WO2017088095A1 - 一种信号肽及其在利用魔芋粉合成l-精氨酸及其高值化的应用 - Google Patents

一种信号肽及其在利用魔芋粉合成l-精氨酸及其高值化的应用 Download PDF

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WO2017088095A1
WO2017088095A1 PCT/CN2015/095286 CN2015095286W WO2017088095A1 WO 2017088095 A1 WO2017088095 A1 WO 2017088095A1 CN 2015095286 W CN2015095286 W CN 2015095286W WO 2017088095 A1 WO2017088095 A1 WO 2017088095A1
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signal peptide
seq
gene
arginine
recombinant
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French (fr)
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饶志明
许正宏
郑俊贤
徐美娟
杨套伟
张显
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江南大学
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Priority to PCT/CN2015/095286 priority Critical patent/WO2017088095A1/zh
Priority to US15/736,544 priority patent/US10351816B2/en
Publication of WO2017088095A1 publication Critical patent/WO2017088095A1/zh

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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
    • 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
    • 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
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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    • 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
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
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    • 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)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • C12N9/2491—Beta-mannosidase (3.2.1.25), i.e. mannanase
    • 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)
    • C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase

Definitions

  • the invention relates to a signal peptide and an application thereof for synthesizing L-arginine by using konjac powder and high value thereof, and belongs to the technical field of genetic engineering and metabolic engineering.
  • Konjac powder mainly containing konjac glucomannan, its source is non-food crop konjac.
  • Konjac is mainly distributed in China, Japan, Sri, Indonesia, Southeast Asia and Africa. It can be interplanted with grain crops or planted in silt or beach. It is a kind of resource that is not competitive with food crops and can be further developed.
  • the use of the Araceae plant According to relevant reports, it is suitable for konjac planting in Yunnan, Guizhou, Sichuan, Hubei, Hunan, and soiled Shaanxi. It has a certain scale, and only konjac planting area in Hubei Province is 410,000 mu.
  • konjac flour is mainly used for some foods, and its hydrolyzed oligosaccharides can be used as functional foods, and the application is not very extensive.
  • Konjac is mainly identified as one of the top ten health foods by the United Nations Health Organization as a dietary fiber food.
  • the daily intake of glucomannan in konjac flour is generally less than 10 g, and thus a large excess of konjac can be used for resource development and utilization.
  • Most microorganisms cannot be directly used, so it is a good application prospect for the modified microorganisms to use the cheaper konjac powder as a carbon source to ferment and produce high value-added products.
  • This laboratory is a strain that has acquired and preserved high-yield L-arginine by multi-stage mutagenesis, related genetic engineering and metabolic engineering technology modification: Corynebacterium cloacae CGMCC No.0890.
  • the high-yield L-arginine-preserved strain preserved by the inventor can only produce L-arginine by using glucose as the main carbon source, and cannot directly use konjac powder as the main carbon source.
  • L-arginine As a semi-essential amino acid, L-arginine has a wide range of applications in the pharmaceutical industry, health care products, food and other fields.
  • the high-yield L-arginine strain was further modified by genetic engineering and metabolic engineering techniques to produce L-arginine by fermentation using konjac flour as a carbon source. It has certain reference application value for simplifying L-arginine production process and saving production cost.
  • L-arginine is hydrolyzed by arginase (EC 3.5.3.1) to produce L-ornithine and urea.
  • L-ornithine as a high-valued product of arginine, is an important non-protein amino acid that promotes protein synthesis and catabolism of sugars and lipids and plays an important role in the detoxification of the liver.
  • L-arginine is catalytically degraded by Arginine deiminase (EC 3.5.3.6) to form L-citrulline And ammonia.
  • L-citrulline as a high-value product of arginine, can be used to maintain Jiankang's lung function, improve mental clarity, help brain nerve cells to store and recall information, and relax human blood vessels. Enhance male sexual function and treat sexual dysfunction.
  • the invention combines the ⁇ -mannanase gene for removing the self-signal peptide with the twelve signal peptides of the two main secretory pathways of Sec and Tat derived from Corynebacterium glutamicum and Bacillus subtilis by genetic engineering technology, and replaces the comparison.
  • Different signal peptides direct the secretion of ⁇ -mannanase gene in different L-arginine-producing bacteria, and the codon-optimized ⁇ -mannanase gene signal peptide, Mannase Signal Peptide (MSP), is secreted.
