Lactic acid-producing engineering bacteria, constructing method thereof and application thereof

A construction method and technology of engineering bacteria, applied in the fields of genetic engineering and fermentation engineering, can solve the problems of high nutritional demand of lactic acid bacteria and restricting the development of L-lactic acid fermentation industry.

Inactive Publication Date: 2010-11-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the development of the L-lactic acid fermentation industry is limited due to the high nutritional requirements of lactic acid bacteria and

Method used

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  • Lactic acid-producing engineering bacteria, constructing method thereof and application thereof
  • Lactic acid-producing engineering bacteria, constructing method thereof and application thereof
  • Lactic acid-producing engineering bacteria, constructing method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 produces the construction of lactic acid engineering bacteria

[0031] 1. Cloning of Bovine LDH gene

[0032] Using the plasmid pLAZ10 (Bianchi, 2010. Efficient Homolactic Fermentation by Kluyveromyces lactis Strains Defective in Pyruvate Utilization and Transformed with the Heterologous LDH Gene. ApplEnviron Microbiol, 67(12): 5621-5625.) as a template, the LDH fragment was amplified by PCR. Using LDH-F and LDH-R with XbaI and ClaI restriction sites as primers, the LDH fragment and plasmid pY13TEF1 (purchased from Delbao Biotechnology Co., Ltd.) were double-digested, purified, and Ligation construction plasmid pY13TEF1-LDH ( figure 1 A). After the ligation product was transformed into JM109 competent cells, the LB plate with ampicillin resistance was spread, cultured overnight at 37°C, and the transformant was picked for verification, as figure 1 b. The positive transformant was inoculated into liquid LB medium, the plasmid was extracted, digested, ver...

Embodiment 2

[0046] Embodiment 2 fermentation produces lactic acid

[0047] Connect a loop of the lactic acid-producing engineered bacteria from the fresh slant to the seed medium (80mL SC / 250mL Erlenmeyer flask), and culture it to the mid-logarithmic phase (24h) at 30°C and 200r / min in a shaker flask, then use 8% inoculum (v / v) into a 1.3L fermentor equipped with 700mL of YNB fermentation medium. During the fermentation process, the pH was maintained at 5.5 with 4mol / L NaOH, the ventilation rate was 1.5L / min, and the dissolved oxygen was controlled at 50%. After 100 hours of fermentation, the lactic acid production reached 15g / L.

[0048] The composition of the seed medium is (g / L): glucose 20, YNB (withoutNH 4 SO 4 ) 1.7, ammonium sulfate 4, the required amount of amino acid.

[0049] The composition of YNB fermentation medium is (g / L): CaCO 3 4.5, YNB (without NH 4 SO 4 ) 1.7, urea 1, ethanol 5, glucose 70, the required amount of amino acids.

Embodiment 3

[0050] The detection of embodiment 3L-lactic acid, ethanol and glucose

[0051] Determination of L-lactic acid and ethanol concentration: Agilent A1200 high performance liquid chromatography (HPLC) instrument was used for determination.

[0052] Chromatographic conditions

[0053] Chromatographic column: Aminex HPX-87H (Bio-Rad)

[0054] Mobile phase: 0.005mol / L -1 h 2 SO 4

[0055] Flow rate: 0.6mL / min -1

[0056] Column temperature: 35°C

[0057] Injection volume: 5μL

[0058] Detector: Differential Refractive Index

[0059] Sample preparation: Centrifuge 5 mL of fermentation broth at 10,000 rpm for 10 min, and transfer the supernatant into a test tube for use in the measurement of L-lactic acid, ethanol, and residual glucose. When measuring L-lactic acid, take 1mL of the supernatant and transfer it into a 10mL volumetric flask, dilute to the mark with deionized water, filter through a 0.45μm filter membrane, and use the filtrate for liquid chromatography analysis....

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Abstract

The invention discloses lactic acid-producing engineering bacteria, a constructing method thereof and application thereof, and belongs to the field of fermentation engineering. In the genetic engineering bacterial strain provided by the invention, gene segments of lactic dehydrogenase (LDH), which are derived from Bovine, are integrated into S.cerevisiae genome by a homologous recombination method, and the pyruvic decarboxylase (PDC1) gene of the bacterial strain is silenced. The engineering bacterial strain obtained by constructing is used for fermenting and producing the lactic acid; after fermentation for 100 hours, the yield of the lactic acid reaches 15g/L, and the yield of ethanol is reduced by 45 percent less than a parent strain, namely 16.2g/L; and the engineering bacteria has a wide application prospect.

Description

technical field [0001] The invention relates to a lactic acid-producing engineering bacterium and a construction method thereof, belonging to the field of genetic engineering. The invention also relates to the application of a lactic acid-producing engineering bacterium, in particular to fermenting and producing lactic acid, which belongs to the field of fermentation engineering. Background technique [0002] As an important platform compound, the demand for L-lactic acid continues to increase with the depletion of petroleum resources. In the L-lactic acid synthesis process, the process of using biomass as raw material and adopting microbial fermentation (lactic acid bacteria and mold) has a wide range of applications. However, the development of the L-lactic acid fermentation industry is limited due to the high nutritional requirements of lactic acid bacteria and the difficulty in tolerance to harsh environmental stresses, as well as the easy formation of mycelium clusters...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/09C12P7/56C12R1/865
Inventor 刘立明赵亮亮陈坚
Owner JIANGNAN UNIV
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