Escherichia coli engineering bacterium for high yield of yellow cochineic acid and construction method of escherichia coli engineering bacterium

Abstract

The invention provides an escherichia coli engineering bacterium for high yield of yellow cochineic acid and a construction method of the escherichia coli engineering bacterium, and a modified chassis cell escherichia coli BAP1-pflB is obtained by performing gene knockout on a pflB gene in an escherichia coli thallus BAP1. And the constructed recombinant plasmid pET30a-antDEFBG + zhuIJ is electrically transformed into escherichia coli BAP1-pflB, so that the engineering bacterium BAP1-pflB-PFK capable of producing the yellow cochineic acid is obtained. Through HPLC (High Performance Liquid Chromatography) detection, after the gene pflB is knocked out, the yield of the yellow cochineic acid is obviously increased. Yellow carmine acid synthesized by the escherichia coli engineering bacteria is a high-grade precursor of carmine acid with high commercial value, and plays an important role in the fields of textiles, skin care products and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology and genetic engineering, and particularly relates to an Escherichia coli engineering bacterium with high yield of kakiimonin acid and a construction method thereof. BACKGROUND

[0002] Kakiimonin acid belongs to anthraquinone derivatives, has a complex ring structure of naphthopyrone and anthrone, contains multiple hydroxyl and carbonyl functional groups, is slightly soluble in water, is easily soluble in organic solvents such as methanol, ethanol and DMSO, has slightly increased solubility under acidic conditions, and is easily ionized under alkaline conditions, and can be applied in the fields of textiles and cosmetics. As a key intermediate for the synthesis of carminic acid, kakiimonin acid plays an important role in the biosynthesis pathway of carminic acid. From the biosynthesis mechanism, the formation of kakiimonin acid is related to multiple enzymes. Escherichia coli can generate kakiimonin acid as an intermediate product by expressing type II polyketide synthase (antDEFBG) from Gentiana triflora, cyclase zhuI and aromatic enzyme zhuJ from Streptomyces sp. R1128, using glucose as a substrate and acetyl coenzyme A and malonyl coenzyme A as precursors. Kakiimonin acid can generate carminic acid under the action of exogenous monooxygenase and C-glucosyltransferase. In Aspergillus nidulans, it is speculated that at least two endogenous enzymes are involved in the biosynthesis of carminic acid, one of which catalyzes the oxidation of kakiimonin acid anthrone to obtain kakiimonin acid. In addition, the endogenous hydroxylase (Cat5) of Saccharomyces cerevisiae can catalyze the hydroxylation of kakiimonin acid to obtain kakiimonin acid, which participates in the synthesis of carminic acid. The improvement of kakiimonin acid yield will also help to improve the yield of carminic acid. At present, the application research of kakiimonin acid is mostly concentrated in the field of participating in the synthesis of carminic acid as an intermediate, aiming to improve the yield of carminic acid by regulating the related biosynthesis pathway, and solve the problems of high cost and limited source in extracting carminic acid from insects. In the future, with the in-depth study of the biosynthesis mechanism, the application of kakiimonin acid in other fields may be expanded, such as the development of new biosynthesis process or the development of new functional compounds based on its structure.

[0003] As a carbon source, glucose is first metabolized by the glycolytic pathway (EMP pathway) to produce pyruvate. In E. coli, the main metabolic direction of pyruvate is to be oxidized and decarboxylated to acetyl-CoA by the pyruvate dehydrogenase complex (PDH), which is an important starting material for the tricarboxylic acid cycle. In addition, there is a branch metabolic pathway catalyzed by pyruvate formate lyase (pflB) that can convert a small amount of pyruvate to acetyl-CoA. Studies have shown that by knocking out the pflB gene through genetic engineering technology, the branch metabolism can be blocked, so that more metabolic flow is directed to the PDH pathway, thereby significantly increasing the accumulation level of intracellular acetyl-CoA, an important precursor substance. This metabolic engineering strategy can effectively promote the biosynthesis of downstream target products by increasing the supply of precursor substances, and ultimately achieve a significant increase in the yield of target products. SUMMARY

[0004] The technical problem to be solved: by gene editing technology to transform E. coli, so that it can produce synthetic xanthurenic acid. The present application specifically knocks out the pflB gene of E. coli BAP1 by Red homologous recombination system, effectively blocks the side branch metabolic pathway of E. coli from pyruvate to acetyl-CoA, thereby optimizing the metabolic flow of the strain, so that the engineering bacteria containing the enzyme gene for synthesizing xanthurenic acid can efficiently synthesize xanthurenic acid.