  • MSP Mannase Signal Peptide
  • a first object of the present invention is to provide a signal peptide MSP having a nucleotide sequence of any one of (a) to (e):
  • a second object of the present invention is a method for enhancing the synthesis of a product of a microorganism using konjac flour by fusing the signal peptide with a ⁇ -mannanase gene, and then ligating the fusion fragment to an expression vector and transforming it into a host microorganism Recombinant microorganisms are obtained therein, and the recombinant microorganism is used to ferment a product in a medium containing konjac powder as a main carbon source;
  • the ⁇ -mannanase gene is derived from Bacillus subtilis, but is not limited to Bacillus subtilis.
  • the ⁇ -mannanase gene is derived from Bacillus subtilis B. subtilis CCTCC M 209200, and the nucleotide sequence is set forth in SEQ ID NO: 3.
  • the product is L-arginine, L-ornithine or L-citrulline.
  • the microorganism is Corynebacterium bluntii, but is not limited to the genus Corynebacterium.
  • the microorganism is Corynebacterium cloacae CGMCC 0890 which produces L-arginine with high yield.
  • the expression vector is pXMJ19, but is not limited to pXMJ19.
  • the microorganism further expresses an arginase gene, and the synthetic product is L-ornithine.
  • the arginase gene is derived from Bacillus cereus, but is not limited to waxy buds. Bacillus.
  • the arginase gene is the gene of gi:22934680 on NCBI.
  • the microorganism further expresses an arginine deiminase gene, and the synthetic product is L-citrulline.
  • the arginine deiminase gene is derived from Bacillus cereus, but is not limited to Bacillus cereus.
  • the arginase gene is the gene of gi: 22939780 on NCBI.
  • the carbon source of the culture medium is a mixed carbon source of konjac powder and glucose, wherein the role of glucose is to accelerate the growth of the pre-fermentation bacteria and to induce the induction of konjac flour, and to synthesize and secrete a large amount.
  • Beta-mannanase used to hydrolyze konjac flour to provide an effective carbon source for the production of related products.
  • the medium initially contains 10 g/L of konjac flour and 50 g/L of glucose, and 90 g/L of konjac flour is added by a fed-batch method in the late stage of fermentation.
  • a third object of the present invention is to provide a recombinant Corynebacterium baumannii characterized in that the recombinant Corynebacterium baumannii expresses a ⁇ -mannanase gene fused to the signal peptide of SEQ ID NO: 1; The signal peptide is fused to the ⁇ -mannanase gene, ligated to an expression vector and transformed into a host bacterium.
  • the recombinant Corynebacterium cloacae is CGMCC 0890/pMSPman, the construction method thereof: the signal peptide of SEQ ID NO: 1 and the ⁇ -mannan derived from B. subtilis CCTCC M 209200
  • the gene fusion was carried out, and then the fusion gene was transformed into the expression vector pXMJ19 to obtain a recombinant plasmid pXMJ19-MSPman, and the recombinant plasmid was transformed into Corynebacterium bluntii, and the obtained recombinant strain was CGMCC 0890/pMSPman. This strain ferments to produce L-arginine.
  • the recombinant Corynebacterium cloacae is CGMCC 0890/pMSPman-pargI
  • the method comprises the steps of: ligating the argI gene derived from Bacillus cereus to pDXW10 to construct a recombinant plasmid, and then recombining The plasmid was transformed into CGMCC 0890/pMSPman. This strain ferments to produce L-ornithine.
  • the recombinant Corynebacterium baumannii is CGMCC 0890/pMSPman-padi
  • the method comprises the steps of: ligating a padi gene derived from Bacillus cereus to pDXW10 to construct a recombinant plasmid, and then recombining The plasmid was transformed into CGMCC 0890/pMSPman. This strain is fermentatively produced to produce L-citrulline.
  • the present invention obtains a novel signal peptide, which mediates a recombinant strain secreting ⁇ -mannanase, and has certain advantages in the production of related products using konjac flour as a substrate, in konjac flour. Utilization, product productivity, and yield are higher than those mediated by other signal peptides. Therefore, recombinant bacteria containing this signal peptide are more advantageous in reducing the process cost of producing L-arginine and its high value product by using cheaper konjac powder as a substrate.
  • the present invention is based on the existing high-yield L-arginine species, and the ⁇ -mannan gene secreted by the signal peptide of the present invention is expressed in different L-arginine-producing bacteria, thereby enabling L-arginine is produced by fermentation using konjac flour and glucose as a mixed carbon source.
  • the recombinant plasmid CGMCC 0890/pMSPman constructed by the present invention optimizes the yield of L-arginine in the 5L tank after fermentation for 96 hours, and the enzyme activity of ⁇ -mannanase in the fermentation broth is 1505 ⁇ 5.7 U/mL. (Carbon source addition method: initial 10g/L konjac flour and 50g/L glucose, and a total of 90g/L konjac powder in the later batch).
