Construction and application of kluyveromyces marxianus Hsp26 engineering bacterium for producing ethyl acetate

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a construction method and application of kluyveromyces marxianus Hsp26 engineering bacteria for producing ethyl acetate. The engineering bacterium is an overexpression bacterium of kluyveromyces marxianus CCTCC (China Center For Type Culture Collection) NO: M2021073. The rice sour soup is produced by fermentation of improved kluyveromyces marxianus Hsp26 engineering bacteria, and the process comprises the following steps: pretreating raw materials, boiling, gelatinizing, liquefying, saccharifying, blending, sterilizing, cooling, inoculating bacteria, and filling into a jar for fermentation. The rice sour soup fermented by the improved kluyveromyces marxianus Hsp26 engineering bacteria is excellent in flavor and taste and high in sensory evaluation; the antioxidant activity is high, and the DPPH clearance rate is as high as 46%-51.5%; the content of ethyl acetate can reach about 195.5 mg / L to 245.5 mg / L, and the content of total acid can reach 0.45 g / 100g to 0.61 g / 100g; and the viable count of the saccharomycetes is up to 1.5-3.2 * 10 < 8 > CFU / mL.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a method for constructing an engineered Kluyveromyces marxianus Hsp26 strain producing ethyl acetate and its application. Background Art

[0002] Guizhou rice sour soup is a liquid flavored food naturally fermented with rice soup as the basic raw material, added with mature old sour soup, with the participation of microorganisms such as yeast, lactic acid bacteria and acetic acid bacteria. "Rice sour soup" is also called "white sour soup". The reason why rice sour soup is favored by consumers is mainly because of its unique geographical environment and climate conditions, as well as the traditional sour soup brewing process and unique raw material components as the base for cooking food, making the dishes more crispy, delicious, refreshing and invigorating. Rice sour soup tastes sweet and flat, and has the effects of replenishing qi, nourishing yin and moistening dryness. In addition, it is rich in a large number of microorganisms and various nutritional metabolites. Eating it regularly can help intestinal digestion, help discharge residual substances, adjust the body's acid-base balance, improve the body's immune function, and scavenge free radicals.

[0003] However, the production of rice sour soup still remains in the traditional process stage of small workshops. The traditional sour soup fermentation process has many steps and a long cycle, and is easily affected by fluctuations in raw material quality, contamination by miscellaneous bacteria and environmental factors. The product quality (such as flavor) is extremely difficult to stabilize, it is difficult to transport and preserve, the sales radius is limited, and there are few extended product developments. Now it has become a key technical "bottleneck" seriously restricting the leapfrog development of the industry or enterprise. Therefore, it is necessary to select or transform excellent microorganisms in rice sour soup, and develop a set of high-quality strains and their fermentation processes suitable for the development of rice sour soup enterprises.

[0004] Regarding the technology for modifying microorganisms, Chinese Patent with publication number CN114874928A discloses a method for improving the stress resistance of cellulose ethanol production strains by overexpressing heat shock proteins, which improves the tolerance of cellulose ethanol production strains to cellulose inhibitors and high temperatures by overexpressing the heat shock protein HSP26 derived from Kluyveromyces marxianus CICC1727-5. However, its purpose is to endow cellulose ethanol production strains with various inhibitor and high temperature resistances. Summary of the Invention

[0005] The researchers of the present invention studied the selection of excellent microorganisms in fermented rice sour soup, and provided a method for constructing an engineered Kluyveromyces marxianus Hsp26 strain producing ethyl acetate and its application, to solve the problems of long production cycle and unstable flavor, taste and quality in the prior art for rice sour soup production. Specifically, it is achieved through the following technical solutions:

[0006] An engineered Kluyveromyces marxianus Hsp26 strain for ethyl acetate production. The engineered strain is constructed by gene editing technology to overexpress the small heat shock protein Hsp26 in Kluyveromyces marxianus. The wild strain of the overexpressing strain, Kluyveromyces marxianus L1-1, has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M2021073 and the deposit date of January 15, 2021.

