TWI640627B - Recombinant yeast and use thereof for preparing biomass alcohol fuels - Google Patents

Abstract

The invention provides a use of a recombinant yeast for producing raw alcohol, comprising: causing yeast to express artificial phytochelatin; contacting the yeast with a raw material, wherein the raw material comprises carbohydrates and heavy metal ions; The artificial phytochelatin binds to the heavy metal ion; and the yeast converts the carbohydrate to ethanol.

Description

Recombinant yeast and use thereof for producing raw alcohol

The present invention is a use of a yeast, in particular, a use of recombinant yeast for the production of raw alcohol.

In the industrial development of bio-alcohol, yeast is currently mainly used for fermentation of biomass to convert sugar in biomass, so the growth of yeast and the ability to produce alcohol significantly affect the production capacity of bio-alcohol. In general, in the conversion reactor of raw alcohol, the yeast is provided with a good growth environment as much as possible. However, if the raw material used is from a biological breeding ground, the raw material may contain a large amount of Heavy metal ions cause the growth of yeast and the conversion of alcohol to be inhibited.

The literature has attempted to emulate the concept of using Escherichia coli (E. coli) to express natural chelate as a heavy metal adsorbent in the prior art, and further use the gene transfer technology to express it by yeast (yeast). The natural phytochelatin, which in turn causes the heavy metal ions to be sequestered before affecting the function of the yeast, to avoid affecting the operation of the process. However, by transposing the expression of the natural phytochelatin gene, there is still a complex structure of natural phytochelatin, and it is impossible to stably translate, fold and modify the functional natural phytochelatin. problem.

In view of this dilemma, the present invention provides a use of a recombinant yeast for producing a raw alcohol comprising an artificial phytochelatin which causes the yeast to express a structure having the chemical formula (1): The yeast is contacted with a raw material, wherein the raw material contains carbohydrates and heavy metal ions, binds the artificial phytochelin to heavy metal ions, and causes the yeast to convert the carbohydrate into ethanol.

Preferably, the yeast line expresses or secretes and expresses artificial phytochelatin on the surface.

Preferably, the heavy metal ions comprise copper ions, lead ions, nickel ions, cadmium ions, cobalt ions, zinc ions, mercury ions, silver ions or any combination thereof.

Preferably, the yeast strain is Saccharomyces cerevisiae.

Preferably, the raw material of the biomass is from a biological breeding ground.

Preferably, the carbohydrate is selected from the group consisting of cellulose, hemicellulose, and lignin.

Preferably, the recombinant yeast strain is further used in conjunction with a biotransformation process.

Further, the present invention provides a recombinant yeast for use in the above-described production of a raw alcohol comprising: an artificial phytochelatin having a structure of the chemical formula (1) expressed or secreted on the surface of the recombinant yeast:

The invention has the following advantages:

1. The selected yeast strain is a eukaryotic organism, which can express the transgenic foreign protein more completely than the simple prokaryotic organism.

2. Compared to intracellular expression, the present invention uses surface display or secretory expression for transfection. The method of surface expression has the following advantages: (1) It can effectively isolate heavy metal ions including cadmium, nickel, copper and lead from chewing outside the yeast cells, and does not enter the yeast body, thus avoiding the influence of yeast metabolism and (2) recovering the heavy metals chelated to the extracellular cells of the yeast, increasing the benefit of using the recombinant yeast of the present invention for the production of bio-alcohol, and further improving the ecological rejuvenation and growth using the present invention. The value of supporting measures between energy and energy.

3. Synthetic phytochelatin (EC) is similar to natural plant chelation. However, the surface expression of artificial phytochelatin in yeast is relatively simple, and it has a stable source and easy industrial production. The advantages of uniform quality are more conducive to the conversion process for raw alcohol. In addition, the recombinant yeast can be used as a technical means to overcome key problems in the current Consolidated Bio-Processing (CBP) process, further strengthen the production process of the raw alcohol, and develop an ecological rehabilitation and biomass. Effective supporting measures between energy sources.

In summary, the recombinant yeast expressing the artificial phytochelatin provided by the present invention can reduce the inhibition of the growth of the yeast by heavy metal ions compared with the unexpressed yeast. Accordingly, the present invention also provides the use of the recombinant yeast to produce a raw material containing heavy metal ions for the production of bio-alcohol.