[0005] Technical scheme: The present application provides a kind of high yield xanthurenic acid E. coli engineering bacteria, the pflB gene of the E. coli engineering bacteria is knocked out in bacterial BAP1, and recombinant plasmid pET30a-antDEFBG+zhuIJ is introduced.

[0006] Further, the knockout of the pflB gene requires the use of three plasmids, namely pKD13 plasmid, pKD46 plasmid and pCP20 plasmid.

[0007] A method for constructing a high-yield xanthurenic acid E. coli engineering bacterium, comprising the following steps: S1. A primer is used to amplify a gene fragment containing kana resistance and FRT sites, which is a pflB knockout component; S2. The pKD46 plasmid is electroporated into E. coli, and then a host bacterium carrying pKD46 is used to prepare competent cells; S3. The pflB knockout component is purified and then electroporated into the competent cells carrying pKD46, and colony PCR is performed on single clone cells to verify and select positive transformants for overnight culture to induce the loss of pKD46 plasmid, thereby obtaining a strain lacking pKD46; S4. Transform pCP20 into the strain losing pKD46, shake culture the screened positive transformant, induce the expression of recombinase FLP, and the FRT site homologously recombines itself to eliminate one FRT site and the resistance gene, subculture the strain, and induce the loss of pCP20 plasmid; S5. Screen the strain by kanamycin and ampicillin to obtain the engineered host strain BAP1-△pflB with the resistance gene eliminated; S6. Transform the recombinant plasmid into the engineered host strain BAP1-△pflB to construct an engineered E. coli strain with high yield of xanthurenic acid.

[0008] Further, the primers in step S1 are a pair of primers homo-pflB-F2 and homo-pflB-R2 containing homologous arms, which are designed according to the pKD13 plasmid and the E. coli genomic sequence information, and the sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0009] Further, the temperature of the overnight culture in step S3 is 37℃; and the sequencing primers for PCR verification are seq-pflB-F and seq-pflB-R, and the sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0010] Further, the temperature of the shake culture in step S4 is 42℃, the rotation speed of the shake culture is 200 rpm, and the shake culture time is 8 h.

[0011] Further, the recombinant plasmid in step S6 is pET30a-antDEFBG+zhuIJ plasmid loaded with cyclase zhuI and aromatic enzyme zhuJ. Beneficial effects

[0012] The application provides an engineered E. coli strain constructed based on a gene editing technology, which is characterized in that the pflB gene of E. coli BAP1 is specifically knocked out by a Red homologous recombination system, the gene modification strategy effectively blocks the side branch metabolic pathway of pyruvic acid to acetyl coenzyme A of E. coli, thereby optimizing the metabolic flow of the strain, and the host strain of the engineered bacteria is a commonly used strain-E. coli BAP1 suitable for industrial fermentation.

[0013] The application utilizes the gene editing technology to transform the recombinant plasmid pET30a-antDEFBG+zhuIJ into the host strain with the pflB gene knocked out to construct an engineered bacteria capable of synthesizing xanthurenic acid, and the pflB gene is knocked out, which improves the metabolism and makes the xanthurenic acid more high-yield, and the yield is increased by 4.3 times compared with the wild type strain. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Agarose gel electrophoresis map of primers homo-PflB-F2 and homo-PflB-R2 for knockout assembly; Figure 2 Agarose gel electrophoresis map of PCR verification for pKD46 plasmid transformation; Figure 3 Agarose gel electrophoresis map of PCR verification for knockout assembly transformation; Figure 4 Growth of positive strain pflB knockout on solid screening medium; Figure 5 Agarose gel electrophoresis map of PCR verification for pET30a-antDEFBG+zhuIJ plasmid transformation; Figure 6 Standard curve of xanthomicrolactonic acid content. DETAILED DESCRIPTION

[0015] The application will be further described below in conjunction with the accompanying drawings and examples, which are illustrative of the application and the application is not limited to the following examples: Example 1

[0016] PCR amplification and purification of knockout assembly: A pair of primers homo-PflB-F2 and homo-PflB-R2 containing homologous arms were used to amplify the gene fragment containing kana resistance and FRT site from pKD13 plasmid, and the target fragment was 1424 bp. The amplified PCR product was gel purified, and the purified product was detected by agarose electrophoresis, and the fragment size was consistent with the theoretical value, as shown in Figure 1 . Example 2

