TWI640633B - Consortium, method of decomposing hemicellulose and method of manufacturing alcohol using the same - Google Patents

Abstract

The invention provides a method for producing raw alcohol, which comprises recombining at least three groups of yeast The bacteria are obtained to obtain at least three groups of recombinant yeasts, and the recombinant yeast comprises hemicellulose transforming bacteria expressing fusion proteins different from each other, and the fusion proteins are hemicelluloses which are different from each other in anchoring proteins. a combination of decomposing enzymes, and the anchoring protein has the ability to anchor to the surface of the hemicellulose-converting bacteria; and a five-carbon sugar-fermenting bacterium expressing a five-carbon sugar fermentation enzyme; co-cultivating the at least three groups of recombinant yeast; Contacting the hemicellulolytic enzyme to obtain a five carbon sugar; and causing the five carbon sugar fermentation enzyme to convert the five carbon sugar into a biomass alcohol.

Description

Mixed bacteria system, hemicellulose decomposition method using the same, and alcohol production method

The present invention relates to a process for the decomposition of hemicellulose. Specifically, the present invention relates to a mixed bacteria system, a hemicellulose decomposition method using the same, and an alcohol production method.

In the industrial development of bio-alcohol, the currently developing combined bio-process (CBP) integrates cellulolytic enzymes and alcohol fermentation into the same reactor, thereby effectively reducing production costs. However, regardless of any process, the raw material must be converted to a monosaccharide by hydrolysis, in order to continue the further fermentation reaction. However, the hemicellulose contained in the raw material has a preferred hydrolysis efficiency in an acidic environment, a high temperature or a high pressure environment in the prior art. However, in the process of CBP, cellulose hydrolysis (including cellulose, hemicellulose, etc.) and alcohol fermentation are carried out in an environment in which yeast can survive, and the above method cannot be used. Therefore, attempts have been made to insert yeast into a highly efficient hemicellulose hydrolase by gene transfer technology, so that the yeast itself has a hemicellulose hydrolysis ability to solve the problem.

However, the five-carbon sugar obtained by the hydrolysis of hemicellulose does not have an effective utilization route, and this situation causes the concentration of the five-carbon sugar in the reaction tank to continuously increase, thereby causing the efficiency of the alcohol process to be low, And the cost increase caused by the need to continuously remove excess five-carbon sugar. Therefore, the prior art also has a sequence in which a fermentation can be carried out using a five-carbon sugar into a yeast strain, so that the yeast can simultaneously have the ability to degrade hemicellulose and utilize a five-carbon sugar. However, due to the insertion of too many foreign genes into the yeast plastids, this has led to the problem of low viability of recombinant yeast. This phenomenon makes the production capacity of raw alcohol in actual application limited, which is one of the urgent problems to be solved.

In view of the above, the present invention provides an adjustable hemicellulose hydrolysis fermentation artificial mixed bacteria system, and establishes yeast strains respectively expressing different hemicellulose hydrolase, so that each yeast strain expresses only one of the hydrolase. In addition, a yeast strain that can utilize a five-carbon sugar for fermentation reaction is more independently established, and the aforementioned plurality of strains are integrated into a single reactor for a CBP process. The mixed bacteria system of the present invention can simultaneously overcome the aforementioned hemicellulose hydrolysis problem, the problem of five-carbon sugar consumption, and the survival rate of recombinant yeast, and provides a high-yield and low-cost bio-alcohol production process.

The invention provides a method for producing raw alcohol, which comprises recombining at least three groups of yeasts to obtain at least three groups of recombinant yeasts, wherein the at least three groups of recombinant yeasts comprise at least two groups of hemicellulose transformed bacteria, each of which expresses each a fusion protein different from each other, the fusion protein between each group is a combination of an anchor protein and a hemicellulose decomposing enzyme different from each other, and the anchor protein has anchor to hemicellulose. The ability to transform the surface of the bacteria; and at least one set of five carbon sugar fermenting bacteria expressing the five carbon sugar fermentation enzyme; cocultivating the at least three groups of recombinant yeast; contacting the hemicellulose with the hemicellulolytic enzyme to obtain five carbons Sugar; and the five-carbon sugar fermentation enzyme converts the five-carbon sugar into bio-alcohol.

Preferably, the gene expressing the hemicellulolytic enzyme comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or a combination thereof.

