Escherichia coli alkali-resistant target spot and application thereof

Abstract

The invention discloses an Escherichia coli alkali-resistant target spot and application thereof, an inhibition model of pH on Escherichia coli E.coli AG1 is established by setting LB culture media with different pH values, and it is found that the inhibition degree of cell growth is positively correlated with the rise of pH. Under the condition that the pH value is equal to 10, the escherichia coli overexpression library ASKA is subjected to high-throughput screening, and 43 alkali-tolerant elements are obtained after multiple rounds of screening. The obtained gene is subjected to KEGG / GO enrichment analysis through an online gene function annotation database DAVID, an EcoCyc database and related literatures are looked up, an alkali tolerance mechanism is predicted, and it is found that the alkali tolerance mechanism is possibly related to biological membrane formation, intracellular pH steady state maintenance, intracellular ROS level reduction and the like. Through overexpression of the genes, the strain can adapt to an environment under alkali stress, the alkali tolerance of the strain is improved, and the strain has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of synthetic biology and metabolic engineering, in particular to Escherichia coli alkali tolerance target and application thereof. BACKGROUND

[0002] Escherichia coli has become one of the most commonly used chassis cells in synthetic biology manufacturing due to its clear genetic background, mature operation tools, rapid growth and other advantages, and has been widely used in the biosynthesis of diamines (such as 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane). However, in the synthesis of diamines, the alkalinity of the product itself will cause the pH value of the fermentation system to continuously rise. This alkaline stress environment will cause serious damage to Escherichia coli cells, disrupt intracellular pH homeostasis, cause oxidative stress, damage biological macromolecules, and ultimately lead to bacterial growth arrest, activity decline, and serious limitation of the final yield, yield and conversion rate of diamines. Therefore, improving the alkali tolerance of Escherichia coli is the key to breaking through the bottleneck of its production of alkaline chemicals.

[0003] At present, the research on the alkali tolerance mechanism of Escherichia coli is not sufficient, and the known mechanisms are mainly limited to a few ion transport proteins (such as NhaA, MdfA) and specific metabolic systems (such as the glutamate-dependent acid resistance system GADR which can also participate in alkali adaptation under specific conditions). The traditional mutagenesis breeding method for breeding resistant strains is long, random and low in efficiency. Therefore, it is of important theoretical value and broad application prospect to systematically mine potential alkali tolerance gene elements in the genome of Escherichia coli, and to construct high-performance alkali-resistant engineering bacteria based thereon and apply them to the synthesis of high-value alkaline chemicals such as diamines. SUMMARY

[0004] The technical problem solved by the present application is to provide an Escherichia coli alkali tolerance target and application thereof. The present application first systematically mines 43 new gene targets related to the alkali tolerance of Escherichia coli through high-throughput screening of a whole-genome overexpression library, especially the combination of metN, prpE, fruK, nikC, mazG and ybiT, which breaks through the limitation of only focusing on a few known ion transport proteins in the past, provides a rich gene resource library, lays a solid foundation for subsequent construction of super engineering bacteria with synergistic effect and better tolerance through multi-gene combination regulation, and provides a strong guarantee for the synthesis of high-value alkaline chemicals such as diamines.

[0005] Technical solution: Escherichia coli alkali-resistant target points, the Escherichia coli alkali-resistant target points include multiple combinations of metN, yagZ, gmhB, prpE, ybcH, rna, ybiT, narH, abgB, wza, ydgH, yodC, yfjW, fruK, nrdB, csdA, ubiH, ygbT, mazG, yggD, hybD, ttdB, yqjI, bcp, gadA, pepQ, yihE, yjcF, phnI, yhbJ, nikC, hemD, ilvE, pyrB, ulaE, yifL, hlyE, yqiG, ptsA, ygcU, ccmH, ampC or ydbJ.

[0006] Preferably, the target points include metN, prpE, fruK, nikC, mazG and ybiT.

[0007] A recombinant plasmid for expressing the above-mentioned plasmid for encoding Escherichia coli alkali-resistant target points.

