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Engineering bacterium capable of producing D-lactate by aid of pentose and hexose synchronously by means of fermentation, and fabrication and application of engineering bacterium

A technology of engineering bacteria and six-carbon sugar, applied in the fields of genetic engineering and fermentation engineering, can solve the problems of low conversion efficiency, low product purity, low fermentation yield, etc., and achieves reduced glucose effect, high product purity, and ethanol. high yield effect

Inactive Publication Date: 2015-11-18
HUBEI UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although there are many D-lactic acid engineering bacteria and production bacteria at present, there are generally the above two problems; although there are a small number of reported modified engineering bacteria that can use mixed sugars for fermentation, it is not that the fermentation yield is low and the conversion efficiency is not high. , that is, a large amount of by-products are generated, and the product purity is not high

Method used

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  • Engineering bacterium capable of producing D-lactate by aid of pentose and hexose synchronously by means of fermentation, and fabrication and application of engineering bacterium
  • Engineering bacterium capable of producing D-lactate by aid of pentose and hexose synchronously by means of fermentation, and fabrication and application of engineering bacterium
  • Engineering bacterium capable of producing D-lactate by aid of pentose and hexose synchronously by means of fermentation, and fabrication and application of engineering bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1 Utilizes the construction of xylose-producing D-lactic acid Escherichia coli engineering bacteria E.coliDX02

[0040] Utilizing xylose to produce D-lactic acid Escherichia coli engineering bacteria E.coliDX02 is the starting strain of Escherichia coli engineering bacteria RM10 that can ferment xylose to produce ethanol. Through homologous recombination technology, the D-lactate dehydrogenase gene (ldhA) The alcohol dehydrogenase gene (adhE) was replaced to obtain the engineering bacterium E.coliDX02.

[0041] E.coliRM10 is derived from the wild strain E.coliB (ATCC11303), which knocked out pyruvate formate lyase (focA-pflB), fumarate reductase (frdBC), acetate kinase (ackA), D-lactate Genes of key enzymes of competitive metabolic pathways such as hydrogenase (ldhA) and partial nuclease genes (rngHSR2), and anaerobic expression of pyruvate dehydrogenase (pflBp6-aceEF-lpd), which can efficiently utilize xylose under anaerobic conditions Homofermentation to p...

Embodiment 2

[0056] Example 2 Construction of high-yield optically pure D-lactic acid engineering bacteria E.coliDX03 that can simultaneously utilize five-carbon sugar and six-carbon sugar to ferment

[0057] On the basis of the xylose-utilizing D-lactic acid engineering bacteria E.coliDX02, the ptsG gene was knocked out by using RED homologous recombination technology, and the D-lactic acid engineering bacteria E. coliDX03.

[0058] The first step, amplifying the ptsG homologous recombination fragment: using the pKD4 plasmid as a template, carrying out PCR amplification with primers ptsG-P1 and ptsG-P2 (knockout primers), the PCR product includes the FRT-kan-FRT sequence on the pKD4 plasmid And ptsG gene open reading frame (ORF) first and last 45bp homologous sequence. The amplification system is: 25 μL of ThermoScientific PCR MasterMix 2X buffer, 20 ng of DNA template, 1 μL of each primer (100 μM), 25 μL of sterile water, and the total volume is 50 μL. The amplification conditions were...

Embodiment 3

[0067] Example 3 Fermentation of engineering bacteria E.coliDX02 and E.coliDX03 to produce D-lactic acid

[0068] Pick a single colony from the plate, inoculate it into an anaerobic tube containing 10 mL of seed culture solution, and culture overnight at 37°C. Take 2 mL of the bacterial liquid and inoculate it into 300 mL of the seed liquid, and cultivate it at 150 r / min at 37°C until the mid-logarithmic growth phase. Inoculate the bacterial liquid into 3L fermentation medium with an inoculum amount of 10% (v / v), place it in a 7L fermenter SartoriusBB-8846880 (SartoriusStedimBiotech, Germany) with an automatic adjustment system, and cultivate and ferment at 150r / min at 37°C , add 3mol / LCa(OH) 2 Control the pH to 7.0. Different combinations of carbon sources: 10% glucose, 10% xylose, 5% glucose + 5% xylose, 6% glucose + 3% xylose + 1% L-arabinose were used as substrates to culture until the end of fermentation. Samples were taken regularly to measure the concentration of bac...

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Abstract

The invention discloses fabrication and application of an engineering bacterium capable of producing D-lactate with high optical purity by the aid of pentose and hexose synchronously by means of fermentation, and belongs to the field of genetic fermentation engineering. Escherichia coli capable of producing alcohol by the aid of the pentose by means of fermentation is used as an original strain, alcohol dehydrogenase genes (adhE) are replaced by D-lactate dehydrogenase genes (idhA) by means of homologous recombination, and encoding genes (ptsG) of enzymes IICB <Glc> of glucose transfer endocytosis are knocked out, so that the engineering bacterium capable of producing the D-lactate with the high optical purity by the aid of the pentose and the hexose synchronously by means of fermentation can be obtained. The fabrication and the application of the engineering bacterium have the advantages that mixed carbon sources are used for lactate fermentation production, accordingly, glucose effects can be reduced, the pentose and the hexose can be synchronously utilized, and the utilization efficiency of the carbon sources in unit time can be improved; carbon metabolic flux is redistributed, accordingly, accumulation of a large quantity of D-lactate which is a target product can be promoted, generation of other byproducts such as acetic acid can be basically prevented, the D-lactate is high in optical purity, and the optical purity of the D-lactate can reach 99.8% at least.

Description

technical field [0001] The invention relates to the construction of an engineering bacterium capable of synchronously utilizing five-carbon sugar and six-carbon sugar to ferment and produce D-lactic acid, and belongs to the field of genetic engineering. The invention also relates to an application of an engineering bacterium capable of synchronously utilizing five-carbon sugar and six-carbon sugar to ferment and produce D-lactic acid, especially fermenting and producing D-lactic acid, which belongs to the field of fermentation engineering. Background technique [0002] D-lactic acid is an important chiral intermediate. Its polymer has high thermal stability and can be made into biodegradable materials. Therefore, its production and application have attracted extensive attention and become a current research hotspot. Due to the advantages of wide source of raw materials, low production cost, high optical purity, and high safety, microbial fermentation has become the main meth...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/56C12R1/19
Inventor 赵筱王金华周胜德王永泽赵锦芳
Owner HUBEI UNIV OF TECH
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