[0007] When Kluyveromyces marxianus L1-1 is cultured on YPD and PDA plates for 3 - 5 days, colonies with a diameter of 2.0 - 5.0 mm, red, oval, smooth and moist surface, and neat and raised edges are formed. When inoculated into rice soup, the pH can be reduced to 3.69. It has a fruity and alcoholic aroma.

[0008] In the present invention, Kluyveromyces marxianus L1-1 and the engineered Kluyveromyces marxianus Hsp26 strain after improvement are inoculated into rice soup raw materials for culture and fermentation, and the inoculation amount is 2.8×10 7 -3.5×10 8 CFU / mL; both have a rich viable cell count during the fermentation of rice soup raw materials. After 24 hours of fermentation, the viable cell number is 1.5 - 3.2×10 8 CFU / mL. This indicates that the improved Hsp26 engineered strain has good growth performance in the rice sour soup fermentation system.

[0009] The construction method of the engineered Kluyveromyces marxianus Hsp26 strain for ethyl acetate production includes the following steps:

[0010] (1) PCR amplification of the promoter PmPGK;

[0011] (2) PCR amplification of the Hsp26 gene;

[0012] (3) One-step cloning of the pEKm-PmPGK-Hsp26 recombinant plasmid;

[0013] (4) Screening of the pEKm-PmPGK-Hsp26 recombinant plasmid;

[0014] (5) Electroporation of the recombinant plasmid overexpression vector into Kluyveromyces marxianus competent cells;

[0015] (6) Screening of the Hsp26 gene overexpressing strain.

[0016] Preferably, the specific steps of step (1) are the selection of promoter PmPGK and PCR amplification of promoter PmPGK. The PCR amplification conditions are as follows: 95°C for 3 min - 5 min, 30 cycles (98°C for 10 sec, 55°C for 5 sec, 72°C for 60 sec), 72°C for 3 min - 5 min, and 12°C for 10 min - 12 min.

[0017] Preferably, the specific steps of step (2) are the amplification of the reaction system and the reverse amplification of the pEKm vector. The PCR amplification conditions of the reaction system are as follows: 95°C for 3 min - 5 min, 30 cycles (98°C for 10 sec, 55°C for 5 sec, 72°C for 60 sec), 72°C for 3 min - 5 min, and 12°C for 10 min - 12 min. The amplification conditions for the reverse amplification of the pEKm vector are as follows: 95°C for 3 min - 5 min, 30 cycles (98°C for 10 sec, 55°C for 5 sec, 72°C for 60 sec), 72°C for 5 min - 8 min, and 12°C for 8 - 12 min.

[0018] Preferably, for step (3), the reverse amplified vector, the PmPGK promoter, and the recovered product of the Hsp26 gene are configured into a system according to the instructions of the ClonExpress II One Step Cloning Kit. Take 200 μL of the incubation solution and spread it on an LB + Zeocin plate, and culture it in an incubator at 37°C for 12 - 16 h.

[0019] Preferably, for step (4), the single colonies grown in the previous step are respectively dissolved in 10 μL of sterile water, and 0.5 μL - 1.0 μL is taken as a template for colony PCR.

[0020] Preferably, for step (5), take 20 ng of the pEKm - PmPGK - Hsp26 recombinant plasmid and put it into 100 μl of competent cells. After electroporation, recover at 30°C for 2 h, and spread it on a solid plate of YPD + Zeocin (500 μg / ml), and culture it in an incubator at 28°C for 48 h - 72 h.

[0021] Preferably, for step (6), pick the single colonies grown on the plate and dissolve them in 50 μL - 100 μL of sterile water respectively, and perform colony PCR to verify the Hsp26 overexpressing strains.

