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Engineering bacterium of fermenting production of optical pure L-lactate by utilizing xylose and construction thereof

An engineering bacteria and optical technology, applied in the fields of microbial fermentation and genetic engineering, to reduce production costs, simplify post-extraction processes, and promote mass accumulation.

Inactive Publication Date: 2012-05-02
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The wild-type liquid phase metabolites of this strain include lactic acid, acetic acid and ethanol. Although lactic acid is the main metabolite, there is still room for improvement in the conversion rate of lactic acid

Method used

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  • Engineering bacterium of fermenting production of optical pure L-lactate by utilizing xylose and construction thereof
  • Engineering bacterium of fermenting production of optical pure L-lactate by utilizing xylose and construction thereof
  • Engineering bacterium of fermenting production of optical pure L-lactate by utilizing xylose and construction thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Construction of an engineering bacterium that can produce optically pure L-lactic acid by fermenting xylose

[0032] An engineering bacterium that can produce optically pure L-lactic acid by xylose fermentation is constructed by inserting a heat-resistant kana resistance coding gene between the genes encoding acetate kinase and phosphotransacetylase to realize acetate kinase and phosphotransferase. Knockout of the gene encoding acetylase can block the branch of acetic acid metabolism, and can preliminarily identify positive recombinants through kana resistance screening. The principle of the specific blocking process is as follows: figure 1 As shown, the specific operation includes the following steps:

[0033] (1) Construction of the Escherichia coli and thermoanaerobic bacillus shuttle vector pBlue-aph carrying the Kana resistance expression cassette: Carry out the whole gene synthesis of the aph gene according to the Kana resistance gene (GenBank: V01547), and inser...

Embodiment 2

[0053] Comparison of engineered bacteria that can ferment xylose to produce optically pure L-lactic acid and wild-type lactic acid

[0054] (1) Seed cultivation

[0055] Insert the engineering bacteria of Example 1 into a 20ml serum bottle containing 10ml of seed culture medium, and cultivate at 55°C and 150r / min for 12h, so that the OD value reaches above 0.8. Inoculate into a 125ml serum bottle containing 50ml seed medium at a ratio of 1:5, culture at 55°C and 150r / min for 12h, and make the OD value reach above 1.0.

[0056] (2) shake flask culture

[0057] The seed solution cultivated in step (1) is connected to a 125ml serum bottle equipped with 50ml fermentation medium according to 10% inoculum amount, and nitrogen gas is filled to a pressure of 0.04MPa. The culture was continued for 24 hours with 10g / L xylose as the substrate.

[0058] The operation of the wild-type strain was the same as above.

[0059] The metabolites were detected by HPLC, and the comparison resul...

Embodiment 3

[0084] Whether the culture medium is sterilized affects the engineering bacterium fermentation product of the present invention

[0085] According to 10% inoculum amount, the seed liquid of embodiment engineering bacteria is inserted in the 5L automatic reaction kettle of NBS company that 3L fermentation medium is housed. Before inoculation, the reaction kettle was first fed with nitrogen for 30 minutes, and then nitrogen was fed for 30 minutes after inoculation to ensure that the fermentation environment was oxygen-free. During the fermentation process, 5mol / L NaOH was used to control the pH value to 6.8.

[0086] Lactic acid, acetic acid, ethanol and sugar concentrations in the fermentation products were determined (Table 2).

[0087] Using 50g / L glucose as substrate for continuous fermentation for 72 hours, the concentration of lactic acid can reach 47.2g / L, the conversion rate can reach 0.95g / g, and the optical purity of L-lactic acid can exceed 99.5%.

[0088] Using 50g...

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Abstract

The invention discloses an engineering bacterium of fermentation production of optical pure L-lactate by utilizing xylose, as well as a construction method and an application thereof. The engineering bacterium is constructed in the following steps of: synthesizing a kar kanamycin resistant gene, and inserting the kar kanamycin resistant gene into a shuttle vector of escherichia coli and thermophilic anaerobic bacillus; amplifying a certain sequence of an acetokinase coding gene, which is a pta-up sequence; inserting the pta-up sequence into the shuttle vector of the escherichia coli and thermophilic anaerobic bacillus to construct a suicide vector 1; amplifying a certain sequence of a phosphotransacetylase coding gene, which is an ack-down sequence; inserting the ack-down sequence into the suicide vector 1 to obtain a suicide vector pPuKAd; converting the suicide vector pPuKAd into the thermophilic anaerobic bacillus, performing resistance screening to obtain the optical pure L-lactate by utilizing fermentation of xylose. The engineering bacterium of the invention is used for fermentation production of lactate, acetic acid is not generated, and a carbon metabolic flux is distributed once again, so that massive accumulation of the target product L-lactate is promoted, and the optical purity of the L-lactate is over 99.5 percent.

Description

technical field [0001] The invention belongs to the field of microbial fermentation and genetic engineering, and specifically relates to an engineering bacterium which lacks an acetic acid metabolic pathway and can produce optically pure L-lactic acid by fermenting xylose, as well as its construction method and application. Background technique [0002] Lactic acid is widely used in food, medicine, cosmetics, leather manufacturing, textile industry and other fields. Lactic acid is also an important chemical platform compound. And due to the attention to biodegradable polylactic acid in recent years, the cheap acquisition of lactic acid has once again attracted the attention of researchers. Compared with chemical synthesis methods, microbial fermentation to produce lactic acid has the advantages of low temperature, low energy consumption and low cost (Datta, R. and M. Henry., Lactic acid: recent advances in products, processes and technologies-a review.Journal of Chemical T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12P7/56C12R1/01C12R1/145
Inventor 李爽王菊芳杨晓锋王小宁于平儒
Owner SOUTH CHINA UNIV OF TECH
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