WO2020228458A1 - 一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用 - Google Patents

一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用 Download PDF

Info

Publication number
WO2020228458A1
WO2020228458A1 PCT/CN2020/084262 CN2020084262W WO2020228458A1 WO 2020228458 A1 WO2020228458 A1 WO 2020228458A1 CN 2020084262 W CN2020084262 W CN 2020084262W WO 2020228458 A1 WO2020228458 A1 WO 2020228458A1
Authority
WO
WIPO (PCT)
Prior art keywords
spo
bacteria
recombinant
amylase
licheniformis
Prior art date
Application number
PCT/CN2020/084262
Other languages
English (en)
French (fr)
Inventor
肖静
李子源
张虎
Original Assignee
齐鲁工业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 齐鲁工业大学 filed Critical 齐鲁工业大学
Priority to KR1020207017367A priority Critical patent/KR102246356B1/ko
Publication of WO2020228458A1 publication Critical patent/WO2020228458A1/zh

Links

Images

Classifications

    • 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
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C12N9/242—Fungal source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • 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
    • C12N2511/00—Cells for large scale production

Definitions

  • the invention relates to a method and application for knocking out spoIIQ and pcf genes to improve fermentation enzyme production of Bacillus licheniformis, and belongs to the technical field of genetic engineering.
  • Bacillus is an important fermentation production strain in the enzyme preparation industry. Among them, Bacillus licheniformis has a strong metabolism and rich enzyme system, which can be used in large-scale fermentation to produce ⁇ -amylase. It is widely used in textile, food, feed, medicine and other industries. application.
  • Bacillus During the enzyme production process of Bacillus, especially after the growth of the bacteria reaches a stable period, the bacteria begin to form spores due to changes in nutritional and environmental conditions.
  • the formation of spores is not conducive to high-density continuous fermentation, and will affect the control of the fermentation process, such as the increase in the viscosity of the fermentation liquid, the increase of foam, etc., which affects the exchange of temperament and easily cause liquid escape, which increases the chance of infection and increases the cost of fermentation; in addition, Bacillus
  • the autolysis of bacteria is often accompanied during the growth process, which is not conducive to the accumulation of biomass, shortens the effective fermentation cycle, and reduces the fermentation production efficiency.
  • Chinese patent document CN108929883A discloses the application of sporulation-related gene spoIIE in affecting the growth of strains and enzyme production.
  • the invention uses insert inactivation to fused partial spoIIE gene fragments with Cmr gene, and the fusion gene spoIIE -Cmr was inserted into the spoIIE gene sequence of Bacillus clausii, which made the spoIIE gene unable to be expressed normally and inactivated.
  • the spoIIE gene-inactivated strain not only reduced the sporulation rate to 0.3%, but also made the engineered strain grow faster than the original strain Speed up, realize the accumulation of cell number in a shorter time and the fermentation production of higher vigor amylase.
  • Chinese patent document CN108949785A discloses the role of the sporulation-related gene spo0A in producing enzymes. By inserting inactivation, part of the spo0A gene fragment is fused with the Cmr gene, and the fusion gene spo0A-Cmr is inserted into the gram In the spo0A gene sequence of Bacillus Rousei, the spo0A gene cannot be normally expressed and inactivated, and the cell metabolism changes. While the sporulation rate of the strain is reduced and the expression of protease is weakened, the amylase activity is increased by 85.8% compared with the original strain. Pectinase activity increased by 235%, and lipase activity increased by 70.0%.
  • the present invention provides a method and application for knocking out spoIIQ and pcf genes to increase the fermentation enzyme production of B. licheniformis.
  • the obtained genetically engineered bacteria B. licheniformis (B. licheniformis) DL-1 ⁇ spoIIQ ⁇ pcf is compared
  • the strain B. licheniformis DL-1 has a significant improvement in fermentation production performance.
  • a recombinant strain with high alpha-amylase production obtained by knocking out the sporulation-related gene spoIIQ and the lysis-related gene pcf in the alpha-amylase-producing microorganism.
  • the nucleotide sequence of the sporulation-related gene spoIIQ is as SEQ ID As shown in NO.1, the nucleotide sequence of the lysis-related gene pcf is shown in SEQ ID NO.2.
  • the ⁇ -amylase producing microorganism is Bacillus licheniformis; further preferably, the Bacillus licheniformis is B. licheniformis DL-1.
  • the specific steps of knocking out the spore-forming gene spoIIQ of the ⁇ -amylase-producing microorganism are as follows:
  • Step 1 Using Bacillus licheniformis genomic DNA as a template, design the primers spoIIQF1, spoIIQR1, spoIIQF2 and spoIIQR2 for the upper and lower homology arms, and carry out PCR amplification to obtain the upper and lower homology arm fragments of spoIIQ. Arm connection to obtain the fusion fragment spoIIQF-spoIIQR;
  • nucleotide sequences of the upstream and downstream homology arm primers spoIIQF1, spoIIQR1, spoIIQF2 and spoIIQR2 are as follows:
  • spoIIQF1 CATCGCCAGTCACTAGGATCCACGCCGAGGACGTAGCCT
  • spoIIQR2 CAGTATTGACTGCAGGCGGCCGCAAGCTTGAACGGCTGCCTATT
  • Step 2 Double-enzyme digestion and purification of knockout pTOPO-Cmr with BamHI and NotI, and ligate the fusion fragment obtained in step 1 with the vector using seamless cloning technology to obtain the recombinant vector pTOPO-Cmr-spoIIQ;
  • Step 3 Prepare competent cells of Bacillus licheniformis, electrotransform the recombinant vector obtained in step 2 into Bacillus licheniformis cells, spread on LB solid medium containing chloramphenicol for preliminary screening, and transfer the preliminary screening strains to those containing The LB liquid medium containing 5% xylose was re-screened to obtain the recombinant B. licheniformis DL-1 ⁇ spoIIQ.
  • the specific steps of knocking out the bacterial autolysis gene pcf in the recombinant bacterium AspoIIQ are as follows:
  • Step I Using Bacillus licheniformis genomic DNA and pHT01 plasmid as templates, design the upstream and downstream primers pcfF, pcfR and Cm r F, Cm r R, PCR amplification to obtain pcf gene fragment and chloramphenicol resistance Cm r gene fragment, Connect the pcf and Cm r gene fragments by overlapping PCR to obtain the fusion fragment pcf-Cm r ;
  • nucleotide sequences of the primers pcfF, pcfR, Cm r F, Cm r R are as follows:
  • Step II The pcf-Cm r obtained in step I was subjected to single enzyme digestion with BamHI, purified, and electrotransformed into recombinant B. licheniformis DL-1 ⁇ spoIIQ cells;
  • Step III Spread the transformed bacteria obtained in Step II on LB solid medium containing chloramphenicol, and screen for chloramphenicol-resistant positive gene-deficient recombinant bacteria to obtain recombinant bacteria B.licheniformis DL -1 ⁇ spoIIQ ⁇ pcf;
  • a method for preparing ⁇ -amylase using the above-mentioned recombinant bacteria with high production of ⁇ -amylase the steps are as follows:
  • step (ii) Enlarged culture of the activated bacteria obtained in step (i) to obtain fermented bacteria
  • step (iii) Inoculating the fermented cells obtained in step (ii) into the fermentation medium, fermenting for 84 hours at 37°C and 300 rpm, separating the solid and liquid, taking the liquid, and purifying to obtain the ⁇ -amylase;
  • the fermentation medium components are as follows:
  • the glycerol tube takes the bacterial solution and streaks it on the LB plate, cultures it at 37°C for 24h, picks a single colony and transfers it to 50mL liquid LB medium, and cultures it at 37°C, 200rpm for 20h Passage twice to prepare activated bacteria.
  • the activated bacteria obtained in the step (i) are transferred to a 100 mL LBPG medium at 37° C. and 200 rpm for expansion culture at a 2% inoculum amount.
  • the LBPG medium components are as follows:
  • the fermentation broth is centrifuged at 4° C. and 12000 rpm for 10 min using a high-speed refrigerated centrifuge to obtain a crude enzyme solution for subsequent enzyme activity determination.
  • sporulation-related gene spoIIQ is different from the known spo gene in the prior art. Knockout of spoIIQ can not directly increase the production of ⁇ -amylase, but when it is combined with promoting bacteria After knocking out the autolytic gene pcf, it can significantly increase the production of ⁇ -amylase;
  • the present invention obtains recombinant bacteria B. licheniformis (B. licheniformis) DL-1 ⁇ spoIIQ ⁇ pcf by means of gene knockout, cultured in LB medium, the spore generation rate of the recombinant bacteria is reduced to 0.16% of the original bacterial strain, and the bacterial growth is more stable , Shake flask fermentation showed that the enzyme activity of recombinant bacteria was 10.9% higher than that of starting bacteria; fermentation in 5L fermentor showed that the enzyme activity of recombinant bacteria was 19.3% higher than that of starting bacteria, which was compared with that of starting bacteria. The activity of amylase was increased by 3.4 times. In the middle and late stages of fermentation, the recombinant bacteria have higher dissolved oxygen in the fermentation broth than the starting bacteria, less foam production, and better fermentation performance than the starting bacteria.
  • spoIIQ upstream homology arm fragment electrophoresis results; in the figure: lanes 1 and 2 are 600bp upstream homology arm fragments, and lane M is Marker;
  • Lanes 1 and 2 are 600bp downstream homology arm fragments, and lane M is Marker;
  • Figure 3 is a bar graph showing the results of determination of the spore formation rate of the starting bacterium B.licheniformis (B.licheniformis) DL-1 and the recombinant bacterium B.licheniformis (B.licheniformis) DL-1 ⁇ spoIIQ in Example 3 of the present invention;
  • Example 4 shows the results of determination of the growth cycle of the starting bacteria and recombinant bacteria in the shake flask fermentation in Example 4 of the present invention
  • Figure 5 is a photo of the initial test results of the starting and recombinant bacteria alpha-amylase activity plate in Example 5 of the present invention.
  • Example 6 is a histogram of the results of determination of ⁇ -amylase enzyme activity in shake flask fermentation of starting bacteria and recombinant bacteria in Example 5 of the present invention