[0022] The Hsp26 reference gene is:

[0023]

[0024] The sequencing sequence of the Hsp26 gene is as follows:

[0025]

[0026] The application of the engineered Kluyveromyces marxianus Hsp26 strain for ethyl acetate production in the fermentation process of rice sour soup includes the following steps:

[0027] (1) Raw material pretreatment: High-speed pulverize glutinous rice, mix it, and sieve it twice through a 80-100 mesh sieve to make rice flour;

[0028] (2) Boiling: Mix the rice flour and water, heat it to boiling, and stir while heating;

[0029] (3) Gelatinization: Put the boiled rice soup into a water bath for gelatinization, and stir while gelatinizing; to prevent local spoilage of the gelatinized liquid due to uneven gelatinization; the gelatinization time is 30-40 min and the gelatinization temperature is 50-60 °C;

[0030] (4) Liquefaction: Add 0.5-2.0% of high-temperature α-amylase based on the mass of the gelatinized rice soup for liquefaction, the liquefaction time is 30-40 min, and the liquefaction temperature is 85-95 °C;

[0031] (5) Saccharification: Add 0.01% of saccharifying enzyme based on the mass of the liquefied rice soup, the saccharification time is 2 h, and the saccharification temperature is 60 °C;

[0032] (6) Blending and sterilization: Add auxiliary materials to the liquefied rice soup, and then perform pasteurization at 95 °C for 10-20 minutes;

[0033] (7) Cooling: Wait for the rice soup to cool to 25-35 °C;

[0034] (8) Inoculation: Inoculate the overexpressed strain of Kluyveromyces marxianus Hsp26 and Kluyveromyces marxianus L1-1 into the rice soup respectively;

[0035] (9) Fermentation in jars: Put the rice soup inoculated with the strains into fermentation jars and ferment in a constant temperature incubator to obtain rice sour soup.

[0036] It should be noted that in the fermentation process of rice sour soup, the raw materials for making rice sour soup can also be cereal grains such as quinoa, coix seed, brown rice, black glutinous rice, millet, red rice, etc.

[0037] The beneficial effects of the present invention are as follows:

[0038] The present invention uses the engineered Kluyveromyces marxianus Hsp26 strain as the fermenting bacterium for fermenting rice sour soup. It has strong acid-producing and aroma-producing properties, and can produce ester aroma components such as ethyl acetate, ethyl propionate, ethyl acetate, and ethanol, which play an important role in enhancing the aroma of rice sour soup. Moreover, the engineered Kluyveromyces marxianus Hsp26 strain has good antioxidant properties in fermenting rice sour soup. Animal experiments show that it can significantly improve the color and brightness of hair. Using different grains (such as white glutinous rice, quinoa, coix seed, brown rice, black glutinous rice, millet, red rice, etc.) as raw materials, after fermentation treatment with the engineered Kluyveromyces marxianus Hsp26 strain, the nutritional value is guaranteed, and it has the effect of regulating the intestinal flora. The rice sour soup fermented with the improved engineered Kluyveromyces marxianus Hsp26 strain has excellent flavor and taste and high sensory evaluation; it has high antioxidant activity, and the DPPH scavenging rate is as high as 46%-51.5%; the ethyl acetate content can reach about 195.5-245.5 mg / L, and the total acid content can reach 0.45-0.61 g / 100 g; the viable yeast count is as high as 1.5-3.2×10 8 CFU / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Shows the pH value changes of rice sour soup fermented by Kluyveromyces marxianus L1-1 and Kluyveromyces marxianus L1-1 Hsp26 overexpression strain, as well as naturally fermented rice sour soup;

[0040] Figure 2 Shows the total acid changes of rice sour soup fermented by Kluyveromyces marxianus L1-1 and Kluyveromyces marxianus L1-1 Hsp26 overexpression strain, as well as naturally fermented rice sour soup;

[0041] Figure 3 Shows the changes in the amino acid nitrogen content of rice sour soup fermented by Kluyveromyces marxianus L1-1 and Kluyveromyces marxianus L1-1 Hsp26 overexpression strain, as well as naturally fermented rice sour soup;

[0042] Figure 4 Shows the changes in the reducing sugar content of rice sour soup fermented by Kluyveromyces marxianus L1-1 and Kluyveromyces marxianus L1-1 Hsp26 overexpression strain, as well as naturally fermented rice sour soup. DETAILED DESCRIPTION OF THE INVENTION

[0043] The following further defines the technical solution of the present invention in combination with specific embodiments, but the scope of protection is not limited to the description made.

