CN109161565B - Method for producing ethanol by using whey - Google Patents
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Abstract
The invention provides a method for producing ethanol by efficiently utilizing whey, which is characterized in that a whey-utilizing saccharomyces cerevisiae engineering bacterium with the preservation number of CGMCC No.11223 is used for standing, culturing and fermenting in a culture medium containing whey or lactose to produce the ethanol. According to the saccharomyces cerevisiae, the glucose repression phenomenon is relieved, and the galactose metabolism regulation of the saccharomyces cerevisiae is eliminated, so that the saccharomyces cerevisiae can grow and ferment to produce ethanol in a culture medium with whey concentration of 120g/L (lactose content of 53.1g/L), the fermentation period is 54h, the lactose utilization rate in whey is 98.7%, and the yield of absolute ethyl alcohol to lactose is 49.7% (equivalent to 92.3% of theoretical yield); simultaneously relieves the repression of glucose to galactose, and leads the glucose and the galactose to be utilized simultaneously.
Description
The application is a divisional application of the following patent applications, the application numbers of the original applications: 2015106320883, filing date: 2015-09-29, title of the invention: a saccharomyces cerevisiae engineering bacterium for producing ethanol by efficiently utilizing whey and a construction method thereof.
Technical Field
The invention belongs to the technical field of bioengineering, relates to breeding of industrial microorganisms, and particularly relates to a method for producing ethanol by efficiently utilizing whey by utilizing a strain of whey-utilizing saccharomyces cerevisiae engineering bacteria.
Background
Whey is an extremely thin liquid remaining after separation of flocs in the manufacture of cheese or casein, and is a by-product of industrially produced cheese and casein, and 9 tons of whey per 1 ton of cheese produced contains 55% of nutrients in milk, but whey has high BOD and COD, and therefore, it imposes a large burden on the environment. At present, the global whey yield is about 16 million tons, only 50 percent of the whey is treated and used for food, feed and the like, the remaining 8 million tons are not effectively utilized and are discharged into the nature, so that not only is the environment polluted, but also huge waste of the resource is caused. Whey contains 20% of dairy protein, and the most lactose, which accounts for about 5% of whey, is the main cause of high BOD and COD.
How to utilize lactose in whey to reduce the pollution of whey to the environment has been a concern in the dairy industry. The utilization of lactose is mainly in the following directions: used in food industry, feed industry and medicine industry. However, with the increasing global energy crisis and the world food safety problems, whey becomes a new main raw material for producing fuel ethanol, and the production of fuel ethanol by using whey also becomes a new hotspot.
At present, the production of fuel ethanol by utilizing whey abroad has been over 40 years of history, and the used strains mainly comprise Kluyveromyces and saccharomyces cerevisiae. Although the Kluyveromyces can utilize whey, the yeast of the genus has low alcohol yield, poor stress resistance and large cell growth amount, and is not suitable for producing ethanol. Although saccharomyces cerevisiae is the best choice for producing ethanol, wild saccharomyces cerevisiae cannot utilize whey as a unique carbon source, so that the research focus of researchers is always how to modify saccharomyces cerevisiae to enable the saccharomyces cerevisiae to ferment whey to produce ethanol. At present, foreign solutions are mainly divided into the following solutions: firstly, a protoplast fusion technology is utilized to fuse saccharomyces cerevisiae and kluyveromyces, and mutant strains which can utilize lactose and can efficiently produce ethanol are screened; secondly, a gene engineering means is adopted to introduce exogenous genes into the saccharomyces cerevisiae body, so that the saccharomyces cerevisiae has the capability of decomposing lactose, and the problem that the saccharomyces cerevisiae cannot utilize whey is solved. The most foreign genes introduced at present are the LAC4 gene (encoding lactalbumin) and the LAC12 gene (encoding lactalbumin) of Kluyveromyces lactis. However, the strains constructed by the two methods have the problems of slow whey utilization, long fermentation period and the like.