  • Fig. 7 is a graph showing the growth measurement results of fermentation starting bacteria and recombinant bacteria in a 5L fermentor in Example 6 of the present invention, wherein (a) is a growth curve and (b) is a dissolved oxygen curve;
  • Fig. 8 is a histogram of the results of the determination of the ⁇ -amylase activity of the fermentation starting bacteria and the recombinant bacteria in the 5L fermentor in Example 6 of the present invention.
  • B. licheniformis (B. licheniformis) DL-1 in the examples was purchased from the American Type Culture Collection, and the strain number is ATCC NO.27811.
  • spoIIQ gene upstream and downstream homology arms spoIIQF, spoIIQR and the fusion fragment spoIIQF-spoIIQR were amplified by PCR.
  • the nucleotide sequences of the primers are as follows:
  • spoIIQF1 CATCGCCAGTCACTAGGATCCACGCCGAGGACGTAGCCT
  • spoIIQR2 CAGTATTGACTGCAGGCGGCCGCAAGCTTGAACGGCTGCCTATT
  • the knockout pTOPO-Cmr was double digested with BamHI and NotI, purified according to the steps provided in the purification kit, and the fusion fragment was connected to the vector by seamless cloning.
  • the connection system is shown in the following table:
  • the recombinant fragment pcf-Cm r was single-enzyme digested with BamHI, and then purified.
  • the recombinant fragment was transformed into spoIIQ gene-deficient bacteria by electrotransformation, resuscitated at 37°C and 180rpm for 1h, coated with chloramphenicol resistant plates, and selected positive recombinants B. licheniformis (B. licheniformis) DL-1 ⁇ spoIIQ ⁇ pcf and B. licheniformis (B. licheniformis) DL-1 ⁇ pcf recombinant bacteria were finally successfully constructed.
  • Example 3 Determination of sporulation rate and enzyme activity of starting bacteria and recombinant bacteria (B.licheniformis) DL-1 ⁇ spoIIQ
  • Formula spore generation rate spore amount/total bacteria amount ⁇ 100%, calculate the spore generation rate of starting bacteria and recombinant bacteria The results are shown in Figure 3.
  • the starting strain B. licheniformis DL-1 has a spore count of 4.62 ⁇ 10 8 CFU/mL, a total of 5.79 ⁇ 10 8 CFU/mL, and a spore generation rate of about 79.82%;
  • the number of spores of B. licheniformis DL-1 ⁇ spoII was 0.51 ⁇ 10 6 CFU/mL, the total number of bacteria was 3.19 ⁇ 10 8 CFU/mL, and the sporulation rate was about 0.16%.
  • the recombinant strain B. licheniformis DL-1 ⁇ spoIIQ ⁇ pcf was constructed on the basis of B. licheniformis DL-1 ⁇ spoIIQ to reduce the autolysis of spore-deficient bacteria.
  • the starting bacteria B. licheniformis (B.licheniformis) DL-1 and the recombinant bacteria B. licheniformis (B.licheniformis) DL-1 ⁇ spoIIQ were fermented for 60h and 48h to reach the maximum enzyme activity, respectively, 391U/mL, 340U/mL; Recombinant lichen Compared with B.licheniformis DL-1 ⁇ spoIIQ and the starting bacteria B.licheniformis DL-1, the fermentation activity of ⁇ -amylase decreased.
  • Example 4 Determination of growth cycle of starting bacteria and recombinant bacteria in shake flask culture
  • the medium involved has the following components:
  • LB medium tryptone 1%, yeast extract 0.5%, NaCl 1%, (all mass fractions) pH 7.0.
  • LBPG medium tryptone 1%, yeast extract 0.5%, NaCl 1%, glucose 0.1%, (all mass fractions) glycerol 2.5% (volume fraction) pH 7.0.
  • the starting strain B. licheniformis DL-1 The starting strain B. licheniformis DL-1, the recombinant strain B. licheniformis DL-1 ⁇ spoIIQ, the recombinant strain B. licheniformis DL-1 ⁇ pcf and the recombinant strain B. licheniformis (B.
  • the starting strain and the recombinant strain were respectively inoculated into 100mL liquid fermentation medium with an inoculum of 2%, cultivated at 37°C and 200rpm. During this period, samples were taken every 12h to determine the ⁇ -amylase activity according to the national standard GB/T24401-2009 method. The results are shown in the figure. 6.
  • B.licheniformis (B.licheniformis) DL-1 ⁇ spoIIQ ⁇ pcf all reached the maximum enzyme activity after 60 hours of fermentation, respectively At 391U/mL, 407U/mL and 434U/mL, B. licheniformis DL-1 ⁇ spoIIQ reached the maximum enzyme activity of 340U/mL at 48h after fermentation, and B. licheniformis DL-1 ⁇ spoIIQ ⁇ pcf was better than B. licheniformis (B.licheniformis) DL-1 enzyme activity increased by 10.9%; B. licheniformis (B.licheniformis) DL-1 ⁇ spoIIQ ⁇ pcf increased by 6.6% compared with the recombinant bacteria B. licheniformis DL-1 ⁇ pcf; further confirm the construction The recombinant strain B.licheniformis DL-1 ⁇ spoIIQ ⁇ pcf can effectively improve the fermentation activity of ⁇ -amylase.
  • ⁇ -amylase activity 1ml of liquid enzyme, liquefying 1mg of soluble starch in 1min under the conditions of 70°C and pH6.0, is 1 unit of enzyme activity, expressed in U/mL.
  • the fermentation medium components are as follows:
  • Example 6 Determination of fermentation performance of starting bacteria and recombinant bacteria in 5L tanks
  • the starting bacteria B. licheniformis (B. licheniformis) DL-1, the recombinant bacteria B. licheniformis (B. licheniformis) DL-1 ⁇ pcf and the recombinant bacteria B. licheniformis (B. licheniformis) DL-1 ⁇ spoIIQ ⁇ pcf were carried out.
  • Activate inoculate 5% into a 5L fermentor.
  • the initial total amount of fermentation broth is 3L, pH7.5, 37°C, and 350rpm.
  • the fermentation cycle is 84h. During the fermentation period, samples are taken every 6-8h to determine the cell concentration OD 600.