[0044] Example 1

[0045] A method for constructing an engineered Kluyveromyces marxianus Hsp26 strain for producing ethyl acetate, comprising the following steps:

[0046] (1) PCR amplification of promoter PmPGK

[0047] The specific steps are the selection of promoter PmPGK and PCR amplification of promoter PmPGK. PCR amplification conditions: 95°C for 3 min, 30 cycles (98°C for 10 sec, 55°C for 5 sec, 72°C for 60 sec), 72°C for 3 min, 12°C for 10 min.

[0048] (2) PCR amplification of Hsp26 gene

[0049] The specific steps are the amplification of the reaction system and the reverse amplification of the pEKm vector. Among them, the PCR amplification conditions of the reaction system are: 95°C for 3 min, 30 cycles (98°C for 10 sec, 55°C for 5 sec, 72°C for 60 sec), 72°C for 3 min, 12°C for 10 min. The amplification conditions for the reverse amplification of the pEKm vector are: 95°C for 3 min, 30 cycles (98°C for 10 sec, 55°C for 5 sec, 72°C for 60 sec), 72°C for 5 min, 12°C for 10 min.

[0050] (3) One-step cloning of pEKm-PmPGK-Hsp26 recombinant plasmid

[0051] Recover the reverse-amplified vector, PmPGK promoter, and Hsp26 gene products and configure the system according to the ClonExpress II OneStep Cloning Kit instructions. Take 200 μL of the incubation solution and spread it on an LB + Zeocin plate, and culture it in a 37°C incubator for 16 h.

[0052] (4) Screening of pEKm-PmPGK-Hsp26 recombinant plasmid

[0053] Dissolve the single colonies grown in the previous step in 10 μL of sterile water respectively, and take 0.5 μL as a template for colony PCR.

[0054] (5) Electroporation of the recombinant plasmid overexpression vector into Kluyveromyces marxianus competent cells

[0055] Take 20 ng of the pEKm-PmPGK-Hsp26 recombinant plasmid and put it into 100 μL of competent cells. After electroporation, recover at 30°C for 2 h, and spread it on a solid plate of YPD + Zeocin (500 μg / ml), and culture it in a 28°C incubator for 48 h.

[0056] (6) Screening of Hsp26 gene overexpression strains

[0057] Pick the single colonies grown on the plate and dissolve them in 50 μL of sterile water respectively, and perform colony PCR to verify the Hsp26 overexpression strains.

[0058] The process of fermenting rice sour soup with the engineered Kluyveromyces marxianus improved by the above steps includes: pretreatment of glutinous rice raw materials, boiling, gelatinization, liquefaction, saccharification, blending and sterilization, cooling, inoculation, and fermentation in jars. Among them, the K. marxianus L1-1 Hsp26 overexpression strain was inoculated into the rice sour soup fermentation tank at an inoculation amount of 3% respectively, and then filled into the fermentation jars and fermented at room temperature.

[0059] Example 2

[0060] Different from Example 1, the K. marxianus L1-1 Hsp26 overexpression strain was inoculated into the rice sour soup fermentation tank at an inoculation amount of 5% respectively, and then filled into the fermentation jars and fermented at room temperature.

[0061] Example 3

[0062] Different from Example 1, the K. marxianus L1-1 Hsp26 overexpression strain was inoculated into the rice sour soup fermentation tank at an inoculation amount of 7% respectively, and then filled into the fermentation jars and fermented at room temperature.

