Construction method and application of an Escherichia coli engineered strain with high production of lactoyl-N-neotetraose

Abstract

The present invention belongs to the field of microbial genetic engineering technology, and discloses a construction method and application of an Escherichia coli engineered strain with high lactoyl-N-neotetraose production. The Escherichia coli engineered strain with high lactoyl-N-neotetraose production of the present invention overexpresses recombinant genes, including β-1,3-N-acetylglucosamine aminotransferase gene lgtA, UDP glucose 4 epimerase gene galE, and β 1,4 galactosyltransferase gene, thereby increasing LNnT production by means of plasmid combination optimization, protein tag co-expression, related gene knockout, and genome integration. The present invention selects an Escherichia coli engineered strain to increase the production of LNnT in shake flask fermentation from 0.67 g / L to 2.66 g / L, and the highest LNnT production in 5L tank fermentation reaches 17.26 g / L.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial genetic engineering, and in particular to a construction method and application of an Escherichia coli engineering strain capable of producing high amounts of lactoyl-N-neotetraose. Background Art

[0002] Lacto-N-neotetraose (LNnT), a key component of human milk oligosaccharides, has important biological functions, including enhancing immunity, regulating intestinal flora, and promoting cell maturation. It can be added to infant formula as a nutritional supplement. Currently, the commercial production of LNnT primarily involves chemical synthesis and biosynthesis. Biosynthesis, however, requires only inexpensive carbon sources and renewable intracellular donors, and offers high economic returns at a lower environmental cost, offering broader application prospects.

[0003] Prior art often uses LNnT synthesis precursors within microorganisms, including the intracellular nucleotides UDP-GlcNAc and UDP-Gal, as well as exogenously added lactose. The synthesis process involves extracellular lactose being transported into the cell by the β-galactoside permease LacY. It then reacts with intracellular UDP-GlcNAc to form LNTII under the catalysis of the heterologously expressed β-1,3-N-acetylglucosaminyltransferase LgtA. LNTII and UDP-Gal are then catalyzed by the heterologously expressed β-1,4-galactosyltransferase LgtB to produce LNnT. In 2002, Bernard Priem et al. first synthesized LNnT and fucosylated LNnT in a recombinant strain of Escherichia coli JM109. This recombinant strain, with the lacZ gene knocked out and the lgtA gene overexpressed, produced approximately 6 g / L of LNTII in a 2-L tank using glycerol as the carbon source. Continuing on this basis, the lgtB gene was overexpressed, and glucose was used as the carbon source. A mixture of LNnT and lactose-N-neohexose (LNnH) exceeding 5 g / L was obtained through high cell density culture. The strain JM109 (pCWlgtA, pBBlgtB) cannot grow on glycerol medium, which may be due to toxicity problems caused by overexpression of the two lgtAB genes, and was overcome by culturing cells with glucose and lowering the culture temperature to 28°C. In 2019, Dong Xiaomin et al. used Bacillus subtilis 168 strain as the initial host and constructed a de novo synthesis pathway for LNnT by co-expressing LacY, LgtA and LgtB. Through various metabolic adjustments, the final strain achieved LNnT production of 2.30 g / L and 5.41 g / L in shake flasks and 3L bioreactors, respectively. In 2022, Zhu Yingying et al. identified a highly efficient β-1,4-galactosyltransferase and, by enhancing the supply of the precursor UDP-galactose, achieved LNnT production of 0.91 g / L and 12.14 g / L in shake flasks and 3L bioreactors, respectively. However, the current yield of LNnT produced by microbial synthesis remains low, unable to meet the needs of large-scale industrial production. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for constructing an engineered Escherichia coli strain that produces high levels of lactoyl-N-neotetraose and its application.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides an engineered Escherichia coli strain with high production of lactoyl-N-neotetraose, wherein the β-galactosidase gene lacZ, the phosphate undecyl glucose phosphotransferase gene wcaj, and the gdp-mannose mannose hydrolase gene nudD are knocked out of the Escherichia coli engineered strain; the recombinant gene includes the β-1,3-N-acetylglucosamine aminotransferase gene lgtA, the UDP glucose 4-dimerase gene galE, and the β1,4-galactosyltransferase gene; the β1,4-galactosyltransferase gene is any one of lgtB, lgtB-2, hpgalT-1, hpgalT-2, lex-1, lex-2, lex-3, lex-4, and lex-5.

