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WO2018171400A1 - Bactérie pour l'ingénierie d'acarbose, procédé de préparation et application associés - Google Patents

Bactérie pour l'ingénierie d'acarbose, procédé de préparation et application associés Download PDF

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WO2018171400A1
WO2018171400A1 PCT/CN2018/077732 CN2018077732W WO2018171400A1 WO 2018171400 A1 WO2018171400 A1 WO 2018171400A1 CN 2018077732 W CN2018077732 W CN 2018077732W WO 2018171400 A1 WO2018171400 A1 WO 2018171400A1
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seq
sequence
gene
acarbose
set forth
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Chinese (zh)
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黄隽
李美红
周军
林甲壇
余贞
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Zhejiang Hisun Pharmaceutical Co Ltd
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Zhejiang Hisun Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/365—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinoplanes (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

  • the invention belongs to the field of bioengineering and relates to an engineering bacteria for producing acarbose and a preparation method and application thereof.
  • the hypoglycemic agent acarbose produced by Actinoplanes sp. is the drug of choice for type 2 diabetes, with annual sales of nearly 2 billion yuan.
  • acarbose mainly has impurity component problems in production, which seriously affects product quality; the most prominent ones are impurities A, B and C components (the structure of each component is shown in Figure 1).
  • impurities A, B and C components the structure of each component is shown in Figure 1.
  • the content of A and B components in the product are all below 0.5wt% (relative to the acarbose content), and products with imperfect impurities cannot enter the sales channel.
  • the contents of components A and B are generally 10 wt% and 3 wt%, respectively, and even for the more excellent strains, the contents of components A and B are Also above 1wt%.
  • subsequent purification steps are required, resulting in complicated processes and increased production costs.
  • the object of the present invention is to provide a new Acarbose engineering bacteria, wherein the content of impurities A and B components in the products produced is reduced; and the production mode and application of the bacteria are also provided to solve the prior art. The above problem.
  • the present invention provides an Acarbose engineering bacteria, which is an acarbose-producing Actinoplanes sp. or a derivative thereof, wherein the following One or two genes are inactivated:
  • "at least X% sequence identity” refers to the percent identity between the amino acid sequences of the two polypeptides to be compared, which is obtained after optimal alignment of the two amino acid sequences.
  • the optimal alignment can be obtained by using any method known in the art, such as the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981), Needleman and Wunsch, J. Mol. Biol. .48:443 (1970) homology permutation algorithm, Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988) similarity search methods and computer implemented programs of these algorithms, as available on the NCBI site Those used by the BLAST P computer software.
  • the sequence and the amino acid sequence of the reference polypeptide have at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence.
  • the polypeptide of identity performs the same biological function as the reference polypeptide in the organism.
  • the sequence has at least 80%, at least 90%, at least 95%, at least 96%, at least the amino acid sequence shown in SEQ ID NO: 3.
  • sequence identity polypeptides perform the same biological function in the organism as the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3, thus the inactivating sequence and SEQ ID NO: 3
  • a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity of the indicated amino acid sequence can be obtained by inactivating the amino acid represented by SEQ ID NO: 3.
  • the polypeptide of the sequence has the same effect, such as reducing or removing the content of acarbose A and B impurity components.
  • acarbose by the actinomycetes is a routine technique in the art, and therefore, those skilled in the art can undoubtedly know which specific strains of the actinomycetes producing acarbose or a derivative thereof can be.
  • the actinomycetes is Actinobacter mobilis SN223/29 or a derivative thereof.
  • the acarbose-producing actinomycetes are Actinoplanes sp. 8-22 or a derivative thereof having the accession number CGMCC No. 7639.
  • the sequence of the gene M is as shown in SEQ ID NO: 4 or is a sequence having at least 80%, 90%, 95% or 99% sequence identity to the sequence set forth in SEQ ID NO: 4, said gene
  • the sequence of N is as set forth in SEQ ID NO: 5 or is a sequence having at least 80%, 90%, 95% or 99% sequence identity to the sequence set forth in SEQ ID NO: 5.
  • the gene M is substantially the bglY gene encoding the bglY protein (protein represented by SEQ ID NO: 2), and the details thereof can be found in Genbank CP003170.1: 5899370-5900680.
  • the gene N is substantially the mpbG gene and encodes the mpbG protein (protein represented by SEQ ID NO: 3).
  • the gene M or the gene N is inactivated as a gene M or a gene N deletion.