[0063] Example 4

[0064] The process of fermenting rice sour soup with the wild Kluyveromyces marxianus L1-1 with independent intellectual property rights owned by the laboratory includes: pretreatment of glutinous rice raw materials, boiling, gelatinization, liquefaction, saccharification, blending and sterilization, cooling, inoculation, and fermentation in jars. Among them, the K. marxianus L1-1 wild strain was inoculated into the rice sour soup fermentation tank at an inoculation amount of 3% respectively, and then filled into the fermentation jars and fermented at room temperature.

[0065] Example 5

[0066] Different from Example 4, the K. marxianus L1-1 wild strain was inoculated into the rice sour soup fermentation tank at an inoculation amount of 5% respectively, and then filled into the fermentation jars and fermented at room temperature.

[0067] Example 6

[0068] Different from Example 4, the K. marxianus L1-1 wild strain was inoculated into the rice sour soup fermentation tank at an inoculation amount of 7% respectively, and then filled into the fermentation jars and fermented at room temperature.

[0069] Control Group 1

[0070] A traditional fermentation process for rice sour soup includes the following processes: raw material pretreatment, boiling, cooling, putting into jars, primary fermentation, and secondary fermentation.

[0071] The pH value changes (measured with a Testo 205 pH meter), total acid changes (measured by referring to the first method in GB / T 12456—2021 "National Food Safety Standard - Determination of Total Acid in Foods"), amino acid nitrogen content changes (measured according to the national food safety standard GB / T 5009.235-2016), and reducing sugar content changes (measured using the DNS method (3,5-dinitrosalicylic acid method)) of the fermented rice sour soup with Kluyveromyces marxianus L1-1 and the Kluyveromyces marxianus L1-1 Hsp26 overexpression strain, as well as the naturally fermented rice sour soup, were measured and data graphs were plotted. The results are respectively as Figures 1-4 shown in the figure. Explanation of the figure: "Over 3%" represents Example 1, "Over 5%" represents Example 2, "Over 7%" represents Example 3, "L1-1 3%" represents Example 4, "L1-1 5%" represents Example 5, "L1-1 7%" represents Example 6, and "Natural" represents Control Group 1.

[0072] Figure 1 It shows that: the pH values of each sample all showed a fluctuating downward trend with the increase of fermentation time, and the total acid content showed an upward trend with the extension of fermentation time. Finally, the pH value of the fermented soup of the Kluyveromyces marxianus Hsp26 engineered strain was the lowest, and the total acid content was the highest, indicating that the fermented rice sour soup with the Kluyveromyces marxianus Hsp26 engineered strain had a better sour taste than that with Kluyveromyces marxianus L1-1 and natural fermentation. Figure 2 It shows that: the amino acid nitrogen content of each sample all showed a fluctuating upward trend with the increase of fermentation time, and the amino acid nitrogen content of the fermented rice sour soup with the Kluyveromyces marxianus Hsp26 engineered strain was significantly higher than that with Kluyveromyces marxianus L1-1 and natural fermentation, indicating that the fermented rice sour soup with the Kluyveromyces marxianus Hsp26 engineered strain had a better flavor than that with Kluyveromyces marxianus L1-1 and natural fermentation. Figure 3 It shows that: the reducing sugar content in the rice sour of each sample all showed a fluctuating downward trend with the extension of fermentation time. Among them, the reducing sugar content of the fermented rice sour soup with the Kluyveromyces marxianus Hsp26 engineered strain decreased the fastest overall, indicating that the microorganisms in it were rapidly utilizing reducing sugars for growth, reproduction, and metabolism to produce other substances. In summary, it shows that the fermented rice sour soup with the Kluyveromyces marxianus Hsp26 engineered strain has good flavor and quality performance.

[0073] It is necessary to point out here that the above examples and control examples are only for further elaboration and understanding of the technical solution of the present invention, and should not be understood as further limiting the technical solution of the present invention. Inventions and creations made by those skilled in the art that do not have prominent substantial features and significant progress still fall within the protection scope of the present invention.

