WO2018171400A1 - Bacterium for engineering acarbose, preparation method therefor, and application thereof - Google Patents

Abstract

Provided are a bacterium for engineering acarbose, a preparation method therefor, and an application thereof. The bacterium for engineering acarbose is an acarbose producing Actinoplanes bacterium or a derivative strain thereof, in which one or both of the following genes are inactivated: (i) a gene M that codes a polypeptide having the sequence shown in SEQ ID NO: 2 or having a sequence identity of at least 80%, 90%, 95%, or 99% with the sequence shown in SEQ ID NO:2; and (ii) a gene N that codes a polypeptide having the sequence shown in SEQ ID NO: 3 or having a sequence identity of at least 80%, 90%, 95%, or 99% with the sequence shown in SEQ ID NO:3. Production of acarbose using the bacterium for engineering acarbose can reduce impurities of a component A and/or a component B in a product.

Description

Acarbose engineering bacteria and preparation method and application thereof Technical field

The invention belongs to the field of bioengineering and relates to an engineering bacteria for producing acarbose and a preparation method and application thereof.

Background technique

According to statistics, the number of people with diabetes in China has reached 110 million, and China has become the country with the most diabetes in the world. In addition, there are about 150 million people in China who are pre-diabetes. For diabetes management alone, China is now investing nearly 173.4 billion yuan per year; direct medical expenditures for diabetes account for 13% of China's medical spending.

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. At present, 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). Under normal circumstances, 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. However, in the existing acarbose produced by the fermentation of actinomycetes, 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%. In order to ensure product quality, subsequent purification steps are required, resulting in complicated processes and increased production costs.

At present, it is used to control the content of the impurity component by controlling the fermentation process, but the method is unstable.

Summary of the invention

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.

In a first aspect, 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:

(i) a gene M of a polypeptide having a coding sequence of SEQ ID NO: 2 or having at least 80%, 90%, 95% or 99% sequence identity to the sequence of SEQ ID NO: 2;

(ii) Gene N of a polypeptide having a coding sequence of SEQ ID NO: 3 or having at least 80%, 90%, 95% or 99% sequence identity to the sequence set forth in SEQ ID NO: 3.

In the present invention, "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.

One skilled in the art can reasonably conclude that in the same genus, 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. For example, in the actinomycetes of the present invention, one skilled in the art can reasonably expect that 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. 97%, at least 98% or at least 99% of the 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.

The production of 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. Preferably, the actinomycetes is Actinobacter mobilis SN223/29 or a derivative thereof. Further preferably, the acarbose-producing actinomycetes are Actinoplanes sp. 8-22 or a derivative thereof having the accession number CGMCC No. 7639.

Preferably, 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. Those skilled in the art will appreciate that a gene having a sequence of at least 80%, 90%, 95% or 99% sequence identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, encoded with SEQ ID NO: 4 Or a polypeptide having the same or similar function as the polypeptide encoded by SEQ ID NO: 5.

When the sequence of the gene M is represented by SEQ ID NO: 4, 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.

When the sequence of the gene N is as shown in SEQ ID NO: 5, the gene N is substantially the mpbG gene and encodes the mpbG protein (protein represented by SEQ ID NO: 3).

In a preferred embodiment, in the Acarbose engineering bacteria, the gene M or the gene N is inactivated as a gene M or a gene N deletion.

Further preferably, in the deletion of the gene M, 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.

It will be understood by those skilled in the art that 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.

Further preferably, the Acarbose engineering bacteria is an actinomycetes engineering strain ΔBY-3 or ΔMG-4, wherein ΔBY-3 is Actinomyces 8-22 and the sequence is SEQ ID The DNA fragment of NO: 13 is formed by homologous recombination, and ΔMG-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. In part, the gene to be inactivated is unable to express the product normally or is not expressed, thereby inactivating the gene in the constructed actinomycetes. In a particular embodiment of the invention, the manner in which homologous recombination is carried out is a junction transfer.