  • the deleted fragment is nucleotides 67 to 1297 of SEQ ID NO: 4; and in the deletion of the gene N, the fragment deleted is the 108th of SEQ ID NO: 5. 1098 nucleotides.
  • gene inactivation refers to a decrease or non-encoding of a polypeptide encoded by a gene, or a loss of the length of the encoded polypeptide resulting in reduced or inactive activity of the polypeptide.
  • Gene deletions including complete and partial deletions of the gene, can result in inactivation of the above genes. Any form of gene inactivation and any form of gene deletion capable of inactivating a gene is within the scope of the present invention.
  • the Acarbose engineering bacteria is an actinomycetes engineering strain ⁇ BY-3 or ⁇ MG-4, wherein ⁇ BY-3 is Actinomyces 8-22 and the sequence is SEQ ID
  • the DNA fragment of NO: 13 is formed by homologous recombination
  • ⁇ MG-4 is formed by homologous recombination of the DNA fragment of Actinobacillus actinomycetes 8-22 and SEQ ID NO: 18.
  • Homologous recombination is a conventional technique in the field of bioengineering.
  • the 5' and 3' ends of the DNA fragment subjected to homologous recombination include the 5' end and the 3' end of the gene to be inactivated, respectively, and lack the intermediate portion of the coding sequence of the gene to be inactivated.
  • the DNA fragment is capable of homologous recombination with the upstream and downstream of the gene to be inactivated in the actinomycetes, thereby recombining the DNA fragment into the genome of the actinomycetes because it lacks the middle of the coding sequence of the gene to be inactivated.
  • the gene to be inactivated is unable to express the product normally or is not expressed, thereby inactivating the gene in the constructed actinomycetes.
  • the manner in which homologous recombination is carried out is a junction transfer.
  • the present invention provides a method for preparing the Acarbose engineering bacteria, the method comprising: displacing one or both of the following genes in an acarbose-producing actinomycete or a derivative thereof live:
  • the acarbose-producing actinomycetes or derivatives thereof have been described above.
  • the Acarbose-producing actinomycetes are Actinobacter mobilis SN223/29 or a derivative thereof; in a further preferred embodiment, the acarbose-producing swimming release line
  • the bacterium is Actinomyces 8-22 or its derivative strain deposited under the number CGMCC No. 7639.
  • inactivation of a gene refers to a decrease or lack of encoding of a polypeptide encoded by a gene, or loss of the length of the encoded polypeptide resulting in reduced or inactive activity of the polypeptide.
  • gene inactivation is a conventional technical means and can be achieved in a variety of ways. For example, gene knockout, mutation, insertional inactivation, homologous recombination, RNA interference, and the like.
  • the present invention inactivates gene M and gene N by means of homologous recombination. Any technical means by which the gene can be inactivated as described above is within the scope of the invention.
  • said inactivating is the homologous recombination of said actinomycetes 8-22 with a DNA fragment of sequence SEQ ID NO: 13 or SEQ ID NO: 18.
  • the use of the Acarbose engineering bacteria of the invention for the preparation of acarbose is provided.
  • an acarbose product obtained by fermenting the Acarbose engineering bacteria of the present invention.
  • the use of the gene M or the polypeptide encoded thereby or the gene N or the polypeptide encoded thereby for the preparation of an Acarbose engineering bacteria having a reduced content of the impurity A component and/or the B component is provided.
  • the information of the gene M or the polypeptide encoded thereby or the gene N or the polypeptide encoded thereby has been described above.
  • the content of the impurity A component and the B component is decreased from 1.4 wt% and 1.31 wt% of the starting bacteria to about 0.5 wt% and about 0.2, respectively.
  • the wt% is very significant, while the acarbose fermentation unit is not affected.
  • Figure 1 Structure of acarbose and impurities A, B, C components and aglycones;
  • Figure 2 Physical map of plasmid pBS-BY334
  • Figure 3 Physical map of plasmid pBS-BYHS
  • Figure 4 Physical map of plasmid pBS-BYHS-AmT
  • Figure 5 Schematic diagram of the deletion of the internal 1231 base bglY gene
  • Figure 6 Physical map of plasmid pBS-MG334
  • Figure 7 Physical map of the vector supAmT
  • Figure 8 Physical map of plasmid SAT-MG512; in the figure, XbaI M indicates that the A base of the XbaI restriction site is methylated and cannot be cleaved by the endonuclease XbaI;
  • Figure 9 Physical map of plasmid SAT-MGHS
  • Figure 10 Schematic representation of the deletion of the internal 991 base mpbG gene.