  • the present invention converts the arginase gene into and expresses it by genetic engineering technology on the basis of the constructed recombinant strain C. conococci CGMCC 0890/pMSPman, and obtains the recombinant Corynebacterium cloacae CGMCC 0890/pMSPman- pargI.
  • the recombinant Corynebacterium cloacae CGMCC 0890/pMSPman-pargI can ferment L-ornithine with konjac flour and glucose as a mixed carbon source.
  • the recombinant Corynebacterium cloacae CGMCC 0890/pMSPman-pargI constructed by the invention is optimized by carbon source to optimize the yield of L-ornithine fermentation in a 5L tank for 96h, and the yield of ⁇ -mannanase in the fermentation broth is 23.5g/L.
  • the activity was 1610 ⁇ 3.8 U / mL. (Carbon source addition method: initial 10g/L konjac flour and 50g/L glucose, and a total of 90g/L konjac powder in the later batch).
  • the present invention converts the arginine deiminase gene into the recombinant bacterium of the genus Corynebacterium CGMCC 0890/pMSPman based on the constructed recombinant strain CGMCC 0890/pMSPman, and obtains the recombinant Corynebacterium cloacae CGMCC 0890. /pMSPman-padi.
  • the recombinant Corynebacterium cloacae CGMCC 0890/pMSPman-padi can ferment L-citrulline with konjac flour and glucose as a mixed carbon source.
  • the recombinant Corynebacterium cloacae CGMCC 0890/pMSPman-padi constructed by the invention is optimized by carbon source to optimize the yield of L-citrulline in a 5L tank for 96h, and the yield of ⁇ -mannanase in the fermentation broth is 26.3g/L.
  • the activity is 1360 ⁇ 6.8 U / mL. (Carbon source addition method: initial 10g/L konjac flour and 50g/L glucose, and a total of 90g/L konjac powder in the later batch).
  • the digested vector and the PCR product were mixed in a certain ratio and ligated overnight under the action of T4 DNA ligase.
  • the ligation product was transferred to E. coli BL21 by CaCl 2 transformation to obtain E. coli BL21 containing the recombinant plasmid.
  • the plasmid was extracted, and the recombinant plasmid was introduced into the relevant strain by electroporation by single-double digestion and PCR verification. Finally, the recombinant bacterial solution containing 15% glycerol was stored in a -70 ° C refrigerator.
  • Acid-producing culture of recombinant bacteria inoculating the recombinant bacteria in fresh LB+0.5% Glucose liquid medium for live The inoculation amount is 1%. The next day was transferred to the fermentation medium at 1% (the substrate was mixed with konjac flour and glucose), and the fermentation broth was collected at the later stage of growth to determine the amino acid content (L-arginine and related amino acids, etc.) using an amino acid automatic analyzer.
  • the fermentation medium has the nutrients required for the growth of microorganisms and is optimized by correlation.
  • Example 1 Primer design of signal peptide primers in tandem with ⁇ -mannanase
  • Signal peptides and gene tandem large fragment primers P1 and P2 (shown as SEQ ID NO: 4 and SEQ ID NO: 5, respectively) were designed based on the relevant gene sequences published on NCBI and the ⁇ -mannanase gene sequence.
  • Example 2 ⁇ -mannanase gene signal peptide replacement and cloning thereof
  • B. subtilis CCTCC M 209200 extracts a chromosome as a template DNA.
  • PCR amplification system 50 ⁇ L: 1 ⁇ L of template, 0.5 ⁇ L of each of the upstream and downstream primers, 4 ⁇ L of dNTP Mix, 5 ⁇ L of 10 ⁇ Ex Taq Buffer, 38.5 ⁇ L of sterilized ddH 2 O, and 0.5 ⁇ L of Ex Taq DNA polymerase.
  • PCR reaction conditions 94 ° C pre-denaturation, 5 min, one cycle; 94 ° C denaturation, 30 s, 56 ° C annealing, 30 s, 72 ° C extension, 1 min 30 s, 30 cycles; 72 ° C, 10 min, one cycle; 4 ° C, 10 min, one Cycling (where the annealing temperature and extension time are adjusted relative to different primers and genes).
  • the PCR product was purified and recovered by a gel recovery kit, and the concentration of the recovered product was examined by electrophoresis. The recovered product was stored in a 1.5 mL centrifuge tube and stored in a -20 ° C refrigerator for later use.
