Patent Application
20230279456
The instant application contains a Sequence Listing in XML format as a file named “3050-YGHY-2022-64.xml”, created on Nov. 11, 2022, last modified on May 24, 2023, of 36.2 kB in size, and which is hereby incorporated by reference in its entirety.
The disclosure relates to genetically engineered bacteria producing lacto-N-neotetraose and a production method thereof, and belongs to the technical fields of metabolic engineering and food fermentation.
The content of human milk oligosaccharides (HMOs) is the third largest in solid components of human milk after lactose and fat, and is 12-13 g/L in mature milk and 22-23 g/L in colostrum, to which cow milk is incomparable (the content of cow milk oligosaccharides is less than 1 g/L). HMOs can tolerate hydrolysis of enzymes in the digestive tract of infants, and then resist infection of pathogenic microorganisms in the gastrointestinal tract and maintain microecological balance of the gastrointestinal tract. Lacto-N-neotetraose, as an important oligosaccharide in HMOs, has biological functions such as enhancing human immunity, regulating intestinal flora, promoting cell maturation and accelerating wound healing. In view of the important biological functions and physiological activity, lacto-N-neotetraose has been allowed to be added into commercial infant formula. However, the amount of lacto-N-neotetraose separated and extracted from natural products is very small, far less than the need for research. Therefore, it is the best choice to obtain lacto-N-neotetraose by artificial synthesis.
At present, the reported synthesis methods of lacto-N-neotetraose mainly include chemical synthesis, enzymatic synthesis and fermentation synthesis. Chemical synthesis has the problems such as complex reaction steps and expensive raw materials, resulting in high production costs and being not conducive to large-scale industrial synthesis. In addition, some toxic reagents used in chemical synthesis make the product unsuitable for food additives. Enzymatic synthesis uses expensive nucleotide sugars as substrates and is not conducive to large-scale production of lacto-N-neotetraose. Biological synthesis may use cheap carbon and nitrogen sources and substrates to achieve the synthesis of lacto-N-neotetraose without affecting the environment. Therefore, preparation of lacto-N-neotetraose by biological synthesis has attracted more and more attention.
The disclosure provides genetically engineered bacteria producing lacto-N-neotetraose, wherein in the genetically engineered bacteria, β-galactosidase gene lacZ is knocked out, and β-1,3-acetylglucosamine transferase gene lgtA, β-1,4-galactosyltransferase gene lgtB, phosphoglucomutase gene pgm, UDP-glucose-4-epimerase gene galE, galactose-1-phosphate uridyltransferase gene galT, galactokinase gene galK and β-galactoside permease gene lacY are overexpressed.
In one embodiment, the β-1,3-acetylglucosamine transferase gene lgtA and β-1,4-galactosyltransferase gene lgtB are both from Neisseria meningitidis; the nucleotide sequence of β-1,3-acetylglucosamine transferase gene lgtA is shown in SEQ ID NO: 1; and the nucleotide sequence of β-1,4-galactosyltransferase gene lgtB is shown in SEQ ID NO: 2.
In one embodiment, the phosphoglucomutase gene pgm, UDP-glucose-4-epimerase gene galE, galactose-1-phosphate uridyltransferase gene galT, galactokinase gene galK, β-galactoside permease gene lacY and β-galactosidase gene lacZ are all from Escherichia coli K-12; the Gene ID of the phosphoglucomutase gene pgm is 945271 (the nucleotide sequence of which is shown in SEQ ID NO: 24); the Gene ID of the UDP-glucose-4-epimerase gene galE is 945354 (the nucleotide sequence of which is shown in SEQ ID NO: 25); the Gene ID of the galactose-1-phosphate uridyltransferase gene galT is 945357 (the nucleotide sequence of which is shown in SEQ ID NO: 26); the Gene ID of the galactokinase gene galK is 945358 (the nucleotide sequence of which is shown in SEQ ID NO: 27); the Gene ID of the β-galactoside permease gene lacY is 949083 (the nucleotide sequence of which is shown in SEQ ID NO: 28); and the Gene ID of the β-galactosidase gene lacZ is 945006 (the nucleotide sequence of which is shown in SEQ ID NO: 23).