[0008] A genetically engineered bacterium, taking Escherichia coli as a starting strain, and expressing a strain containing the above-mentioned alkali-resistant target points.

[0009] Preferably, the alkali-resistant pH of the genetically engineered bacterium is 10.

[0010] The above-mentioned Escherichia coli alkali-resistant target points or genetically engineered bacterium in the application of Escherichia coli fermentation to produce diamines.

[0011] Beneficial effects: Compared with the prior art, the Escherichia coli alkali-resistant target points and the application thereof have the following advantages: 1. Novel target points and strong systematicity: The present application first systematically mines 43 new gene target points related to Escherichia coli alkali tolerance through high-throughput screening of whole genome overexpression library, which covers multiple key physiological pathways such as biofilm formation, pH homeostasis maintenance, oxidative stress response and energy metabolism. It breaks through the limitation of only focusing on a few known ion transport proteins in the past, and provides a rich gene resource library.

[0012] 2. Significant improvement in tolerance: Through multiple combinations, it is proved that under the extreme alkali stress of pH=10, overexpression of the gene target points of the present application can make the growth of Escherichia coli up-regulated by more than 3 times, especially the combination of metN, prpE, fruK, nikC, mazG and ybiT target points, which has significant and repeatable effect.

[0013] 3. Possesses synergistic combination potential: The gene target library provided by this invention lays a solid foundation for the subsequent construction of super engineered bacteria with synergistic effects and better tolerance through multi-gene combination regulation. Applying it to engineered bacteria producing diamines can effectively alleviate product inhibition, providing a reliable technical solution for improving production intensity and final yield. Attached Figure Description

[0014] Figure 1 For different pH pairs E. coli The impact on AG1 growth; Figure 2 These are the initial screening results; Figure 3 This is the result of the second screening; Figure 4 GO enrichment analysis for 43 different targets; Figure 5 KEGG enrichment analysis for 43 different targets; Figure 6 Intracellular ATP levels were measured in strains expressing different target sites: metN, prpE, fruK, nikC, mazG, and ybiT. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of protection of the present invention. Unless otherwise specified, the reagents, equipment, and methods used in the present invention are commercially available reagents, equipment, and methods conventionally used in this technical field.

[0016] Example 1: Construction of an Alkali Tolerance Inhibition Model Escherichia coli AG1 (ME5305) (purchased from https: / / shigen.nig.ac.jp / ecoli / strain / resource / askaClone / about) was inoculated into LB broth containing 50 µg / mL chloramphenicol and incubated overnight at 37°C and 200 rpm. The bacterial cells were collected by centrifugation, resuspended in sterile water, and the initial OD was adjusted. 600 The culture medium was inoculated into 96-well plates containing 50 µg / mL chloramphenicol and 0.1 mM IPTG at different pH values ​​(7.0 to 10.0, gradient 0.5). The plates were incubated at 37°C and 800 rpm, and the OD was monitored using a microplate reader. 600 .

[0017] The results are as follows Figure 1 As shown, bacterial growth was significantly inhibited when pH ≥ 9.5, with the most severe inhibition at pH = 10. Therefore, pH = 10 was selected as the stress condition for subsequent screening.

[0018] Example 2: High-throughput screening of ASKA libraries and acquisition of alkali tolerance targets 1. Initial screening A total of 4123 ASKA library strains (all single-gene overexpression strains, purchased from https: / / shigen.nig.ac.jp / ecoli / strain / resource / askaClone / about) stored at -80℃ were thawed in 96-well plates. 10 μL of each strain was transferred to a new 96-well plate containing 90 μL of LB medium (containing 50 µg / mL chloramphenicol) and cultured overnight at 37℃ and 800 rpm as the seed culture. Another 10 μL of the seed culture was transferred to a 96-well plate containing 140 μL of selection medium (LB, pH=10, containing 50 µg / mL chloramphenicol and 0.1 mM IPTG) and cultured for 12 h under the same conditions. OD600 was then measured. Using the AG1 strain carrying the empty vector pCA24N as a control, the upregulation rate of each strain was calculated (OD600 of the experimental group). 600 - Control group OD 600 ) / Control group OD 600 Clones with upregulation rates > 3 were selected, resulting in a total of 214 potential targets. Figure 2 ).