  • the product recovered by PCR in Example 2 was ligated to the cloning vector pMD18-T, the ligation system was solution I 5 ⁇ L, the target gene was 4.8 ⁇ L, and the pMD18-T plasmid was 0.2 ⁇ L, which was ligated overnight at 16 °C.
  • the ligation product was transformed into E.coil JM109, coated on LB plate containing 100 ug/mL ampicillin, cultured overnight at 37 ° C, and single colony was picked into 10 mL liquid LB medium containing 100 ug/mL ampicillin, and shaker was shaken at 37 ° C. After overnight culture, the plasmid was extracted and named as pMD18-T-MSPman. After successful ligation by PCR and restriction enzyme digestion, the bacterial solution was added to glycerol and stored in a -70 °C refrigerator.
  • Example 4 Electroporation of recombinant plasmid pXMJ19-MSPman to Corynebacterium cloacae CGMCC 0890
  • LBG medium medium composition: 0.5% yeast extract, 1% peptone, 1% NaCl, 0.5% glucose
  • the cells were cultured for 64 hours in a constant temperature incubator at 30 ° C, and a colony of normal colony morphology was picked and cultured.
  • the cultured monoclonal was subjected to plasmid extraction, enzyme digestion verification and PCR verification to obtain a positive recombinant strain CGMCC 0890/pMSPman, and stored in a -70 ° C refrigerator.
  • Example 5 Determination of ⁇ -mannanase activity by recombinant Clostridium cloacae CGMCC 0890/pMSPman
  • the recombinant C. falciparum CGMCC 0890/pMSPman seed solution was inoculated to konjac powder medium, and fermented at 30 ° C, 600 r / min culture conditions, 96 h L-arginine yield was 45 ⁇ 0.9 g / L, fermentation
  • the enzyme activity of the ⁇ -mannanase in the liquid was 1505 ⁇ 5.7 U/mL.
  • the initial medium contains the initial konjac flour 10, glucose 50, yeast powder 8, (NH 4 ) 2 SO 4 20, K 2 HPO 4 1.5, MgSO 4 ⁇ 7H 2 O 0.5, FeSO 4 ⁇ 7H 2 O 0.02, MnSO 4 H 2 O 0.02, CaCO 3 30. PH 7.0 ⁇ 7.2; after 12h of fermentation, the konjac powder was added in batches, and the total dosage was 90g/L.
  • nucleotide sequence is SEQ ID NO: 2
  • fermentation shows: L-arginine production
  • the enzyme activity of ⁇ -mannanase in the fermentation broth was 1050 ⁇ 3.8 U/mL at 31 ⁇ 3.7 g/L.
  • nucleotide sequence of the nucleotide sequence of SEQ ID NO: 1 is replaced with a surface protein signal peptide PS such as: Corynebacterium glutamicum (nucleotide sequence such as SEQ ID NO) :6) or the B. subtilis-derived ⁇ -amylase signal peptide AE (the nucleotide sequence is shown in SEQ ID NO: 7), and the fermentation shows that the yield of L-arginine is 26 ⁇ 1.5g, respectively. /L and 23 ⁇ 1.2 g/L, the enzyme activities of ⁇ -mannanase in the fermentation broth were 867 ⁇ 3.7 U/mL and 732 ⁇ 5.9 U/mL, respectively.
  • a surface protein signal peptide PS such as: Corynebacterium glutamicum (nucleotide sequence such as SEQ ID NO) :6) or the B. subtilis-derived ⁇ -amylase signal peptide AE (the nucleotide sequence is shown in SEQ ID NO: 7)
  • the signal peptide of SEQ ID NO: 1 of the present invention can significantly improve the ability of the recombinant bacteria to synthesize the product L-arginine using konjac powder, compared with other signal peptides, and the product can be increased by up to 45.2. %-96%, while the enzyme activity of ⁇ -mannanase in the fermentation broth increased by 43%-105%.
  • Example 7 Determination of recombinant L-arginine by recombinant C. aureus CGMCC 0890/pMSPman
  • the L-arginine content in the fermentation broth was initially determined by the mouthwash reagent method.
  • the fermentation supernatant was diluted 500 times, 1 mL was placed in a colorimetric tube, 4 mL of 0.375 mol/L NaOH solution was added, and 1 mL of the color developer was added thereto, and then shaken in a 30 ° C water bath for 20 minutes, and measured by a spectrophotometer at 520 nm.
  • the absorbance value of each tube was calculated based on the standard curve for the L-arginine content in the fermentation broth. It is accurately determined by an automatic amino acid analyzer.