Preferably, the gene expressing the fusion protein comprises SEQ ID NO: 6 and SEQ ID NO: 7.

Preferably, the five carbon sugar fermentation enzyme comprises xylose reductase, xylulokinase or a combination thereof.

Preferably, the method for producing raw alcohol is to reconstitute six groups of recombinant yeast, co-culture six groups of recombinant yeast, to make hemicellulose contact the hemicellulolytic enzyme, and to make five-carbon sugar fermentation enzyme to five-carbon sugar Converted to raw alcohol.

Preferably, the six groups of recombinant yeasts comprise five sets of hemicellulose transforming bacteria which are expressed by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO, respectively. : 5 expressed hemicellulolytic enzyme; and a set of the five carbon sugar fermenting bacteria expressing a five carbon sugar fermentation enzyme comprising xylose reductase, xylulokinase or a combination thereof.

Preferably, the ratio of the total cell number of the hemicellulose-converting bacteria to the number of cells of the five-carbon sugar-fermenting bacteria is 4:1.

Preferably, in the hemicellulose-transformed bacteria, the ratio of the number of cells expressing the genes of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 is 10 ~20:5~20:5~15:1~15:30~79.

In addition, the present invention further provides a method for decomposing hemicellulose used in the method for producing raw alcohol, which comprises recombining at least three groups of yeasts to obtain at least three groups of recombinant yeasts, wherein the recombinant yeast comprises: at least two Group of hemicellulose-transformed bacteria, which express between groups a fusion protein different from each other, and the fusion protein between each group is a combination of an anchoring protein and a hemicellulose decomposing enzyme different from each other, and the anchor protein has an anchorage to the surface of the hemicellulose conversion bacteria. Ability to co-culture recombinant yeast; and to expose hemicellulose to hemicellulolytic enzyme, which decomposes hemicellulose into five carbon sugars.

Specifically, the method for producing a biomass alcohol provided above is a mixed system obtained by applying the hemicellulose decomposition method provided by the present invention, which comprises at least three groups of recombinant yeast co-cultured, wherein the recombinant yeast comprises at least two a group of hemicellulose-transformed bacteria expressing a fusion protein different from each other, which is a combination of an anchoring protein and a hemicellulolytic enzyme different from each other, and the anchoring protein has an anchor to half. The ability of the cellulose to transform the surface of the bacteria; and at least one group of five carbon sugar fermenting bacteria expressing a five carbon sugar fermentation enzyme.

In summary, the present invention employs the aforementioned hemicellulose decomposition method to provide a mixed bacteria system comprising a plurality of sets of recombinant yeasts each expressing a different hemicellulolytic enzyme, and having a certain number of ratios therebetween Good hemicellulose decomposition efficiency.

The existing bio-alcohol production process can be enhanced by the mixed bacteria system of the present invention. In the past, recombination caused a group of yeasts to express only one degrading enzyme, which was considered to be inefficient. However, the improvement of insertion of multiple exogenous genes resulted in low viability and improved efficiency. The present invention skips the existing technical bias, and even if only one group of yeast expresses only one decomposing enzyme, the effective use of the mixed bacteria system can achieve the efficiency improvement that cannot be achieved by the conventional technology, and overcome the production capacity limitation of the raw alcohol.

In addition, the present invention further proves that the hemicellulolytic enzyme is immobilized on the surface of the yeast, and is not fixed to the outside evenly secreting the hemicellulolytic enzyme, and has better production efficiency of the raw alcohol, and the existing one can be made. The process of raw alcohol has been further improved.

S10, S20, S21, S22, S23, S30, S40‧‧

Y, YAH‧‧‧ yeast

YF‧‧‧ five carbon sugar fermentation bacteria

YT‧‧‧ hemicellulose conversion bacteria

HE‧‧‧ hemicellulolytic enzyme

AP‧‧‧ Anchored Protein

FP‧‧‧ fusion protein

CS‧‧‧ mixed bacteria system

Figure 1 presents a flow chart of an embodiment of the invention.

Fig. 2 is a view showing the structure of a hemicellulose-converting bacterium according to an embodiment of the present invention.

Figure 3 is a graph showing the growth of a five-carbon sugar-fermenting bacterium and an unrecombinant yeast in a cellulose-rich medium in an exemplary embodiment of the present invention.