For Lac+For the saccharomyces cerevisiae engineering strain, lactose in whey is decomposed into glucose and galactose before being metabolized. In the case of Saccharomyces cerevisiae, there is a great difference in the utilization of galactose and glucose, which includes both the regulation of galactose metabolism itself and the inhibition of galactose utilization by glucose, i.e., the glucose repression phenomenon. Galactose as Saccharomyces cerevisiae "Alternative carbon source ", whose metabolic rate in the oxygen-depleted state is only 1/3 of the glucose metabolic rate. Galactose needs to be metabolized into glucose-6-phosphate through the Leloir pathway before entering the glycolysis pathway to be utilized by yeast. The enzymes required for the Leloir pathway are encoded by GAL genes, which belong to the classical expression regulation genes of Saccharomyces cerevisiae. Four gene products are involved in such expression regulation of the GAL gene system, and they are the GAL4 gene on chromosome XVI encoding a Gal4 protein that binds to the upstream sequence sites of the above five genes and activates transcription of these genes, respectively; and Gal80 protein encoded by GAL80 gene located on chromosome XIII can be directly combined with Gal4 protein, so as to inhibit the transcriptional activation function of Gal 4; and GAL3 gene located on chromosome IV encodes GAL3 protein that can produce allosteric effects with ATP and galactose under the action of galactose inducing signal, and the produced allosteric effector binds to GAL80 protein to release GAL4 protein, but the mechanism of action of galactose inducing signal is not clear at present. In addition to the above three regulatory proteins, recent studies have shown that the GAL6 protein encoded by GAL6 gene located on chromosome XIV also belongs to one of GAL regulatory genes, and that the GAL6 protein affects the stability of mRNA transcribed from GAL family genes.
In addition to the regulation process of GAL gene expression itself, its expression is also inhibited by glucose. In yeast, the expression of many genes is controlled and regulated by glucose, repression of glucose involves many factors including glucose signaling, intermediate regulators, specific transcriptional repression and activator proteins, etc., and the complex interaction between the Mig1 protein complex and GAL-activator protein (Gal4) is considered as a major step in repressing galactose metabolism by glucose.
In s.cerevisiae, neutral trehalase is encoded by the NTH1 and NTH2 genes, of which NTH1 encodes the protein that plays a major role. The glucose repressor complex is a complex consisting of three proteins, of which only the MIG1 protein encoded by MIG1 binds to the gene of the enzyme required for galactose utilization, inhibiting its transcription. In the galactose metabolic regulation pathway, there are two regulatory proteins acting as negative regulation expression, Gal80 (encoded by Gal80 gene) and Gal6 (encoded by Gal6 gene).
Zhongjing et al (Lac)+The influence of GAL80 gene knockout on whey utilization in Saccharomyces cerevisiae engineering bacteria discloses the construction of a Saccharomyces cerevisiae engineering strain capable of efficiently utilizing whey, the cloning of GAL80 gene of AY5 and the construction of knockout plasmid pUC-GABCUP. PCR amplifying homologous recombination fragment GA-CUPl-GB and Lac using said plasmid as template+Saccharomyces cerevisiae AY51024M is a receptor strain, GAL80 gene in the receptor strain is knocked out by using a homologous recombination method, and a strain AY51024M-G which can relieve Gal80 inhibition and can utilize whey is obtained.
At present, no report that the regulation of galactose metabolism is released while LAC4 and LAC12 genes are overexpressed at home and abroad is available.
Disclosure of Invention
The invention aims to provide a method for producing ethanol by efficiently utilizing whey by utilizing a strain of whey-utilizing saccharomyces cerevisiae engineering bacteria.
The method is characterized in that a saccharomyces cerevisiae strain with the preservation number of CGMCC No.11223 is subjected to static culture and fermentation in a culture medium containing whey or lactose to produce ethanol, and the fermentation period is 54 h.
Preferably, the composition of the culture medium is: whey powder 120g/L or lactose 53.1g/L, (NH)4)2SO4 5g/L，MgSO4·7H2O1 g/L and the solvent is water.
Preferably, the temperature of the fermentation is 30 ℃.
The whey-utilizing Saccharomyces cerevisiae engineering bacteria (Saccharomyces cerevisiae) provided by the invention is specifically AY5MG, which is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of CGMCC No. 3 of West Lu No.1 of the sunward area in Beijing, the institute of microbiology in China academy of sciences, zip code 100101) at 8-11 th month in 2015, the preservation number is CGMCC No.11223, and the classification names are as follows: saccharomyces cerevisiae.