  • Example 8 Primer design of recombinant C. congener CGMCC 0890/pMSPman-pargI
  • Primers p1 and p2 designed according to the relevant gene sequences published on NCBI and the arginase gene sequence (sequences are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively)
  • the primers designed in Example 8 were used to obtain the argI gene PCR product with EcoRI and SacI restriction sites, and purified by kit.
  • the purified PCR product and the expression vector plasmid pDXW10 were digested with EcoRI and SacI enzymes, respectively, and recovered by gel, and ligated overnight at 16 °C.
  • the ligation product was transformed into E.coil JM109, plated on LB plates containing 100 ug/mL kanamycin, cultured overnight at 37 ° C, and single colonies were picked to 10 mL liquid LB medium containing 100 ug/mL kanamycinmycin.
  • the plasmid was extracted by shaking at 37 ° C overnight, and named as pDXW10-argI. After successful ligation by PCR and restriction enzyme digestion, the bacterial solution was added to glycerol and stored in a -70 ° C refrigerator.
  • Example 10 Recombinant plasmid pDXW10-argI was electrotransferred to Corynebacterium cloacae CGMCC 0890/pMSPman
  • the starting strain for electroporation used was Corynebacterium cloacae CGMCC 0890/pMSPman, and the labeled antibiotics were card. Natamycin.
  • the remaining positive recombinant strain CGMCC 0890/pMSPman-pargI obtained in Reference Example 4 was used.
  • Example 11 Enzyme activity assay of arginase in recombinant C. congener CGMCC 0890/pMSPman-pargI cells
  • a 0.2 M substrate L-arginine (0.2 M carbonate buffer, pH 9.0) was prepared, 0.9 ml of the substrate solution was taken, 0.1 ml of the enzyme solution was added, and the reaction was carried out at 40 ° C for 10 min.
  • the enzyme reaction solution was diluted to a corresponding multiple, and 1 ml of the diluted reaction solution was taken, and the content of L-ornithine in the reaction solution was measured by Chinard colorimetric method.
  • the enzyme activity defines the amount of enzyme required to catalyze the conversion of 1 umol L-arginine to L-ornithine in 1 min.
  • Example 12 Fermentation medium for recombinant Corynebacterium cloacae CGMCC 0890/pMSPman-pargI
  • the recombinant C. falciparum CGMCC 0890/pMSPman-pargI seed solution was inoculated into konjac powder medium, and fermented at 30 ° C, 600 r / min culture conditions, 96 h L-ornithine yield was 23.5 g / L, fermentation
  • the enzyme activity of the ⁇ -mannanase in the liquid was 1610 ⁇ 3.8 U/mL.
  • the initial medium contains the initial konjac flour 10, glucose 50, yeast powder 8, (NH 4 ) 2 SO 4 20, K 2 HPO 4 1.5, MgSO 4 ⁇ 7H 2 O 0.5, FeSO 4 ⁇ 7H 2 O 0.02, MnSO 4 H 2 O 0.02, CaCO 3 30. PH 7.0 ⁇ 7.2; after 12h of fermentation, the konjac powder was added in batches, and the total dosage was 90g/L.
  • nucleotide sequence is SEQ ID NO: 2
  • the fermentation showed that the yield of L-ornithine was 21 ⁇ 0.8 g/L, and the enzyme activity of ⁇ -mannanase in the fermentation broth was 1025 ⁇ 5.8 U/mL.
  • nucleotide sequence of the nucleotide sequence of SEQ ID NO: 1 is replaced with a surface protein signal peptide PS such as: Corynebacterium glutamicum (nucleotide sequence such as SEQ ID NO) :6) or the B. subtilis-derived ⁇ -amylase signal peptide AE (the nucleotide sequence is shown in SEQ ID NO: 7), and the fermentation shows that the yield of L-ornithine is 17 ⁇ 1.5g, respectively. /L and 15 ⁇ 1.3 g/L, the enzyme activities of ⁇ -mannanase in the fermentation broth were 865 ⁇ 6.7 U/mL and 752 ⁇ 3.9 U/mL, respectively.
  • a surface protein signal peptide PS such as: Corynebacterium glutamicum (nucleotide sequence such as SEQ ID NO) :6) or the B. subtilis-derived ⁇ -amylase signal peptide AE (the nucleotide sequence is shown in SEQ ID NO: 7)
  • the signal peptide of SEQ ID NO: 1 of the present invention can significantly improve the ability of the recombinant bacteria to synthesize the product L-ornithine using konjac powder as compared with other signal peptides.
  • Example 13 Determination of L-ornithine in recombinant B. coli CGMCC 0890/pMSPman-pargI fermentation broth
  • the L-ornithine content in the fermentation broth was initially determined by the Chinard colorimetric method, and the relevant samples were accurately determined by an amino acid analyzer.