  • the pathway for the production of the A component of the acarbose impurity is also similar to the pathway for the production of the C component: a specific enzyme catalyzes the sucrose to obtain maltulose, which is infiltrated into the synthetic pathway of acarbose. In the end, the impurity A component is finally formed. As long as the gene encoding this particular enzyme is inactivated, it is theoretically possible to block the synthesis of the malt ketose, thereby reducing or eliminating the impurity A component.
  • actinomycete genome based on the amino acid sequence of AglB (SEQ ID NO: 1), and screened for the bglY gene ( Genbank CP003170.1:5899370-5900680) and mpbG gene (SEQ ID NO:5), the amino acid sequence and the sucrose isomerase gene are similar to 45% and 44%, respectively. Further tests confirmed whether the two genes were inactivated. It can block the production of the impurity A component.
  • Digestion of DNA using TaKaRa endonuclease, end-filling (BKL kit using TaKaRa, product number: 6127A), ligation (Solution I using TaKaRa, product number 6022Q) and PCR reaction (PCR assay use) TaKaRa's Taq enzyme, product number R001; amplification of the target gene fragment using TaKaRa's PrimeSTAR enzyme, product number R010Q) are well known in the art, and can be found in the corresponding product specification; The well-known CaCl 2 method.
  • the Actinoplanes sp. 8-22 used in the present invention is deposited at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee (No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing).
  • CGMCC General Microbiology Center
  • the actinomycetes of CGMCC No.7639 are deposited on May 24, 2013.
  • the actinomycetes 8-22 are not sporulated, and the aerial hyphae grow tightly, and the color is from orange to brownish yellow, producing pigment.
  • Example 1-1 Construction of recombinant plasmid pBS-BYHS-AMT for inactivating the bglY gene
  • a primer of BYDMAF3 (SEQ ID NO: 6)/BY3R54 (SEQ ID NO: 7) was amplified from Actinobacillus actinomycetes 8-22 genomic DNA to obtain a fragment of about 3.1 kb BY334 (SEQ ID NO: 23). After the fragment was phosphorylated, it was inserted into the HincII site of the vector pBluKS (Genebank X52331.1) to obtain plasmid pBS-BY334 (the insertion direction of the fragment is shown in Fig. 2);
  • the fragment HHCPCR (SEQ ID NO: 24) was amplified by using HisunHF (SEQ ID NO: 9) / HisunCR (SEQ ID NO: 10) as a primer and a single strand (SEQ ID NO: 8) as a template.
  • the plasmid pBS-BY334 was re-extracted after transformation into E. coli JM110 to remove methylation at base A, thereby allowing the ClaI cleavage site to be cleaved by the endonuclease ClaI.
  • the plasmid was digested with HindIII/ClaI, and ligated with the same digested fragment HHCPCR to obtain plasmid pBS-BY334HS;
  • Plasmid pIJ773 Plasmid pIJ773 (plasmid pIJ773 in the literature Gust B, Kieser T and Chater K, F. Technology: PCR-targeting system in Streptomyces coelicolor. detailed in John Innes Centre. 2002) After digestion with XbaI, the gene containing the ampicillin resistance gene aac(3)IV and the junction transfer initiation site oriT was recovered. A fragment of kb (SEQ ID NO: 26) was blunt-ended with the BLK kit; this fragment was inserted into the DraI site of plasmid pBS-BYHS to obtain recombinant plasmid pBS-BYHS-AmT (see Figure 4).
  • the bglY gene of this plasmid lost its original biological activity by deleting the internal 1231 bases (Fig. 5).
  • the gene sequence for homologous recombination with Actinobacillus actinomycetes 8-22 is set forth in SEQ ID NO: 13, which comprises the 5' end portion of the bglY gene, the 3' end portion sequence and the partial sequence of the fragment HHCPCR.
  • Example 1-2 Construction of recombinant plasmid SAT-MGSH for inactivating mpbG gene
  • the primer MG3F63 (SEQ ID NO: 14) / MG3R64 (SEQ ID NO: 15) was amplified from Actinobacillus actinomycetes 8-22 genomic DNA to obtain a fragment of about 3.5 kb MG334 (SEQ ID NO: 27). After the fragment was phosphorylated, it was inserted into the HincII site of the vector pBluKS to obtain plasmid pBS-MG334 (the insertion direction of the fragment is shown in Fig. 6);
  • the fragment HHCPCR was amplified by using HisunHF (SEQ ID NO: 9)/HisunCR (SEQ ID NO: 10) as a primer and a single strand (SEQ ID NO: 8) as a template.
