阅读说明：本技术 一种重组大肠杆菌及其在筛选赤藓糖醇生产菌株中的应用 (recombinant escherichia coli and application thereof in screening erythritol production strains ) 是由 张娟 邱学良 李江华 堵国成 陈坚 于 2019-11-07 设计创作，主要内容包括：本发明公开了一种重组大肠杆菌及其在筛选赤藓糖醇生产菌株中的应用,属于微生物技术领域。本发明的筛选赤藓糖醇生产菌株的方法所使用的重组大肠杆菌能很好的对不同浓度的赤藓糖醇进行正相关感应,因此,本发明的筛选赤藓糖醇生产菌株的方法具有灵敏度高的优势。在工业上发酵生产赤藓糖醇多采用高浓度葡萄糖作为发酵底物,而本发明的筛选赤藓糖醇生产菌株的方法可克服高浓度葡萄糖的干扰,在高浓度葡萄糖的干扰下,本发明的筛选赤藓糖醇生产菌株的方法所使用的重组大肠杆菌仍能很好的对不同浓度的赤藓糖醇进行正相关感应,并且,相关性较无葡萄糖干扰时更高,因此,本发明的筛选赤藓糖醇生产菌株的方法具有抗干扰能力强的优势。(The invention discloses recombinant escherichia coli and application thereof in screening erythritol producing strains, and belongs to the technical field of microorganisms.)

The recombinant plasmids are characterized in that pET-22b (+) plasmids are used as expression vectors of the recombinant plasmids to express genes and marker genes for coding transcription regulatory factors, and nucleotide sequences of the genes for coding the transcription regulatory factors are shown in SEQ ID No. 1.

2. The recombinant plasmids of claim 1, wherein the recombinant plasmid uses pET-22b (+) plasmid as expression vector to express the gene coding the transcription regulatory factor, the marker gene and the transcription regulatory factor binding sequence, and the transcription regulatory factor binding sequence is shown as SEQ ID No. 5.

3. The recombinant plasmids of claim 1 or 2, wherein the recombinant plasmid is obtained by inserting a marker gene downstream of the promoter of pET-22b (+) plasmid T7 and replacing the nucleotide sequence of the Lac I gene on pET-22b (+) plasmid with the nucleotide sequence of a gene encoding a transcription regulatory factor and the nucleotide sequence of the Lac O gene with a transcription regulatory factor binding sequence.

4. The recombinant plasmid of any one of claims 1-3 of , wherein the marker gene is a gene encoding a fluorescent protein.

5. The recombinant plasmids of claim 4, wherein the nucleotide sequence of the gene encoding the fluorescent protein is shown in SEQ ID No. 2.

recombinant Escherichia coli, characterized in that, the recombinant Escherichia coli contains the recombinant plasmid of any of claims 1-5, .

7. The recombinant Escherichia coli as claimed in claim 6, wherein the recombinant Escherichia coli has Escherichia coli (Escherichia coli) BL21(DE3) as expression host.

8, A method for screening erythritol-producing strains, comprising inoculating the recombinant Escherichia coli of claim 6 or 7 to a fermentation supernatant of a strain to be screened, and culturing the strain to be screened to obtain a culture solution, wherein erythritol-producing ability of the strain to be screened can be confirmed based on an expression level of a marker gene in the culture solution.

9. The method for screening of erythritol-producing strains according to claim 8, wherein the medium is a glucose-containing medium.

10. Use of the recombinant plasmid according to any of claims 1-5 or the recombinant escherichia coli according to claim 6 or 7 or the method for screening erythritol-producing strains according to claim 8 or 9 for screening erythritol-producing strains.

Technical Field

The invention relates to recombinant escherichia coli and application thereof in screening erythritol producing strains, belonging to the technical field of microorganisms.