Abstract

一种敲除spo Ⅱ Q和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用，本发明通过敲除spo Ⅱ Q、pcf基因，获得重组菌地衣芽孢杆菌(B.licheniformis)DL-1Δspo Ⅱ QΔpcf，芽孢形成率降低至0.16％，摇瓶发酵培养重组菌α-淀粉酶酶活提高了10.9％，5L发酵培养时重组菌α-淀粉酶酶活提高了19.3％。

Description

一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用 技术领域
本发明涉及一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用，属于基因工程技术领域。
背景技术
芽孢杆菌是酶制剂行业重要的发酵生产菌株，其中地衣芽孢杆菌(Bacillus licheniformis)代谢旺盛，酶系丰富，能规模化发酵生产α-淀粉酶等，在纺织、食品、饲料、医药等行业有广泛应用。
芽孢杆菌发酵产酶过程中，尤其在菌体生长到达稳定期后，由于营养条件及环境条件的改变，菌体开始形成芽孢。芽孢的生成不利于高密度连续发酵，且会影响发酵过程控制，如发酵液粘度增加、泡沫增多等，影响气质交换，容易造成逃液等，增加染菌机会，增加发酵成本；此外，芽孢杆菌在生长过程中常伴有菌体自溶现象，不利于生物量的积累，缩短有效的发酵周期，降低发酵生产效率。
中国专利文件CN108929883A(申请号201810886538.5)公开了芽孢形成相关基因spoⅡE在影响菌株生长及产酶中的应用，该发明通过插入式失活的方式，将spoⅡE部分基因片段与Cmr基因融合，融合基因spoⅡE-Cmr插入克劳氏芽孢杆菌spoⅡE基因序列中，使spoⅡE基因无法正常表达而失活，spoⅡE基因失活菌株不仅可以使芽孢生成率显著降低至0.3％，并且使工程菌株生长速度比原始菌株明显加快，实现了在更短时间内细胞数量的积累和更高活力的淀粉酶的发酵生产。
中国专利文件CN108949785A(申请号201810886539.X)公开了芽孢形成相关基因spo0A在产酶方面的作用，通过插入式失活的方式，将spo0A部分基因片段与Cmr基因融合，融合基因spo0A-Cmr插入克劳氏芽孢杆菌spo0A基因序列中，使spo0A基因无法正常表达而失活，细胞代谢发生变化，在菌株芽孢生成率降低和蛋白酶削弱表达的同时，使淀粉酶酶活较之出发菌株提高85.8％，果胶酶酶活提高235％，脂肪酶酶活提高70.0％。
上述技术方案均发现芽孢形成相关基因spo家族通过敲除后，可以提高淀粉酶的酶活，因此，寻找更多的具有相关功能的基因，对于后续研究和制备淀粉酶具有非常显著的意义。
发明内容
本发明针对现有技术的不足，提供一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用，所得到的基因工程菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf较之出发菌株地衣芽孢杆菌(B.licheniformis)DL-1在发酵生产性能方面有显著提高。
本发明技术方案如下：
一种高产α-淀粉酶的重组菌，将产α-淀粉酶微生物中的芽孢形成相关基因spoⅡQ和溶菌相关基因pcf敲除后获得，所述的芽孢形成相关基因spoⅡQ核苷酸序列如SEQ ID NO.1所示，溶菌相关基因pcf核苷酸序列如SEQ ID NO.2所示。
根据本发明优选的，所述产α-淀粉酶微生物为地衣芽孢杆菌；进一步优选的，所述地衣芽孢杆菌为地衣芽孢杆菌(B.licheniformis)DL-1。
上述高产α-淀粉酶的重组菌的制备方法，步骤如下：
(1)敲除产α-淀粉酶微生物的芽孢形成基因spoⅡQ，获得重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ；
(2)敲除步骤(1)制得的重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ中菌体自溶基因pcf，获得重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf；
根据本发明优选的，所述步骤(1)中，敲除产α-淀粉酶微生物的芽孢形成基因spoⅡQ的具体步骤如下：
步骤1：以地衣芽孢杆菌基因组DNA为模板，设计上、下游同源臂引物spoⅡQF1、spoⅡQR1以及spoⅡQF2、spoⅡQR2，进行PCR扩增，获得spoⅡQ上、下游同源臂片段，然后通过重叠PCR将同源臂连接获得融合片段spoⅡQF-spoⅡQR；
所述上、下游同源臂引物spoⅡQF1、spoⅡQR1以及spoⅡQF2、spoⅡQR2的核苷酸序列如下所示：
spoⅡQF1：CATCGCCAGTCACTAGGATCCACGCCGAGGACGTAGCCT
spoⅡQR1:TTACCGTGCTTGGCATTATCCCTCGCTGAACATGTCCCGCTTT
spoⅡQF2:AAAGCGGGACATGTTCAGCGAGGGATAATGCCAAGCACGGTAA
spoⅡQR2:CAGTATTGACTGCAGGCGGCCGCAAGCTTGAACGGCTGCCTATT
步骤2：利用BamHⅠ与NotⅠ对敲除体pTOPO-Cmr进行双酶切、纯化，利用无缝克隆技术将步骤1获得的融合片段与载体连接，获得重组载体pTOPO-Cmr-spoⅡQ；
步骤3：制备地衣芽孢杆菌感受态细胞，将步骤2获得的重组载体电转化入地衣芽孢杆菌菌体中，涂布含氯霉素LB固体培养基上进行初筛，初筛菌株转接至含5％木糖的LB液体培养基复筛，制得重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ。
根据本发明优选的，所述步骤(2)中，敲除重组菌ΔspoⅡQ中菌体自溶基因pcf的具体步骤如下：
步骤I：以地衣芽孢杆菌基因组DNA以及pHT01质粒为模板，设计上下游引物pcfF、pcfR以及Cm rF、Cm rR，PCR扩增获得获得pcf基因片段以及氯霉素抗性Cm r基因片段，通过重叠PCR将pcf、Cm r基因片段连接获得融合片段pcf-Cm r；
所述引物pcfF、pcfR以及Cm rF、Cm rR核苷酸序列如下：
pcfF CGCGGATCCTGTATAAGCCCTATCAAGATG
pcfR TTACCGTGCTTGGCATTATCCCTCGCTGAACATGTCCCGCTTT
Cm rF CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTCAC
Cm rR_CGCGGATCCTAGTGACTGGCGATGCTGTCGG
步骤II：对步骤I获得的pcf-Cm r利用BamHⅠ进行单酶切、纯化后电转化至重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ菌体中；
步骤III：将步骤II获得的转化菌涂布含氯霉素的LB固体培养基上，筛选具有氯霉素抗性的阳性基因缺失重组菌，即得重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf；
一种利用上述高产α-淀粉酶的重组菌制备α-淀粉酶的方法，步骤如下：
(i)将高产α-淀粉酶的重组菌进行活化，制得活化菌体；