The saccharomyces cerevisiae engineering bacteria AY5MG of the invention grow and ferment in a culture medium with whey concentration of 120g/L (lactose content of 53.1g/L) to produce ethanol, the fermentation period is 54h, and the utilization rate of lactose in whey is 98.7%; the yield of absolute ethanol to lactose was 49.7% (corresponding to 92.3% of the theoretical yield).
The construction method of the whey-utilizing saccharomyces cerevisiae engineering bacteria respectively expresses a lactose lyase gene LAC4 and a lactose permease gene LAC12 through a strong promoter PGK1, and simultaneously knocks out MIG1, NTH1 and GAL6 genes, so that the glucose repression phenomenon is relieved, the galactose metabolism regulation of saccharomyces cerevisiae is eliminated, and the high-tolerance saccharomyces cerevisiae engineering bacteria capable of efficiently utilizing lactose to produce ethanol is obtained.
The construction method of the saccharomyces cerevisiae engineering strain specifically comprises the following steps:
1) taking a saccharomyces cerevisiae genome as a template, and connecting the upstream and downstream fragments GAL6A and GAL6B of GAL6 gene amplified by PCR with a pUC19 plasmid to obtain a plasmid pUC-G6 AB;
2) connecting a copper resistance gene CUP1 derived from the Yep-C plasmid with pUC-G6AB to obtain a plasmid pUC-G6AB-CUP 1;
3) by utilizing a PCR amplification technology, a homologous recombination fragment GAL6A-CUP1-GAL6B of GAL6 gene is amplified by taking plasmid pUC-G6AB-CUP1 as a template;
4) introducing GAL6A-CUP1-GAL6B homologous recombination fragments into a-type and alpha-type haploids of a saccharomyces cerevisiae receptor strain by adopting a lithium acetate conversion method to obtain a saccharomyces cerevisiae genetic engineering haploid strain after homologous recombination; the acceptor strain is a genetic engineering bacterium obtained by respectively expressing a whey lyase gene LAC4 and a whey permease gene LAC12 through a strong promoter PGK1 and knocking out a MIG1 gene and an NTH1 gene by adopting a method disclosed by a patent CN 102604849B.
5) And fusing the purified saccharomyces cerevisiae a-type and alpha-type recombinant haploids, and screening by using a resistance plate and a sporulation experiment to obtain the saccharomyces cerevisiae engineering bacteria (diploid) capable of utilizing whey.
The invention also provides a verification sequence specially used for identifying the galactose metabolism negative regulator removal, the gene sequence takes G6A-U and G6B-D as primers, the genome of the saccharomyces cerevisiae engineering strain utilizing whey as a template, and the sequencing of an amplified fragment is a specific sequence, as shown in the sequence table 1.
The size of the primers amplified from G6A-U and G6B-D can be used to determine whether the gene has been deleted.
The invention has the advantages and positive effects that:
according to the invention, on the basis of the original whey utilization capability of the strain, the saccharomyces cerevisiae engineering strain AY5MG which removes galactose metabolism regulation and can quickly and efficiently utilize whey to produce ethanol is obtained by blocking galactose metabolism regulation negative repression element genes.
Compared with the initial Saccharomyces cerevisiae (recipient bacterium AY-510B24M Saccharomyces cerevisiae CGMCC No.5843), the Saccharomyces cerevisiae engineering bacterium AY5MG (with the preservation number of CGMCC No.11223) capable of utilizing whey, which is obtained by the invention, can utilize whey more quickly to grow and ferment in a culture medium with whey concentration of 120g/L (lactose content of 53.1g/L) to produce ethanol; the fermentation period is 54h, and the utilization rate of lactose in whey is 98.7%; the yield of absolute ethyl alcohol to lactose is 49.7 percent (which is equal to 92.3 percent of the theoretical yield); meanwhile, the galactose metabolism regulation of the saccharomyces cerevisiae is relieved, so that the whey can be quickly utilized and can be normally fermented when the whey contains 19% (v/v) of ethanol or the fermentation temperature is 39 ℃. The saccharomyces cerevisiae engineering bacteria have no special requirements on fermentation equipment and conditions, and can be used in equipment and conditions of common wineries, so that the saccharomyces cerevisiae engineering bacteria have wide application prospect and can provide possibility for producing fuel ethanol by taking whey as a raw material.