Background

Erythritol is a sugar alcohol with the smallest relative molecular mass found in nature, not only has all the excellent functions of sugar alcohol products, such as preventing dental caries and being suitable for diabetics, but also has the characteristics of extremely low calorie (less than or equal to 1.66 kJ.g) and extremely high tolerance (no side effect), has good food processing adaptability and physiological health-care function, and is currently used as a raw material of novel functional health-care foods in the fields of food, medicine, cosmetics, chemical industry and the like by .

The erythritol producing strains include fungi, bacteria and the like, and many of them produce polyols such as xylitol, ethanol, glycerol and the like in addition to erythritol. Currently, erythritol producing strains used in commerce are mainly an Aureobasidium sp.variant in Japan and Candida magnoliae in Korea. In view of the important application value and the huge market demand of erythritol, the search for erythritol producing strains with excellent fermentation performance is still the basis of all research and development work.

The existing method for screening erythritol producing strains mainly comprises the steps of culturing strains to be screened firstly, and then determining the content of erythritol in fermentation liquor by a High Performance Liquid Chromatography (HPLC) method, wherein the method needs complex sample treatment, has a long detection period, is not suitable for rapid detection of erythritol fermentation liquor, and is difficult to realize high-throughput screening of erythritol producing strains.

Disclosure of Invention

[ problem ] to

The invention aims to provide efficient erythritol production strain screening methods.

[ solution ]

In order to solve the problems, the invention provides recombinant plasmids, the recombinant plasmids take pET-22b (+) plasmids as expression vectors, express genes for coding transcription regulatory factors and marker genes, and the nucleotide sequences of the genes for coding the transcription regulatory factors are shown in SEQ ID No. 1.

In embodiments of the present invention, the recombinant plasmid uses pET-22b (+) plasmid as expression vector to express the gene coding transcription regulatory factor, marker gene and transcription regulatory factor binding sequence, and the transcription regulatory factor binding sequence is shown in SEQ ID No. 5.

In embodiments of the present invention, the recombinant plasmid is obtained by inserting a marker gene downstream of the promoter of pET-22b (+) plasmid T7, and replacing the nucleotide sequence of the LacI gene on pET-22b (+) plasmid with the nucleotide sequence of a gene encoding a transcription regulatory factor, and the nucleotide sequence of the lacO gene with a transcription regulatory factor binding sequence.

In embodiments of the invention, the marker gene is a gene encoding a fluorescent protein.

In embodiments of the present invention, the nucleotide sequence of the gene encoding fluorescent protein is shown in SEQ ID No. 2.

The invention also provides recombinant escherichia coli, which contains the recombinant plasmid.

In embodiments of the present invention, the recombinant E.coli has E.coli (Escherichia coli) BL21(DE3) as an expression host.

The invention also provides a method for screening erythritol producing strains, which comprises the steps of inoculating the recombinant escherichia coli to fermentation supernatant of a strain to be screened for culture to obtain a culture solution, and confirming the erythritol producing capacity of the strain to be screened according to the expression quantity of the marker gene in the culture solution.

In embodiments of the invention, the medium is a glucose-containing medium.

In embodiments of the present invention, the concentration of glucose in the culture medium is 10-300 g/L.

The invention also provides the application of the recombinant plasmid or the recombinant escherichia coli or the method for screening the erythritol producing strain in screening the erythritol producing strain.

[ advantageous effects ]

(1) The invention provides recombinant plasmids for screening erythritol producing strains, which contain genes encoding transcription regulatory factors and marker genes, wherein recombinant escherichia coli for screening erythritol producing strains can be obtained after the recombinant plasmids are introduced into escherichia coli (Escherichia coli) BL21(DE3), and when the recombinant escherichia coli is inoculated into fermentation supernatant of strains to be screened for culture, if the strains to be screened can produce erythritol, the erythritol produced by the strains to be screened can relieve the inhibition of the transcription regulatory factor eryD in the recombinant plasmids on the marker genes, so that the marker genes can be expressed, and further the expression of the marker genes can be detected in culture solution obtained by co-culture.