  • Example 14 Primer design of recombinant Corynebacterium cloacae CGMCC 0890/pMSPman-padi
  • Example 16 Recombinant plasmid pDXW10-adi was electrotransferred to Corynebacterium cloacae CGMCC 0890/pMSPman
  • the starting strain for electroporation used was Corynebacterium cloacae CGMCC 0890/pMSPman, the labeled antibiotic was kanamycin, and the recombinant plasmid was pDXW10-adi.
  • the remaining positive recombinant strain CGMCC 0890/pMSPman-padi obtained in Reference Example 4 was used.
  • Example 17 Enzyme activity assay of arginase in recombinant C. acuminata CGMCC 0890/pMSPman-padi cells
  • a 0.2 M substrate L-arginine (0.2 M carbonate buffer, pH 9.0) was prepared, 0.9 ml of the substrate solution was taken, 0.1 ml of the enzyme solution was added, and the reaction was carried out at 40 ° C for 10 min.
  • the enzyme reaction solution was diluted to a corresponding multiple, and 1 ml of the diluted reaction solution was taken, and the content of L-citrulline in the reaction liquid of the amino acid analyzer was measured.
  • the enzyme activity defines the amount of enzyme required to catalyze the conversion of 1 umol L-arginine to L-citrulline in 1 min.
  • Example 18 Fermentation medium for recombinant Corynebacterium cloacae CGMCC 0890/pMSPman-padi
  • the recombinant C. falciparum CGMCC 0890/pMSPman-padi seed solution was inoculated to konjac powder medium, and fermented at 30 ° C, 600 r / min culture conditions, 96 h L-citrulline yield was 26.3 g / L, fermentation
  • the enzyme activity of the ⁇ -mannanase in the liquid was 1360 ⁇ 6.8 U/mL.
  • the initial medium contains the initial konjac flour 10, glucose 50, yeast powder 8, (NH 4 ) 2 SO 4 20, K 2 HPO 4 1.5, MgSO 4 ⁇ 7H 2 O 0.5, FeSO 4 ⁇ 7H 2 O 0.02, MnSO 4 H 2 O 0.02, CaCO 3 30. PH 7.0 ⁇ 7.2; after 12h of fermentation, the total supplement amount of konjac powder was 90g/L.
  • nucleotide sequence of SEQ ID NO: 1 is replaced with the original signal peptide of the ⁇ -mannanase gene (nucleotide sequence is SEQ ID NO: 2)
  • the fermentation showed that the yield of L-citrulline was 22 ⁇ 0.9 g/L, and the enzyme activity of ⁇ -mannanase in the fermentation broth was 1002 ⁇ 4.7 U/mL.
  • nucleotide sequence of the nucleotide sequence of SEQ ID NO: 1 is replaced with a surface protein signal peptide PS such as: Corynebacterium glutamicum (nucleotide sequence such as SEQ ID NO) :6) or subtilis
  • a surface protein signal peptide PS such as: Corynebacterium glutamicum (nucleotide sequence such as SEQ ID NO) :6) or subtilis
  • the bacterial-derived ⁇ -amylase signal peptide AE (the nucleotide sequence is shown in SEQ ID NO: 7), the fermentation showed that the yield of L-citrulline was 19 ⁇ 1.3 g/L and 16 ⁇ 0.7 g/L, respectively.
  • the enzyme activities of ⁇ -mannanase in the fermentation broth were 832 ⁇ 7.7 U/mL and 763 ⁇ 6.9 U/mL, respectively.
  • the signal peptide of SEQ ID NO: 1 of the present invention can significantly improve the ability of the recombinant bacteria to synthesize the product L-citrulline using konjac powder as compared with other signal peptides.
  • Example 19 Determination of L-citrulline in recombinant B. coli CGMCC 0890/pMSPman-padi fermentation broth
  • the L-citrulline content in the fermentation broth was accurately determined by an amino acid analyzer.

