CN103725718B - Method for synthesizing acetoin and derivative thereof through biological method - Google Patents

Abstract

The invention discloses a method for biologically synthesizing acetoin and its derivatives, belonging to the technical field of molecular biology. The invention introduces the gene of pyruvate decarboxylase and the gene with acetoin synthase activity into host bacteria to obtain recombinant bacteria, uses the recombinant bacteria to ferment and produce acetoin, and further produces 2,3-butanediol on the basis of acetoin. The invention successfully utilizes the recombined bacterial strain to ferment acetoin and 2,3-butanediol, and solves the problem that the consumption of acetolactate, an intermediate product of the natural production route, affects the production of acetoin and 2,3-butanediol.

Description

A method for biologically synthesizing acetoin and its derivatives

technical field

The invention relates to a method for synthesizing acetoin, specifically, carrying out molecular biological transformation on Escherichia coli to synthesize acetoin, and further synthesizing 2,3-butanediol on the basis of acetoin, belonging to molecular The field of biological technology.

Background technique

Acetoin (alias: methyl acetylmethanol, 3-hydroxy-2-butanone) has a strong creamy and fat-like aroma, and has a pleasant milky aroma after being highly diluted. It is mainly used to configure flavors and is an important food Additives and drug synthesis raw materials. Traditionally, acetoin can be obtained by reacting 2,3-butanedione with zinc under acidic conditions, or fermented from carbohydrates with fungi such as Aspergillus or Penicillium. 2,3-Butanediol is mainly used as spices and organic synthesis reagents, and it is also a potential biofuel, which can be obtained from carbohydrates fermented by Bacillus subtilis.

Although the production of acetoin and 2,3-butanediol by engineered microorganisms from carbohydrates has been reported, these methods are all based on a natural anabolic pathway: acetolactate synthase catalyzes two acetone Acid condensation into acetolactate; acetolactate decarboxylase catalyzes the decarboxylation of acetolactate to acetoin; acetoin reductase catalyzes the reduction of acetoin to 2,3-butanediol. This natural pathway uses acetolactate as a key metabolic intermediate, but acetolactate is also used in microbial metabolism to synthesize amino acids, terpenoids, etc., so it will be consumed in large quantities, which will affect acetoin and 2,3-butanedi Alcohol production.

Contents of the invention

The invention provides a method for synthesizing acetoin, by introducing a pyruvate decarboxylase gene and an acetoin synthase active gene into a host bacterium to obtain a recombinant bacterium, and using the recombinant bacterium to ferment and produce acetoin, further on the basis of acetoin The method for producing 2,3-butanediol.

Technical scheme of the present invention is as follows:

(1) Clone the pyruvate decarboxylase gene and the gene with acetoin synthase activity;

(2) Linking the gene obtained in step (1) to a plasmid vector, respectively constructing recombinant plasmids containing the pyruvate decarboxylase gene and the acetoin synthase gene;

(3) Introducing the recombinant plasmid in step (2) into the host bacterium to obtain the recombinant bacterium;

(4) Using the recombinant bacteria in step (3) to ferment and produce acetoin.

The specific steps of the above method are as follows:

(1) Clone the pyruvate decarboxylase gene and the gene with acetoin synthase activity respectively;

(2) connecting the gene obtained in step (1), the pyruvate decarboxylase gene to the pET28a plasmid, and the gene with acetoin synthase activity to the pACYduet1 plasmid;

(3) introducing the two recombinant plasmids obtained in step (2) into Escherichia coli to obtain recombinant Escherichia coli;

(4) Using the recombinant Escherichia coli obtained in step (3) to ferment and produce acetoin.

In the production of above-mentioned acetoin, described pyruvate decarboxylase gene is derived from Zymomonas mobilis or Fusobacterium acetobutylicum; The described gene with acetoin synthase activity is acoABCL of acetoin dehydrogenase, pyruvate Any one of dehydrogenase E1 subunit PDHA1 gene and LOC100516695 gene, acetolactate synthase ILV2 gene or YerE enzyme yerE gene.

According to the method described above, the yerE gene of pyruvate decarboxylase PDC gene and bacteria Yersinia pseudotuberculosis is used to produce acetoin, and the specific steps are as follows:

(1) Cloning the pyruvate decarboxylase PDC gene of Zymomonas mobilis and the yerE gene of Yersiniapseudotuberculosis respectively;

(2) connecting the PDC gene described in step (1) to the pET28a plasmid to obtain a new plasmid pJXL65, and connecting the yerE gene to the pACYduet1 plasmid to obtain a new plasmid pJXL63;

(3) Electric shock transformation of the plasmid vectors pJXL65 and pJXL63 described in step (2) into Escherichia coli BL21 (DE3) to obtain recombinant bacteria;

(4) Using the recombinant bacteria in step (3) to ferment and produce acetoin.