(2) The invention provides recombinant escherichia coli capable of being used for screening erythritol producing strains, wherein the recombinant escherichia coli contains recombinant plasmids capable of expressing genes encoding transcription regulation factors and marker genes, when the recombinant escherichia coli is inoculated into fermentation supernatant of strains to be screened for culture, if the strains to be screened can produce erythritol, the erythritol produced by the strains to be screened can relieve the inhibition of the transcription regulation factors eryD in the recombinant plasmids on the marker genes, so that the marker genes can be expressed, and the expression of the marker genes can be detected in culture solution obtained by co-culture.

(3) The invention provides a method for screening erythritol producing strains of species, which comprises the steps of inoculating recombinant escherichia coli containing a gene for coding a transcription regulatory factor and a recombinant plasmid of a marker gene into a fermentation supernatant of a strain to be screened for culture, wherein if the strain to be screened can produce erythritol, the erythritol produced by the strain to be screened can relieve the inhibition of the transcription regulatory factor eryD in the recombinant plasmid on the marker gene, so that the marker gene can be expressed, and further the expression of the marker gene can be detected in a culture solution obtained by co-culture.

(4) The recombinant escherichia coli used in the method for screening the erythritol producing strain can well perform positive correlation induction on erythritol with different concentrations, so that the method for screening the erythritol producing strain has the advantage of high sensitivity.

(5) The method for industrially fermenting and producing the erythritol mostly adopts high-concentration glucose as a fermentation substrate, the interference of the high-concentration glucose can be overcome, the recombinant escherichia coli used by the method for screening the erythritol producing strain can still well perform positive correlation induction on the erythritol with different concentrations under the interference of the high-concentration glucose, and the correlation is higher than that of the erythritol without the glucose interference, so that the method for screening the erythritol producing strain has the advantage of strong anti-interference capability.

(6) The trend of the result of detecting the expression amount of the marker gene in the fermentation broth obtained by co-culture by using the method of the present invention and the trend of the result of detecting the content of erythritol in the fermentation broth obtained by co-culture by using the liquid chromatography is , so the method of screening erythritol producing strains of the present invention has the advantage of high accuracy.

Drawings

FIG. 1: plasmid map of plasmid pET-22b (+).

FIG. 2: plasmid map of recombinant plasmid pET-22b (+) -eryD-1.

FIG. 3: plasmid map of recombinant plasmid pET-22b (+) -eryD-2.

FIG. 4: influence of erythritol induction on fluorescence intensity in fermentation broth obtained by fermentation of recombinant Escherichia coli E.coli BL21(DE3)/pET-22b (+) -eryD-3.

FIG. 5: influence of erythritol induction concentration on fluorescence intensity of fermentation liquor obtained by fermentation of recombinant Escherichia coli E.coli BL21(DE3)/pET-22b (+) -eryD-3.

FIG. 6: influence of glucose concentration on fluorescence intensity of fermentation broth obtained by fermentation of recombinant Escherichia coli E.coli BL21(DE3)/pET-22b (+) -eryD-3.

Detailed Description

Coli (Escherichia coli) BL21(DE3) referred to in the examples below was purchased from Biotechnology engineering (Shanghai) Ltd; the pET-22b (+) plasmid referred to in the examples below was purchased from Nanjing Kinsry.

The media involved in the following examples are as follows:

LB liquid medium: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder and 100ng/mL of ampicillin.

LB solid medium: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder, 20g/L of agar and ng/mL of ampicillin.

YPD liquid medium: tryptone 20g/L, yeast powder 10g/L, glucose 20 g/L.

YPD solid Medium: tryptone 20g/L, yeast powder 10g/L, glucose 20g/L and agar 20 g/L.

The detection methods referred to in the following examples are as follows:

the method for detecting the erythritol content comprises the following steps:

the erythritol and glucose in the supernatant of the fermentation broth were accurately determined by high performance liquid chromatography (1260Infinity, Agilent, USA) with mobile phase of dilute sulfuric acid (5mm), column of amine ○ RHPX-87H ion exclusion column, column temperature of 40 deg.C, flow rate of 0.6mL/min, detector of 1260 RID.