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Abstract

提供了一种信号肽及其在利用魔芋粉合成L-精氨酸及其高值化中的应用，所述信号肽的核苷酸序列为SEQ ID NO:1。本发明将所述信号肽与来源 B.subtilis CCTCC M 209200的β-甘露聚糖酶融合，在产L-精氨酸的钝齿棒状杆菌中表达，所得重组钝齿棒状杆菌能利用魔芋粉为碳源，生产L-精氨酸；在此基础上进一步构建两株分别产L-鸟氨酸和L-瓜氨酸的菌株。

Description

[根据细则26改正06.01.2016]　一种信号肽及其在利用魔芋粉合成L-精氨酸及其高值化的应用 技术领域
本发明涉及一种信号肽及其在利用魔芋粉合成L-精氨酸及其高值化的应用，属于基因工程和代谢工程技术领域。
背景技术
魔芋粉，主要含魔芋葡甘露聚糖，其来源是非粮食作物魔芋。魔芋主要分布于中国、日本、缅甸、越南、印度尼西亚，东南亚及非洲等地区，可与粮食作物套种，也可种植于淤泥或者滩地，是一种不与粮食作物争地且资源丰富，可进一步开发利用的天南星科植物。据相关报道在我国云南、贵州、四川、湖北和湖南的西部、陕西南部等地均适宜魔芋种植，目前已有一定的规模，仅湖北省魔芋种植面积就达41万亩。目前魔芋粉主要用于部分食品，其水解低聚糖可作为功能性食品，应用还不是很广泛。魔芋主要作为膳食纤维食品被联合国卫生组织确定为十大保健食品之一。但是由于人体本身无法代谢分解魔芋粉中的葡甘露聚糖每天的摄取量一般不超过10g，因而还有大量过剩的魔芋可用于资源的开发利用。大部分微生物无法直接利用，所以改造微生物利用更为廉价的魔芋粉作为碳源发酵生产高附加值产品具有良好的应用前景。现国内外已有报道，改造酿酒酵母直接以淀粉为碳源生产乙醇，导入α-淀粉酶基因使谷氨酸棒状杆菌利用可溶性生产L-赖氨酸的相关报道，未见有以魔芋粉底物的相关微生物改造。
本研究室是前期工作通过多级诱变、相关基因工程和代谢工程技术改造已获得并保藏高产L-精氨酸的菌种：钝齿棒状杆菌CGMCC No.0890。
发明人保藏的高产L-精氨酸的菌种只能以葡萄糖为主要碳源发酵生产L-精氨酸，不能直接利用魔芋粉作为主要碳源。
L-精氨酸作为半必需氨基酸，在医药工业，保健品、食品等领域皆有广泛的应用。通过基因工程和代谢工程技术进一步改造高产L-精氨酸菌种，使其能利用魔芋粉作为碳源发酵生产L-精氨酸。对简化L-精氨酸生产工艺、节约生产成本有一定的参考应用价值。
L-精氨酸经精氨酸酶(Arginase,EC 3.5.3.1)催化水解，可生成L-鸟氨酸和尿素。L-鸟氨酸作为精氨酸的高值化产品是一种重要的非蛋白质氨基酸，可促进蛋白质的合成以及糖类和脂质的分解代谢并对肝脏的解毒功能具有重要的保障作用。
L-精氨酸经精氨酸脱亚胺酶(Arginine deiminase,EC 3.5.3.6)催化降解，可生成L-瓜氨酸 和氨气。L-瓜氨酸作为精氨酸的高值化产品可用于维护建康的肺功能，提高脑力清晰度，有助于脑神经细胞贮藏与调回讯息同时可使人的血管获得松弛，用于增强男性性功能，以及治疗性功能障碍。
在获得能利用廉价碳源魔芋粉生产L-精氨酸的菌株上，进一步通过基因工程和代谢工程的技术手段，可获得能利用魔芋粉作为碳源生产L-精氨酸的高值化产品L-鸟氨酸和L-瓜氨酸的优势菌种，对节约粮食和生产成本的生产工艺有一定的参考价值。
发明内容
本发明通过基因工程技术将去除自身信号肽的β-甘露聚糖酶基因与来源谷氨酸棒状杆菌和枯草芽孢杆菌的Sec和Tat两个主要分泌途径的十二条信号肽分别融合，替换对比不同信号肽引导分泌β-甘露聚糖酶基因在不同L-精氨酸高产菌的效果，得到经密码子优化的β-甘露聚糖酶基因信号肽——Mannase Signal Peptide(MSP)为分泌胞外酶效果较好的一种新发现信号肽。
本发明的第一个目的是提供一种信号肽MSP，其核苷酸序列是(a)～(e)中任意一项:
(a)SEQ ID NO:1所示的DNA；
(b)包含SEQ ID NO:1所示的碱基序列、并且具有信号肽活性的DNA；