(ii)将步骤(i)制得的活化菌体经扩大培养，制得发酵菌体；
(iii)将步骤(ii)制得的发酵菌体接种至发酵培养基，经37℃，300rpm条件下发酵84h，经固液分离，取液体，纯化，制得α-淀粉酶；
所述发酵培养基组份如下：
胰蛋白胨10g/L、酵母浸粉5g/L、可溶性淀粉20g/L氯化钠10g/L、葡萄糖1g/L、甘油2.5％(体积分数)，pH 7.0。
根据本发明优选的，所述步骤(i)中，甘油管取菌液在LB平板划线，37℃培养24h，挑取单菌落转接至50mL的液体LB培养基，37℃、200rpm培养20h传代两次制得活化菌体。
根据本发明优选的，所述步骤(ii)中，将步骤(i)获得的活化菌体按2％接种量，转接至100mL LBPG培养基37℃、200rpm进行扩培。
所述LBPG培养基组份如下：
胰蛋白胨10g/L、酵母浸粉5g/L、氯化钠10g/L、葡萄糖1g/L、甘油2.5％(体积分数)，pH 7.0。
根据本发明优选的，所述步骤(iii)中，利用高速冷冻离心机将发酵液在4℃、12000rpm条件下离心10min获得粗酶液，用于后续酶活测定。
有益效果
1、发明人通过研究发现芽孢形成相关基因spoⅡQ不同于现有技术中已知的spo族基因的特性，敲除spoⅡQ后并不能直接提高α-淀粉酶的产量，但当其同时与具有促进菌体自溶基因pcf敲除后，可以显著提升α-淀粉酶的产量；
2、本发明通过基因敲除的方式获得重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf，在LB培养基培养，重组菌芽孢生成率降低至出发菌菌株的0.16％，菌体生长更加稳定，摇瓶发酵显示重组菌较出发菌α-淀粉酶酶活提高了10.9％；5L发酵罐发酵显示，重组菌较出发菌α-淀粉酶酶活提高了19.3％，较摇瓶发酵时α-淀粉酶酶活提高了3.4倍。在发酵中后期，重组菌较出发菌发酵液中的溶氧高，泡沫产量少，发酵性能较出发菌有一定提高。
附图说明
图1、本发明实施例1中琼脂糖凝胶电泳结果图片；
其中，(a)、spoⅡQ上游同源臂片段电泳结果图片；图中：泳道1、2为600bp的上游同源臂片段，泳道M为Marker；
(b)、spoⅡQ下游同源臂片段电泳结果图片；图中：泳道1、2为600bp的下游同源臂 片段，泳道M为Marker；
(c)、融合片段spoⅡQF-spoⅡQR的电泳结果图片；图中：泳道1、2为1200bp的目的片段，泳道M为Marker；
图2、本发明实施例2中琼脂糖凝胶电泳结果图片；
其中，(a)、pcf基因片段电泳结果图片；图中：泳道1、2、3为418bp的pcf部分基因片段，泳道M为Marker；
(b)、Cm r基因片段电泳结果图片；图中：泳道1、2、3为1245bp的Cm r基因片段，泳道M为Marker；
(c)、融合片段pcf-Cm r的电泳结果图片；图中：泳道1、2、3为1663bp的目的片段，泳道M为Marker；
图3为本发明实施例3中摇瓶培养出发菌地衣芽孢杆菌(B.licheniformis)DL-1与重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ芽孢形成率测定结果柱状图；
图4为本发明实施例4中摇瓶发酵出发菌与重组菌菌体生长周期测定结果；
图5为本发明实施例5中出发菌与重组菌α-淀粉酶活力平板初测结果照片；
其中，(a)、出发菌地衣芽孢杆菌(B.licheniformis)DL-1结果图片，(b)、重组菌地衣芽孢杆菌(B.licheniformis)DL-1spoⅡQ结果图片，(c)重组菌地衣芽孢杆菌(B.licheniformis)DL-1Δpcf结果图片，(d)、重组菌地衣芽孢杆菌(B.licheniformis)DL-1spoⅡQΔpcf结果图片；
图6为本发明实施例5中出发菌与重组菌摇瓶发酵α-淀粉酶酶活测定结果柱状图；
图7为本发明实施例6中5L发酵罐发酵出发菌与重组菌生长测定结果曲线图其中(a)为生长曲线(b)为溶氧曲线；
图8为本发明实施例6中5L发酵罐发酵出发菌与重组菌α-淀粉酶酶活测定结果柱状图。
具体实施方式
下面结合实施例对本发明的技术方案做进一步阐述，但本发明所保护范围不限于此。
生物材料来源：
实施例中的地衣芽孢杆菌(B.licheniformis)DL-1购自美国模式培养物集存库，菌种编号ATCC NO.27811。
实施例1重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ的构建
以地衣芽孢杆菌基因组为模板，进行spoⅡQ基因上、下游同源臂spoⅡQF、spoⅡQR以及融合片段spoⅡQF-spoⅡQR PCR扩增，引物核苷酸序列如下：
spoⅡQF1：CATCGCCAGTCACTAGGATCCACGCCGAGGACGTAGCCT
spoⅡQR1:TTACCGTGCTTGGCATTATCCCTCGCTGAACATGTCCCGCTTT
spoⅡQF2:AAAGCGGGACATGTTCAGCGAGGGATAATGCCAAGCACGGTAA
spoⅡQR2:CAGTATTGACTGCAGGCGGCCGCAAGCTTGAACGGCTGCCTATT
扩增产物以1％琼脂糖凝胶电泳检测，600bp及1200bp左右出现特异性条带，结果见 图1(a)、(b)、(c)。
利用BamHⅠ与NotⅠ对敲除体pTOPO-Cmr进行双酶切，按照纯化试剂盒提供的步骤进行纯化，利用无缝克隆将融合片段与载体连接，连接体系如下表所示:
Figure PCTCN2020084262-appb-000001
利用电转化将重组载体转入地衣芽孢杆菌菌体，37℃、180rpm复苏1h，涂布含氯霉素抗性平板筛选发生一次同源单交换菌株，将成功发生一次同源交换菌株转接至含5％木糖的LB液体培养基37℃、200rpm传代3次，每代24h筛选发生第二次同源单交换菌株，最终成功构建重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ。
实施例2重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf、地衣芽孢杆菌(B.licheniformis)DL-1Δpcf的构建
由于重组菌地衣芽孢杆菌(B.licheniformis)DL-1Δpcf与重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf构建过程与方法相同因此只以重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf为例进行说明；
以地衣芽孢杆菌基因组DNA以及pHT01质粒为模板，PCR扩增pcf、Cm r以及重组片段pcf-Cm r，使用引物核苷酸序列如下：
pcfF CGCGGATCCTGTATAAGCCCTATCAAGATG
pcfR TTACCGTGCTTGGCATTATCCCTCGCTGAACATGTCCCGCTTT
Cm rF CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTCAC
Cm rR CGCGGATCCTAGTGACTGGCGATGCTGTCGG
扩增产物以1％琼脂糖凝胶电泳检测，分别在600bp、1200bp、1600bp左右出现特异性条带，结果见图2(a)、(b)、(c)。成功获得pcf-Cm r重组片段。