[0019] 2. Secondary screening The 214 strains obtained from the initial screening were subjected to three rounds of replicate experiments, with inoculation and culture conditions identical to the initial screening, and three biological replicates for each strain. Ultimately, 43 gene targets that showed stable performance and significantly superior results compared to the control were selected in the replicate experiments. Figure 3 It should be noted that the sequence information for these 43 targets can be found at https: / / shigen.nig.ac.jp / ecoli / pec_w3110 / .

[0020] Example 3: Functional Verification and Mechanism Analysis of Some Alkali Tolerance Targets 1. Enrichment Analysis: A list of 43 target genes was submitted to the DAVID database for GO and KEGG enrichment analysis. GO analysis results showed ( Figure 4 These genes were significantly enriched in functional categories such as "outer cell membrane," "dipeptidase activity," "protein binding," "ATP binding," and "oxidoreductase activity." KEGG analysis results showed ( Figure 5), which are significantly enriched in the pathways of "metabolic pathways", "pyrimidine metabolism", "cofactor biosynthesis", "ABC transporters", etc. This indicates that the alkali tolerance mechanism is a complex multi-factor synergistic process. In the growth and metabolic activities of cells, ATP is mainly involved in the transport and metabolism of intracellular substances. Both GO and KEGG enrichment analysis show ATP-related genes, so ATP-related genes (metN, prpE, fruK, nikC, mazG, ybiT) are selected for further functional verification.

[0021] 2. Intracellular ATP level determination: select overexpression strains of some target points metN, prpE, fruK, nikC, mazG, ybiT Take 5 μL of glycerol-preserved bacterial liquid and inoculate in each well of a 96-well plate containing 145 μL of LB medium, and place it in a microplate shaker for overnight culture to prepare the seed liquid. Take 100 uL and inoculate in 5 mL of screening medium, and measure the OD 600 after 4 h of culture. Take a certain amount of bacterial liquid into a centrifuge tube, so that the total OD 600 of the bacterial liquid in the centrifuge tube is 3. Centrifuge at 4°C, 12000 rpm for 2 min, collect the bacterial cells, and then determine the intracellular ATP level. The intracellular ATP level is determined using the ATP detection kit from Biyun Tian.

[0022] The results are shown in Figure 6 Compared with the control, the intracellular ATP level of these strains is significantly reduced, indicating that they function by hydrolyzing ATP (such as substance transport, signal transduction, etc.), thereby activating the alkali tolerance mechanism of the strains.

Claims

1. An Escherichia coli alkali-tolerance target characterized in that, The E. coli alkali-resistant target points include a plurality of combinations of metN, yagZ, gmhB, prpE, ybcH, rna, ybiT, narH, abgB, wza, ydgH, yodC, yfjW, fruK, nrdB, csdA, ubiH, ygbT, mazG, yggD, hybD, ttdB, yqjI, bcp, gadA, pepQ, yihE, yjcF, phnI, yhbJ, nikC, hemD, ilvE, pyrB, ulaE, yifL, hlyE, yqiG, ptsA, ygcU, ccmH, ampC or ydbJ.

2. The E. coli alkali-tolerance target of claim 1, wherein, The target points include metN, prpE, fruK, nikC, mazG and ybiT.

3. A recombinant plasmid, characterized in that, A plasmid expressing an E. coli alkali-resistant target point as claimed in claim 1.

4. A genetically engineered bacterium, taking E. coli as a starting strain, and expressing a strain containing the recombinant plasmid as claimed in claim 3. 5.The genetically engineered bacterium of claim 4, characterized in that, The genetically engineered bacterium has an alkali-resistant pH of 10.

6. Use of the E. coli alkali-resistant target point as claimed in claim 1 or the genetically engineered bacterium as claimed in claim 4 in E. coli fermentation to produce a diamine.

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