In one embodiment, the genetically engineered bacteria express the gene lacY with pETDuet-1 plasmid, express the genes pgm, galE, galT and galK sequentially with pRSFDuet-1 plasmid, and express the genes lgtA and lgtB sequentially with pCDFDuet-1 plasmid.
The disclosure further provides a method for producing lacto-N-neotetraose, including the following steps:
In one embodiment, carbon source in the fermentation medium is one or more of glucose, galactose and glycerin.
Preferably, the carbon source is glycerin or a mixture of glycerin and galactose.
More preferably, the carbon source is 20 g/L glycerin, or a mixture of 10 g/L glycerin and 10 g/L galactose.
In one embodiment, the fermentation system further contains 13.5 g/L potassium dihydrogen phosphate, 4.0 g/L diammonium hydrogen phosphate, 1.7 g/L citric acid, 1.4 g/L magnesium sulfate heptahydrate and 10 ml/L trace metal elements, the trace metal elements including 10 g/L ferrous sulfate, 2.25 g/L zinc sulfate heptahydrate, 1.0 g/L anhydrous copper sulfate and 2.0 g/L calcium chloride dihydrate, and the pH of the fermentation system being 6.8.
In one embodiment, the seed medium in the method is an LB liquid medium, and the seeds are cultured at 35° C.-40° C. and 200 rpm-250 rpm for 10 h-14 h in a shake flask.
In one embodiment, the fermentation broth in the method is specifically prepared by inoculating the seed solution into a fermentation system, culturing the seed solution at 35° C.-40° C. and 200 rpm-250 rpm until the OD600 is 0.6-0.8, then adding IPTG with a final concentration of 0.4 mM and lactose with a final concentration of 8 g/L-10 g/L, and inducing culture at 22° C.-30° C. and 200 rpm-250 rpm for 42 h-48 h.
The disclosure further provides application of the genetically engineered bacteria in producing lacto-N-neotetraose.
Through exogenous expression of lgtA and lgtB, reasonable combination and regulation of overexpression of lacY, pgm, galE, galT and galK in a lacto-N-neotetraose synthesis pathway, knockout of lacZ expression in the lacto-N-neotetraose synthesis pathway of an E. coli host, and optimization of a carbon source of a fermentation medium, the disclosure achieves the objectives of regulating the carbon flux of a metabolic pathway and improving the yield of lacto-N-neotetraose. In a shake flask experiment, the yield of lacto-N-neotetraose produced by E. coli increased from 304 mg/L to 1031 mg/L, laying a foundation for industrial production of the lacto-N-neotetraose.
The specific implementation of the disclosure is further described in the following examples and figures. Plasmids, PCR reagents, restriction endonucleases, plasmid extraction kits, DNA gel extraction kits, and the like used in the following examples are commercial products, and are specifically operated according to the kit instructions. The embodiments of the disclosure are not limited thereto, and other unspecified experimental operations and process parameters are carried out according to conventional technology.
Plasmids and DNA products are sequenced by Talen Biotechnology (Shanghai) Co., Ltd.
E. coli competent cells are prepared using a kit of Shanghai Sangon Biotechnology Co., Ltd.
An LB liquid medium contains 10 g/L peptone, 5 g/L yeast extract, and 10 g/L sodium chloride.
An LB solid medium contains 10 g/L peptone, 5 g/L yeast extract powder, 10 g/L sodium chloride, and 15 g/L agar powder.
The lacto-N-neotetraose in the embodiments of the disclosure is measured by HPLC, specifically:
1 mL of fermentation broth is boiled at 100° C. for 10 min and centrifuged at 13400 rpm for 10 min, the supernatant is filtered with a 0.22 μM membrane, and the yield of lacto-N-neotetraose is detected by HPLC. HPLC detection uses a differential refraction detector, the chromatographic column is Rezex ROA organic acid (Phenomenex, USA), the column temperature is 50° C., the mobile phase is a 5 mM H2SO4 aqueous solution, the flow rate is 0.6 mL/min, and the injection volume is 10 μL.