In a second aspect, 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:

(i) a gene M of a polypeptide having a coding sequence of SEQ ID NO: 2 or having at least 80%, 90%, 95% or 99% sequence identity to the sequence of SEQ ID NO: 2;

(ii) a gene N of a polypeptide having a coding sequence of SEQ ID NO: 3 or having at least 80%, 90%, 95% or 99% sequence identity to the sequence of SEQ ID NO: 3;

The acarbose-producing actinomycetes or derivatives thereof have been described above. Preferably, 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.

The sequences of the genes M and N have been described above.

As noted above, 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. It is known to those skilled in the art that in the field of bioengineering, 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.

Further preferably, 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.

In a third aspect, the use of the Acarbose engineering bacteria of the invention for the preparation of acarbose is provided.

In a fourth aspect, there is provided an acarbose product obtained by fermenting the Acarbose engineering bacteria of the present invention.

In a fifth aspect, 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. Among them, the information of the gene M or the polypeptide encoded thereby or the gene N or the polypeptide encoded thereby has been described above.

In the acarbose product produced by the Acarbose engineering bacteria provided by the present invention, 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. By using the Acarbose engineering bacteria of the invention to produce acarbose, the content of the impurity components A and B in the product is significantly reduced, the product quality is ensured, the subsequent purification step or the purification step is relatively simple, and the process steps are reduced. , reducing costs.

DRAWINGS

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.

detailed description

According to the literature (Appl Microbiol Biotechnol. 2008, 80: 767-778), there are two ways to produce a component of acarbose impurity: one is the malto-oligosaccharide-based trehalose synthase encoded by the treY gene of acarbose. The catalytic reaction directly obtains the C component; the second is the synthesis of trehalose by the trehalase encoded by the treS gene, and then enters the synthetic route to obtain the C component. From this we hypothesize that 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.

Therefore, how to screen for an enzyme gene related to the synthesis of maltoxose is the key to reducing or removing the component A of the impurity. However, the biosynthetic pathways for maltoxose have so far been reported in the literature, and it is impossible to obtain an enzyme gene related to maltosynthesis synthesis by the prior art. The gene related to the maltulose that can be retrieved from the NCBI website (https://www.ncbi.nlm.nih.gov/nuccore/?term=maltulose) is the agl gene, which encodes an alpha-glucosidase. According to the literature (Appl Environ Microbiol. 2009, 75(4): 1135-1143; Caries Res. 2003, 37(6): 410-415; J Biol Chem. 2006, 281(26): 17900-17908; J Biol Chem .2001, 276(40): 37415-37425) reported that alpha-glucosidase is primarily involved in the metabolism of maltoulose, rather than in synthesis. The genome of Actinoplanes sp. SE50/110 has been sequenced (Genbank CP003170.1). We searched for sucrose isomerase aglB (Genbank AF337811.1) from NCBI and compared it with the A. 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.

Based on the above analysis, the inventors conducted the following experiments and completed the present invention. The invention will be described below with reference to specific embodiments and drawings, but the content of the invention is not limited thereto. Unless otherwise stated, the reagents used in the following examples are commercially available from conventional chemical reagent stores; the methods used and their parameters can be accomplished by those skilled in the art based on the prior art or common general knowledge. For ease of description, the methods involved in this patent are listed below:

Isolation of the Actinomyces faecalis genome (described in the example as "extracted genomic DNA"): 200 μl of Actinobacillus actinomycetes 8-22 cryopreservation solution (applicant) was inoculated into 30 ml TSB medium (Bacto) TM Tryptic Soy Broth. BD, Cat. No. 211825), cultured at 28 ° C, 28 ° C for 48 hr, centrifuged in a 50 ml centrifuge tube at 4000 rpm for 10 minutes, and the supernatant was removed. 30 ml of sucrose-Tris buffer (10.3% sucrose, 10 mM Tris) was used for precipitation. After washing twice with -HCl, pH 8.0, it was suspended in 5 ml of sucrose-Tris buffer. 20 μl of a 100 mg/ml lysozyme solution was added, and a water bath at 37 ° C for 2 hr. Add 500 μl of 10% SDS solution and gently invert until essentially clear. 5 ml of a solution of phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.0) was added, and after gently inverting several times, it was centrifuged at 4000 rpm for 10 minutes. 4 ml of the upper layer solution was taken, and 4 ml of a phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.0) solution was added, and the mixture was gently inverted several times, and then centrifuged at 4000 rpm for 10 minutes. 3 ml of the upper layer solution was taken, 300 μl of 3 mol/L HAc/NaAc buffer (pH 5.3), and 3 ml of isopropanol were added, and after gently inverting several times, the pellet of the agglomeration was picked up into a 1.5 ml centrifuge tube with a pipette tip. The precipitate was washed twice with 70% ethanol and dried at room temperature. After dissolving by adding 500 μl of Tris-HCl (pH 8.0), total DNA of Actinobacillus actinomycetes 8-22 was obtained.

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). 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) 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);

b) 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;

c) amplification of a fragment of about 3.1 kb BY512 (SEQ ID NO: 25) from the genomic DNA of Actinobacillus actinomycetes 8-22 with primer BY5F51 (SEQ ID NO: 11) / BY5R52 (SEQ ID NO: 12), The fragment was digested with XbaI and inserted into the XbaI site of the vector pBS-BY334HS to obtain the plasmid pBS-BYHS (see Figure 3 for the insertion direction of the fragment);

technology:PCR-targeting system in Streptomyces coelicolor.John Innes Centre.2002中有详 细说明)以XbaI酶切后，回收包含氨普霉素抗性基因aac(3)IV和接合转移起始位点oriT的1.3kb的片段(SEQ ID NO:26)，并以BLK试剂盒补平末端；该片段插入到质粒pBS-BYHS的DraI位点，得到重组质粒pBS-BYHS-AmT(见图4)。该质粒的bglY基因因为缺失了内部的1231个碱基而失去原有的生物活性(图5)。用于与游动放线菌8-22进行同源重组的基因序列见SEQ ID NO:13，其包含bglY基因的5’端部分序列、3’端部分序列和片段HHCPCR的部分序列。 d) 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

a) 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);

b) 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;

c) 1229 bp fragment containing pUC ori (SEQ ID NO: 28) was excised from cosmids supcos-1 (Stratagene, Inc.) with HincII+DraI; 1271 bp aac3 was cleaved from pIJ773 with BstBI+XbaI ( IV) and a fragment of oriT (1-1271 of SEQ ID NO: 26), and blunt-ended with a BLK kit. After the above two fragments are ligated, the vector supAmT is obtained (Fig. 7).

d) 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);

e) 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

technology:PCR-targeting system in Streptomyces coelicolor.John Innes Centre.2002中有详细说明)：取重组质粒1μl加入到100μl大肠杆菌ET12567(pUZ8002)感受态细胞(以CaCl ₂法制备)中，冰上放置30分钟后，42℃热击90秒，再迅速放到冰上冷却1分钟，加入900μl LB培养，37℃水浴50分钟。取 100μl涂布于含25μg/ml氯霉素(Cm)、50μg/ml卡那霉素(Km)、50μg/ml安普霉素(Am)的固体LB培养上，37℃培养过夜，长出转化子ET12567(pUZ8002,PBS-BYHS-AmT)和ET12567(pUZ8002,SAT-MGSH)。 a) Transformation of the recombinant plasmids PBS-BYHS-AmT and SAT-MGSH into E. coli ET12567 (pUZ8002) (in the literature Gust B, Kieser T and Chater K, F.