Drawings
FIG. 1: the construction process of plasmid pUC-G6AB-CUP 1;
FIG. 2: a verified electrophoretogram of plasmid pUC-G6AB-CUP 1.
FIG. 3: and (5) verifying the positive recombination saccharomyces cerevisiae haploid.
FIG. 4: and constructing a route map of the whey utilization type saccharomyces cerevisiae engineering bacteria.
FIG. 5: and (3) comparing the fermentation conditions of the engineering bacteria and the parents in the simulated whey decomposition liquid, wherein (A) is the parent AY-510B24M, and (B) is the engineering bacteria AY5 MG.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
The recipient strain used in the present invention is Lac from any source+A saccharomyces cerevisiae diploid strain.
Example 1: construction of whey saccharomyces cerevisiae gene engineering bacteria for removing galactose metabolism regulation
(1) Construction of genetically engineered Strain
1) Using Saccharomyces cerevisiae AY5(Saccharomyces cerevisiae CGMCC No 2.1364) genome as template, using G6A-U and G6A-D as primers, amplifying fragment GAL6A with 646bp upstream length of GAL6 gene complete sequence by PCR, similarly using G6B-U and GB6-D as primers, amplifying fragment GAL6B with 665bp downstream length of GAL6 gene complete sequence (the primer sequence and the enzyme cutting site are shown in Table 1), and then connecting GAL6A, GAL6B and pUC19 plasmid to obtain plasmid pUC-G6 AB;
2) using Yep-C plasmid as a template, and using Cup-U, Cup-D1 and Cup-D2 as primers to amplify 1410bp copper resistance gene CUP1 to be connected with plasmid pUC-G6AB to obtain plasmid pUC-G6AB-CUP1 (the construction flow is shown in figure 1);
3) the constructed pUC-G6AB-CUP1 plasmid is taken as a template, G6A-U, G6B-D is taken as a primer, and a homologous recombination fragment GAL6A-CUP1-GAL6B is amplified by utilizing a PCR amplification technology;
TABLE 1 primer sequences and cleavage sites used in plasmid construction
FIG. 2 is an electrophoresis chart showing the procedure of constructing the plasmid pUC-G6AB-CUP 1. Wherein, Lane 1 is GAL6A fragment amplified using AY-510B24M as a template; lane 2 shows the GAL6A fragment amplified from the pUC-G6AB-CUP1 plasmid as a template; lane 3 shows GAL6B fragment amplified using AY-510B24M as a template; lane 4 shows the GAL6B fragment amplified from the pUC-G6AB-CUP1 plasmid as a template; lane 5 is the fragment of CUP1 amplified using Yep-C as template; lane 6 CUP1 fragment amplified using pUC-G6AB-CUP1 plasmid as template; lane 7 is the amplified GAL6A-CUP1-GAL6B gene fragment using pUC-G6AB-CUP1 plasmid as template, and Lane M is DL5000DNA marker.
4) The GAL6A-CUP1-GAL6B homologous recombination fragment is introduced into Lac by using a lithium acetate transformation method+Obtaining homologous recombined saccharomyces cerevisiae gene engineering haploid strains by the a-type haploid and the alpha-type haploid of the saccharomyces cerevisiae; lac used in this example+The Saccharomyces cerevisiae is specifically Saccharomyces cerevisiae AY-510B24M (Saccharomyces cerevisiae with preservation number of CGMCC No 5843) disclosed in patent CN102604849B
FIG. 3 shows the PCR verification results of a and alpha type positive recombination haploid. Wherein, the Lane 1 is GAL6A-CUP1-GAL6B gene segment amplified from a type a recombinant haploid; lane 2 shows GAL6A-CUP1-GAL6B gene fragment amplified from the alpha-type recombinant haploid; lane 3 shows the complete GAL6 fragment amplified with the same primers as AY-510B24M as a template; lane 4 is DL5000DNA marker. Lanes 1, 2 show that the GAL6 gene in the parent has been replaced by GAL6A-CUP1-GAL6B homologous recombination fragments.
5) And fusing the purified saccharomyces cerevisiae a-type and alpha-type recombinant haploids, and screening by using a resistance plate and a sporulation experiment to obtain the engineering bacteria (diploid) capable of utilizing whey.
