Acetic acid leakage type high-yield pyruvate bacterium and its breeding method and producing pyruvic acid by said bacterium via fermentation process

Abstract

The present invention belongs to the field of bioengineering technology. The bacterium of the present invention is a kind of Torulopsis glabrata, WSH-, obtained with WSH-IP303 as starting strain and through NTG mutagenesis and breeding selection in culture medium with added acetic acid as replenishing carbon source. Compared with the starting strain, LQ307 has mush reduced pyruvate decarboxylase, and strong and stable pyruvate producing capacity. The yield of pyruvate reaches 46.2 g / L, 21% higher than that of starting strain when using acetic acid as replenishing carbon source and through shaking bottle culture for 48 hr; the yield of pyruvate may reach 68.7 g / L the glucose converting rate may reach 0.651 g / g through fermentation in a 5 L fermenting tank for 64 hr.

Description

technical field

[0001] The invention relates to an acetic acid leakage type high-yield pyruvate bacterium, a breeding method thereof and a fermentation method for producing pyruvate by using the acetic acid leakage type, which belongs to the technical field of bioengineering. Background technique

[0002] Pyruvic acid is an important intermediate product in sugar metabolism. Pyruvate is an important precursor for the synthesis of various amino acids, vitamins and other useful substances, and is widely used in industries such as medicine, daily chemicals, agricultural chemicals, feed and food. Although as a chemical product, pyruvic acid has already been industrialized, the tartaric acid dehydration and decarboxylation method is still used to produce pyruvic acid. The price of acid products remains high, and the promotion and application are naturally limited. The production of pyruvate by fermentation has many advantages, such as low cost of raw materials, wide range of so...

Examples

Embodiment 1

[0056] Embodiment 1: produce pyruvic acid with WSH-LQ307 fermentation method

[0057] Connect a ring of LQ-307 strains from a fresh slant into the seed medium (50ml / 500ml Erlenmeyer flask), cultivate at 30°C and 200rpm for 24h, then insert into the fermentation medium with 10% inoculum (v / v) . The composition of the seed medium and the fermentation medium is as described in this specification, and 6 g / L sodium acetate is added to the fermentation medium.

[0058] Shake flask culture: the liquid volume of the fermentation medium in the 500ml Erlenmeyer flask is 50ml, and the rotation speed is 200rpm. The fermentation time was 48h, and the yield of pyruvate was 46.2g / L.

Embodiment 2

[0059] Embodiment 2: Synthesize pyruvate in 5L fermenter with WSH-LQ307

[0060] Slope seed culture and fermentation inoculation conditions are all the same as in Example 1, and the volume of the fermentation medium (adding 6g / L sodium acetate) in the 5L fermenter is 3L. The ventilation rate is 3L / min, the stirring speed is 700rpm in the first 16h, and it is reduced to 500rpm after 16h, the pH is controlled at about 5.0 with 5mol / LKOH or 5mol / L NaOH, the fermentation temperature is 30°C, and the fermentation equipment is produced by Korean Fermentation Tank Company. A fully automatic fermentation tank model KBT-5L. The fermentation time is 64h. The yield of pyruvate was 68.7g / L, and the conversion rate to glucose was 0.651g / g.

Embodiment 3

[0061] Embodiment 3: synthesize pyruvate with WSH-LQ307 in 300L fermenter

[0062] Slope seed cultivation and fermentation inoculation conditions are all the same as in Example 1, and the 30L automatic fermenter (Changzhou Shuguang Chemical Factory) used for seed cultivation is the KF-30L automatic fermenter produced by Korea Fermenter Company. The reactor used for fermentation is a 300L upper mechanical stirring tank (Changzhou Shuguang Chemical Factory), with a liquid capacity of 200L, an aeration rate of 1L / L min, a stirring speed of 250rpm for 0-16h, and 220rpm after 16h, using industrial alkali ( The mass concentration is about 300g / L), the pH is controlled to be 5.0, and the fermentation temperature is 30°C. The fermentation time is 65h. The yield of pyruvate was 63.8g / L, and the conversion rate to glucose was 0.588g / g.

technical field

[0001] The invention relates to an acetic acid leakage type high-yield pyruvate bacterium, a breeding method thereof and a fermentation method for producing pyruvate by using the acetic acid leakage type, which belongs to the technical field of bioengineering. Background technique

[0002] Pyruvic acid is an important intermediate product in sugar metabolism. Pyruvate is an important precursor for the synthesis of various amino acids, vitamins and other useful substances, and is widely used in industries such as medicine, daily chemicals, agricultural chemicals, feed and food. Although as a chemical product, pyruvic acid has already been industrialized, the tartaric acid dehydration and decarboxylation method is still used to produce pyruvic acid. The price of acid products remains high, and the promotion and application are naturally limited. The production of pyruvate by fermentation has many advantages, such as low cost of raw materials, wide range of so...

Examples

Embodiment 1

[0056] Embodiment 1: produce pyruvic acid with WSH-LQ307 fermentation method

[0057] Connect a ring of LQ-307 strains from a fresh slant into the seed medium (50ml / 500ml Erlenmeyer flask), cultivate at 30°C and 200rpm for 24h, then insert into the fermentation medium with 10% inoculum (v / v) . The composition of the seed medium and the fermentation medium is as described in this specification, and 6 g / L sodium acetate is added to the fermentation medium.

[0058] Shake flask culture: the liquid volume of the fermentation medium in the 500ml Erlenmeyer flask is 50ml, and the rotation speed is 200rpm. The fermentation time was 48h, and the yield of pyruvate was 46.2g / L.

Embodiment 2

[0059] Embodiment 2: Synthesize pyruvate in 5L fermenter with WSH-LQ307

[0060] Slope seed culture and fermentation inoculation conditions are all the same as in Example 1, and the volume of the fermentation medium (adding 6g / L sodium acetate) in the 5L fermenter is 3L. The ventilation rate is 3L / min, the stirring speed is 700rpm in the first 16h, and it is reduced to 500rpm after 16h, the pH is controlled at about 5.0 with 5mol / LKOH or 5mol / L NaOH, the fermentation temperature is 30°C, and the fermentation equipment is produced by Korean Fermentation Tank Company. A fully automatic fermentation tank model KBT-5L. The fermentation time is 64h. The yield of pyruvate was 68.7g / L, and the conversion rate to glucose was 0.651g / g.

Embodiment 3

[0061] Embodiment 3: synthesize pyruvate with WSH-LQ307 in 300L fermenter

[0062] Slope seed cultivation and fermentation inoculation conditions are all the same as in Example 1, and the 30L automatic fermenter (Changzhou Shuguang Chemical Factory) used for seed cultivation is the KF-30L automatic fermenter produced by Korea Fermenter Company. The reactor used for fermentation is a 300L upper mechanical stirring tank (Changzhou Shuguang Chemical Factory), with a liquid capacity of 200L, an aeration rate of 1L / L min, a stirring speed of 250rpm for 0-16h, and 220rpm after 16h, using industrial alkali ( The mass concentration is about 300g / L), the pH is controlled to be 5.0, and the fermentation temperature is 30°C. The fermentation time is 65h. The yield of pyruvate was 63.8g / L, and the conversion rate to glucose was 0.588g / g.

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