Technology: PCR-targeting system in Streptomyces coelicolor. detailed in John Innes Centre. 2002): 1 μl of recombinant plasmid was added to 100 μl of E. 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 μl of LB, and incubated at 37 ° C for 50 minutes. 100 μl of the solution was applied to a solid LB culture containing 25 μg/ml chloramphenicol (Cm), 50 μg/ml kanamycin (Km), and 50 μg/ml apramycin (Am), and cultured overnight at 37 ° C to grow. Transformants ET12567 (pUZ8002, PBS-BYHS-AmT) and ET12567 (pUZ8002, SAT-MGSH).

b) Culture of E. coli ET12567 (pUZ8002, PBS-BYHS-AMT) and ET12567 (pUZ8002, SAT-MGSH): Pick a single transformant single colony in 3 ml containing 25 μg/ml Cm, 50 μg/ml Km and 50 μg/ml Am The liquid LB medium was cultured overnight at 37 ° C, 250 rpm, and 300 μl 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 μl of the bacterial solution was taken, centrifuged to remove the supernatant, and then suspended in 500 μl of 2×YT medium, and then water-cooled at 37° C. for 20 minutes, and naturally cooled, and set aside.

d) Engagement transfer: 500 μl of the bacterial solution of b) was added to 500 μl of the bacterial solution of c), and 800 μl of the supernatant was removed by centrifugation. The cells were suspended in the remaining supernatant and applied to 2 CMC medium. After incubation at 28 ° C for 16-20 h, it was covered with 1 ml of sterile water containing 625 μg of Am and 500 μg of nalidixic acid (Nal), and cultured at 28 ° C for 4-8 days to grow transformants.

Example 3: Screening and culture of engineering bacteria for actinomycetes with bglY gene or mpbG gene deletion

a) One transformant was picked and streaked on YMS medium containing 25 μg/ml Am and 25 μg/ml Nal, and cultured at 28 ° C for 4-6 days. The grown colonies were continuously cultured on YMS medium containing no antibiotics at 28 ° C for 2 passages, and then single colonies were streaked on YMS medium containing no antibiotics, and cultured at 28 ° C for 4-6 days.

b) The single colonies obtained in a) were spotted on a YMS medium containing and without 25 μg/ml Am with a toothpick, and cultured at 28 ° C for 4-6 days. Colonies grown on YMS medium containing no 25 μg/ml Am and grown on YMS medium without Am were selected for amplification culture on YMS medium without antibiotics.

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:

15 μl/tube was dispensed, and the colonies screened in Example 3-b) were picked up with a toothpick as a template for PCR reaction, and 0.2 μl of 8-22 genomic DNA and recombinant plasmid PBS-BYHS-AMT were used as negative control and positive control respectively. . The PCR reaction procedure is:

95 ° C × 10 minutes,

(94 ° C × 30 seconds, 68 ° C × 2 minutes) × 30 cycles,

72 ° C × 2 minutes,

16 ° C × 1 second.

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 ΔBY-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:

15 μl/tube was dispensed, and the colonies screened in Example 3-b) were picked up with a toothpick as a template for PCR reaction, and 0.2 μl of 8-22 genomic DNA and recombinant plasmid SAT-MGSH were used as negative control and positive control, respectively. The PCR reaction procedure is:

95 ° C × 10 minutes,

(94 ° C × 30 seconds, 68 ° C × 2 minutes) × 30 cycles,

72 ° C × 2 minutes,

16 ° C × 1 second.

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 ΔMG-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 bacteriological actinomycetes △BY-3 and △MG-4, which were deficient in the bglY gene and the mpbG gene, were cultured on YMS medium for 28-d°C at -28 days, and the colonies with an area of about 1 cm ^{2 were} excavated into 30 ml of seed culture. Base (soybean cake powder 3.0%, corn starch 1.0%, glucose 1.0%, glycerol 2.0%, CaCO ₃ 0.20%, pH natural), cultured at 28 ° C, 220 rpm for 24-30 h, transferred to fermentation culture with 2 ml inoculum Base (glucose 3.0%, maltose 5.0%, soybean cake powder 1.2%, K ₂ HPO ₄ 0.10%, CaCl ₂ ·H ₂ O 0.35%, FeCl ₃ 0.05%, CaCO ₃ 0.3%, sodium glutamate 0.1%), Incubate at 28 ° C, 220 rpm for 7-8 days. After 5 ml of the fermentation broth was filtered, 1 ml of the filtrate was taken, 4 ml of anhydrous methanol was added, and after soaking overnight, it was filtered. 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 ΔBY-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 ΔBY-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 ΔMG-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

Strain A component content B component content Acarbose fermentation unit Starting bacteria 8-22 1.4wt% 1.31wt% 5001mg/L △BY-3 0.53wt% 0.20wt% 4807mg/L △MG-4 0.42wt% 0.21wt% 5887mg/L

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.