[0007] The present invention screened β1,4 galactosyltransferases from different sources for synthesizing LNnT, and successfully screened a β1,4 galactosyltransferase from Helicobacter pylori that can efficiently synthesize LNnT from LNTⅡ and UDP-galactose, significantly improving the yield of LNnT.

[0008] As a preferred embodiment of the high-yield lactoyl-N-neotetraose Escherichia coli engineered strain of the present invention, the expression vector of the recombinant gene adopts at least one of pBluescript II SK(+), pCDFDuet-1, pETDuet-1, and pRSFDuet-1.

[0009] The present invention further uses vectors with different copy numbers, pBluescript II SK(+), pCDFDuet-1, pETDuet-1, and pRSFDuet-1, to optimize the plasmid combination of β-1,3-N-acetylglucosamine aminotransferase LgtA, UDP-glucose 4-dimerase GalE, and β 1,4-galactosyltransferase Hp-GalT, to obtain a combination that further improves the yield of LNnT.

[0010] Preferably, the expression vector of the β-1,3-N-acetylglucosamine aminotransferase gene lgtA and the UDP-glucose 4-dimerase gene galE is pRSFDuet-1.

[0011] Preferably, the expression vector of the β1,4 galactosyltransferase gene hpgalT-1 is pETDuet-1.

[0012] As a preferred embodiment of the high-yield lactoyl-N-neotetraose Escherichia coli engineered strain of the present invention, a fusion tag is inserted into the N-terminus of the β 1,4 galactosyltransferase gene hpgalT-1; the fusion tag is at least one of MBP, SUMO, TrxA, Fh8, and hisProp.

[0013] The present invention further selects five fusion tags MBP, SUMO, TrxA, Fh8, and hisProp to co-express β-1,4-galactosyltransferase Hp-GalT, among which the β-1,4-galactosyltransferase Hp-GalT connected with Fh8 can better improve the yield of LNnT.

[0014] As a preferred embodiment of the high-yield lactoyl-N-neotetraose Escherichia coli engineered strain described in the present invention, the glucose-1-phosphatase gene agp, the trehalose-6-phosphate synthase gene otsA, the UDP glucose 6-dehydrogenase gene ugd, the biofilm PGA synthase gene pgaCD, the glucosamine-6-phosphate deaminase gene nagB, the UDP-N-acetylglucosamine undecylphosphate N-acetylglucosamine phosphotransferase gene wecA, the UDP-N-acetylglucosamine 2-dimerase gene wecB, the mannose-6-phosphate isomerase gene manA, and the NADP+-dependent glucose-6-phosphate dehydrogenase gene zwf are also knocked out.

[0015] The present invention further knocked out glucose-1-phosphatase Agp, trehalose-6-phosphate synthase OtsA, UDP glucose 6-dehydrogenase Ugd, biofilm PGA synthase PgaCD, glucosamine-6-phosphate deaminase NagB, UDP-N-acetylglucosamine undecylphosphate N-acetylglucosamine phosphotransferase WecA, UDP-N-acetylglucosamine 2-dimerase WecB, mannose-6-phosphate isomerase ManA, and NADP+-dependent glucose-6-phosphate dehydrogenase Zwf in the genome to further enhance LNnT synthesis.

[0016] As a preferred embodiment of the Escherichia coli engineered strain with high lactoyl-N-neotetraose production of the present invention, the genome of the Escherichia coli engineered strain has the galETKM gene cluster integrated into the galM site and the lacZΔm15 gene integrated into the knocked-out lacZ site.

[0017] The present invention further inserts UDP glucose 4-epimerase GalE, galactose-1-phosphate uridyltransferase GalT, galactokinase GalK and galactose-1-epimerase GalM into the Escherichia coli genome and complements the defective β-galactosidase LacZΔm15, thereby increasing the accumulation of UDP-galactose and thus the production of LNnT.