Figure 4 is a diagram showing a comparison group and an implementation group in an exemplary embodiment of the present invention.

Figure 5 is a graph showing yeast density vs. time for a control group, a comparison group, and an implementation group in an exemplary embodiment of the present invention.

Figure 6 is a graph showing the concentration versus time of reducing sugars in the medium of the control group, the comparison group and the implementation group in an exemplary embodiment of the present invention.

Fig. 7 is a graph showing the relationship between the yeast density of the implementation group, the ethanol concentration in the medium, and the concentration of reducing sugars in the medium in an exemplary embodiment of the present invention.

The hemicellulose decomposition method provided by the present invention mainly comprises the steps of dividing the yeast into several groups, wherein the genes of different hemicellulolytic enzymes and anchoring proteins can be fused and inserted into the genome of the yeast to make these groups. Do not secrete fusion proteins that express different hemicellulolytic enzymes. The expressed fusion protein has a portion that anchors the protein, and when it comes into contact with the surface of the yeast, it is inserted onto the surface of the yeast, and the hemicellulolytic enzyme is immobilized on the surface of the yeast. On the other hand, the remaining group can insert a foreign gene to express an enzyme having a five-carbon sugar fermentation function. Next, all of the above groups of yeasts were co-cultured to form a mixed system. When adding hemicellulose-containing raw material to the mixing system, Hemicellulose can be broken down into five-carbon sugars by hemicellulolytic enzymes, and five-carbon sugars can be converted into biomass alcohol by enzymes having a five-carbon sugar fermentation function. Applying the mixed bacteria system to the combined biotransformation process not only removes the five-carbon sugar that could not be effectively utilized, but also reduces the cost and improves the yield of the raw alcohol.

In order to make the above-mentioned objects, technical features, and gains after actual implementation more obvious, a more detailed description will be given below with reference to the corresponding drawings in the preferred embodiments.

In an embodiment of the invention, the flow is as shown in FIG.

In step S10, a recombinant yeast having a five-carbon sugar fermentation function is established, which is hereinafter referred to as a five-carbon sugar fermentation bacterium YF. The five-carbon sugar fermentation bacterium YF is established by the yeast Y expressing a five-carbon sugar fermentation enzyme by inserting a foreign gene capable of expressing a five-carbon sugar fermentation enzyme into the genome of the unrecombined yeast Y. The yeast Y selected in this step may be any yeast commonly used for alcohol fermentation, for example, Schizosaccharomyces genus, Saccharomycodes genus, Hanseniaspora genus yeast, and the like. Preferably, the five carbon sugar fermentation enzyme may be xylose reductase (XI), xylulose kinase (XK) or other enzymes having similar functions. Preferably, the five carbon sugar fermentation enzyme is expressed in the yeast.

In step S20, a recombinant yeast having a function of converting hemicellulose to a five-carbon sugar is established, which is hereinafter referred to as hemicellulose-transformed bacteria YT. Step S20 further includes steps S21 to S23.

In step S21, a foreign gene expressing an anchor protein AP, a foreign gene having a secretory signal, and a foreign gene expressing a hemicellulolytic enzyme HE are fused to express an anchor protein AP and a hemicellulose decomposing enzyme HE. When the state of fusion can be expressed together, it is called fusion protein FP. Preferably, the gene expressing the anchor protein AP may be a lectin, alpha lectin or various genes commonly used in surface expression techniques. Preferably, the exogenous gene expressing the hemicellulolytic enzyme HE comprises Abf (SEQ ID NO: 1), Axe1 (SEQ ID NO: 2), Bx11 (SEQ ID NO: 3) obtained from Trichoderma reesei. ), Glr1 (SEQ ID NO: 4) and Xyn2 (SEQ ID NO: 5) genes, It can express arabinofuranosidase GH52 (arabinofuranosidase GH52, GH52), acetylxylan esterase CE5 (CE5), β-xylosidase GH3, GH3, α-glucuronidase GH67 (α-D-glucuronidase GH67, GH67) and endoxylanase GH11 (GH11).

In step S22, the foreign gene expressing the fusion protein FP is inserted into the yeast Y, and the yeast Y has the ability to express the fusion protein FP which binds to the anchor protein AP and the hemicellulolytic enzyme HE to obtain the yeast YAH. The type of the yeast Y used in this step may be the same as or different from the type of the yeast Y used in the step S10, and even if a different type of yeast Y is used, it is a yeast commonly used for alcohol fermentation. Preferably, the fusion protein secretes expression such that it is excreted from the yeast cells.

In step S23, the fusion protein FP secreted and expressed in step S22 comprises a portion of the anchor protein AP and a portion of the hemicellulose decomposing enzyme HE, and when the portion of the anchor protein AP contacts the surface of the yeast YAH, it is inserted into In the surface of the yeast YAH, the part of the hemicellulose decomposing enzyme HE is exposed outside the cell. In summary, the hemicellulolytic enzyme HE binds to the surface of the yeast YAH by anchoring protein AP, and the morphology is similar to that of the fibrous body. Hereinafter, the yeast YAH into which the fusion protein FP has been inserted is called hemicellulose conversion bacteria. YT.

Next, in step S30, the five carbon sugar fermentation bacterium YF obtained in step S10 and the hemicellulose conversion bacteria YT obtained in step S23 are co-cultured to form a mixed system CS. The mixed bacteria system CS can be any environment capable of coexisting and continuously growing the five-carbon sugar fermentation bacteria YF and the hemicellulose conversion bacteria YT, and can be produced by the mixed bacteria system CS after the addition of the raw material.

In step S40, the raw material is added to the mixed system CS formed in step S30. Specifically, the raw material of the raw material may be any raw material having cellulose, such as barley, wheat, oats, Grain, sugar beet, sweet sorghum, cassava, and sweet potato; or non-food raw materials such as straw, rice straw, corn stalks, etc.; or agricultural, urban and construction waste such as kitchen waste, newspapers, sawdust, waste Wood, etc.; or fast-growing cellulosic crops such as Miscanthus, Pennisetum, and switchgrass; or readily harvestable materials such as seaweed.

The present invention will be described below with an exemplary embodiment in combination with the above steps S10, S20 to S23, S30 and S40.

Corresponding to step S10, the pRH384 plastid and the pRH385 plastid (reference: Hector RE, Dien BS, Cotta MA, Mertens JA: Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24. Biotechnology for biofuels 2013,6(1):84) The genes expressing xylose reductase and xylulose kinase were excised and recombined into the genome of S. cerevisiae BY4742 yeast by gene transfer technology. The S. cerevisiae BY4742 yeast can ferment five-carbon sugar through the XI-XK pathway to produce alcohol as a five-carbon sugar fermentation bacterium YF.

Corresponding to step S20, step S21 is performed. SEQ ID NO. 1 to SEQ ID NO: 5 were previously amplified by Trichoderma ressei by PCR, and then SEQ ID NO: 7 and SEQ ID NO. 1 to SEQ ID NO: 5, respectively. One of the fusions and fusion of SEQ ID NO: 6 to obtain the fusion gene FG, wherein SEQ ID NO: 7 is amplified from S. cerevisiae by PCR, and the expressed protein Agα1 is used as anchor protein AP, SEQ ID NO :6 is also amplified by S. cerevisiae by PCR, the expressed protein MFa1 is provided as a secretion signal, and the proteins expressed by SEQ ID NO: 1 to SEQ ID NO: 5 are used as a hemicellulose fermentation enzyme HE.

Corresponding to step S22, the fusion gene FG having SEQ ID NO: 1 to SEQ ID NO: 5 is inserted into the genome of five groups of S. cerevisiae EBY100 yeast, respectively, to have a secreted expression with fusion protein FP and hemicellulolytic enzyme. The ability of HE to fuse protein FP as yeast YAH.

Corresponding to step S23, the fusion protein FP secreted by the yeast YAH in the step S22 is discharged from the yeast YAH, and when the anchor protein AP, that is, the Agα1 portion is in contact with the surface of the yeast YAH, the yeast YAH is inserted. In the surface and in combination with it. As a result, the hemicellulolytic enzyme HE was immobilized on the surface of the yeast YAH via the anchor protein AP, and the whole was used as the hemicellulose transforming bacteria YT, and its structure was as shown in Fig. 2 .

Corresponding to step S30, the obtained five-carbon sugar fermentation bacterium YF and the hemicellulose conversion bacterium YT are pre-cultured for one day, and the individual bacterial cell concentration is measured, and then all the groups are co-cultured at any ratio as a mixed system CS. . The preferred ratio of co-cultivation will be defined in the paragraphs described below with the experimental results.

Corresponding to step S40. The raw material is added to the mixed system CS, and the hemicellulose in the raw material is decomposed by the hemicellulolytic enzyme HE on the surface of the hemicellulose conversion bacteria YT to form a raw alcohol.

Hereinafter, the efficiency gain of the hemicellulose decomposing enzyme HE immobilized on the yeast surface for hemicellulose decomposition in the present invention will be explained by the results obtained by the above exemplary examples, and it is confirmed that the yeast only expresses one kind in the present invention. Whether the mixed system formed by the exogenous hemicellulolytic enzyme HE has a better hemicellulose decomposition efficiency than the mixed system formed by expressing a plurality of exogenous hemicellulose decomposing enzymes HE.

As shown in the following Tables 1 and 3, the five-carbon sugar fermentation bacterium YF expressing the XI and XK obtained in the above-described exemplary embodiment and the non-recombinant yeast Y (BY4742) were cultured. In the xylose-rich medium, the growth rate of both was observed and compared with the growth rate of the YRH1114 cell line in the reference (Hector RE, et al., 2013). The results showed that the growth of the five-carbon sugar fermentation bacterium YF was much better than that of the non-recombinant yeast. This phenomenon can be caused by the difference in the concentration of xylose present in the medium. The five-carbon sugar fermentation bacterium YF can effectively utilize the xylose in the medium, so that the concentration is gradually reduced, so that the growth condition is not affected, but the unreconstituted yeast Y is This effect is not achieved and the growth situation is poor.

On the other hand, as shown in Fig. 4, in order to confirm the mixing or not, and whether the hemicellulose decomposing enzyme HE is fixed on the cell surface or not, it will affect the fermentation efficiency of the five-carbon sugar, and design comparison group A, A', B , C and implementation group D. Comparative group A used yeast Y into five groups and inserted into SEQ ID NO: 1 to SEQ ID NO: 5 to its genome, respectively, to secrete five hemicellulolytic enzymes HE, half of this group. The cellulolytic enzyme HE was not fused with the anchoring protein AP, and the strains expressing the five hemicellulolytic enzymes HE were separately cultured; the comparison group A' was the yeast YT expressing the five different hemicellulolytic enzymes HE, respectively. However, the culture was performed separately; the comparison group B used the same recombinant yeast as the comparison group A, and also expressed the hemicellulose decomposing enzyme HE which was not fused with the anchor protein AP, but the strains which express the five hemicellulolytic enzymes HE, respectively. Co-culture; the comparison group C used hemicellulose-transformed strain YT, and co-cultured the strains expressing the five hemicellulolytic enzymes HE respectively; the group D used the same hemicellulose-transformed strain YT as the comparison group C, The strain expressing the five hemicellulolytic enzymes HE was co-cultured, but additionally added with the five-carbon sugar fermentation bacterium YF.

As shown in Table 2, specific activity, and exhibits a dynamic parameter comparative group A and A 'of Five hemicellulolytic enzymes HE comparison, where V _max is the maximum reaction rate; K _M is the Michaelis constant. As a result, it was found that the specific activity of each hemicellulolytic enzyme HE was higher in the comparison group A' having the anchor protein AP than in the comparison group A having no anchor protein AP. The maximum reaction rate between the two comparison groups also showed this trend, indicating that the comparison group A' with the anchor protein AP was present, that is, the hemicellulose decomposing enzyme HE was immobilized on the cell surface with better decomposition efficiency.

On the other hand, as shown in the following Table 3, the difference in specific activity between the comparative group A, B, and C when the hemicellulose-converting bacteria YT expressing various hemicellulolytic enzymes HE had a specific ratio was exhibited. The hemicellulose-containing material used to test the hemicellulolytic enzyme HE is beech xylan and wheat arabinoxylan. The raw material is a group of beech xylan, and the number of yeasts expressing the Abf1, Axe1, Bx11, Glr1, and Xyn2 genes is about 0.14:0.16:0.11:0.12:0.47; and the raw material is the group of wheat arabinoxylan. The ratio between the five yeasts was about 0.17:0.07:0.10:0.04:0.62. The results showed that in the case of supplying two kinds of raw materials, the specific activity was that the comparison group C was larger than the comparison group B, and the comparison group B was compared. It is larger than the comparison group A, and the difference magnification of the comparison groups A and C is about 25 to 67%. It was confirmed that immobilization of hemicellulose decomposing enzyme HE on the cell surface did enhance its activity.