(2) Specific sequence of genetic engineering strain
The obtained genetic engineering strain AY5MG chromosome contains a section of specific sequence, and identification of negative control gene GAL6 removal can be carried out after PCR amplification sequencing.
The primer sequences for specific fragment amplification are respectively as follows:
G6A-U:5'-AAAGAATTCGCGGAAAGGCAGGCAATA-3’
G6B-D:5'-ATCAAGCTTATGGTAGCCGAATGAATGAAAT-3’
the gene sequence of the specific fragment is shown in a sequence table 1.
Example 2: research on fermentation performance of engineering bacteria for producing fuel ethanol by utilizing whey
Respectively inoculating AY5MG and its parent AY-510B24M into 20mL glucose culture solution, and culturing at 30 deg.C overnight for 12 h; after centrifugal washing, transferring all bacteria liquid into 200mL whey culture medium, and standing, culturing and fermenting at 30 ℃. The whey culture medium is (g/L): whey powder 120 (lactose content 53.1g/L), (NH)4)2SO4 5，MgSO4·7H2O1, and metering to 1L by using water. Sampling every 24h during fermentation, and recording the weight loss; after fermentation, stopping culturing and weighing; the residual sugar concentration, alcohol concentration and dry weight of the fermentation broth were measured to characterize the overall properties, and the results are shown in Table 2.
TABLE 2 fermentation Performance of Saccharomyces cerevisiae recipient and engineered bacteria in whey
Note: the data shown are the average of the results of three replicates.
Example 3: research on glucose repression phenomenon of whey utilization type saccharomyces cerevisiae engineering bacteria and starting strain whey decomposition products
Respectively inoculating the engineering bacteria and the receptor bacteria into 5mL of YEPD culture solution, and culturing at 30 ℃ overnight for 12 h; the whole strain was transferred to 20mL of galactose medium and cultured at 30 ℃ for 24 hours. Preparing a simulated whey decomposition product culture medium: glucose 3g, galactose 3g, (NH)4)2SO4 0.5g，MgSO4·7H20.1g of O, 0.2g of yeast powder, 0.1g of peptone and KH2PO40.3g, distilled water 100 mL. Inoculating according to the inoculation amount of 10 percent, and standing and culturing at 30 ℃. Samples were taken at regular intervals during the fermentation and the results are shown in FIG. 5 for different sugar concentrations. Lactose in whey is decomposed into glucose and galactose in the saccharomyces cerevisiae engineering strain firstly, and then enters respective metabolic pathways. However, in the case of Saccharomyces cerevisiae, the presence of glucose severely inhibits galactose utilization, a phenomenon known as glucose repression. From FIG. 5It can be seen that the removal of GAL6 gene can also slow down the glucose repression phenomenon of AY51024M, shortening the fermentation period in mixed sugar.
Example 4: comparing the tolerance of the obtained engineering bacteria with that of the recipient bacteria
Respectively inoculating the engineering bacteria and the receptor bacteria into 6 mL/tube YEPD for primary culture, performing static culture at 30 ℃ for 24h, inoculating 0.5mL of seed liquid into YEPD culture media with different alcohol concentrations, and performing static culture at 30 ℃ to observe gas production. The temperature tolerance experiment includes inoculating seed liquid 0.5mL into YEPD culture medium, culturing at different temperatures, and observing gas production. The alcohol tolerance and temperature tolerance are shown in Table 4, and the specific medium components are shown in Table 3.
TABLE 3 media ingredient contents of different alcohol concentrations
TABLE 4 comparison of alcohol tolerance and temperature tolerance of engineering and recipient bacteria
Note: "+" is growth, "-" is not long; the results shown are the average of three parallel experiments.
Sequence listing
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<120> a method for producing ethanol using whey
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Claims (3)
1. A method for producing ethanol by whey is characterized in that a saccharomyces cerevisiae strain with the preservation number of CGMCC No.11223 is subjected to static culture and fermentation in a culture medium containing whey or lactose to produce ethanol, and the fermentation period is 54 h.
2. The method of claim 1, wherein the composition of the medium is: whey powder 120g/L or lactose 53.1g/L, (NH)4)2SO4 5g/L，MgSO4·7H2O1 g/L and the solvent is water.
3. The method of claim 1 or 2, wherein the temperature of the fermentation is 30 ℃.
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