The above-mentioned recombinant bacteria preferably use glucose as a raw material to ferment and produce acetoin.

The present invention also provides a method for synthesizing 2,3-butanediol, which is to introduce the pyruvate decarboxylase gene, the gene with acetoin synthase activity and the gene with acetoin reductase activity into the host bacterium to obtain the recombinant bacterium , using recombinant bacteria to ferment and produce 2,3-butanediol, the main steps are as follows:

(1) Clone the pyruvate decarboxylase gene, the gene with acetoin synthase activity and the gene with acetoin reductase activity;

(2) Linking the gene with acetoin synthase activity, pyruvate decarboxylase gene and acetoin reductase activity gene to two plasmids that are compatible in the same host cell;

(3) Transforming the recombinant plasmid obtained in step (2) into Escherichia coli to obtain recombinant bacteria;

(4) Using the recombinant bacteria in step (3) to ferment and produce 2,3-butanediol.

The specific steps of the above method are as follows:

(1) Clone the pyruvate decarboxylase gene, the gene with acetoin synthase activity and the gene with acetoin reductase activity; the pyruvate decarboxylase gene is derived from Zymomonas mobilis or Fusobacterium acetobutylicum; The gene with acetoin synthase activity is any one of acetoin dehydrogenase acoABCL, pyruvate dehydrogenase E1 subunit PDHA1 gene and LOC100516695 gene, acetolactate synthase ILV2 gene or YerE enzyme yerE gene; The gene with acetoin reductase activity is any one of the 2,3-butanediol dehydrogenase bdhA gene or the alcohol dehydrogenase adh gene;

(2) Ligate the gene with acetoin synthase activity to pACYduet1 plasmid to obtain plasmid pJXL63, connect the pyruvate decarboxylase gene obtained in step (1) to pJXL63 plasmid to obtain recombinant plasmid pJXL78, and connect the gene with acetoin reductase activity to the recombinant plasmid pJXL79 obtained from the pET28a plasmid vector;

(3) Transforming the recombinant plasmids pJXL78 and pJXL79 obtained in step (2) into Escherichia coli to obtain recombinant bacteria;

(4) Using the recombinant bacteria in step (3) to ferment and produce 2,3-butanediol.

According to the above method, the specific steps for synthesizing 2,3-butanediol using pyruvate decarboxylase PDC gene and 2,3-butanediol dehydrogenase bdhA gene are as follows:

(1) Clone the pyruvate decarboxylase PDC gene of Zymomonas and the 2,3-butanediol dehydrogenase bdhA gene of Bacillus subtilis; (2) The new plasmid pJXL63 obtained by linking the yerE gene to the pACYduet1 plasmid vector, Connecting the PDC gene obtained in step (1) to the pJXL63 plasmid to obtain the recombinant plasmid pJXL78, and connecting the bdhA gene to the pET28a plasmid vector to obtain the recombinant plasmid pJXL79;

(3) Electrically transform the recombinant plasmids pJXL78 and pJXL79 obtained in step (2) into Escherichia coli BL21 (DE3) to obtain recombinant bacteria;

(4) Using the recombinant bacteria in step (3) to ferment and produce 2,3-butanediol.

The above-mentioned recombinant bacteria preferably use glucose as a raw material to ferment and produce 2,3-butanediol. The invention also provides methods and materials for the biological production of acetoin and 2,3-butanediol. Specifically, the invention provides nucleic acid molecules, polypeptides, host cells and methods for the production of acetoin and 2,3-butanediol. The nucleic acid molecules mentioned herein can be used to genetically engineer host cells with the ability to produce acetoin and 2,3-butanediol. The polypeptides mentioned herein can be used to produce acetoin and 2,3-butanediol in a cell-free system. The host cells mentioned here can be used to produce acetoin and 2,3-butanediol in a culture system.

The invention provides cells having pyruvate decarboxylase activity and/or acetoin synthase activity and/or methylacetoin reductase activity, and methods of producing products such as those described herein by culturing these cells. In some embodiments the cells themselves contain the desired enzyme activity eg Zymomonas mobilis, yeast. In other embodiments, cells contain exogenous nucleic acids encoding these enzymes.

In some embodiments, the cells further contain diol dehydratase activity and/or alcohol dehydrogenase activity. These cells can further convert 2,3-butanediol to butanone and 2-butanol.

The invention successfully utilizes the recombinant bacterial strain to ferment acetoin and 2,3-butanediol, and solves the problem that the consumption of acetolactate, an intermediate product in the natural production pathway, affects the production of acetoin and 2,3-butanediol.

Description of drawings

The structural formula of each compound mentioned in Fig. 1 the present invention;

(1. Acetolactate, 2. Acetaldehyde, 3. Pyruvate, 4. Acetoin, 5. 2,3-Butanediol).