[0017] Preparation of E. coli competent cells: Single colony of E. coli BAP1 was picked and cultured in LB medium at 37°C for 12 h, with a shaking speed of 200 rpm. 1 mL of the culture was added to 50 mL of LB medium, and the culture was continued until OD600 = 0.3-0.5. The bacterial cells were cooled on ice for 30 min, then transferred to a pre-cooled 50 mL sterile centrifuge tube at 4°C, and centrifuged at 5000 rpm for 15 min. The supernatant was discarded, and the bacterial cells were resuspended and washed twice with ice-cold sterile high-purity water. The supernatant was removed by centrifugation at 5000 rpm, and the bacterial cells were resuspended with 0.5 mL of pre-cooled sterile high-purity water. After aliquoting, the bacterial cells were ready for use. Example 3

[0018] Host bacteria competent cells carrying pKD46 plasmid: The 1 μL pKD46 plasmid was added to the E. coli BAP1 competent cells for electrotransformation to obtain E. coli BAP1-pKD46, and a single colony grown on an ampicillin LB plate was used for colony PCR verification. Agarose gel electrophoresis detection showed that the band size was consistent with the expected size, as shown in Figure 2 . Example 4

[0019] Knockout assembly transformation into E. coli carrying pKD46 plasmid and disappearance of pKD46: The purified knockout assembly was added to the competent cells of E. coli BAP1-pKD46 for electrotransformation, and positive clones were screened on a kanamycin-resistant LB plate at 37°C. Colony PCR verification was performed on a single colony grown on the plate to screen for positive transformants, as shown in Figure 3 . The positive transformants were cultured in kanamycin-containing LB liquid medium at 42°C and 200 rpm for 8 h, and then transferred to overnight culture at 37°C to remove the pKD46 plasmid. Example 5

[0020] Introduction and disappearance of pCP20 plasmid: The pCP20 plasmid was introduced into the E. coli BAP1 competent cells from which the pKD46 plasmid was removed. After positive transformants were grown, they were cultured at 42°C and 200 rpm for 8 h, and then transferred to overnight culture at 37°C. The culture was streaked on LB plates without antibiotics, ampicillin-resistant LB plates, and kanamycin-resistant LB plates, respectively. Only the strain that did not grow on the ampicillin-resistant and kanamycin-resistant LB plates was the positive strain in which pflB was knocked out, as shown in Figure 4 , to obtain a successfully modified chassis cell. Example 6

[0021] Recombinant plasmid pET30a-antDEFBG+zhuIJ transformation: The successfully modified chassis cell and wild-type E. coli BAP1 were prepared into competent cells to obtain the modified E. coli BAP1-△pflB competent cells and wild-type E. coli BAP1 competent cells. The pET30a-antDEFBG+zhuIJ plasmid was electrotransformed into the E. coli BAP1-△pflB and wild-type E. coli BAP1 hosts. The plasmid was added to 40 μL E. coli BAP1-△pflB competent cells and wild-type E. coli BAP1 competent cells, respectively, and placed in a 1 mm electroporation cup. The cells were kept still on ice for 5 min, and then electrotransformed under the conditions of 1.8 kV, 25 μF, and 200Ω. After transformation, the cells were plated on LB plates containing kana for screening, and positive strains were obtained by colony PCR verification, as shown in Figure 5), named as BAP1-△pflB-PFK and BAP1-PFK, respectively. Example 7

[0022] Shaking flask fermentation of E. coli engineering strain The obtained E. coli BAP1-△pflB-PFK and control strain wild-type E. coli BAP1-PFK were streaked on LB plates containing kana and placed in a 37 °C constant temperature incubator for overnight culture. Single colonies of the recombinant strain and the control strain were picked from the new activation plate and inoculated into a shaking flask containing fresh LB. After overnight culture at 37 °C and 200 rpm, the activated seed was obtained. The activated seed was inoculated into fresh R2 medium and cultured at 30 °C and 200 rpm until the OD600 reached 0.6-0.8. Then, IPTG with a final concentration of 0.5 mM was added, and glucose (20 g / L) and ascorbic acid (0.45 g / L) were added at the same time. The expression was induced and cultured at 30 °C and 200 rpm for 24 h. Index test

[0023] Production determination of canthaxanthin acid The fermentation broth sample was pretreated and extracted by solid phase extraction. The shaking flask culture of the strain was centrifuged at 4000 g for 30 min, and the supernatant was added to a SPE (HLB) column activated in advance with methanol. The supernatant was loaded, and after loading, 6 mL of 5% methanol water was used for cleaning, 12 mL of methanol was used for elution, nitrogen was used for drying, and an appropriate amount of 1% formic acid methanol was used for redissolution. The sample was concentrated by 50 times, filtered with a 0.22 μm nylon filter, and used.