LacZ was knocked out from E. coli BL21 (lacZ has a gene ID of 945006 and is shown in SEQ ID NO: 23) using a CRISPR-Cas9 gene knockout system by the following specific steps (the primer sequences involved are shown in Table 1):
Recombinant bacteria are constructed specifically by the following steps (the primer sequences involved are shown in Table 2):
After fermentation, the yields of lacto-N-neotetraose of different engineered strains were 304 mg/L, 508 mg/L, 595 mg/L, 654 mg/L, 749 mg/L and 837 mg/L respectively. Compared with A0, the yield of lacto-N-neotetraose of strain A5 increased by 175.3%. Therefore, the expression of the endogenous genes pgm and galE-galT-galK of E. coli related to UDP-galactose synthesis can increase the yield of lacto-N-neotetraose. The metabolic pathway of lacto-N-neotetraose of the strain A5 is shown in
The fermentation method was as follows: the constructed genetically engineered strains A0-A5 were inoculated into LB liquid media, and the strains were cultured at 37° C. and 200 rpm for 12 h in a shake flask to obtain seed solutions; the seed solutions were inoculated into 50 ml of fermentation media at an inoculation volume of 2 mL/100 mL, and the seed solutions were cultured at 37° C. and 200 rpm in a shake flask until the OD600 was 0.6; and IPTG with a final concentration of 0.4 mM was added, lactose was added until the lactose concentration was 10 g/L, and culture was induced at 25° C. and 200 rpm for 48 h to obtain the fermentation broths.
The above fermentation medium contains 20 g/L glycerin, 13.5 g/L potassium dihydrogen phosphate, 4.0 g/L diammonium hydrogen phosphate, 1.7 g/L citric acid, 1.4 g/L magnesium sulfate heptahydrate and 10 ml/L trace metal elements, the trace metal elements including 10 g/L ferrous sulfate, 2.25 g/L zinc sulfate heptahydrate, 1.0 g/L anhydrous copper sulfate and 2.0 g/L calcium chloride dihydrate, and the pH of the fermentation medium being 6.8.
E. coli BL21 (DE3) with lacZ gene knocked out,
E. coli BL21 (DE3) with lacZ gene knocked out,
E. coli BL21 (DE3) with lacZ gene knocked out,
E. coli BL21 (DE3) with lacZ gene knocked out,
E. coli BL21 (DE3) with lacZ gene knocked out,
E. coli BL21 (DE3) with lacZ gene knocked out,
The engineered strain A5 with high lacto-N-neotetraose yield obtained in Example 2 was fermented in fermentation media with different combinations of carbon sources by the following specific fermentation method:
The constructed genetically engineered strain A5 was inoculated into an LB liquid medium, and the strain was cultured at 37° C. and 200 rpm for 12 h in a shake flask to obtain a seed solution; the seed solution were inoculated into 50 ml of fermentation media at an inoculation volume of 2 mL/100 mL, and the seed solution was cultured at 37° C. and 200 rpm in a shake flask until the OD600 was 0.6; and IPTG with a final concentration of 0.4 mM was added, lactose was added until the lactose concentration was 10 g/L, and culture was induced at 25° C. and 200 rpm for 48 h to obtain the fermentation broths.
The above fermentation media contain carbon sources (see Table 4 for combinations and contents of the carbon sources), 13.5 g/L potassium dihydrogen phosphate, 4.0 g/L diammonium hydrogen phosphate, 1.7 g/L citric acid, 1.4 g/L magnesium sulfate heptahydrate and 10 ml/L trace metal elements, the trace metal elements including 10 g/L ferrous sulfate, 2.25 g/L zinc sulfate heptahydrate, 1.0 g/L anhydrous copper sulfate and 2.0 g/L calcium chloride dihydrate, and the pH of the fermentation media being 6.8.
Table 4 shows the results of the yields of lacto-N-neotetraose of the strain A5 in the fermentation media of different combinations of carbon sources. From the table, when the combination of carbon sources in the medium was 10 g/L galactose+10 g/L glycerin, the yield of lacto-N-neotetraose was increased by 23.2% compared to that fermented by using 20 g/L glycerin as the carbon source at the very start.
Although the disclosure has been disclosed as above in preferred embodiments, it is not intended to limit the disclosure. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the disclosure. Therefore, the protection scope of the disclosure should be defined by the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021104470824 | Apr 2021 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/087318 | Apr 2022 | US |
| Child | 18061585 | US |