In addition, the reutilization ability of the hemicellulose decomposing enzyme HE immobilized on the surface of the hemicellulose transforming bacteria YT via the anchor protein AP was also tested in the comparison group C and the implementation group D. After 3~4 times of re-use, the activity of hemicellulose decomposing enzyme HE decreased with the initial activity, but the decrease was less than 10% to the 4th repetition, indicating that the present invention will The immobilization of the hemicellulolytic enzyme HE on the surface of the hemicellulose-transformed bacteria YT did not adversely affect its ability to be reused.

In Fig. 5, Comparative Group C and Implementation Group D are presented, and the unreconstituted yeast Y is used as a control group to indicate growth within 120 hours in the xylose-rich medium. The culture solution containing no bacterial solution is used as a blank solution, and the OD600 value of the culture-containing culture solution after the culture is measured to trace the yeast density, which is a conventional measurement method commonly used in laboratories. The results showed that the density of the yeast in the group D was higher than that in the group C, and the yeast Y in the unreconstituted group was the group with the lowest density, and it was found that the hemicellulose decomposing enzyme HE was immobilized to the yeast. The surface does enhance the growth of the yeast.

In Fig. 6, the comparison group C and the implementation group D are presented, and the unrecombined yeast Y is used as the control group to express the xylan accumulated in the medium in the xylose-rich medium for 120 hours. Comparison of reduced reducing sugar content. The results showed that the unreconstituted yeast Y had the least amount of reducing sugars in the medium, and the main reason was that the unreacted yeast Y could not reduce the xylan; Group C had the most reducing sugar content in the medium, indicating that the hemicellulose decomposing enzyme HE was immobilized on the surface of the yeast to achieve a good hemicellulose decomposition effect to reduce xylan; while the implementation group D had the same as in the comparison group C. The hemicellulose conversion bacteria YT has the same hemicellulose decomposition effect as the comparison group C, and should have the same reducing sugar content as the comparison group C. However, since the implementation group D more co-cultures the five carbon sugar fermentation bacteria YF, The reducing sugar is effectively utilized and converted into alcohol, so the amount of reducing sugar accumulated in the medium is small.

In Fig. 7, the change in the ethanol content in the culture group D culture in a xylose-rich medium for 120 hours was presented. As can be seen from the figure, the ethanol content increased significantly with time, and the ethanol content of up to 14000 mg/L was reached at 120 hours, indicating that the mixed bacteria system in Group D had good alcohol production capacity.

Further, in the mixed system provided by the present invention, the number of yeasts expressing the Abf1, Axe1, Bx11, Glr1, and Xyn2 genes has a preferable effect at a specific ratio, and has different specific ratios corresponding to different hemicellulose raw materials. When the hemicellulose raw material is beech xylan, the number of yeasts expressing Abf1, Axe1, Bx11, Glr1, and Xyn2 genes is preferably 10-20:5-20:5-15:1-15:30-79. More preferably 12~16:10~20:7~13:10~15:36~51, more preferably 13~15:12~18:9~13:11~14:40~55. When the hemicellulose raw material is wheat arabinoxylan, the number of yeasts expressing Abf1, Axe1, Bx11, Glr1, and Xyn2 genes is preferably 10-20:5~20:5~15:1~15:30-79 More preferably, it is 12~19:5~15:7~13:2~10:43~74, and more preferably 15~18:6~13:8~12:3~8:49~63.

In summary, the results obtained confirm that the mixed bacteria system of the present invention, the hemicellulose decomposition method and the alcohol production method thereof can provide high hemicellulolytic enzyme HE activity. Further, by using the hemicellulose decomposition method of the present invention to produce a quality alcohol, the decomposition efficiency can be greatly improved. Moreover, In the hemicellulose decomposition method and the alcohol production method of the present invention, a better mixed ratio of yeasts expressing each hemicellulolytic enzyme HE is found, and a more efficient mixed bacteria system is provided, which is capable of the present invention. It does break through the limitations of the existing joint biotransformation process.

Although the present invention has specifically described the hemicellulose decomposition method, the alcohol production method, and the mixed bacteria system therefor in the above embodiments, those of ordinary skill in the art to which the present invention pertains should understand that it is possible not to violate the present invention. Modifications and variations of the embodiments are made in the spirit and spirit of the invention. Therefore, the scope of protection of the present invention should be as described in the appended claims.

<110> National Tsinghua University Changchun Artificial Resin Factory Co., Ltd. Changchun Petrochemical Co., Ltd.

<120> Mixed bacteria system, hemicellulose decomposition method using the same, and alcohol production method

<160> 7

<210> 1

<211> 1440

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<223> yeast mating type [alpha] factor secretion signal sequence (Yeast Mating Factor α Secretion Signal Sequence , MFa1)

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Claims (6)

A method for producing raw alcohol, comprising: recombining at least three groups of yeasts to obtain at least three groups of recombinant yeasts, wherein the at least three groups of recombinant yeasts comprise: at least two groups of hemicellulose-transformed bacteria, each of which is expressed in each group a fusion protein different from each other, the fusion protein being a combination of an anchor protein and a half cellulolytic enzyme different from each other, and the anchor protein has an anchor to the at least two groups of hemicellulose The ability to transform the surface of the bacteria; and at least one group of five carbon sugar fermenting bacteria expressing a five carbon sugar fermentation enzyme; cocultivating the at least three groups of recombinant yeast; contacting half of the cellulose with the hemicellulolytic enzyme to obtain a a five carbon sugar; and the five carbon sugar fermentation enzyme converting the five carbon sugar into a primary alcohol; wherein the gene expressing the hemicellulolytic enzyme comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. SEQ ID NO: 4, SEQ ID NO: 5 or a combination thereof; wherein the gene expressing the fusion protein comprises SEQ ID NO: 6 and SEQ ID NO: 7; wherein the five carbon sugar fermentation enzyme comprises xylose reductase, Xylulose kinase or a combination thereof. The production method according to claim 1, wherein the six groups of the yeast are recombined to obtain six groups of the recombinant yeast, comprising: five groups of the hemicellulose transformed bacteria, respectively expressed by SEQ ID NO: 1. , the hemicellulolytic enzyme expressed by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; and a set of the five carbon sugar fermenting bacteria expressing xylose reductase, wood Ketokinase or a combination thereof. The production method as described in claim 2, wherein the ratio of the total cell number of the hemicellulose-converting bacteria to the total cell number of the five-carbon sugar-fermenting bacteria is from 2:1 to 8:1. The production method as described in claim 2, wherein the hemicellulose-transformed bacteria express SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID The ratio of the number of cells in NO: 5 is 10~20:5~20:5~15:1~15:30~79. A method for decomposing hemicellulose, comprising: recombining at least three groups of yeasts to obtain at least three groups of recombinant yeasts, wherein the at least three groups of recombinant yeasts comprise: at least two groups of hemicellulose-transformed bacteria, each of which is expressed in each group a fusion protein different from each other, the fusion protein being a combination of an anchor protein and a half cellulolytic enzyme different from each other, and the anchor protein has an anchor to the at least two groups of hemicellulose The ability to transform the surface of the bacteria; co-cultivating the at least three groups of recombinant yeast; and contacting half of the cellulose with the hemicellulolytic enzyme to decompose the hemicellulose into a five-carbon sugar; wherein the hemicellulolytic enzyme is expressed a gene comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or a combination thereof; wherein the gene expressing the fusion protein comprises SEQ ID NO: 6 and SEQ ID NO:7; wherein the five carbon sugar fermentation enzyme comprises xylose reductase, xylulokinase or a combination thereof. A mixed bacteria system comprising: at least three groups of recombinant yeast co-cultured, wherein the at least three groups of recombinant yeasts comprise: at least two sets of hemicellulose-transforming bacteria, which express a fusion protein different from each other, The fusion protein is a combination of an anchor protein and a half cellulolytic enzyme different from each other, and the anchor protein has the ability to anchor to the surface of the at least two groups of hemicellulose conversion bacteria; and at least one a group of five carbon sugar fermenting bacteria expressing a five carbon sugar fermentation enzyme; wherein the gene expressing the hemicellulolytic enzyme comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 SEQ ID NO: 5 or a combination thereof; wherein the gene expressing the fusion protein comprises SEQ ID NO: 6 and SEQ ID NO: 7; wherein the five carbon sugar fermentation enzyme comprises xylose reductase, xylulokinase or a combination thereof .