利用BamHⅠ对重组片段pcf-Cm r单酶切，之后进行纯化，利用电转化将重组片段转入spoⅡQ基因缺失菌，37℃、180rpm复苏1h，涂布含氯霉素抗性平板，筛选阳性重组菌，最终成功构建地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf、地衣芽孢杆菌(B.licheniformis)DL-1Δpcf重组菌。
实施例3出发菌与重组菌(B.licheniformis)DL-1ΔspoⅡQ芽孢形成率和酶活测定
接种环甘油管取出发菌地衣芽孢杆菌(B.licheniformis)DL-1与重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ菌液一环划线于LB平板，37℃培养24h，挑取平板上的 单菌落接种于50mL的液体LB培养基，37℃、200rpm培养20h传代两次；取第2代活化的菌液按2％的接种量将出发菌株与重组菌转接至100mL液体LB培养基，37℃，200rpm摇瓶培养36h，取出发菌及重组菌菌液各2mL，于80℃水浴中热处理15min，梯度稀释后涂布LB固体培养基，37℃培养20h，平板菌落计数，根据公式芽孢生成率＝芽孢量/总菌量×100％，计算出发菌及重组菌芽孢生成率。结果见图3，出发菌株地衣芽孢杆菌(B.licheniformis)DL-1芽孢数为4.62×10 8CFU/mL，菌体总数为5.79×10 8CFU/mL，芽孢生成率约为79.82％；重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡ芽孢数为0.51×10 6CFU/mL，菌体总数为3.19×10 8CFU/mL，芽孢生成率约为0.16％。
敲除基因spoⅡQ可以获得芽孢缺失菌株，但芽孢缺失工程菌菌体易自溶，生物量较出发菌株降低。在地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ基础上构建重组菌株地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf降低芽孢缺失菌的菌体自溶。
出发菌地衣芽孢杆菌(B.licheniformis)DL-1与重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ分别发酵60h与48h达到最大酶活，分别为391U/mL、340U/mL；重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ与出发菌地衣芽孢杆菌(B.licheniformis)DL-1相比较，α-淀粉酶发酵活力有所下降。
实施例4摇瓶培养出发菌与重组菌生长周期的测定
出发菌地衣芽孢杆菌(B.licheniformis)DL-1及重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ、重组菌地衣芽孢杆菌(B.licheniformis)DL-1Δpcf、重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf按实施例1的方法进行活化，取活化的菌液2mL接种于100mL LBPG液体培养基37℃、200rpm摇瓶培养，期间每隔4-6h取样测定菌体浓度OD 600，并绘制生长曲线，结果见图4。所述涉及到的培养基，其组分如下：
LB培养基：胰蛋白胨1％，酵母浸粉0.5％，NaCl 1％，(均为质量分数)pH7.0。
LBPG培养基：胰蛋白胨1％，酵母浸粉0.5％，NaCl 1％，葡萄糖0.1％，(均为质量分数)甘油2.5％(体积分数)pH7.0。
实施例5出发菌与重组菌摇瓶发酵菌体发酵产酶的测定
出发菌株地衣芽孢杆菌(B.licheniformis)DL-1、重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ、重组菌地衣芽孢杆菌(B.licheniformis)DL-1Δpcf和重组菌株地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf接种于固体培养基，37℃培养48h，平板滴加碘液并观察透明圈大小；结果见图5(a)、(b)、(c)、(d)，淀粉水解透明圈直径大小分别为2.7cm、2.2cm、3.1cm、3.4cm，初步鉴定重组菌地衣芽孢杆菌(B.licheniformis)DL-1Δpcf和重组菌株地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf发酵产α-淀粉酶的能力高于出发菌。出发菌株与重组菌株分别以2％的接种量接种于100mL液体发酵培养基，37℃、200rpm培养，期间每隔12h取样按照国标GB/T24401-2009方法测定α-淀粉酶酶活，结果见图6。出发菌地衣芽孢杆菌(B.licheniformis)DL-1、重组菌地衣芽孢杆菌(B.licheniformis)DL-1Δpcf与地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf都是在发酵60h达到最大酶活，分别 为391U/mL、407U/mL和434U/mL，地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQ在发酵48h达到最大酶活340U/mL，地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf较地衣芽孢杆菌(B.licheniformis)DL-1酶活提高了10.9％；地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf较重组菌地衣芽孢杆菌(B.licheniformis)DL-1Δpcf酶活提高了6.6％；进一步确定构建的的重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf能有效提高α-淀粉酶发酵活力。
α-淀粉酶活力定义：1ml液体酶，于70℃、pH6.0条件下，1min液化1mg可溶性淀粉，即为1个酶活力单位，以U/mL表示。
所述发酵培养基组分如下：
可溶性淀粉2％、胰蛋白胨1％、酵母浸粉0.5％、氯化钠1％、葡萄糖0.1％、甘油2.5％(均为质量分数)pH 7.0。
实施例6出发菌与重组菌5L罐菌体发酵性能的测定
按实施例1中的方法对出发菌地衣芽孢杆菌(B.licheniformis)DL-1、重组菌地衣芽孢杆菌(B.licheniformis)DL-1Δpcf及重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf进行活化，按5％接种至5L发酵罐中，发酵液初始总量为3L，pH7.5，37℃，350rpm条件下进行发酵，发酵周期84h，期间每隔6-8h取样测定菌体浓度OD 600，每隔12h取样进行α-淀粉酶酶活测定，菌体生长曲线见图7。与摇瓶发酵相比，5L罐发酵时菌体浓度更高，且菌体生长更加稳定；α-淀粉酶酶活测定结果见图8，重组菌地衣芽孢杆菌(B.licheniformis)DL-1Δpcf、重组菌地衣芽孢杆菌(B.licheniformis)DL-1ΔspoⅡQΔpcf酶活达到1397U/mL、1488U/mL，较出发菌α-淀粉酶酶活分别提高12.1％、19.3％。