Fig. 2 is a schematic diagram of the synthetic metabolic pathway of producing acetoin by biological method;

(1. Pyruvate decarboxylase, 2. Acetoin synthase).

Fig. 3 is a schematic diagram of the synthetic metabolic pathway of producing 2,3-butanediol by biological method;

(1. Pyruvate decarboxylase, 2. Acetoin synthase, 3. Acetoin reductase).

Fig. 4 Schematic diagram of the structure of plasmid pJXL65.

Fig. 5 Schematic diagram of the structure of plasmid pJXL63.

Fig. 6 Schematic diagram of the structure of plasmid pJXL78.

Fig. 7 Schematic diagram of the structure of plasmid pJXL79.

The acetoin generated in Fig. 8 is detected with a gas chromatography-mass spectrometer to generate a TIC diagram.

The acetoin generated in Fig. 9 is detected with a mass spectrogram by gas chromatography-mass spectrometry.

detailed description

Figure 1-3 depicts the biological synthesis of methylacetoin and its derivatives: pyruvate decarboxylase catalyzes the decarboxylation of pyruvate to acetaldehyde; an enzyme with acetoin synthase activity catalyzes acetaldehyde and pyruvate The reaction produces acetoin; acetoin reductase catalyzes the reduction of acetoin to 2,3-butanediol. The decarboxylase, synthase, and reductase activities mentioned here widely exist in various cells. These enzymatic activities inherent in cells can be utilized. Enzymes can also be cloned from another species and introduced into cells. These enzymes may also be engineered enzymes. This modification can be achieved by mutagenesis screening or directed evolution.

Many proteins with thiamine pyrophosphate as a coenzyme have been found to have the acetoin synthase activity shown in Figures 1-3. For example, the E1 subunit of acetoin dehydrogenase, the E1 subunit of animal pyruvate dehydrogenase, acetolactate synthase, transketolase, YerE enzyme. These proteins can all be used in the present invention. Examples of sources of these proteins and nucleic acids encoding them are as follows:

Table 1 Proteins with acetoin synthase activity and their genes

Many proteins have been found to have the pyruvate decarboxylase activity shown in Figures 1-3. These proteins and the sources of nucleic acids encoding them are exemplified as follows

Table 2 Pyruvate decarboxylase and its genes

Many proteins have been found to have the acetoin reductase activity shown in Figures 1-3. These proteins and sources of nucleic acids encoding them are exemplified as follows:

Table 3 Proteins with acetoin reductase activity and their genes

Nucleic acids encoding these enzymes can be constructed on suitable vectors such as pET28a, pACYduet1, and transformed into cells. It is also possible to use the enzyme activity of the cell itself, such as the 2,3-butanediol dehydrogenase activity of Bacillus subtilis. In the latter case the catalytic activity of the enzyme can be increased by increasing the copy number of the gene. The method for constructing said these cells is a method commonly used in biology, and the specific steps can refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al ., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Enzyme expression was verified by conventional northren hybridization. The ability of the cells to produce acetoin, 2,3-butanediol, etc. was demonstrated by testing fermentation products. Detection of these products was performed using gas chromatography and mass spectrometry techniques.

Example 1

The PDC gene of Zymomonas mobilis was cloned between the NdeI and BglII sites of the pET28a (purchased from Novagen) plasmid vector, and the resulting new plasmid was called pJXL65 (Figure 4). Zm6PDC in Fig. 4 is the pyruvate decarboxylase coding gene of Zymomonas mobilis. The yerE gene of Yersinia pseudotuberculosis was cloned between the NdeI and KpnI restriction sites of the pACYduet1 (purchased from Novagen) plasmid vector, and the resulting new plasmid was called pJXL63 (Figure 5). Both pJXL65 and pJXL63 were transformed into Escherichia coli BL21(DE3) (purchased from Invitrogen) by electric shock. Culture the constructed cells in 100ml LB glucose medium (dissolve 10g sodium chloride, 10g trypsin powder and 5g yeast powder in 0.5L water for steam sterilization, dissolve 20g glucose in 0.5L water for steam sterilization, after sterilization and cooling mix the two in equal proportions). The culture temperature was maintained at 30°C. When the cells grow to a certain stage, usually the exponential growth stage, add IPTG (final concentration of 50mg/L) to induce the expression of exogenous enzymes, so that the cells can produce acetoin, and the synthesized acetoin is detected by gas chromatography-mass spectrometry (See Figure 8, 9). The peak at 7.049 points in Figure 8 is the peak of acetoin, and the mass spectrum fragmentation pattern in Figure 9 is consistent with the molecular structure of acetoin. The concentration of acetoin was 10.2g/L.