S10, S20, S31, S32‧‧‧ steps

Figure 1 is a flow chart of an embodiment of the present invention.

Fig. 2 is a schematic view showing the structure of a recombinant yeast according to an embodiment of the present invention.

Figure 3 is a photograph of a fluorescent microscope according to an embodiment of the present invention.

Figure 4 is an adsorption analysis diagram of an embodiment of the present invention.

Figure 5 is a graph showing cell growth of an embodiment of the present invention.

Fig. 6A and Fig. 6B are diagrams showing the analysis of biomass alcohol yield and yield according to an embodiment of the present invention.

Fig. 7 is a graph showing the analysis of the yield of biomass alcohol according to another embodiment of the present invention.

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 manufacturing process is as shown in FIG.

In step S10, a recombinant yeast is established because the yeast has a cell wall having a sufficient thickness and stability, and the protein on which the surface is expressed is stable, and the post-translational modification is good, and is suitable for expressing a complex protein. The method for expressing a foreign gene by yeast includes intracellular expression, secretory expression and surface expression. The way of intracellular expression is that the exogenous protein is expressed and accumulated in the cytoplasm of the yeast; the way of secretory expression is to secrete the exogenous protein outside the cell; and the way of surface expression is different from the former two, which is A method of immobilizing an exogenous protein. The method of surface expression is to bind the gene of the exogenous protein to the gene of the specific anchor molecule, and then transfer it to the host cell, so that the exogenous protein is translated together with the anchor molecule, and the secretion is expressed in After cytoplasmic, the exogenous protein is anchored to the surface of the host cell along with the anchor molecule.

In this embodiment, surface expression is selected to establish recombinant yeast. Specifically, the yeast may be any yeast commonly used for alcohol fermentation, for example, Schizosaccharomyces, Saccharomycodes, Hanseniaspora, etc., preferably beer yeast, which is certified as GRAS (generally recognized as safe) The organism is recognized as a safe organism by the FDA; and the anchor molecule can be selected from the commonly used anchors including a lectin (eg, Aga1, Aga2), α-agglutinin (eg, Agα1), and flocculant (FlolP). An anchoring molecule having an affinity when any of the artificial phytochelins corresponding to the recombinant yeast of the present invention. In addition, the recombinant yeast can also express a five-carbon sugar decomposing enzyme at the same time according to the demand, so that it has the decomposition ability of five carbon sugars.

In addition, metal chelating proteins commonly used for chelation of metals include metallothioneins (MTs) and phytochelins. Among them, the ability of phytochelatin to chelate heavy metals Better than metallothionein. However, when yeast is used to express phytochelatin, since phytochelatin is an enzyme-synthesizing peptide, it is not easily expressed on yeast and is not easily synthesized in one step in a solution. Therefore, in this case, an artificial plant chelator which is similar in structure to phytochelatin after yeast translation is used. It can be known from the following chemical formula (1) and chemical formula (2) that the chemical structure of the translated artificial phytochelatin contains an alpha bond, and the translated phytochelatin is a gamma bond, and the difference in structure is neither It will cause the impact of heavy metal adsorption capacity, and can greatly reduce the difficulty of the process.

In step S20, a raw material containing a carbohydrate and a heavy metal ion is added. In particular, the raw material of the raw material may be derived from an ecological breeding ground contaminated by heavy metals. Heavy metals can contain copper ions, Lead ion, nickel ion, cadmium ion, cobalt ion, zinc ion, mercury ion, silver ion or any combination thereof. The type of raw material can be any raw material having cellulose. Because the plants used in biological rejuvenation will make different choices depending on the type of heavy metal pollution, no matter which plant is selected, as long as it has cellulose, it can be used as raw material for raw materials. The raw material of the raw material may also be a common plant contaminated by heavy metals, such as barley, wheat, oats, rice, sugar beets, sweet sorghum, cassava, and sweet potato; or non-food 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, switchgrass; or readily harvestable materials such as seaweed Wait.