[0024] The production of canthaxanthin acid was determined by HPLC. The chromatographic column was ZORBAX Eclipse Plus C18 column (4.6 x 150 mm, 5 μm), and the HPLC detection was performed at 40 °C. The flow rate was 1 mL / min, the injection volume was 10 μL, the mobile phase A was 0.1% formic acid aqueous solution, the mobile phase B was 0.1% formic acid acetonitrile, and the ultraviolet detection wavelength was 254 nm. The elution conditions were as follows: 0-20 min, 10%-100% mobile phase B; 20-25 min, 100% mobile phase B; 25-27 min, 100%-10% mobile phase B; 28-30 min, 10% mobile phase B.

[0025] The results are shown in Table 1. Canthaxanthin acid was detected in both E. coli engineering strain BAP1-△pflB-PFK and wild-type E. coli BAP1-PFK, with a yield of 0.8 mg / L and 0.15 mg / L, respectively. The yield of canthaxanthin acid in BAP1-△pflB-PFK was about 4.3 times higher than that in the wild-type control strain BAP1-PFK.

[0026] Table 1 HPLC detection of xanthoerythronate production

[0027] The above description is only the preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical solutions of the present application, still falls within the scope of protection of the technical solutions of the present application.

Claims

1. A high-yield engineered Escherichia coli strain producing flavonoid acid, characterized in that: The engineered Escherichia coli strain had the pflB gene knocked out in the BAP1 cell and the recombinant plasmid pET30a-antDEFBG+zhuIJ was introduced.

2. The engineered Escherichia coli strain with high production of flavonoid acid according to claim 1, characterized in that: The knockout of the pflB gene requires the use of three plasmids: pKD13, pKD46, and pCP20.

3. A method for constructing an engineered *Escherichia coli* strain that produces high levels of flavonoid acid, characterized in that... The steps are as follows: S1. A gene fragment containing kana resistance and FRT sites is amplified using primers, which is the pflB knockout component; S2. The pKD46 plasmid was electroporated into E. coli, and competent cells were prepared using host bacteria carrying pKD46. S3. After purifying the pflB knockout component, electroporate it into competent cells carrying pKD46. Single-clone cells are verified by colony PCR. Positive transformants are screened and cultured overnight to induce the loss of pKD46 plasmid, thus obtaining strains that have lost pKD46. S4. Transform pCP20 into a strain that has lost pKD46, screen for positive transformants, culture them in a shaker, and then passage them to obtain strains that have lost the pCP20 plasmid. S5. The engineered host strain BAP1-△pflB with eliminated resistance genes was obtained by screening strains with kanamycin and ampicillin; S6. Transform the recombinant plasmid into the engineered host bacterium BAP1-△pflB to construct an engineered Escherichia coli strain that produces high levels of flavonoids.

4. The method for constructing a high-yield flavonoid-producing engineered Escherichia coli strain according to claim 3, characterized in that: In step S1, the primers are a pair of primers, homo-pflB-F2 and homo-pflB-R2, designed based on the pKD13 plasmid and E. coli genome sequence information, containing homologous arms. The sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

5. The method for constructing an engineered Escherichia coli strain with high production of flavonoid acid according to claim 3, characterized in that: In step S3, the overnight culture temperature is 37°C; the sequencing primers used for PCR verification are seq-pflB-F and seq-pflB-R, with sequences shown in SEQ ID NO.3 and SEQ ID NO.

4.

6. The method for constructing a high-yield flavonoid-producing engineered Escherichia coli strain according to claim 3, characterized in that: In step S4, the temperature of the shaker culture is 42℃, the rotation speed of the shaker culture is 200 rpm, and the shaking time is 8 h.

7. The method for constructing a high-yield (high-flavonoid) engineered *Escherichia coli* strain according to claim 3, characterized in that: In step S6, the recombinant plasmid is the pET30a-antDEFBG+zhuIJ plasmid loaded with cyclase zhuI and aromatase zhuJ.

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