[0018] In a second aspect, the present invention provides a method for constructing an engineered Escherichia coli strain that produces high levels of lactoyl-N-neotetraose, comprising the following steps:

[0019] (1) constructing a recombinant expression vector of the recombinant gene;

[0020] (2) knocking out the β-galactosidase gene lacZ, the phosphate undecyl glucose phosphotransferase gene wcaj, and the gdp-mannose mannose hydrolase gene nudD using the CRISPER method to obtain gene-knockout Escherichia coli;

[0021] (3) Transfecting the recombinant expression vector into the gene-knockout Escherichia coli and fermenting the recombinant expression vector to obtain the engineered Escherichia coli strain.

[0022] In a third aspect, the present invention uses the engineered Escherichia coli strain in the production of lactoyl-N-neotetraose.

[0023] As a preferred embodiment of the application of the present invention, the production includes fermentation culture in a fermenter or shake flask.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This study has developed an engineered strain using a highly efficient β1,4-galactosyltransferase from Helicobacter pylori to enhance LNnT synthesis. LNnT production is further enhanced through plasmid combination optimization and protein tag co-expression. Furthermore, UDP-galactose synthesis is enhanced by knockout of some related genes and genomic integration, thereby increasing LNnT production. The engineered strain achieved LNnT production in shake flasks from 0.67 g / L to 2.66 g / L, with a peak yield of 17.26 g / L in 5-L fermentation tanks. This study demonstrates significant results, breaking the bottleneck in LNnT synthesis and possessing high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure shows the production statistics of LNnT and LNTII of engineered Escherichia coli containing different β1,4 galactosyltransferases;

[0027] Figure 2The figure shows the statistical graph of the production of LNnT and LNTII of engineered Escherichia coli containing different combinations of plasmids;

[0028] Figure 3 The figure shows the production statistics of LNnT and LNTII of engineered Escherichia coli strains containing different protein tags;

[0029] Figure 4 Statistical graph of the production of LNnT and LNTII in Escherichia coli edited with different genes. DETAILED DESCRIPTION

[0030] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The yields of LNnT and LNTⅡ were determined by high performance liquid chromatography in the examples.

[0032] The culture medium for shake flask fermentation in the embodiment is: 30g / L glycerol, 12g / L glucose, 2g / L yeast powder, 10g / L tryptone, 14.3g / L Na2HPO4·12H2O, 3.1g / L KH2PO4, 2.0g / L NH4Cl, 1.0g / L (NH4)2HPO4, 1.7g / L citric acid, 15mg / L CaCl2 and 2.2g / L C6H5Na·2H2O, 2g / L MgSO4·7H2O, 10mg / L vitamin B1 and 10mL / L trace elements (25g / L FeCl3·6H2O, 2g / L CaCl2·2H2O, 2.0g / L ZnCl2, 1.9g / L CuSO4·5H2O, 0.42g / L MnCl·H2O, 2g / L Na2B4O7·10H2O and 2g / L Na2MoO4·2H2O) were cultured at 37°C and induced at 28°C with 0.2mM IPTG. The 5L jar culture medium was the same as above.

[0033] The plasmid cloning primer pairs in the examples are shown in Table 1.

[0034] Table 1 Plasmid cloning primer pairs

[0035]

[0036]

[0037] The gene editing primer pairs in the examples are shown in Table 2.

[0038] Table 2 Gene editing primer pairs

[0039]

[0040]

[0041] Example 1: Construction of LNnT biosynthetic Escherichia coli strain

[0042] (1) The β-1,3-N-acetylglucosamine aminotransferase LgtA (Genbank: NP_274923) from Neisseria meningitidis MC58 and the UDP-glucose diastereomerase GalE (Genbank: NP_415280.3) from Escherichia coli K12 MG1655 were cloned into pCDFDuet-1 and named pCDF-lgtA-galE for the synthesis of LNTⅡ. The β-1,4-galactosyltransferase lgtB (Genbank: CAX50884.1) from Neisseria meningitidis MC58 was cloned into pBluescript II SK(+) and named pBS-lgtB for the synthesis of LNnT.