  • the fragment was digested with HindIII/ClaI, and ligated with the same plasmid pBS-MG334 to obtain plasmid pBS-MG334HS;
  • a primer of MG5F61 (SEQ ID NO: 16) / MG5R62 (SEQ ID NO: 17) was amplified from Actinobacillus actinomycetes 8-22 genomic DNA to obtain a fragment of about 3.1 kb MG512PCR (SEQ ID NO: 29).
  • the fragment was digested with XbaI and inserted into the XbaI site of the vector SupAmT (Fig. 7) to obtain plasmid SAT-MG512 (the insertion direction of the fragment is shown in Fig. 8);
  • Plasmid pBS-MG334HS was digested with XbaI, and a fragment of about 3.6 kb was recovered; this fragment was inserted into the XbaI site of plasmid SAT-MG512 to obtain recombinant plasmid SAT-MGHS (the insertion direction of the fragment is shown in Fig. 9).
  • the mpbG gene of this plasmid lost its original biological activity by deleting the internal 991 bases (Fig. 10).
  • the sequence for homologous recombination with Actinobacillus actinomycetes is shown in SEQ ID NO: 18, which comprises the 5' end portion of the mpbG gene, the 3' end portion sequence and the fragment HHCPCR partial sequence.
  • Example 2 Recombinant plasmids containing bglY gene and mpbG gene deletion PBS-BYHS-AMT and SAT-MGSH were transformed into host actinomycetes 8-22
  • coli ET12567 (pUZ8002) competent cells (prepared by CaCl 2 method), placed on ice 30 After a minute, it was heat-shocked at 42 °C for 90 seconds, then quickly placed on ice for 1 minute, added to 900 ⁇ l of LB, and incubated at 37 ° C for 50 minutes. 100 ⁇ l of the solution was applied to a solid LB culture containing 25 ⁇ g/ml chloramphenicol (Cm), 50 ⁇ g/ml kanamycin (Km), and 50 ⁇ g/ml apramycin (Am), and cultured overnight at 37 ° C to grow. Transformants ET12567 (pUZ8002, PBS-BYHS-AmT) and ET12567 (pUZ8002, SAT-MGSH).
  • E. coli ET12567 (pUZ8002, PBS-BYHS-AMT) and ET12567 (pUZ8002, SAT-MGSH): Pick a single transformant single colony in 3 ml containing 25 ⁇ g/ml Cm, 50 ⁇ g/ml Km and 50 ⁇ g/ml Am
  • the liquid LB medium was cultured overnight at 37 ° C, 250 rpm, and 300 ⁇ l of the bacterial solution was inoculated into 30 ml of liquid LB medium containing Cm, Km, Am, and cultured at 37 ° C, 250 rpm for 4-6 h, to an OD600 of 0.4-0.6.
  • the bacterial solution was collected, centrifuged, washed twice with LB medium, and finally suspended in 3 ml of LB medium for use.
  • c) Preparation of host bacteria 8-22 bacterial solution The hyphae of the downstream actinomycetes 8-22 were scraped from the plate, and cultured in 30 ml TSB medium at 28 ° C for 24-40 hours until the bacterial liquid turned black. 3 ml of the bacterial solution was transferred to 30 ml of TSB medium, and cultured at 28 ° C for 6 hours. 500 ⁇ l of the bacterial solution was taken, centrifuged to remove the supernatant, and then suspended in 500 ⁇ l of 2 ⁇ YT medium, and then water-cooled at 37° C. for 20 minutes, and naturally cooled, and set aside.
  • Example 3 Screening and culture of engineering bacteria for actinomycetes with bglY gene or mpbG gene deletion
  • Example 4-1 Identification of engineered bacteria of actinomycetes with bglY gene deletion
  • the amplified cultured strain was screened by a PCR method. Prepare the reaction solution according to the following ratio:
  • the PCR product with a size of 1496 bp is a back mutation, that is, its genotype is the same as that of the original strain 8-22; the PCR product size is 437 bp, which is a LIVE-activated actinomycete strain with PBS-BYHS-AMT gene deletion.
  • the strain ⁇ BY-3 was screened by this method. The strain was sequenced and identified, and the sequencing result was consistent with SEQ ID NO: 13, indicating that the strain is the expected strain of the present invention.