Claims (9)

  1. 一种高产α-淀粉酶的重组菌，将产α-淀粉酶微生物中的基因spo ⅡQ和基因pcf敲除后获得，所述的基因spo ⅡQ核苷酸序列如SEQ ID NO.1所示，基因pcf核苷酸序列如SEQ ID NO.2所示；
    所述产α-淀粉酶微生物为地衣芽孢杆菌(B.lichenifsrmis)DL-1，来源于美国模式培养物集存库，菌种编号ATCC NO.27811。
  2. 权利要求1所述高产α-淀粉酶的重组菌的制备方法，其特征在于，步骤如下：
    (1)敲除产α-淀粉酶微生物的基因spo ⅡQ，获得重组菌地衣芽孢杆菌(B.lichenifsrmis)DL-1Δspo ⅡQ；
    (2)敲除步骤(1)制得的重组菌地衣芽孢杆菌(B.lichenifsrmis)DL-1Δspo IIQ中基因scf，获得重组菌地衣芽孢杆菌(B.lichenifsrmis)DL-1Δspo IIQΔscf。
  3. 权利要求2所述高产α-淀粉酶的重组菌的制备方法，其特征在于，所述步骤(1)中，敲除产α-淀粉酶微生物的芽孢形成基因spo IIQ的具体步骤如下：
    步骤1：以地衣芽孢杆菌基因组DNA为模板，设计上、下游同源臂引物spo IIQF1、spo IIQR1以及spo IIQF2、spo IIQR2，进行PCR扩增，获得spo IIQ上、下游同源臂片段，然后通过重叠PCR将同源臂连接获得融合片段spo IIQF-spo IIQR；
    所述上、下游同源臂引物spo IIQF1、spo IIQR1以及spo IIQF2、spo IIQR2的核苷酸序列如下所示：
    spo IIQF1：CATCGCCAGTCACTAGGATCCACGCCGAGGACGTAGCCT；
    spo IIQs1:TTACCGTGCTTGGCATTATCCCTCGCTGAACATGTCCCGCTTT；
    spo IIQF2:AAAGCGGGACATGTTCAGCGAGGGATAATGCCAAGCACGGTAA；
    spo IIQR2:CAGTATTGACTGCAGGCGGCCGCAAGCTTGAACGGCTGCCTATT；
    步骤2：利用BamH Ⅰ与Nst Ⅰ对敲除体pTOPO-Cmr进行双酶切、纯化，利用无缝克隆技术将步骤1获得的融合片段与载体连接，获得重组载体pTOPO-Cmr-spo IIQ；
    步骤3：制备地衣芽孢杆菌感受态细胞，将步骤2获得的重组载体电转化入地衣芽孢杆菌菌体中，涂布含氯霉素LB固体培养基上进行初筛，初筛菌株转接至含5％木糖的LB液体培养基复筛，制得重组菌地衣芽孢杆菌(B.lichenifsrmis)DL-1Δspo IIQ。
  4. 权利要求2所述高产α-淀粉酶的重组菌的制备方法，其特征在于，所述步骤(2)中，敲除重组菌Δspo IIQ中菌体自溶基因scf的具体步骤如下：
    步骤I：以地衣芽孢杆菌基因组DNA以及pHT01质粒为模板，设计上下游引物scfF、scfR以及Cm rF、Cm rR，PCR扩增获得获得scf基因片段以及氯霉素抗性Cm r基因片段，通过重叠PCR将scf、Cm r基因片段连接获得融合片段scf-Cm r；
    所述引物scfF、scf R以及Cm rF、Cm rR核苷酸序列如下：
    scfF CGCGGATCCTGTATAAGCCCTATCAAGATG
    scfR TTACCGTGCTTGGCATTATCCCTCGCTGAACATGTCCCGCTTT
    Cm rF CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTCAC
    Cm rR_CGCGGATCCTAGTGACTGGCGATGCTGTCGG
    步骤II：对步骤I获得的scf-Cm r利用BamH Ⅰ进行单酶切、纯化后电转化至重组菌地衣芽孢杆菌(B.lichenifsrmis)DL-1Δspo IIQ菌体中；
    步骤III：将步骤II获得的转化菌涂布含氯霉素的LB固体培养基上，筛选具有氯霉素抗性的阳性基因缺失重组菌，即得重组菌地衣芽孢杆菌(B.lichenifsrmis)DL-1Δspo IIQΔscf。
  5. 一种利用权利要求1所述高产α-淀粉酶的重组菌制备α-淀粉酶的方法，其特征在于，步骤如下：
    (i)将高产α-淀粉酶的重组菌进行活化，制得活化菌体；
    (ii)将步骤(i)制得的活化菌体经扩大培养，制得发酵菌体；
    (iii)将步骤(ii)制得的发酵菌体接种至发酵培养基，经37℃，300rpm条件下发酵84h，经固液分离，取液体，纯化，制得α-淀粉酶；
    所述发酵培养基组份如下：
    胰蛋白胨10g/L、酵母浸粉5g/L、可溶性淀粉20g/L氯化钠10g/L、葡萄糖1g/L、甘油2.5％(体积分数)，pH 7.0。
  6. 如权利要求5所述的方法，其特征在于，所述步骤(i)中，甘油管取菌液在LB平板划线，37℃培养24h，挑取单菌落转接至50mL的液体LB培养基，37℃、200rpm培养20h传代两次制得活化菌体。
  7. 如权利要求5所述的方法，其特征在于，所述步骤(ii)中，将步骤(i)获得的活化菌体按2％接种量，转接至100mL LBPG培养基37℃、200rpm进行扩培。
  8. 如权利要求7所述的方法，其特征在于，所述LBPG培养基组份如下：
    胰蛋白胨10g/L、酵母浸粉5g/L、氯化钠10g/L、葡萄糖1g/L、甘油2.5％(体积分数)，pH 7.0。
  9. 如权利要求5所述的方法，其特征在于，所述步骤(iii)中，利用高速冷冻离心机将发酵液在4℃、12000rpm条件下离心10min获得粗酶液，用于后续酶活测定。
PCT/CN2020/084262 2019-05-10 2020-04-10 一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用 WO2020228458A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020207017367A KR102246356B1 (ko) 2019-05-10 2020-04-10 spoⅡQ와 pcf 유전자 녹아웃을 통해 바실러스 리케니포르미스 발효 효소 생산을 향상시키는 방법 및 응용

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910389741.6A CN110055204B (zh) 2019-05-10 2019-05-10 一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用
CN201910389741.6 2019-05-10

Publications (1)

Publication Number Publication Date
WO2020228458A1 true WO2020228458A1 (zh) 2020-11-19

Family

ID=67322921

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/084262 WO2020228458A1 (zh) 2019-05-10 2020-04-10 一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用

Country Status (3)