[0043] (2) The β-galactosidase gene lacZ, the undecyl-glucose phosphate phosphotransferase gene wcaj, and the gdp-mannose mannose hydrolase gene nudD of Escherichia coli BL21(DE3) were knocked out to obtain the strain BL21(DE3)ΔLWD (BL21(DE3)ΔlacZΔwcajΔnudD).

[0044] (3) The plasmids pCDF-lgtA-galE and pBS-lgtB were transformed into the laboratory-constructed strain BL21(DE3)ΔLWD (BL21(DE3)ΔlacZΔwcajΔnudD) and named L1006.

[0045] The strain L1006 was subjected to shake flask fermentation experiments, and the strain produced 0.67 g / L LNnT and 3.3 g / L LNTⅡ in 48 h.

[0046] Example 2: Screening for efficient β1,4 galactosyltransferase to synthesize LNnT

[0047] (1) β1,4 galactosyltransferases from different sources were selected and their gene sequences were obtained from NCBI, including lgtB-2 from Neisseria meningitidis 8013 (Genbank: CAX50884.1), hpgalT-1 from Helicobacter pylori (Genbank: BAA88524.1), hpgalT-2 from Helicobacter pylori 26695 (Genbank: AAD07876.1), lex-1 from Aggregatibacter aphrophilus W10433 (Genbank: AKU62975.1), lex-2 from Aggregatibacter actinomycetemcomitans NUM4039 (Genbank: BAS48030.1), and lex-3 from Aggregatibacter aphrophilus W10433 (Genbank: AKU62975.1). lex-3 of W10433 (Genbank: AKU62973.1), lex-4 of Aggregatibacter aphrophilus ATCC 19415 (Genbank: OBY55126.1), and lex-5 of Aggregatibacter aphrophilus NJ8700 (Genbank: ACS98560.1) were synthesized by the company after codon optimization. Primers were designed to clone the above genes into pBluescript II SK(+) and named them pBS-lgtB-2, pBS-hpgalT-1, pBS-hpgalT-2, pBS-lex-1, pBS-lex-2, pBS-lex-3, pBS-lex-4, and pBS-lex-5, respectively.

[0048] (2) The plasmid pCDF-lgtA-galE prepared in Example 1 and the above-mentioned plasmids pBS-lgtB-2, pBS-hpgalT-1, pBS-hpgalT-2, pBS-lex-1, pBS-lex-2, pBS-lex-3, pBS-lex-4, and pBS-lex-5 were respectively transformed into the strain BL21(DE3)ΔLWD and named L1007, L1008, L1009, L1010, L1011, L1012, L1013, and L1014, respectively.

[0049] The strains L1006, L1007, L1008, L1009, L1010, L1011, L1012, L1013 and L1014 were fermented in shake flasks for 48 h (see Figure 1), L1008 had the highest yield of 0.96 g / L LNnT and 1.92 g / L LNTⅡ. It was determined that hpgalT-1 from Helicobacter pylori could produce LNnT more efficiently.

[0050] Example 3: Plasmid combination optimization of LNnT synthesis

[0051] (1) Plasmid combination optimization of β-1,3-N-acetylglucosamine aminotransferase LgtA (Genbank: NP_274923), UDP-glucose 4-dimerase GalE (Genbank: NP_415280.3), and β-1,4-galactosyltransferase Hp-GalT-1 (Genbank: BAA88524.1) was performed using vectors pBluescript II SK(+), pCDFDuet-1, pETDuet-1, and pRSFDuet-1 with different copy numbers. LgtA and GalE were cloned into pCDFDuet-1, pETDuet-1, and pRSFDuet-1 to obtain plasmids pCDF-lgtA-galE, pET-lgtA-galE, and pRSF-lgtA-galE, respectively. Hp-GalT-1 was cloned into SK(+), pETDuet-1, and pRSFDuet-1 to obtain plasmids pBS-hpgalT1, pET-hpgalT1, and pRSF-hpgalT1, respectively.