Claims (10)

An Acarbose engineering bacteria, which is an acarbose-producing Actinoplanes sp. or a derivative thereof, wherein one or two of the following genes are inactivated: (i) a gene M of a polypeptide having a coding sequence of SEQ ID NO: 2 or having at least 80%, 90%, 95% or 99% sequence identity to the sequence of SEQ ID NO: 2; (ii) a gene N of a polypeptide having a coding sequence of SEQ ID NO: 3 or having at least 80%, 90%, 95% or 99% sequence identity to the sequence of SEQ ID NO: 3; Preferably, the Acarbose-producing Actinomycetes is Actinobacter mobilis SN223/29 or a derivative thereof; further preferably, the Acarbose-producing Actinomycetes is a deposit number Actinobacteria 8-22 or a derivative thereof derived from CGMCC No. 7639; Preferably, 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 Acarbose engineering bacteria according to claim 1, wherein the gene M or the gene N is inactivated as a gene M or a gene N deletion in the Acarbose engineering bacteria. The Acarbose engineering bacteria according to claim 2, wherein, in the deletion of the gene M, the deleted fragment is nucleotides 67 to 1297 of SEQ ID NO: 4; and the gene N is deleted, deleted The fragment is nucleotides 108-1098 of SEQ ID NO:5. The Acarbose engineering bacteria according to claim 3, wherein the Acarbose engineering bacteria is a swimming actinomycete ΔBY-3 or ΔMG-4, wherein ΔBY-3 is a swimming release Cyclosporin 8-22 is formed by homologous recombination with the DNA fragment of SEQ ID NO: 13, and ΔMG-4 is the same as the DNA fragment of SEQ ID NO: 18. The source is formed by reorganization. A method for preparing an Acarbose engineering bacteria, the method comprising: inactivating one or both of the following genes in apocalin-producing actinomycetes or a derivative thereof: (i) a gene M of a polypeptide having a coding sequence of SEQ ID NO: 2 or having at least 80%, 90%, 95% or 99% sequence identity to the sequence of SEQ ID NO: 2; (ii) a gene N of a polypeptide having a coding sequence of SEQ ID NO: 3 or having at least 80%, 90%, 95% or 99% sequence identity to the sequence of SEQ ID NO: 3; Preferably, the Acarbose-producing Actinomycetes is Actinobacter mobilis SN223/29 or a derivative thereof; further preferably, the Acarbose-producing Actinomycetes is a deposit number Actinobacteria 8-22 or a derivative thereof derived from CGMCC No. 7639; Preferably, 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 method according to claim 5, wherein the method of inactivating the gene M or the gene N includes, but is not limited to, gene knockout, mutation, insertion inactivation, homologous recombination, RNA interference. The method according to claim 6, wherein the inactivating is homologous recombination of the Actinobacter mobilis 8-22 with a DNA fragment of the sequence SEQ ID NO: 13 or SEQ ID NO: 18. Use of the Acarbose engineering bacteria according to any one of claims 1 to 4 for the preparation of acarbose. An acarbose product obtained by culturing the Acarbose engineering bacteria according to any one of claims 1 to 4. The use of the gene M or the polypeptide encoded thereby or the gene N or the polypeptide encoded thereby for preparing an Acarbose engineering bacteria having a reduced content of the impurity A component and/or the B component, wherein the gene M coding sequence is SEQ ID NO a polypeptide represented by 2 or having at least 80%, 90%, 95% or 99% sequence identity to the sequence of SEQ ID NO: 2, the gene N coding sequence being as set forth in SEQ ID NO: 3 or with SEQ ID NO a polypeptide having an amino acid sequence of at least 80%, 90%, 95% or 99% sequence identity; Preferably, 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.

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