Country Link
KR (1) KR102246356B1 (zh)
CN (1) CN110055204B (zh)
WO (1) WO2020228458A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108823185A (zh) * 2018-06-25 2018-11-16 安徽新熙盟生物科技有限公司 高酶活发酵液的培养方法及提取耐酸α-淀粉酶的方法
CN114276952A (zh) * 2021-12-06 2022-04-05 湖北工业大学 一种提高地衣芽胞杆菌芽胞形成效率的方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109868253B (zh) * 2019-01-11 2020-03-13 齐鲁工业大学 抑制菌体自溶的地衣芽孢杆菌工程菌及其构建方法和应用
CN110055204B (zh) * 2019-05-10 2020-04-10 齐鲁工业大学 一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用
CN112159786B (zh) * 2020-11-04 2022-03-01 河北科技大学 一种Cr(Ⅵ)还原菌株C6及其培养条件和应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006073514A2 (en) * 2004-08-25 2006-07-13 Tufts University Compositions, methods and kits for repressing virulence in gram positive bacteria
CN1926431A (zh) * 2004-01-09 2007-03-07 诺维信股份有限公司 地衣芽孢杆菌染色体
CN1930289A (zh) * 2004-03-05 2007-03-14 花王株式会社 变异芽孢杆菌属细菌
WO2019002218A2 (en) * 2017-06-25 2019-01-03 Snipr Technologies Limited MODIFICATION OF MICROBIAL POPULATIONS AND MODIFICATION OF MICROBIOTA
CN110055204A (zh) * 2019-05-10 2019-07-26 齐鲁工业大学 一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006296268A (ja) * 2005-04-19 2006-11-02 Kao Corp 組換え微生物
CN108949785B (zh) * 2018-08-06 2020-03-06 齐鲁工业大学 芽孢形成相关基因spo0A在产酶中的应用
CN108949784B (zh) * 2018-08-06 2020-01-10 齐鲁工业大学 芽孢形成相关基因sigmaF在产酶中的应用
CN108929883B (zh) * 2018-08-06 2020-05-01 齐鲁工业大学 芽孢形成相关基因spoⅡE在影响菌株生长及产酶中的应用
CN108913645B (zh) * 2018-08-08 2019-06-28 绿康生化股份有限公司 敲除malR的地衣芽胞杆菌菌株在杆菌肽生产中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1926431A (zh) * 2004-01-09 2007-03-07 诺维信股份有限公司 地衣芽孢杆菌染色体
CN1930289A (zh) * 2004-03-05 2007-03-14 花王株式会社 变异芽孢杆菌属细菌
WO2006073514A2 (en) * 2004-08-25 2006-07-13 Tufts University Compositions, methods and kits for repressing virulence in gram positive bacteria
WO2019002218A2 (en) * 2017-06-25 2019-01-03 Snipr Technologies Limited MODIFICATION OF MICROBIAL POPULATIONS AND MODIFICATION OF MICROBIOTA
CN110055204A (zh) * 2019-05-10 2019-07-26 齐鲁工业大学 一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANA B ABECASIS ET AL.: "A Genomic Signature and the Identification of New Sporulation Genes", J BACTERIOL, vol. 195, no. 9, 31 May 2013 (2013-05-31), XP055753403, ISSN: 0021-9193, DOI: 20200624091229 *
G E MCDONNELL ET AL.: "Genetic Control of Bacterial Suicide: Regulation of the Induction of PBSX in Bacillus Subtilis", J BACTERIOL, vol. 176, no. 18, 30 September 1994 (1994-09-30), XP055753407, ISSN: 0021-9193, DOI: 20200624162255A *
ZHANG, HU ET AL.: "Non-official translation: Study on Autolytic Gene Knockout in Bacillus Licheniformis", BASIC SCIENCES, CHINA MASTER’S THESES FULL-TEXT DATABASE, no. 9, 15 September 2019 (2019-09-15), ISSN: 1674-0246, DOI: 20200624162601PX *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108823185A (zh) * 2018-06-25 2018-11-16 安徽新熙盟生物科技有限公司 高酶活发酵液的培养方法及提取耐酸α-淀粉酶的方法
CN114276952A (zh) * 2021-12-06 2022-04-05 湖北工业大学 一种提高地衣芽胞杆菌芽胞形成效率的方法
CN114276952B (zh) * 2021-12-06 2023-08-18 湖北工业大学 一种提高地衣芽胞杆菌芽胞形成效率的方法

Also Published As

Publication number Publication date
KR102246356B1 (ko) 2021-04-29
CN110055204A (zh) 2019-07-26
CN110055204B (zh) 2020-04-10
KR20200132837A (ko) 2020-11-25

Similar Documents

Publication Publication Date Title
WO2020228458A1 (zh) 一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用
JPH06505875A (ja) 組換え宿主によるエタノール生産
CN102676480A (zh) 一种应用自诱导培养基和双温度调控策略生产胞外普鲁兰酶的方法
CN104593308A (zh) 一种基因工程菌及其构建方法和在生产木糖醇中的应用
CN103898035A (zh) 生产β-丙氨酸的重组大肠杆菌菌株及其构建方法和应用
US20230272443A1 (en) N-Acetylglucosamine-Producing Bacterial Strain As Well As Method Of Construction And Use Thereof
CN107904223A (zh) 一种褐藻胶裂解酶、分泌褐藻胶裂解酶的宿主细胞及其应用
CN105647822A (zh) 一株过表达来源于寄生疫霉的ω-3脱饱和酶的重组高山被孢霉、其构建方法及应用
CN105624051B (zh) 基于进化工程构建的木糖发酵酵母菌株及构建方法
CN102226166A (zh) 高效表达普鲁兰酶的基因工程菌株及其构建方法
He et al. Direct production of ethanol from raw sweet potato starch using genetically engineered Zymomonas mobilis
CN102851266B (zh) 一株耐热α-淀粉酶及其基因工程菌的构建方法
WO2020134427A1 (zh) sll0528基因在提高集胞藻PCC6803乙醇耐受性中的应用
CN104560845A (zh) 一种表达嗜热木聚糖酶的重组枯草芽孢杆菌的构建方法
CN106636238A (zh) 一种微生物利用酒糟发酵生产γ‑氨基丁酸的方法
CN105296523A (zh) 一种信号肽及其在利用淀粉产γ-氨基丁酸重组菌中的应用
CN109868253B (zh) 抑制菌体自溶的地衣芽孢杆菌工程菌及其构建方法和应用
CN105112348B (zh) 一种高产普鲁兰酶的重组短小芽孢杆菌及其应用
CN107083375B (zh) 一种中温α-淀粉酶及其基因和应用
CN107475140B (zh) 一株在酸性条件下发酵速度提高的高产普鲁兰酶的重组巴斯德毕赤酵母突变体
CN111893107A (zh) 一株异源表达纤维素酶基因egⅳ的毕赤酵母工程菌株及应用
CN110643648A (zh) 一种高效利用淀粉生产l-亮氨酸的方法
CN105586365B (zh) 一种发酵生产混合醇的方法
CN111334446A (zh) 一株耐高温糖化酵母菌株及其应用
CN112779174B (zh) 一株敲除Cln3基因的酿酒酵母基因工程菌及其构建方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20806401

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20806401

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC