WO2024114637A1 - Bactérie modifiée pour la production d'acarbose, son procédé de construction et son utilisation - Google Patents

Bactérie modifiée pour la production d'acarbose, son procédé de construction et son utilisation Download PDF

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WO2024114637A1
WO2024114637A1 PCT/CN2023/134740 CN2023134740W WO2024114637A1 WO 2024114637 A1 WO2024114637 A1 WO 2024114637A1 CN 2023134740 W CN2023134740 W CN 2023134740W WO 2024114637 A1 WO2024114637 A1 WO 2024114637A1
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mall
acarbose
seq
gene
supamt
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Chinese (zh)
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余贞
庞天鹏
杨小虎
胡建红
孙鹏
金玲月
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浙江海正药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/045—Actinoplanes

Definitions

  • the invention belongs to the field of biotechnology and relates to a strain for producing acarbose.
  • Acarbose is an ideal drug for the treatment of type 2 diabetes.
  • acarbose by microbial fermentation is the main production method at present, but it is accompanied by the generation of a variety of impurities.
  • the limit of impurity A in the pharmacopoeia is 0.6%.
  • Impurity A is an isomer of acarbose, and its structure is very close to that of acarbose.
  • a multi-step chromatography process is required to remove it during production, which makes the purification process of acarbose complicated and the yield is low.
  • Patent CN108624544B discloses Actinomycetes ⁇ MG-4, and the impurity A content in its fermentation product is 0.42%, which is within 0.6%, but is still relatively close to the content limit specified in the pharmacopoeia. Therefore, it is of great significance to further use genetic engineering technology to transform Actinomycetes to increase the yield of acarbose, reduce the content of impurity A, and thus reduce costs.
  • the applicant of the present invention further modified the actinomycetes on the basis of patent CN108624544B, and obtained acarbose high-yield strain, which increased the acarbose fermentation unit and reduced the content of impurity A.
  • the present invention provides an engineered bacterium for producing acarbose, wherein the engineered bacterium is an acarbose-producing actinomycete in which the malL gene is inactivated, wherein the acarbose-producing actinomycete is ⁇ MG-4 in CN108624544B, and the amino acid sequence encoded by the malL gene is shown in SEQ ID NO: 2.
  • the inactivation of the maL gene refers to the deletion of the maL gene. Further preferably, the deletion is a complete gene deletion or a partial gene deletion.
  • the nucleotide sequence of the maIL gene is as shown in SEQ ID NO:1, and the inactivation is the deletion of nucleotides at positions 111 to 1669 in the sequence shown in SEQ ID NO:1.
  • the engineered bacteria is formed by homologous recombination between the actinomycetes ⁇ MG-4 and fragment P, wherein the nucleotide sequence of fragment P is as shown in SEQ ID NO:3.
  • a method for constructing an engineered bacterium that produces acarbose comprising inactivating the malL gene in the actinomycete ⁇ MG-4, wherein the amino acid sequence encoded by the malL gene is as shown in SEQ ID NO:2; preferably, the nucleotide sequence of the malL gene is as shown in SEQ ID NO:1.
  • the construction method includes: homologous recombination of the actinomycetes ⁇ MG-4 and fragment P, wherein the nucleotide sequence of fragment P is as shown in SEQ ID NO:3.
  • the homologous recombination is accomplished by co-culturing hyphae of Actinomycetes ⁇ MG-4 and recombinant bacteria containing fragment P to perform conjugation transfer.
  • the fragment P described in the present invention comprises homology arm T1 and homology arm T2 from the 5' end to the 3' end, and homology arm T1 and homology arm T2 can undergo homologous recombination with the malL gene, thereby inactivating the malL gene.
  • the homology arm T1 consists of 110 bp nucleotides at the 5' end of the malL gene coding sequence and the upstream fragment of the malL gene
  • the homology arm T2 consists of 32 bp nucleotides at the 3' end of the malL gene coding sequence and the downstream fragment of the malL gene.
  • the fragment P comprises the 5' end and 3' end sequences of the malL gene coding sequence at the homology arm T1 and the homology arm T2, respectively, but lacks a part of the coding sequence in the middle of the malL gene.
  • T1 and T2 undergo homologous recombination with the upstream and downstream of the malL gene of the starting actinomycetes, the fragment P will be recombined into the genome of the actinomycetes; because it lacks a part of the sequence of the malL gene, the malL gene cannot express a normal product or does not express, thereby interrupting and inactivating the malL gene in the constructed actinomycetes.
  • the method of the present invention is completed by constructing a plasmid and a recombinant bacterium containing a DNA fragment P, and utilizing the recombinant bacterium to homologously recombine with the starting actinomycete.
  • Those skilled in the art can select a suitable original plasmid and strain according to experimental conditions to construct a suitable plasmid and recombinant bacterium containing a DNA fragment P.
  • the plasmid containing fragment P is SupAmT-malL-UD, and its construction process comprises:
  • the vector SupCosI was double-digested with HicII+DraI, and then ligated with the plasmid pIJ773 double-digested with XbaI+BstBI to obtain the vector SupAmT;
  • the recombinant bacterium containing fragment P is ET12567 (pUZ8002, SupAmT-malL-UD), and its construction process is: transforming the plasmid SupAmT-malL-UD into Escherichia coli ET12567 (pUZ8002).
  • the method for constructing an engineered bacterium for producing acarbose comprises conjugating and transferring the recombinant bacterium ET12567 (pUZ8002, SupAmT-malL-UD) with ⁇ MG-4 to obtain the fragment P by homologous recombination with the malL gene.
  • the present invention provides use of an engineered bacterium for producing acarbose in preparing acarbose.
  • the present invention provides a method for preparing acarbose, comprising fermenting the engineered bacteria for producing acarbose of the present invention.
  • the present invention provides the use of fragment P, a recombinant plasmid or a recombinant bacterium comprising the fragment P in constructing an engineered bacterium for producing acarbose, and the nucleotide sequence of the fragment P is shown in SEQ ID NO:3.
  • the present invention provides an application of the maL gene in constructing an acarbose engineering bacterium with reduced impurity component A, wherein the amino acid sequence encoded by the maL gene is shown in SEQ ID NO: 2.
  • the nucleotide sequence of the maL gene is shown in SEQ ID NO: 1.
  • the key to the present invention is to prevent the protein with the amino acid sequence shown in SEQ ID NO:2 from being normally expressed, thereby reducing the content of impurity A and increasing the yield of acarbose. Therefore, any nucleotide sequence encoding the sequence shown in SEQ ID NO:2 is regarded as the maL gene. In the present invention, the nucleotide sequence of the maL gene is shown in SEQ ID NO:1.
  • nucleotide fragments having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 and encoding an amino acid fragment as shown in SEQ ID NO: 2 are all within the scope of the maIL gene of the present invention.
  • the invention interrupts and inactivates the malL gene through homologous recombination, obtains an engineered bacterium for producing acarbose, and ferments the obtained acarbose fermentation unit, which is 24.7% higher than that of the starting strain actinomycetes ⁇ MG-4; the content of impurity A component is reduced from 0.42% to about 0.06%.
  • the invention uses genetic engineering technology to break through the blindness of traditional strain selection, obtains a strain with good stability, greatly increases the yield of acarbose, reduces production costs, ensures product quality, and improves economic benefits.
  • Figure 1 Schematic diagram of the construction process of the recombinant plasmid SupAmT-malL-UD for double exchange of the malL gene
  • Figure 2 Schematic diagram of the structure and restriction sites of the recombinant plasmid SupAmT-malL-U;
  • Figure 3 Enzyme digestion electrophoresis detection diagram of constructed plasmid SupAmT-malL-U: wherein M is a DNA marker (Fermentas#SM0333), and lanes 1 and 2 are electrophoretic diagrams of plasmid SupAmT-malL-U after double digestion with XbaI and HindIII, and double digestion with PstI and KpnI, respectively;
  • Figure 4 Schematic diagram of the structure and restriction sites of the recombinant plasmid SupAmT-malL-UD;
  • FIG. 5 Enzyme digestion electrophoresis detection diagram of the constructed recombinant plasmid SupAmT-malL-UD: M is DNA ladder, and lanes 1 to 4 are the electrophoresis diagrams of plasmid SupAmT-malL-UD after double digestion with BglII, XbaI and SphI, and double digestion with ApaI, XhoI and SphI, respectively;
  • FIG. 6 Schematic diagram of the double exchange principle and restriction sites of malL gene
  • Figure 7 Electrophoretic diagram of PCR screening of malL gene interrupted recombinants: wherein lanes 1 to 6 are the strains to be tested, Actinomycetes aquaticus ⁇ malL-1# to ⁇ malL-6#; lane 7 is the plasmid SupAmT-malL-UD (positive control); lane 8 is the starting strain Actinomycetes aquaticus ⁇ MG-4 (negative control); M is a DNA marker;
  • Figure 8 HPLC spectrum of acarbose standard
  • Figure 9 HPLC profile of acarbose, a fermentation product of the starting bacteria Actinomycetes ⁇ MG-4;
  • Figure 10 HPLC spectrum of acarbose, a fermentation product of the engineered bacterium Actinomycetes ⁇ malL-3#.
  • gene inactivation refers to the phenomenon that the gene cannot be expressed normally or the expression activity is reduced due to factors such as mutation, frameshift, deletion, etc. in the coding sequence.
  • the causes or methods leading to gene inactivation include but are not limited to: gene deletion, gene mutation, gene frameshift, antisense technology, homologous recombination, etc.
  • gene deletion refers to the phenomenon that DNA or RNA lacks nitrogenous bases or gene fragments, resulting in the inability to express genes normally.
  • homologous recombination refers to the recombination between non-sister chromatids or between or within DNA molecules containing homologous sequences on the same chromosome. It is a conventional method in the art to use homologous recombination methods to create gene mutations, study protein structure and function, and create transgenic organisms.
  • conjugative transfer refers to the process by which plasmid DNA is transferred from the donor to the recipient by direct contact between the donor and recipient cells.
  • the reagents and instruments used in the following methods are commonly used reagents and instruments in the art and can be obtained commercially; the methods used are conventional methods in the art, and those skilled in the art can undoubtedly perform the methods and obtain corresponding results based on the contents described in the embodiments.
  • the tool enzymes used were purchased from Dalian TakaRa; DNA molecular weight markers were purchased from Thermo Fisher Scientific; gel recovery kits, PCR product recovery kits, plasmid extraction kits, etc. were purchased from Corning Biotechnology Co., Ltd.; seamless cloning kits were purchased from Sangon Biotech (Shanghai) Co., Ltd.; the usage instructions refer to the product manual.
  • Plasmid SupAmT is an E. coli-Streptomyces shuttle plasmid, which is obtained by double restriction digestion of vector SupCosI (Stratagene, USA)/HicII+DraI and plasmid pIJ773/XbaI+BstBI and then ligation.
  • Plasmid pIJ773 is described in detail in the document Gust B, Kieser T and Chater K, F. technology: PCR-targeting system in Streptomyces coelicolor. John Innes Centre. 2002, and is specifically disclosed in CN108624544B.
  • the starting strain of the present invention is actinomycetes ⁇ MG-4, which is obtained according to the preparation method of CN108624544B.
  • the construction process of the actinomycetes ⁇ MG-4 is as follows: the actinomycetes 8-22 with the deposit number of CGMCC No.7639 undergoes homologous recombination with the DNA fragment shown in the sequence number SEQ ID NO:11 to obtain ⁇ MG-4.
  • Example 1 Construction of the recombinant plasmid SupAmT-malL-UD for interrupting the malL gene
  • malLU-F gatcttcacctagatccttttTCGCGTTCCGGCCAGCACCAGTC (SEQ ID NO: 4)
  • malLU-R tccgaagttcctattctctagaAGGTGGCCGATGATCCCGCGCAG (SEQ ID NO: 5)
  • the PCR product was recovered with a PCR product recovery kit and then seamlessly spliced with the vector SupAmT cut with XbaI using a seamless cloning kit to obtain the recombinant plasmid SupAmT-malL-U.
  • Plasmid SupAmT-malL-U The structure and restriction sites of plasmid SupAmT-malL-U are shown in Figure 2.
  • the restriction electrophoresis of plasmid SupAmT-malL-U is shown in Figure 3. It was verified that the plasmid was constructed correctly.
  • the PCR product was recovered using a PCR product recovery kit and then seamlessly spliced with SupAmT-malL-U cut with XbaI to obtain the recombinant plasmid SupAmT-malL-UD.
  • the structure and restriction sites of the recombinant plasmid SupAmT-malL-UD are shown in Figure 4.
  • the constructed plasmids were double-digested with BglII, XbaI and SphI, and double-digested with ApaI, XhoI and SphI to check whether they were correct.
  • the restriction electrophoresis of the plasmid SupAmT-malL-UD is shown in Figure 5. It was verified that the plasmid was constructed correctly.
  • Example 2 Transformation of the malL gene-interrupted recombinant plasmid SupAmT-malL-UD into the host bacterium Actinomycetes ⁇ MG-4
  • Cm chloramphenicol
  • Km 50 ⁇ g/ml kanamycin
  • Am 50 ⁇ g/ml apramycin
  • E. coli ET12567 (pUZ8002, SupAmT-malL-UD): Pick a single colony of the transformant and place it in 3 ml of liquid LB medium containing 25 ⁇ g/ml Cm, 50 ⁇ g/ml Km and 50 ⁇ g/ml Am. Cultivate overnight at 37°C and 220 rpm. Inoculate 300 ⁇ l of the bacterial solution in 30 ml of liquid LB medium containing Cm, Km and Am. Cultivate at 37°C and 220 rpm for 4-6 hours until the OD600 is between 0.4 and 0.6. Collect the bacterial solution, centrifuge it, wash it twice with LB medium, and finally suspend it with 3 ml LB medium for later use.
  • ⁇ MG-4 culture liquid scrape the hyphae of the motile actinomycetes ⁇ MG-4 from the plate, and culture in 30ml TSB medium at 28°C until the culture liquid turns black. Take 3ml of the culture liquid and transfer it to 30ml TSB medium, and culture it at 28°C for 6h. Take 500 ⁇ l of the culture liquid, centrifuge and remove the supernatant, then suspend it in 500 ⁇ l of 2 ⁇ YT medium, and water bath at 37°C for 20min. Cool it for use to obtain spore suspension.
  • Conjugation transfer Take 500 ⁇ l of the bacterial solution from b) and add it to 500 ⁇ l of the spore suspension from c), mix well and centrifuge to remove 800 ⁇ l of the supernatant. Use the remaining supernatant to suspend the bacteria and spread on STY plates (3% sucrose, 0.5% tryptone, 0.5% yeast extract powder, 0.1% acid hydrolyzed casein, 0.1% dipotassium hydrogen phosphate, 0.05% potassium chloride, 0.005% ferrous sulfate, 2% agar).
  • Tmp 500 ⁇ g Am and 500 ⁇ g trimethoprim
  • Example 3 Screening, cultivation and identification of engineered actinomycetes with interrupted malL gene
  • YMS medium (0.4% yeast extract, 0.4% soluble starch, 1.0% malt extract powder, 0.0005% cobalt chloride, 2% agar) containing 50 ⁇ g/ml Am and 50 ⁇ g/ml Tmp, and culture it at 28°C for 5-6 days. After the growing colonies are cultured continuously at 28°C for 2 generations on YMS medium without antibiotics, streak a single colony on YMS medium without antibiotics and culture it at 28°C for 5-6 days.
  • b) Use a toothpick to spot the single colony obtained in a) on YMS medium with and without 50 ⁇ g/ml Am, and culture at 28°C for 5-6 days. Select the colony that does not grow on YMS medium with 50 ⁇ g/ml Am but grows on YMS medium without Am, and amplify it on YMS medium without antibiotics.
  • malL-F3 GGCGAACAGGGTGACGTCCA (SEQ ID NO: 8)
  • malL-R3 GGCCTGGAGACCGACCTGCT (SEQ ID NO: 9)
  • the PCR product size of 2173 bp is a reverse mutation; that is, its genotype is the same as the starting strain ⁇ MG-4; the PCR product size of 620 bp is an engineered actinomycete with interrupted malL gene.
  • Figure 7 is an electrophoresis diagram of PCR screening. The 3# strain was selected and named ⁇ malL-3#. The strain was sequenced and identified. The sequencing results are shown in SEQ ID NO: 10. The PCR electrophoresis results and sequencing results show that the strain is the strain expected by the present invention.
  • Example 4 Fermentation test of the engineered actinomycete ⁇ malL-3# with interrupted malL gene
  • the mycelium was inoculated into 30 ml seed medium (3.0% soybean meal, 1.0% corn starch, 1.0% glucose, 2.0% glycerol, 0.20% CaCO 3 , natural pH), and cultured at 28°C, 250rpm for about 42 hours.
  • the HPLC method is as follows: chromatographic column: 5 ⁇ m amino column (Yuexu Technology, 250 ⁇ 4.6 mm); mobile phase: 0.6 g potassium dihydrogen phosphate, 0.35 g disodium hydrogen phosphate, 1000 ml water, 3000 ml acetonitrile; wavelength 210 nm; flow rate 1.0 ml/min.
  • chromatographic column 5 ⁇ m amino column (Yuexu Technology, 250 ⁇ 4.6 mm)
  • mobile phase 0.6 g potassium dihydrogen phosphate, 0.35 g disodium hydrogen phosphate, 1000 ml water, 3000 ml acetonitrile; wavelength 210 nm; flow rate 1.0 ml/min.
  • the fermentation product of the starting strain ⁇ MG-4 was detected in the same way for comparison.
  • test results are shown in Figures 8, 9, 10 and Table 1.
  • the standard products were purchased from China Food and Drug Inspection Institutes.
  • Table 1 Comparison of the content of acarbose and impurity A in the fermentation broth of engineered strain ⁇ malL-3# and starting strain ⁇ MG-4
  • the acarbose content in the fermentation broth sample of the starting strain ⁇ MG-4 was 5878 mg/L, and the impurity A content was 0.42%; the acarbose content in the fermentation broth sample of the engineered bacteria ⁇ malL-3# after the malL gene was interrupted was 7331 mg/L, and the impurity A content was 0.06%; compared with the starting strain, the acarbose fermentation unit increased by 24.7%, and the impurity A content was also greatly reduced.

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Abstract

L'invention concerne des bactéries modifiées pour produire de l'acarbose, et une utilisation de celles-ci. Les bactéries modifiées pour produire de l'acarbose sont obtenues par inactivation d'un gène malL chez actinoplanes produisant de l'acarbose ; après fermentation, l'unité de fermentation de l'acarbose obtenue est augmentée de 24,7% par rapport à celle d'une souche d'origine, la teneur en composant d'une impureté A est réduite de 0,42% à environ 0,06%, et le rendement en acarbose de la souche obtenue est amélioré.
PCT/CN2023/134740 2022-11-28 2023-11-28 Bactérie modifiée pour la production d'acarbose, son procédé de construction et son utilisation WO2024114637A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106566796A (zh) * 2016-10-28 2017-04-19 上海交通大学 阿卡波糖生产菌Actinoplanes spp.的遗传操作体系
CN108624544A (zh) * 2017-03-20 2018-10-09 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
WO2021073900A1 (fr) * 2019-10-16 2021-04-22 Bayer Aktiengesellschaft Procédés pour la formation améliorée d'acarbose

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106566796A (zh) * 2016-10-28 2017-04-19 上海交通大学 阿卡波糖生产菌Actinoplanes spp.的遗传操作体系
CN108624544A (zh) * 2017-03-20 2018-10-09 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
WO2021073900A1 (fr) * 2019-10-16 2021-04-22 Bayer Aktiengesellschaft Procédés pour la formation améliorée d'acarbose

Non-Patent Citations (2)

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Title
DATABASE Protein 19 May 2021 (2021-05-19), ANONYMOUS: "MULTISPECIES: alpha-glucosidase [unclassified Actinoplanes]", XP093175436, retrieved from NCBI Database accession no. WP_014693768.1 *
SCHAFFERT LENA, SCHNEIKER BEKEL SUSANNE; DYMEK SASKIA; DROSTE JULIAN; PERSICKE MARCUS; BUSCHE TOBIAS; BRANDT DAVID; PÜHLER ALFRED;: "Essentiality of the Maltase AmlE in Maltose Utilization and Its Transcriptional Regulation by the Repressor AmlR in the Acarbose-Producing Bacterium Actinoplanes sp. SE50/110", FRONTIERS IN MICROBIOLOGY, FRONTIERS MEDIA, LAUSANNE, vol. 10, 29 October 2019 (2019-10-29), Lausanne , pages 2448, XP093175432, ISSN: 1664-302X, DOI: 10.3389/fmicb.2019.02448 *

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