[0052] (2) The plasmid combinations pCDF-lgtA-galE+pET-hpgalT1, pCDF-lgtA-galE+pRSF-hpgalT1, pET-lgtA-galE+pRSF-hpgalT1, pRSF-lgtA-galE+pBS-hpgalT1, and pRSF-lgtA-galE+pET-hpgalT1 were transformed into the strain BL21(DE3)ΔLWD and named L1015, L1016, L1017, L1018, and L1019, respectively.

[0053] The strains L1008, L1015, L1016, L1017, L1018 and L1019 were fermented in shake flasks for 48 h (see Figure 2 ), with L1019 producing the highest yields of 1.51 g / L LNnT and 0.49 g / L LNTⅡ. The plasmid combination pRSF-lgtA-galE+pET-hpgalT1 was determined to be more efficient in producing LNnT.

[0054] Example 4: Screening for efficient β 1,4 galactosyltransferase synthesis of LNnT

[0055] (1) The fusion tags MBP, SUMO, TrxA, Fh8, and hisProp were cloned into the N-terminus of the hpgalT1 gene in the pET-hpgalT1 vector prepared in Example 3 for expression, respectively, to obtain plasmids pET-MBP-hpgalT1, pET-SUMO-hpgalT1, pET-TrxA-hpgalT1, pET-Fh8-hpgalT1, and pET-hisProp-hpgalT1, respectively.

[0056] (2) The plasmid pRSF-lgtA-galE prepared in Example 3 and the above plasmids were transformed into the strain BL21(DE3)ΔLWD and named L1020, L1021, L1022, L1023, and L1024, respectively.

[0057] The strains L1019, L1020, L1021, L1022, L1023 and L1024 were fermented in shake flasks for 48 h (see Figure 3 ), with L1023 producing the highest yields of 1.97 g / L LNnT and 1.09 g / L LNTⅡ. The Fh8 fusion tag was determined to enhance the conversion of LNTⅡ to LNnT by the β1,4-galactosyltransferase Hp-GalT-1, resulting in higher LNnT yields.

[0058] Example 5: Construction of gene knockout strain and its production of LNnT

[0059] (1) The CRISPER method was used to knock out the target genes. Primers were designed to knock out the glucose-1-phosphatase gene agp, trehalose-6-phosphate synthase gene otsA, UDP glucose 6-dehydrogenase gene ugd, biofilm PGA synthase gene pgaCD, glucosamine-6-phosphate deaminase gene nagB, UDP-N-acetylglucosamine undecylphosphate N-acetylglucosamine phosphotransferase gene wecA, UDP-N-acetylglucosamine 2-dimerase gene wecB, mannose-6-phosphate isomerase gene manA, and NADP+-dependent glucose-6-phosphate dehydrogenase gene zwf. The strain BL21(DE3)ΔLWDΔagpΔotsAΔugdΔpgaCDΔnagBΔwecAΔwecBΔmanAΔzwf was obtained and named BL21(DE3)ΔLWD10.

[0060] (2) The fermentation plasmid pRSF-lgtA-galE prepared in Example 3 and pET-Fh8-hpgalT1 prepared in Example 4 were transformed into BL21(DE3)ΔLWD10 to obtain strain L10025.

[0061] Strain L1025 was fermented in shake flasks for 48 h (see Figure 4 ), L1025 had a yield of 2.3 g / L LNnT and 2.32 g / L LNTⅡ, which was 16.8% higher than that of the original strain L1023.

[0062] Example 6: Construction of gene-integrated strain and its production of LNnT

[0063] (1) The CRISPER-transposition method was used to integrate the gene into the genome. The galETKM gene cluster (obtained from Escherichia coli K12 MG1655) was integrated into the galM site of the genome; the lacZΔm15 gene (obtained from Escherichia coli K12 MG1655) was integrated into the knocked-out lacZ site, and the primers were designed and sent to the company for synthesis.

[0064] (2) After the galETKM gene was transposed into BL21(DE3)ΔLWD10, it was named BL21(DE3)ΔLWD11. After the lacZΔm15 gene was transposed into BL21(DE3)ΔLWD11, it was named BL21(DE3)ΔLWD12.

[0065] (3) The fermentation plasmid pRSF-lgtA-galE prepared in Example 3 and pET-Fh8-hpgalT1 prepared in Example 4 were transformed into BL21(DE3)ΔLWD12 to obtain strain L1226.

[0066] The strain L1226 was fermented in shake flasks for 48 h (see Figure 4 ), L1226 had a yield of 2.66 g / L LNnT and 1.69 g / L LNTⅡ, which was 15.7% higher than that of the original strain L1025.

[0067] L1226 was fermented in a 5 L tank, and the final yield obtained was 17.26 g / L LNnT and 20.19 g / L LNTⅡ.

[0068] This study screened a highly efficient β-1,4-galactosyltransferase from Helicobacter pylori that can enhance LNnT synthesis. LNnT production was further enhanced through plasmid combination optimization and protein tag co-expression. Furthermore, UDP-galactose synthesis was enhanced by knocking out some related genes and integrating them into the genome, thereby increasing LNnT production. The engineered strain achieved a maximum LNnT yield of 2.66 g / L in shake flasks and 17.26 g / L in 5L tanks. This study demonstrates significant results, breaking the bottleneck in LNnT synthesis and possessing high application value.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An engineered Escherichia coli strain that produces high levels of lactoyl-N-neotetraose, characterized in that: The Escherichia coli engineered strain is depleted of the β-galactosidase gene lacZ , undecyl-glucose phosphate phosphotransferase gene wcaj and GDP-mannose mannose hydrolase genes nudD The Escherichia coli engineered strain overexpresses a recombinant gene, the recombinant gene including a β-1,3-N-acetylglucosamine aminotransferase gene lgtA , UDP-glucose 4-epimerase gene galE and β-1,4-galactosyltransferase gene; the β-1,4-galactosyltransferase gene is hpgalT-1 ; The β-1,3-N-acetylglucosamine aminotransferase gene lgtA The Genbank accession number is NP_274923; the UDP-glucose 4-dimerase gene galE The Genbank accession number is NP_415280.3; the β-1,4-galactosyltransferase gene is hpgalT-1 The Genbank accession number is BAA88524.1; The β-1,4-galactosyltransferase gene hpgalT-1 The expression vector is pETDuet-1; The β-1,4-galactosyltransferase gene hpgalT-1 A fusion tag Fh8 is inserted into the N-terminus; The β-1,3-N-acetylglucosamine aminotransferase gene lgtA and UDP-glucose 4-epimerase gene galE The expression vector is pRSFDuet-1; The E. coli engineered strain also knocks out the glucose-1-phosphatase gene agp , trehalose-6-phosphate synthase gene otsA 、UDP glucose 6-dehydrogenase gene ugd , biofilm PGA synthase gene pgaCD , glucosamine-6-phosphate deaminase gene nagB UDP-N-acetylglucosamine undecylphosphate N-acetylglucosamine phosphotransferase gene wecA 、UDP-N-acetylglucosamine 2-dimerase gene weCb , mannose-6-phosphate isomerase gene manA and NADP+-dependent glucose-6-phosphate dehydrogenase genes zwf .

2. The engineered Escherichia coli strain with high lactoyl-N-neotetraose production according to claim 1, characterized in that The genome of the Escherichia coli engineered strain has a galETKM gene cluster integrated into the galM site, and a lacZΔm15 gene integrated into the knocked-out lacZ site.

3. A method for constructing an engineered Escherichia coli strain that produces high levels of lactoyl-N-neotetraose according to claim 1 or 2, characterized in that: The following steps are involved: (1) constructing a recombinant expression vector of the recombinant gene; (2) Knockout of the β-galactosidase gene using the CRISPER method lacZ , undecyl-glucose phosphate phosphotransferase gene wcaj and GDP-mannose mannose hydrolase genes nudD , gene knockout Escherichia coli was obtained; (3) Transfecting the recombinant expression vector into the gene-knockout Escherichia coli to obtain the engineered Escherichia coli strain.

4. Use of the engineered Escherichia coli strain according to claim 1 or 2 in the production of lactoyl-N-neotetraose.

5. The use according to claim 4, characterized in that The production includes fermentation culture in fermenters or shake flasks.

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