - Google Patents

一种能降解利用餐厨废弃物的基因重组酿酒酵母 Download PDF

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WO2015100856A1
WO2015100856A1 PCT/CN2014/073890 CN2014073890W WO2015100856A1 WO 2015100856 A1 WO2015100856 A1 WO 2015100856A1 CN 2014073890 W CN2014073890 W CN 2014073890W WO 2015100856 A1 WO2015100856 A1 WO 2015100856A1
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gene
saccharomyces cerevisiae
recombinant
expression vector
acid protease
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PCT/CN2014/073890
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French (fr)
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刘泽寰
方龙
闫凯
康小龙
郑阳阳
刘人怀
林蒋海
肖文娟
李晶博
龚映雪
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广东启智生物科技有限公司
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Priority to LU92808A priority Critical patent/LU92808B1/xx
Priority to JP2016544561A priority patent/JP6532139B2/ja
Priority to EP14877182.7A priority patent/EP3091070B1/en
Priority to US15/109,018 priority patent/US10584359B2/en
Priority to AU2014375928A priority patent/AU2014375928B2/en
Publication of WO2015100856A1 publication Critical patent/WO2015100856A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase

Definitions

  • the present invention relates to the field of genetic engineering and fermentation engineering, and more particularly to a genetically modified Saccharomyces cerevisiae capable of degrading the use of kitchen waste.
  • Kitchen waste is a nutrient-rich renewable biomass rich in organic matter such as starch, sugar, protein, fat, etc., of which organic matter accounts for more than 95% of dry matter.
  • Kitchen waste also contains trace elements such as vitamins, nitrogen, phosphorus, sulfur, potassium, calcium and magnesium. The nutrients are complete and can be reused by organisms. Due to the high water content and rich nutrients of the kitchen waste, the micro-organisms will be rapidly metabolized and metabolized by various organic and inorganic salts under normal temperature conditions, causing the kitchen waste to rot and odor, pollute the environment, and bring trouble to the treatment.
  • Saccharomyces cerevisiae 03 ⁇ 4cc1 ⁇ 2rowjC£W cerew iK is the first choice for industrial ethanol fermentation. It has the ability to efficiently convert glucose into ethanol, but it lacks enzymes that effectively degrade starch to produce glucose, and enzymes that degrade proteins into peptides and amino acids. Under natural conditions, starch and protein in kitchen waste cannot be directly used as a carbon source and a nitrogen source to ferment ethanol.
  • Saccharomyces cerevisiae can use its secreted amylase and protease to degrade starch and protein in kitchen waste into available carbon sources - glucose and nitrogen sources - peptides and amino acids, thereby It can realize the industrialization purpose of producing fuel ethanol by fermentation of kitchen waste.
  • the technical problem to be solved by the present invention is to provide a genetically modified Saccharomyces cerevisiae which can degrade the utilization of kitchen waste in order to overcome the above-mentioned deficiencies of the prior art.
  • Another technical problem to be solved by the present invention is to provide a method for constructing a recombinant recombinant Saccharomyces cerevisiae which can degrade the use of kitchen waste.
  • a genetically modified Saccharomyces cerevisiae capable of degrading the use of kitchen waste is an a-amylase (AMY) gene, a glucoamylase (GA) gene, an acid protease (acid protease, The AP) gene was constructed by transfecting the S. cerevisiae expression vector into Saccharomyces cerevisiae and obtaining the correct secretory expression.
  • AY a-amylase
  • GA glucoamylase
  • the AP was constructed by transfecting the S. cerevisiae expression vector into Saccharomyces cerevisiae and obtaining the correct secretory expression.
  • the focus of the present invention is to transfer the ⁇ -amylase gene, the saccharification enzyme gene and the acid protease gene into Saccharomyces cerevisiae and secrete the expression thereof, and the tool used is a Saccharomyces cerevisiae expression vector, as a preferred embodiment, the Saccharomyces cerevisiae
  • the expression vector is a S. cerevisiae multi-gene co-expression vector; the Saccharomyces cerevisiae multi-gene co-expression vector can simultaneously transfer the ⁇ -amylase gene, the saccharification enzyme gene and the acid protease gene into Saccharomyces cerevisiae.
  • the S. cerevisiae multi-gene co-expression vector can simultaneously transfer the ⁇ -amylase gene, the saccharification enzyme gene and the acid protease gene into Saccharomyces cerevisiae.
  • the S. cerevisiae multi-gene co-expression vector can simultaneously transfer the ⁇ -amylase gene,
  • cerevisiae multi-gene co-expression vector is the vector pScIKP (see ZL 200810029630.6 for the preparation method), and other types of Saccharomyces cerevisiae polygene co-expression vectors can of course be used.
  • a method for constructing a genetically modified Saccharomyces cerevisiae capable of degrading the use of kitchen waste comprises the following steps:
  • the ⁇ -amylase gene, the saccharification enzyme gene and the acid protease gene sequence are respectively obtained by PCR amplification; the nucleotide residue C of the 1566th glucoamylase gene is artificially mutated into ⁇ , the 1155-position nucleus of the acid protease gene The nucleotide C residue is artificially mutated into hydrazine;
  • the ⁇ -amylase gene, the glucoamylase gene and the acid protease gene are integrated into a Saccharomyces cerevisiae expression vector to construct a recombinant multi-gene co-expression vector;
  • the recombinant multi-gene co-expression vector obtained above was cleaved with a restriction enzyme, linearized, and transformed into S. cerevisiae to construct a recombinant Saccharomyces cerevisiae.
  • the S2 constructing the recombinant multi-gene co-expression vector comprises the following steps:
  • the Saccharomyces cerevisiae expression vector, the ⁇ -amylase gene, the saccharification enzyme gene, and the acid protease gene are respectively cleaved by a restriction endonuclease; 512.
  • the ⁇ -amylase gene, the saccharification enzyme gene, and the acid protease gene are respectively introduced into the Saccharomyces cerevisiae expression vector to form three recombinant single gene vectors;
  • the complete ⁇ -amylase gene expression cassette, the saccharification enzyme gene expression cassette, and the acid protease gene expression cassette containing the vector promoter and the terminator fragment were excised from the three recombinant single gene vectors using restriction endonucleases, respectively. And then accessed into the same S. cerevisiae expression vector one by one in the form of a tandem expression cassette amy-ga-ap.
  • the restriction endonuclease described in S3 is ⁇ 1.
  • the transformation described in S3 is carried out using an electrotransformation method, a freezing method or a chemical reagent method.
  • the restriction enzymes for cleavage of the ⁇ -amylase gene, the saccharification enzyme gene, and the acid protease gene in S 11 are both Sa HI and 5) ⁇ 1; the restriction enzyme used in S13; For the same tail enzymes N1 ⁇ 2 I and ⁇ 1.
  • the ⁇ -amylase gene is an ⁇ -amylase gene of Aspergillus oryzae; the saccharification enzyme gene is a glucoamylase gene of Aspergillus niger; and the acid protease gene is an acid protease of Aspergillus niger. gene.
  • the ⁇ -amylase gene is an ⁇ -amylase gene of Aspergillus oryzae CICC 40344 (purchased from the China Industrial Microbial Culture Collection Management Center); the glucoamylase gene is black Aspergillus niger CICC 40179 (purchased from the China Industrial Microbial Culture Collection Management Center) saccharification enzyme gene; the acid protease gene is an acid protease gene of Aspergillus niger CICC 40179.
  • the nucleotide sequence of the ⁇ -amylase gene of Aspergillus oryzae CICC 40344 is shown in SEQ ID NO. 1, and the nucleotide sequence of the glucoamylase gene of Aspergillus niger CICC 40179 is shown in SEQ ID N0.2 (the 1566 position thereof) Nucleotide residue C (cytosine) has been artificially mutated to T (thymine), and the nucleotide sequence of the acid protease gene of Aspergillus niger CICC 40179 is shown in SEQ ID N0.3 (the nucleotide at position 1155) Residue C (cytosine) has been artificially mutated to T (thymine)).
  • the present invention has the following beneficial effects:
  • the present invention converts the enzyme gene which degrades starch and protein into Saccharomyces cerevisiae to achieve secretory expression, thereby enabling the recombinant yeast of the present invention to secrete amylase and protease, thereby efficiently degrading starch and protein in kitchen waste to be available.
  • a carbon source and a nitrogen source such as glucose, a polypeptide, and an amino acid are further fermented by the recombinant yeast into ethanol.
  • the difficulty of the present invention is how to successfully transfer the three genes of ⁇ -amylase gene, glucoamylase gene and acid protease gene into Saccharomyces cerevisiae through the Saccharomyces cerevisiae co-expression vector while achieving secretory expression.
  • we must first construct a recombinant vector containing the expression cassettes of amy, ga, and ap. In the process of constructing the recombinant three-gene co-expression vector, it is required that the same tail enzyme Nhe cannot be present in each gene sequence.
  • the cleavage site of I and ft a I because if these two cleavage sites are present, the sequence will be cleaved by Nhe I and Xba I during splicing into a tandem expression cassette and cannot be accessed with the complete gene sequence.
  • Co-expression vector for which we mutate the nucleotide residue C (cytosine) at position 1566 of the glucoamylase gene to T (thymidine) without changing the amino acid coding, resulting in the original one in the glucoamylase gene sequence.
  • the Me cleavage site was destroyed; the nucleotide residue C at position 1155 of the acid protease gene was
  • Figure 1 Flow chart of the construction of recombinant S. cerevisiae multi-gene expression vector pScIKP-amy-ga-ap.
  • a is an amplified fragment of ⁇ -amylase gene
  • b is a fragment of glucoamylase gene amplification
  • c is an amplified fragment of acid protease gene.
  • Figure 3 Enzyme activity assay of recombinant yeast a-amylase and glucoamylase (iodine fumigation staining).
  • Figure 4 Recombinant yeast acid protease activity test (casein color method).
  • Fig. 5 shows the fermentation liquid chromatogram of the recombinant yeast using the kitchen waste for ethanol fermentation.
  • Saccharomyces cerevisiae AS2.489 was purchased from the Institute of Microbial Culture Collection of the Chinese Academy of Sciences.
  • the vector pScIKP was constructed and preserved by the Molecular Biology Research Center of Jinan University.
  • the construction method can refer to the patent ZL 200810029630.6.
  • Example 1 Cloning of ct-amylase gene glucoamylase gene g a and acid protease gene ap refers to the Aspergillus oryzae spergiU oryzae ⁇ -amylase gene awj (accession number XM_001821384) published in GenBank, Aspergillus niger spergiU niger saccharification enzyme gene (login No. XM 001390493.1) and the sequence of the acid protease gene (accession number 00_001401056.2), design primers using Oligo 6 primer design software, and add appropriate restriction sites:
  • a j gene amplification primer A j gene amplification primer:
  • the total R A of Aspergillus oryzae CICC 40344 was extracted, and the reverse transcription PCR amplification reaction was carried out, and the PCR amplification product of the amy gene was ligated to the pGEM-T Easy vector (purchased from Promega) and verified by sequencing.
  • the PCR reaction conditions of the amy gene are: 94 °C 5 min
  • the total R A of Aspergillus niger CICC 40179 was extracted, and the reverse transcription PCR amplification reaction was carried out.
  • the PCR amplification products of the ga gene and the gene were ligated to the pGEM-T Easy vector, respectively, and verified by sequencing.
  • the PCR reaction conditions of the gene are:
  • the nucleotide sequence of the ⁇ -amylase gene amy of Aspergillus oryzae CICC 40344 is shown in SEQ ID N0.1, and the nucleotide sequence of the glucoamylase gene ga of Aspergillus niger CICC 40179 is shown in SEQ ID N0.2 (its 1566)
  • the nucleotide residue C (cytosine) has been artificially mutated to T (thymine)
  • the acid protease gene ap nucleotide sequence of Aspergillus niger CICC 40179 is shown in SEQ ID N0.3 (the 1155 position thereof) Nucleotide residue C (cytosine) has been artificially mutated to T (thymine)).
  • Example 2 Three enzyme gene construction co-expression recombinant vector
  • the amy, ga and ap coding sequences obtained in Example 1 were digested with restriction endonucleases Bamli I and Spe I from the pGEM-T Easy vector, respectively, and ligated to the vector digested with the same double enzyme.
  • pScIKP recombinant plasmids pScIKP-amy, pScIKP-ga and pScIKP-ap were obtained.
  • pScIKP-ga was digested with Nhe I and I to obtain a g a gene expression cassette fragment containing the PGK promoter and terminator.
  • the pScIKP-am was digested with Nhe I to linearize it.
  • pScIKP-amy-ga The two were ligated with T4 DNA ligase (using the principle that Nhe I and I are homologous enzymes) to obtain the recombinant plasmid pScIKP-amy-ga.
  • pScIKP-ap was digested with Nhe I and Xba I to obtain an ap gene expression cassette fragment containing the PGK promoter and terminator, which was ligated to pScIKP-amy-ga linearized with Nhe I.
  • the recombinant plasmid pScIKP-amy-ga-ap was obtained.
  • the sensitivity of the resistance screening marker G418 was determined for Saccharomyces cerevisiae AS2.489. It was found that the yeast had been inhibited from growing on the YPD plate with a G418 concentration of 150 ⁇ / ⁇ 1, so it was screened. Transformants can be screened with concentrations of G418 over 150 ⁇ ⁇ / ⁇ 1.
  • the three-gene co-expression recombinant plasmid pScIKP-amy-ga-ap obtained in Example 2 was linearized with restriction endonuclease Apa I and then transferred into S. cerevisiae AS2.489 by electroporation transformation at a concentration of G418. After culturing on a YPD agar plate of 200 ⁇ ⁇ / ⁇ 1 for 3 to 4 days, the colonies which can grow normally are the transformants transfected with the above recombinant plasmid. Colony PCR using primers specific to each of the three enzyme genes successfully amplified the respective gene fragments (see Figure 2), and verified that the three enzyme genes were indeed transferred into and integrated into the S. cerevisiae genome.
  • Example 4 Enzyme activity assay of amylase and protease secreted by recombinant Saccharomyces cerevisiae
  • the G418-resistant transformant colonies obtained in Example 3 were inoculated onto a YNBS plate (YNB 6.7 g/1, soluble starch 10 g/1, agar powder 15 g/1) containing 1% soluble starch at 30 °. After incubation for 72 h in a C incubator, the plate was fumigated with iodine vapor to observe the presence or absence of a hydrolyzed transparent circle. As a result, as shown in Fig. 3, a clear starch hydrolyzate transparent circle was observed around the colony, indicating that the transformant can be degraded by using the starch in the medium as a carbon source for growth.
  • the G418-resistant transformant colonies obtained in Example 3 were inoculated to a YPD solid medium (0.5 g yeast extract, 2 g peptone, 1.5 g agar plus 1% casein solution to 100 ml) containing 1% casein. 3 to 4 days.
  • a YPD solid medium 0.5 g yeast extract, 2 g peptone, 1.5 g agar plus 1% casein solution to 100 ml
  • the protease can degrade the casein, so that the transformant colony capable of secreting the protease can form a transparent casein hydrolyzed circle around the colony.
  • Jelly medium YPD medium (yeast extract 10 g / l, tryptone 20 g / l, glucose 20 g / l), after autoclaving, spare. Jelly culture for recombinant yeast.
  • Fermentation medium Kitchen waste, collected from the rest of the dining halls of a university in Guangzhou. After removing the garbage from the collected kitchen waste, the kitchen waste is pulverized by a special pulverizing waste disposing processor, thoroughly mixed and mixed, and then filled into a 1 L large-capacity triangular flask, and sterilized at 121 ° C for 20 min. Handle the backup. Fermentation for recombinant yeast. Its physical and chemical properties were determined as follows: moisture content 73.8%, dry matter content 26.2%, starch content 9.7%, protein content 1.0%, soluble sugar content 4.4%, other ingredients content 11.1%, H 6.1.
  • the recombinant Saccharomyces cerevisiae strain was activated, it was transferred to 25 ml YPD wine medium at 2% inoculum, cultured at 30 °C, 200 rpm for 24 h, and then inoculated into 200 ml YPD medium at 10% inoculum.
  • the seed is expanded and cultured at 30 °C and 200 rpm until the logarithmic growth phase.
  • the number of cells reaches 0.8 ⁇ 1.2x10 s /mL, the germination rate is about 20%, and the mortality rate is less than 1%, which is the mature sign of the wine.
  • the cultured seed liquid was transferred to the above-mentioned sterilized kitchen waste by 10% of the volume of the fermentation medium to start fermentation.
  • the culture conditions were 30 ° C, 250 rpm, natural pH, aeration culture for 4 h ; then changed to 30 ° C, 150 rpm, natural pH, anaerobic fermentation for 60 h.
  • Samples were taken every 12 h during fermentation, and the ethanol yield of the fermentation broth was measured by high performance liquid chromatography (see Figure 5). The results showed that the highest peak of ethanol production in recombinant yeast occurred at about 52 h, and the highest ethanol concentration reached 66 g/L.
  • the conversion rate between waste and ethanol reaches the level of lg ethanol produced per 4 g of kitchen waste (dry weight).

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Abstract

提供了一种能降解利用餐厨废弃物的基因重组酿酒酵母。该基因重组酿酒酵母是将α-淀粉酶基因（a-amylase）、糖化酶（glucoamylase）基因、酸性蛋白酶（acidprotease）基因通过酿酒酵母多基因共表达载体同时转入酿酒酵母并获得正确的分泌表达而构建成的。

Description

一种能降解利用餐厨废弃物的基因重组酿 技术领域
本发明涉及遗传工程和发酵工程领域， 更具体地，涉及一种能降解利用餐厨 废弃物的基因重组酿酒酵母。
背景技术
目前我国车用燃料乙醇几乎都是用粮食玉米等为原料生产，大量生产燃料乙 醇势必会带来车与人争粮的问题，直接推动粮食价格的不断上涨， 并且会有引发 食物短缺的危机。解决方式是尽量由非粮的可再生生物质为原料生产乙醇。餐厨 废弃物是一种数量巨大的可再生生物质资源， 在我国每年的餐厨废弃物产量在
6000万吨以上， 大部分被用来喂猪、 填埋、 焚烧， 甚至制造 "地沟油"， 造成了 严重的环境污染， 少部分得以合理利用， 比如制造堆肥和沼气等等， 但经济效益 低下。因此，用这些餐厨废弃物作为原料来生产燃料乙醇将会是一个颇具前景的 发展方向， 既可以变废为宝， 又可以缓解粮食与能源危机。
餐厨废弃物是一种营养丰富的可再生生物质， 富含淀粉、 糖类、 蛋白质、脂 肪等有机物，其中有机物含量占干物质的 95%以上。餐厨废弃物同时还含有维生 素、 氮、 磷、 硫、 钾、 钙、 镁等微量元素， 营养元素齐全， 能够被生物再利用， 具有再利用价值。 由于餐厨废弃物含水量高、 营养物质丰富， 在常温条件下微生 物会利用各类有机物和无机盐快速繁殖进行代谢， 使餐厨废弃物腐烂发臭，污染 环境， 为处理带来麻烦。
酿酒酵母 0¾cc½rowjC£W cerew iK是工业上进行乙醇发酵的首选菌种， 具 有将葡萄糖高效转化为乙醇的能力， 但其缺乏有效降解淀粉生成葡萄糖的酶，以 及缺乏降解蛋白质成为多肽和氨基酸的酶，在自然条件下不能直接利用餐厨废弃 物中的淀粉和蛋白质作为碳源和氮源来发酵产生乙醇。因此， 若需将餐厨废弃物 作为原料发酵生产乙醇，必须将能降解淀粉和蛋白质的酶基因导入酿酒酵母并实 现分泌表达， 以遗传工程手段弥补酿酒酵母降解利用餐厨废弃物的能力缺陷，使 得基因重组酿酒酵母可以利用自身分泌的淀粉酶和蛋白酶将餐厨废弃物中的淀 粉和蛋白质降解成为可以利用的碳源——葡萄糖和氮源——多肽和氨基酸，从而 可实现利用餐厨废弃物发酵生产燃料乙醇的工业化目的。
发明内容
本发明所要解决的技术问题是， 为了克服现有技术的上述不足，提供一种能 降解利用餐厨废弃物的基因重组酿酒酵母。
本发明所要解决的另一技术问题是，提供一种能降解利用餐厨废弃物的基因 重组酿酒酵母的构建方法。
一种能降解利用餐厨废弃物的基因重组酿酒酵母，该基因重组酿酒酵母是将 α -淀粉酶 （ a -amylase, AMY) 基因、 糖化酶 （glucoamylase, GA) 基因、 酸性 蛋白酶（acid protease, AP)基因通过酿酒酵母表达载体同时转入酿酒酵母并获得 正确的分泌表达而构建成的。
本发明的重点在于将 α -淀粉酶基因、 糖化酶基因和酸性蛋白酶基因转入酿 酒酵母中并使其分泌表达，所用的工具为酿酒酵母表达载体，作为一种优选方案， 所述的酿酒酵母表达载体为酿酒酵母多基因共表达载体；酿酒酵母多基因共表达 载体可以实现将 α -淀粉酶基因、 糖化酶基因和酸性蛋白酶基因同时转入酿酒酵 母中。 作为一种优选方案， 所述酿酒酵母多基因共表达载体为载体 pScIKP (制 备方法见专利 ZL 200810029630.6) ， 当然也可以使用其他类型的酿酒酵母多基 因共表达载体。
一种能降解利用餐厨废弃物的基因重组酿酒酵母的构建方法， 包括如下步 骤：
51.通过 PCR扩增分别得到 α -淀粉酶基因、糖化酶基因和酸性蛋白酶基因序 列； 将糖化酶基因的 1566位的核苷酸残基 C人工突变成为 Τ， 酸性蛋白酶基因 的 1155位的核苷酸残基 C人工突变成为 Τ;
52.将 α -淀粉酶基因、 糖化酶基因和酸性蛋白酶基因接入酿酒酵母表达载体 中， 构建重组多基因共表达载体；
53.将上述构建得到的重组多基因共表达载体用限制性内切酶切割， 使之线 性化后转化到酿酒酵母中， 构建基因重组酿酒酵母。
作为一种优选方案， S2构建重组多基因共表达载体包括如下步骤：
S11.用限制性内切酶分别切割酿酒酵母表达载体、 α -淀粉酶基因、 糖化酶 基因、 酸性蛋白酶基因； 512.将 α -淀粉酶基因、 糖化酶基因、 酸性蛋白酶基因分别接入酿酒酵母表 达载体， 形成三个重组单基因载体；
513.将含有载体启动子和终止子片段的完整的 α -淀粉酶基因表达盒、 糖化 酶基因表达盒和酸性蛋白酶基因表达盒使用限制性内切酶分别从三种重组单基 因载体上切下，然后以串联表达盒 amy-ga-ap的形式逐个接入同一酿酒酵母表达 载体中。
作为一种优选方案， S3中所述的限制性内切酶为^ ^ 1。
作为一种优选方案， S3中所述的转化是使用电转化法、 冷冻法或化学试剂 法进行转化。
作为一种优选方案， S 11中用于切割 α -淀粉酶基因、糖化酶基因、酸性蛋白 酶基因的限制性内切酶均为 Sa H I和 5)^ 1; S13中使用的限制性内切酶为同尾 酶 N½ I和 ΧΒσ 1。
作为一种优选方案， 所述的 α -淀粉酶基因为米曲霉的 α -淀粉酶基因； 所述 的糖化酶基因为黑曲霉的糖化酶基因；所述的酸性蛋白酶基因为黑曲霉的酸性蛋 白酶基因。
作为一种最优选方案， 所述的 α -淀粉酶基因为米曲霉 spergillus oryzae) CICC 40344 (购自中国工业微生物菌种保藏管理中心） 的 α -淀粉酶基因； 所述 的糖化酶基因为黑曲霉 (Aspergillus niger) CICC 40179 (购自中国工业微生物菌 种保藏管理中心） 的糖化酶基因； 所述的酸性蛋白酶基因为黑曲霉 (Aspergillus niger) CICC 40179的酸性蛋白酶基因。
米曲霉 CICC 40344的 α -淀粉酶基因的核苷酸序列如 SEQ ID NO. l所示，黑 曲霉 CICC 40179的糖化酶基因的核苷酸序列如 SEQ ID N0.2所示 （其 1566位 的核苷酸残基 C (胞嘧啶）已被人工突变成为 T (胸腺嘧啶）），黑曲霉 CICC 40179 的酸性蛋白酶基因核苷酸序列如 SEQ ID N0.3所示 （其 1155位的核苷酸残基 C (胞嘧啶） 已被人工突变成为 T (胸腺嘧啶））。
与现有技术相比， 本发明具有如下有益效果：
餐厨废弃物富含淀粉和蛋白质等营养物质， 如果不经过降解， 无法被酿酒酵 母当做碳源和氮源所利用， 因此，普通的酿酒酵母无法直接利用餐厨废弃物发酵 生产乙醇。 鉴于此， 为了使酿酒酵母能够利用餐厨废弃物作为原料来发酵乙醇， 本发明将降解淀粉和蛋白质的酶基因转入酿酒酵母中实现分泌表达，从而使得本 发明的重组酵母能够分泌淀粉酶和蛋白酶，因此能够高效地降解餐厨废弃物中的 淀粉和蛋白质成为可利用的葡萄糖、多肽和氨基酸等碳源和氮源物质，进而可被 重组酵母发酵成为乙醇。
本发明的难点在于如何将 α -淀粉酶基因、 糖化酶基因和酸性蛋白酶基因这 三种基因成功的通过酿酒酵母共表达载体一起转入酿酒酵母同时实现分泌表达。 为了克服这一困难， 我们必须先构建同时含有 amy、 ga、 ap三个基因表达盒的重 组载体， 而在重组三基因共表达载体的构建过程中， 要求各个基因序列里不能有 同尾酶 Nhe I和 fta I的酶切位点， 因为如果存在这两个酶切位点， 则序列在拼 接成串联表达盒的过程中会被 Nhe I和 Xba I切断而无法以完整的基因序列接入 共表达载体， 为此我们在不改变氨基酸编码的情况下将糖化酶基因第 1566位的 核苷酸残基 C (胞嘧啶） 突变成为 T (胸腺嘧啶）， 导致糖化酶基因序列中原先 的一个 Me l酶切位点被破坏掉；又将酸性蛋白酶基因第 1155位的核苷酸残基 C
(胞嘧啶）突变成为 τ (胸腺嘧啶）， 导致酸性蛋白酶基因序列中一个原先的 aa
I酶切位点被破坏掉。 于是最终获得了含有三个完整基因序列表达盒的重组共表 达载体 pScIKP-amy-ga-ap。
附图说明
图 1重组酿酒酵母多基因表达载体 pScIKP-amy-ga-ap的构建流程图。
图 2阳性转化子的 PCR鉴定结果， a为 α-淀粉酶基因扩增片段， b为糖化酶 基因扩增片段， c为酸性蛋白酶基因扩增片段。
图 3重组酵母 a -淀粉酶和糖化酶的酶活检验 （碘熏蒸染色法）。
图 4重组酵母酸性蛋白酶酶活检验 （酪素显色法）。
图 5重组酵母利用餐厨废弃物进行乙醇发酵的发酵液相色谱检测图。
具体实施方式
下面结合说明书附图和具体实施例来进一步解释本发明，但实施例对发明不 做任何形式的限定。
酿酒酵母 AS2.489购自中国科学院微生物所菌种保藏中心， 载体 pScIKP为 暨南大学分子生物研究中心构建并保存， 其构建方法可参照专利 ZL 200810029630.6ο 实施例 1 ct -淀粉酶基因 糖化酶基因 ga和酸性蛋白酶基因 ap的克隆 参照 GenBank上公布的米曲霉 spergiU oryzae α-淀粉酶基因 awj (登 录号 XM_001821384 )， 黑曲霉 spergiU niger 糖化酶基因 (登录号 XM 001390493.1 ) 和酸性蛋白酶基因 的序列 （登录号 ΧΜ_001401056.2 ) ， 用 Oligo 6引物设计软件设计引物， 同时添加上合适的酶切位点：
a j基因的扩增引物：
上游弓 I物： 5'-GGATCCATGATGGTCGCGTGGTGGTCTGTA-3'
个
BamH I
下游引物： '-ACTAGTTCACGAGCTACTACAGATCTTGC
个
Spe I
^基因的扩增引物：
上游弓 I物： 5'-GGATCCATGTCGTTCCGATCTCTACTC-3'
个
BamH I
下游弓 I物： 5'-ACTAGTCTACCGCCAGGTGTCAGT-3'
†
Spe I
基因的扩增引物：
上游弓 I物： 5'-GGATCCATGGTCGTCTTCAGCAAAACC-3'
个
BamH I
下游引物： 5'-ACTAGTCTAAGCCTGAGCGGCGAATC-3'
个
Spe I
提取米曲霉 CICC 40344总 R A， 进行反转录 PCR扩增反应， 将 amy基因 的 PCR扩增产物连接到 pGEM-T Easy载体（购自 Promega公司）上，测序验证。
其中 amy基因的 PCR反应条件为： 94 °C 5 min
94 °C 30 s
53.3 °C 30 s ^循环 30次
2°C 100 s
72 °C 10 min
提取黑曲霉 CICC 40179的总 R A， 进行反转录 PCR扩增反应， 将 ga基因 和 基因的 PCR扩增产物分别连接到 pGEM-T Easy载体上， 测序验证。
其中 基因的 PCR反应条件为：
94 °C 5 min
94 °C 30 s
57 °C 30 s ^循环 30次
72 °C 100 s 其中 基因的 PCR反应条件为:
Figure imgf000008_0001
72 °C 10 min
米曲霉 CICC 40344的 α-淀粉酶基因 amy的核苷酸序列如 SEQ ID N0.1所示， 黑曲霉 CICC 40179的糖化酶基因 ga的核苷酸序列如 SEQ ID N0.2所示（其 1566 位的核苷酸残基 C (胞嘧啶） 已被人工突变成为 T (胸腺嘧啶））， 黑曲霉 CICC 40179的酸性蛋白酶基因 ap核苷酸序列如 SEQ ID N0.3所示（其 1155位的核苷 酸残基 C (胞嘧啶） 已被人工突变成为 T (胸腺嘧啶））。
实施例 2 三种酶基因构建共表达重组载体
三种酶基因共表达重组质粒的构建流程如图 1所示。
将实施例 1得到的 amy、 ga和 ap编码序列用限制性内切酶 Bamli I和 Spe I 分别从 pGEM-T Easy载体上双酶切切下， 分别连接到用同样双酶酶切过的载体 pScIKP上， 获得重组质粒 pScIKP-amy、 pScIKP- ga和 pScIKP-ap。 用 Nhe I禾 Π I双酶切 pScIKP-ga， 得到含有 PGK启动子和终止子的 ga 基因表达盒片段。 用 Nhe I单酶切 pScIKP- amy使其线性化。 将两者用 T4 DNA 连接酶连接（利用 Nhe I和 I是同尾酶的原理），获得重组质粒 pScIKP-amy-ga。 利用同样的原理， 用 Nhe I和 Xba I双酶切 pScIKP-ap， 得到含有 PGK启动子和 终止子的 ap基因表达盒片段， 与用 Nhe I单酶切后线性化的 pScIKP-amy-ga相 连， 最终得到重组质粒 pScIKP-amy-ga-ap。
实施例 3 重组酵母转化子的筛选与验证
在酿酒酵母进行电转化之前， 对酿酒酵母 AS2.489进行抗性筛选标记 G418 的敏感性测定，发现在 G418浓度为 150 μ§/ηι1的 YPD平板上酵母已经被抑制而 不能生长， 因而在筛选转化子的时候可以用超过 150 μ§/ηι1的 G418的浓度来筛 选。
将实施例 2得到的三基因共表达重组质粒 pScIKP-amy-ga-ap用限制性内切 酶 Apa I线性化后， 用电穿孔转化法转入酿酒酵母 AS2.489中， 在 G418的浓度 为 200 μ§/ηι1的 YPD琼脂平板上培养 3〜4d后， 挑取能够正常生长的菌落即为 转有上述重组质粒的转化子。 以三种酶基因各自特异性的引物进行菌落 PCR， 均能成功扩增出各自的基因片段 （见图 2)， 验证了三种酶基因确实已经转入并 整合入酿酒酵母基因组中。
实施例 4 重组酿酒酵母分泌的淀粉酶和蛋白酶的酶活性检验
将实施例 3得到的具有 G418抗性的转化子菌落接种至含有 1%可溶性淀粉 的 YNBS平板 （YNB 6.7 g/1, 可溶性淀粉 10 g/1, 琼脂粉 15 g/1) 上， 在 30 °C培 养箱中孵育 72 h后， 用碘蒸气熏蒸平板， 观察有无水解透明圈。 结果如图 3所 示， 菌落周围能观察到明显的淀粉水解透明圈，表明转化子可以降解利用培养基 中的淀粉作为碳源进行生长。
将实施例 3得到的具有 G418抗性的转化子菌落接种到含 1%酪素的 YPD固 体培养基 （0.5g酵母提取物、 2g蛋白胨、 1.5g琼脂加 1%酪素溶液到 100ml) 上 培养 3〜4天。 结果如图 4所示， 蛋白酶能降解酪素， 故能分泌蛋白酶的转化子 菌落在菌落周围能形成透明的酪素水解圈。
实施例 5 重组酵母发酵餐厨废弃物制备乙醇
( 1 ) 培养基组成 酒母培养基： YPD培养基 （酵母提取物 10 g/l， 胰蛋白胨 20 g/l， 葡萄糖 20 g/l)， 高压灭菌后备用。 用于重组酵母的酒母培养。
发酵培养基： 餐厨废弃物， 收集自广州市某高校食堂师生用餐后剩余物。将 收集回来的餐厨废弃物除去杂物后使用垃圾专用粉碎处理器将餐厨废弃物粉碎， 充分搅拌混匀后， 分装入 1 L大容量三角瓶中， 121 °C、 20 min灭菌处理备用。 用于重组酵母的发酵。 其理化性质经测定后如下： 水分含量 73.8%， 干物质含量 26.2%,淀粉含量 9.7%，蛋白含量 1.0%，可溶性糖含量 4.4%，其他成分含量 11.1%， H 6.1。
(2) 发酵过程
将重组酿酒酵母菌株活化后， 以 2%接种量转接至 25 ml YPD酒母培养基， 30 °C、 200 rpm培养 24 h， 然后将菌液以 10%接种量接种到 200 ml YPD培养基 进行种子扩大培养， 30°C、 200 rpm培养至对数生长期， 当细胞数目达到 0.8〜 1.2xlOs/mL左右， 出芽率 20%左右， 死亡率 1%以下， 即为酒母成熟标志。
按发酵培养基体积的 10%将培养好的种子液转接至上述灭菌后的餐厨废弃 物中， 开始发酵。 培养条件为 30 °C， 250 rpm, 自然 pH， 通气培养 4h; 然后改 为 30 °C， 150 rpm, 自然 pH， 厌氧发酵 60 h。 发酵期间每 12 h取样， 用高效液 相色谱法检测发酵液乙醇产量 （结果见图 5 )， 结果发现重组酵母产乙醇最高峰 出现在 52 h左右， 最高乙醇浓度达到 66 g/L， 餐厨废弃物-乙醇之间的转化率达 到每 4 g餐厨废弃物 （干重） 产生 l g乙醇的程度。 通过以上结果可知本发明构 建的重组酿酒酵母能降利用解餐厨废弃物， 并将其变废为宝地转化成为乙醇，所 以发明人将其命名为 "噬污酵母 1号"。

Claims

1. 一种能降解利用餐厨废弃物的基因重组酿酒酵母， 其特征在于， 该基因 重组酿酒酵母是将 α -淀粉酶基因、 糖化酶基因和酸性蛋白酶基因通过酿酒酵母 表达载体同时转入酿酒酵母并获得正确的分泌表达而构建成的。
2. 根据权利要求 1所述的基因重组酿酒酵母， 其特征在于， 所述的酿酒酵 母表达载体为酿酒酵母多基因共表达载体。
3. 根据权利要求 2所述的基因重组酿酒酵母， 其特征在于， 所述酿酒酵母 多基因共表达载体为载体 pScIKP。
4. 权利要求 1至 3任一项所述基因重组酿酒酵母的构建方法， 其特征在于， 包括如下步骤：
51.通过 PCR扩增分别得到 α -淀粉酶基因、糖化酶基因和酸性蛋白酶基因序 列； 将糖化酶基因的 1566位的核苷酸残基 C人工突变成为 Τ， 酸性蛋白酶基因 的 1155位的核苷酸残基 C人工突变成为 Τ;
52.将 α -淀粉酶基因、 糖化酶基因和酸性蛋白酶基因接入酿酒酵母表达载体 中， 构建重组多基因共表达载体；
53.将上述构建得到的重组多基因共表达载体用限制性内切酶切割， 使之线 性化后转化到酿酒酵母中， 构建基因重组酿酒酵母。
5. 根据权利要求 4所述的构建方法， 其特征在于， S2构建重组多基因共表 达载体包括如下步骤：
51 1.用限制性内切酶分别切割酿酒酵母表达载体、 α -淀粉酶基因、 糖化酶 基因、 酸性蛋白酶基因；
512.将 α -淀粉酶基因、 糖化酶基因、 酸性蛋白酶基因分别接入酿酒酵母表 达载体， 形成三个重组单基因载体；
513.将含有载体启动子和终止子片段的完整的 α -淀粉酶基因表达盒、 糖化 酶基因表达盒和酸性蛋白酶基因表达盒使用限制性内切酶分别从三种重组单基 因载体上切下，然后以串联表达盒 amy-ga-ap的形式逐个接入同一酿酒酵母表达 载体中。
6. 根据权利要求 4所述的构建方法， 其特征在于， S3中所述的限制性内切 酶为^ ¾r l。
7. 根据权利要求 4所述的构建方法， 其特征在于， S3中所述的转化是使用 电转化法、 冷冻法或化学试剂法进行转化。 8. 根据权利要求 5所述的构建方法， 其特征在于， S11中用于切割酿酒酵母 表达载体、 α-淀粉酶基因、 糖化酶基因、 酸性蛋白酶基因的限制性内切酶均为 awHI和 S^I; S13中使用的限制性内切酶为同尾酶 Mel和 δαΙ。
9. 根据权利要求 4、 5或 8所述的构建方法， 其特征在于， 所述的 α-淀粉酶 基因为米曲霉的 α-淀粉酶基因； 所述的糖化酶基因为黑曲霉的糖化酶基因； 所 述的酸性蛋白酶基因为黑曲霉的酸性蛋白酶基因。
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