Claims

1. An engineered Kluyveromyces marxianus Hsp26 strain for producing ethyl acetate, characterized in that, The engineered bacteria are overexpressing bacteria of the small heat shock protein Hsp26 constructed by using gene editing technology in Kluyveromyces marxianus. The wild strain of the overexpressing strain, Kluyveromyces marxianus L1-1, has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M2021073 and the deposit date of January 15, 2021.

2. The construction method of Kluyveromyces marxianus Hsp26 engineering bacteria for producing ethyl acetate according to claim 1, characterized in that, It includes the following steps: (1) PCR amplify the promoter PmPGK; (2) PCR amplify the Hsp26 gene; (3) One-step clone the pEKm-PmPGK-Hsp26 recombinant plasmid; (4) Screen the pEKm-PmPGK-Hsp26 recombinant plasmid; (5) Electrotransform the overexpression vector of the recombinant plasmid into Kluyveromyces marxianus competent cells; (6) Screen the Hsp26 gene overexpressing strain.

3. The construction method of a Kluyveromyces marxianus Hsp26 engineered bacterium for producing ethyl acetate as described in claim 2, characterized in that, In the step (1), the PCR amplification conditions are: 95°C for 3 min - 5 min, 30 cycles (98°C for 10 sec, 55°C for 5 sec, 72°C for 60 sec), 72°C for 3 min - 5 min, 12°C for 10 min - 12 min.

4. The construction method of a Kluyveromyces marxianus Hsp26 engineered strain for producing ethyl acetate according to claim 2, characterized in that, For the step (2), the specific steps are the amplification of the reaction system and the reverse amplification of the pEKm vector. Among them, the PCR amplification conditions of the reaction system are: 95°C for 3 min - 5 min, 30 cycles (98°C for 10 sec, 55°C for 5 sec, 72°C for 60 sec), 72°C for 3 min - 5 min, 12°C for 10 min - 12 min; the amplification conditions of the reverse amplification of the pEKm vector are: 95°C for 3 min - 5 min, 30 cycles (98°C for 10 sec, 55°C for 5 sec, 72°C for 60 sec), 72°C for 5 min - 8 min, 12°C for 8 - 12 min.

5. Application of Kluyveromyces marxianus Hsp26 engineered bacteria for producing ethyl acetate in the fermentation process of rice sour soup, characterized in that, It includes the following process: (1) Raw material pretreatment: High-speed crush glutinous rice, mix, and sieve twice through 80 - 100 mesh to make rice flour; (2) Boiling: Mix the rice flour and water, heat to boiling, and stir while heating; (3) Gelatinization: Put the boiled rice soup into a water bath for gelatinization, and stir while gelatinizing; to prevent local deterioration of the gelatinized liquid due to uneven gelatinization; the gelatinization time is 30 - 40 min, and the gelatinization temperature is 50 - 60°C; (4) Liquefaction: Add 0.5 - 2.0% of high-temperature α-amylase based on the mass of the gelatinized rice soup for liquefaction. The liquefaction time is 30 - 40 min, and the liquefaction temperature is 85 - 95°C; (5) Saccharification: Add 0.01% of saccharifying enzyme to the liquefied rice soup. The saccharification time is 2 h, and the saccharification temperature is 60°C; (6) Blending and sterilization: Add auxiliary materials to the liquefied rice soup, and then perform pasteurization at 95°C for 10 - 20 minutes; (7) Cooling: Wait for the rice soup to cool to 25 - 35°C; (8) Inoculation: Inoculate the Kluyveromyces marxianus Hsp26 overexpressing bacteria and Kluyveromyces marxianus L1-1 into the rice soup respectively; (9) Filling the vat for fermentation: Put the rice soup inoculated with the strain into a fermentation vat, place it in a constant temperature incubator for fermentation, and then the sour rice soup is obtained.

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