In step S31, the recombinant yeast is contacted with the raw material and the raw alcohol is produced, and at the same time, the heavy metal ion is chelated to the outside of the yeast cell in step S32. The process of producing yeast alcohol by yeast is a well-known technique of those having ordinary knowledge in the art, and therefore will not be described herein.

In an embodiment of the present invention, a schematic diagram of the structure of the recombinant yeast is shown in Fig. 2. Among them, the yeast uses Saccharomyces genus Saccharomyces cerevisiae, and the anchoring molecule uses Agα1, which is a cell wall-anchored protein. The sequence of the artificial phytochelatin (EC20) (SEQ ID NO: 2) is combined with α- agglutinin (Agα1) (SEQ ID NO: 3), and inserted into the genome of Saccharomyces cerevisiae to make the artificial phytochelatin and Agα1 is secreted and expressed by a secretion signal (MFα1) (SEQ ID NO: 1) and anchored to the surface of the cell wall of S. cerevisiae for surface expression. Moreover, the Agα1 gene in the genome of S. cerevisiae remains intact, so as not to impair the structure and cell-to-cell signal exchange between the cell wall of S. cerevisiae, thereby avoiding the ability of yeast to produce bio-alcohol. In addition, the sequestration of heavy metal ions and artificial phytochelatins is carried out outside the yeast cells, so the growth of the yeast is not affected by the heavy metal inhibitory effect.

In an embodiment of the present invention, first, using the polymerase chain reaction (PCR) the α agglutinin (Agα1) and secretion signal (MF [alpha]) amplification from beer yeast. A gene encoding an artificial plant chelator (EC20) is synthesized by annealing. Finally, the three genes are rejoined to the expression plastid of the yeast by restriction enzymes and ligase. Thereafter, the plastid is firstly transformed into E. coli and then transformed into yeast, and the artificial phytochelin (EC20) is expressed on the surface of the yeast so that it can adsorb heavy metals on the surface.

In the following, an analysis of the use of the recombinant yeast which expresses the artificial phytochelatin on the surface of the raw material to produce the raw alcohol is carried out to prove the use of the recombinant yeast of the present invention and the production of the raw alcohol, thereby achieving effective production of the raw alcohol. the goal of.

The unrecombinant yeast of the present invention is a brewer's yeast which has not been subjected to any of the wild type. As shown in Fig. 3, the recombinant yeast expressing the artificial phytochelatin on the surface was labeled with Alexa Fluor 488 fluorescent dye. Fig. 3 (A) and (B) are photographs of recombinant yeast and unrecombinant yeast in a bright field of the microscope, respectively, showing the forms of recombinant yeast and unrecombinant yeast. Figure 3 (C) and (A) show the results of wavelength observation of the excitation of Alexa Fluor 488 fluorescent dye in the same field of view, and the excited fluorescence is clearly observed, indicating that the artificial phytochelatin is indeed expressed on the surface of the recombinant yeast. . Figure 3 (D) and (B) show the results of the wavelength of the excited Alexa Fluor 488 fluorescent dye in the same field of view. No fluorescent signal was observed, indicating that the artificial phytochelatin was not expressed on the yeast. .

As shown in Fig. 4 and Table 1, the above examples were carried out to analyze the adsorption capacity. The heavy metal cadmium (Cd) was selected as the pollution source, and the adsorption capacity of the recombinant yeast and the unrecombined yeast was analyzed by inductively coupled plasma emission spectrometer (ICP-AES) to explore and measure the mode of adsorption of cadmium metal. The Langmuir equation (qm=KbC _e /(1+KC _e ), where qm is the adsorption amount, C _e is the equilibrium concentration, b is the maximum adsorption amount, K is the adsorption equilibrium constant of the adsorbate) and the Freundlich equation (qm) =KC ^1/n , where K and n are constants) Fitting the data. It can be seen that the effect of fitting by the Langmuir equation is better, and the adsorption capacity of the recombinant yeast is more than twice that of the unrecombinant yeast.

As shown in Fig. 5, the above examples were followed to analyze the cell growth ability. It can be seen that in the cadmium-free environment, the growth of recombinant yeast and non-recombinant yeast is not much different. However, in the presence of 50 μM cadmium, the growth of recombinant yeast was significantly better than that of unrecombinant yeast. Representing recombinant yeast can reduce the inhibition of heavy metal growth on yeast.