Example 2

The PDC gene of Zymomonas mobilis was cloned between the NcoI and BamHI restriction sites of the pJXL63 plasmid, and the resulting new plasmid was called pJXL78 (Figure 6). The bdhA gene of Bacillus subtilis was cloned between the NcoI and BamHI restriction sites of the pET28a (purchased from Novagen) plasmid vector, and the resulting new plasmid was called pJXL79 (Figure 7). Both pJXL78 and pJXL79 were transformed into Escherichia coli BL21(DE3) (purchased from Invitrogen) by electric shock. Culture the constructed cells in 100ml LB glucose medium (dissolve 10g sodium chloride, 10g trypsin powder and 5g yeast powder in 0.5L water for steam sterilization, dissolve 20g glucose in 0.5L water for steam sterilization, after sterilization and cooling mix the two in equal proportions). The culture temperature was maintained at 30°C. When the cells grow to a certain stage, usually the exponential growth stage, add IPTG (final concentration 50mg/L) to induce the expression of exogenous enzymes, so that the cells can produce 2,3-butanediol. The concentration of 2,3-butanediol was 9.5g/L.

Claims (6)

1. a kind of method that bioanalysis synthesizes 3-hydroxy-2-butanone, it is characterised in that step is as follows：

(1) Pyruvate Decarboxylase Gene PDC genes and YerE enzyme yerE genes are cloned respectively；

(2) gene obtained by step (1) is connected on plasmid vector, builds contain Pyruvate Decarboxylase Gene and second idol respectively The recombinant plasmid of relation by marriage synthase gene；

(3) recombinant plasmid in step (2) is imported into Escherichia coli, obtains recombination bacillus coli；

(4) the recombination bacillus coli fermenting and producing 3-hydroxy-2-butanone obtained using step (3).

2. method according to claim 1, it is characterised in that comprise the following steps that：

(1) Pyruvate Decarboxylase Gene PDC genes and YerE enzyme yerE genes are cloned respectively；

(2) by the gene obtained by step (1), Pyruvate Decarboxylase Gene is connected on pET28a plasmids, is made recombinant plasmid PJXL65, is connected on pACYduet1 plasmids with 3-hydroxy-2-butanone synthase activity gene, is made recombinant plasmid pJXL63；

(3) two recombinant plasmids that step (2) is obtained are imported into Escherichia coli, obtains recombination bacillus coli；

(4) the recombination bacillus coli fermenting and producing 3-hydroxy-2-butanone obtained using step (3).

3. the either method according to claim 1-2, it is characterised in that the Pyruvate Decarboxylase Gene is sent out from motion Ferment monad or Clostridium acetobutylicum.

4. the either method according to claim 1-2, it is characterised in that comprise the following steps that：

(1) the Pyruvate Decarboxylase Gene PDC genes and bacterium Yersinia of zymomonas mobilis are cloned respectively The yerE genes of pseudotuberculosis；

(2) step (1) the PDC genes are connected on pET28a plasmids, the novel plasmid pJXL65 for obtaining, yerE genes is connected It is connected on pACYduet1 plasmid vectors, the novel plasmid pJXL63 for obtaining；

(3) the plasmid vector pJXL65 and pJXL63 described in step (2) together Electroporation are entered into e. coli bl21 (DE3) In, obtain recombinant bacterium；

(4) using the recombinant bacterium fermenting and producing 3-hydroxy-2-butanone in step (3).

5. it is a kind of synthesize 2,3-butanediol method, it is characterised in that comprise the following steps that：

(1) pyruvate decarboxylase PDC genes, YerE enzyme yerE genes and 2,3-butanediol dehydrogenase bdhA genes are cloned respectively；

(2) by pyruvate decarboxylase PDC genes, YerE enzyme yerE genes and 2,3-butanediol dehydrogenase bdhA genes are connected respectively To can be compatible in same host cell two plasmids on；

(3) step (2) is obtained into recombinant plasmid transformed enter Escherichia coli and obtain recombinant bacterium；

(4) using the recombinant bacterium fermenting and producing 2,3- butanediols in step (3).

6. method according to claim 5, it is characterised in that comprise the following steps that：

(1) the 2,3- butanediol dehydrogenases of the pyruvate decarboxylase PDC genes of clone's fermentation single cell bacterium and bacillus subtilis BdhA genes；

(2) yerE genes are connected to pACYduet1 plasmid vectors and obtain recombinant plasmid pJXL63, by step (1) gained PDC bases Recombinant plasmid pJXL78 is obtained because being connected to pJXL63 plasmids, bdhA genes are connected to the recombinant plasmid that pET28a plasmids are obtained pJXL79；

(3) step (2) is obtained during recombinant plasmid pJXL78 and pJXL79 Electroporation enters e. coli bl21 (DE3), is obtained Recombinant bacterium；

(4) using the recombinant bacterium fermenting and producing 2,3- butanediols in step (3).