  • Example 4-2 Identification of engineered bacteria of actinomycetes with mpbG gene deletion
  • the amplified cultured strain was screened by a PCR method. Prepare the reaction solution according to the following ratio:
  • the PCR product with a size of 1447 bp was a back mutation, that is, its genotype was the same as that of the original strain 8-22; the PCR product with a size of 625 bp was the ACT-activated actinomycete strain with the SAT-MGSH gene deletion.
  • the strain ⁇ MG-4 was screened by this method. The strain was sequenced and identified, and the sequencing result was consistent with SEQ ID NO: 18, indicating that the strain is the expected strain of the present invention.
  • Example 5 Fermentation test of bacteriological actinomycetes ⁇ BY-3 and ⁇ MG-4 with bglY gene or mpbG gene deletion
  • the filtrate was taken for HPLC to check the content of acarbose and A and B components impurities.
  • the HPLC method was an amino column, and the mobile phase was KH 2 PO 4 0.87 g, K 2 HPO 4 0.46 g, acetonitrile 2550 ml, and H 2 O 1450 ml.
  • the detection wavelength was 210 nm and the flow rate was 1 ml/min.
  • the fermentation products of the starting strains 8-22 were tested in the same manner for comparison.
  • the HPLC results are shown in Tables 1 to 4. The results showed that compared with the starting strain 8-22, the contents of the impurity components A and B of the actinomycetes ⁇ BY-3 and ⁇ MG-4, which were deficient in the bglY gene or the mpbG gene, were significantly decreased.
  • Table 1 HPLC results of the starting strain 8-22 fermentation samples.
  • the peak times of impurity A, impurity B and acarbose were 27.886 min, 25.031 min and 30.702 min, respectively.
  • Table 2 HPLC results of the strain ⁇ BY-3 fermentation sample.
  • the peak times of impurity A, impurity B and acarbose were 28.155 min, 25.165 min and 30.733 min, respectively.
  • Table 3 HPLC results of the strain ⁇ MG-4 fermentation sample.
  • the peak times of impurity A, impurity B and acarbose were 28.837 min, 25.767 min and 31.421 min, respectively.
  • Table 4 Comparative analysis of the contents of impurities A and B in fermentation products of starting strains and genetically engineered bacteria
  • the data in Table 4 indicates that the content of the impurity A component and the impurity B component can be reduced by the inactivating gene bglY or mpbG, which indicates that the production of the impurity A component and the impurity B component may be correlated with each other. It is also indicated that these two genes are likely to be key genes that determine the impurity A component and the impurity B component. At the same time, the inactivation of the gene did not affect the fermentation unit of acarbose.

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Abstract

La présente invention concerne une bactérie pour l'ingénierie d'acarbose, un procédé de préparation et une application associés. La bactérie pour l'ingénierie d'acarbose est une bactérie Actinoplanes produisant de l'acarbose ou une souche dérivée de celle-ci, dans laquelle un ou les deux gènes suivants sont inactivés : (i) un gène M qui code pour un polypeptide ayant la séquence représentée dans SEQ ID NO : 2 ou ayant une identité de séquence d'au moins 80 %, 90 %, 95 %, ou 99 % avec la séquence représentée dans SEQ ID NO : 2; et (ii) un gène N qui code pour un polypeptide ayant la séquence représentée dans SEQ ID NO: 3 ou ayant une identité de séquence d'au moins 80 %, 90 %, 95 %, ou 99 % avec la séquence présentée dans SEQ ID NO : 3. La production d'acarbose à l'aide de la bactérie pour l'ingénierie d'acarbose peut réduire les impuretés d'un composant A et/ou d'un composant B dans un produit.
PCT/CN2018/077732 2017-03-20 2018-03-01 Bactérie pour l'ingénierie d'acarbose, procédé de préparation et application associés Ceased WO2018171400A1 (fr)

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WO2021073900A1 (fr) * 2019-10-16 2021-04-22 Bayer Aktiengesellschaft Procédés pour la formation améliorée d'acarbose
CN112592878B (zh) * 2020-12-25 2022-08-26 上海交通大学 增强正调控蛋白基因表达以提高阿卡波糖发酵水平的方法
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CN103305544A (zh) * 2013-07-05 2013-09-18 浙江海正药业股份有限公司 阿卡波糖工程菌及其制备方法和应用

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