As shown in Figures 6A and 6B, the above examples were followed for analysis of biomass alcohol yield and yield. For the trees that rehabilitated the land, and using gas chromatography (GC) to explore the effect of heavy metal cadmium on the efficacy of yeast production of alcohol. As shown in Figure 6A, in the cadmium-free environment, the yield of alcohol produced by recombinant yeast and non-recombinant yeast is not much different. As the cadmium concentration increases, the ability of recombinant yeast to produce alcohol is better than that of unreconstituted yeast. The situation of ability is becoming more apparent. Representing recombinant yeast is free from the inhibition of yeast growth by heavy metals and is effective in producing bio-alcohol. Figure 6B is a graph showing the yield analysis based on the concentration and time relationship of Figure 6A. It shows that the yield of alcohol produced by recombinant yeast and non-recombinant yeast is not much different in the cadmium-free environment. As the cadmium concentration increases, Recombinant yeast to make alcohol The more powerful the ability to produce alcohol than the unreconstituted yeast is, the more obvious it is. Representing recombinant yeast is free from the inhibition of yeast growth by heavy metals and is effective in producing bio-alcohol.

As shown in Fig. 7, the above examples were carried out to analyze the inhibition of different types of heavy metals on the production of bio-alcohol by recombinant yeast and non-recombinant yeast. It can be seen that the recombinant yeast is superior to the unrecombined yeast in the environment containing 150 μM copper, 150 μM nickel or 150 μM lead, and the recombinant yeast can be inhibited from the growth of yeast by heavy metals, and is effective. Produce raw alcohol.

In summary, it is confirmed that the use of a recombinant yeast of the present invention for producing raw alcohol can effectively reduce the inhibition of heavy metal growth on yeast, and improve the predicament of yeast production of raw alcohol in the presence of heavy metal environment. Improve the production and yield of bio-alcohol in the presence of heavy metal ions, and develop an effective supporting measures between ecological remediation and biomass energy.

Although the present invention has specifically described the use of a recombinant yeast of the present invention for the production of bio-alcohol in the above-described embodiments, it will be understood by those of ordinary skill in the art to which the present invention pertains, without departing from the technical principles of the present invention. Modifications and changes to the examples are made under the spirit. 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> Recombinant yeast and its use for the production of raw alcohol

<160> 3

<210> 1

<211> 267

<212> DNA

<213> Saccharomyces cerevisiae

<220>

<223> Secretion signal (MF α 1) (Secretion signal)

<400> 1

<210> 2

<211> 126

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial plant chelation (EC20) has the function of chelation of metal ions.

<400> 2

<210> 3

<211> 963

<212> DNA

<213> Saccharomyces cerevisiae

<220>

<223> α- agglutinin (Ag α 1)

<400> 3

Claims (9)

A use of a recombinant yeast for producing a biomass alcohol, comprising: causing a yeast to express an artificial phytochelatin having a structure of the formula (1) by using an α- agglutinin: The yeast is contacted with a raw material, wherein the raw material comprises a carbohydrate and a heavy metal ion; the artificial plant chelator is bound to the heavy metal ion; and the yeast is converted to ethanol. The use of claim 1, wherein the yeast cell surface expresses the artificial phytochelatin. The use of claim 1, wherein the yeast strain secretes and expresses the artificial phytochelatin. The use according to any one of the preceding claims, wherein the heavy metal ion is selected from the group consisting of copper ions, lead ions, nickel ions, cadmium ions, cobalt ions, zinc ions, mercury ions, silver ions. Or any combination thereof. The use according to any one of the preceding claims, wherein the yeast strain is Saccharomyces cerevisiae. The use of any of claims 1 to 3, wherein the raw material is from a biological breeding ground. The use according to any one of the preceding claims, wherein the carbohydrate is selected from the group consisting of cellulose, hemicellulose, and lignin. The use of any of claims 1 to 3, wherein the recombinant yeast strain is further used in a combined biotransformation process. A recombinant yeast comprising: expressing an artificial phytochelatin having a structure of the formula (1) on the surface of the recombinant yeast: Wherein, the recombinant yeast further comprises: α lectin, and anchoring the artificial plant chelator to the recombinant yeast.