(c)包含在SEQ ID NO:1所示的碱基序列中缺失、取代或添加至少1个碱基所得的碱基序列、并且具有信号肽活性的DNA；
(d)与SEQ ID NO:1所示的碱基序列在严紧条件下杂交、并且具有信号肽活性/来源于枯草芽孢杆菌属细菌的DNA。
本发明的第二个目的是一种提高微生物利用魔芋粉合成产物的方法，是将所述信号肽与β-甘露聚糖酶基因融合，然后将融合片段连接到表达载体上并转化到宿主微生物内得到重组微生物，利用重组微生物在以魔芋粉为主要碳源的培养基中发酵生产产物；
在本发明的一种实施方式中，所述β-甘露聚糖酶基因来源于枯草芽孢杆菌属，但不限于枯草芽孢杆菌属。
在本发明的一种实施方式中，所述β-甘露聚糖酶基因来源于枯草芽孢杆菌B.subtilis CCTCC M 209200，核苷酸序列如SEQ ID NO:3所示。
在本发明的一种实施方式中，所述产物为L-精氨酸、L-鸟氨酸或L-瓜氨酸。
在本发明的一种实施方式中，所述微生物为钝齿棒状杆菌，但不限于钝齿棒状杆菌属。
在本发明的一种实施方式中，所述微生物为高产L-精氨酸的钝齿棒状杆菌CGMCC 0890。
在本发明的一种实施方式中，所述表达载体为pXMJ19，但不限于pXMJ19。
在本发明的一种实施方式中，所述微生物还表达精氨酸酶基因，合成产物为L-鸟氨酸。
在本发明的一种实施方式中，所述精氨酸酶基因来源于蜡样芽胞杆菌，但不限于蜡样芽 胞杆菌属。
在本发明的一种实施方式中，所述精氨酸酶基因是NCBI上gi:22934680的基因。
在本发明的一种实施方式中，所述微生物还表达精氨酸脱亚胺酶基因，合成产物为L-瓜氨酸。
在本发明的一种实施方式中，所述精氨酸脱亚胺酶基因来源于蜡样芽胞杆菌，但不限于蜡样芽胞杆菌属。
在本发明的一种实施方式中，所述精氨酸酶基因是NCBI上gi:22939780的基因。
在本发明的一种实施方式中，所述培养基的碳源为魔芋粉和葡萄糖混合碳源，其中葡萄糖的作用是有利于加快发酵前期菌体生长并结合魔芋粉的诱导，大量合成并分泌β-甘露聚糖酶，用于水解魔芋粉提供有效碳源进行相关产品的生产。
在本发明的一种实施方式中，所述培养基中初始含有10g/L魔芋粉和50g/L葡萄糖，在发酵后期通过分批补料方法补加90g/L魔芋粉。
本发明的第三个目的是提供一种重组钝齿棒状杆菌，其特征在于，所述重组钝齿棒状杆菌表达与SEQ ID NO:1的信号肽融合的β-甘露聚糖酶基因；所述信号肽与β-甘露聚糖酶基因融合后，连接到表达载体上并转化到宿主菌内。
在本发明的一种实施方式中，所述重组钝齿棒状杆菌是CGMCC 0890/pMSPman，其构建方法：将SEQ ID NO:1的信号肽与B.subtilis CCTCC M 209200来源的β-甘露聚糖酶基因融合，然后将融合基因转化到表达载体pXMJ19上得到重组质粒pXMJ19-MSPman，再将重组质粒转化到钝齿棒状杆菌中，得到的重组菌即为CGMCC 0890/pMSPman。该菌株发酵生产L-精氨酸。
在本发明的一种实施方式中，所述重组钝齿棒状杆菌是CGMCC 0890/pMSPman-pargI，其构建方法：将来源于蜡样芽孢杆菌的argI基因连接到pDXW10上构建重组质粒，然后将重组质粒转化到CGMCC 0890/pMSPman中得到的。该菌株发酵生产L-鸟氨酸。
在本发明的一种实施方式中，所述重组钝齿棒状杆菌是CGMCC 0890/pMSPman-padi，其构建方法：将来源于蜡样芽孢杆菌的padi基因连接到pDXW10上构建重组质粒，然后将重组质粒转化到CGMCC 0890/pMSPman中得到的。该菌株发酵生产L-瓜氨酸。
本发明的有益效果：
(1)本发明得到了一种新的信号肽，以该信号肽介导分泌β-甘露聚糖酶的重组菌，在利用魔芋粉做底物生产相关产品具有一定的优势，在魔芋粉的利用率、产品的生产效率及产量上都比其它信号肽介导的高。因此，含有此信号肽的重组菌在利用更为廉价魔芋粉为底物降低生产L-精氨酸及其高值化产品的工艺成本上更具优势。
(2)本发明在已有高产L-精氨酸菌种的基础上，通过本发明信号肽引导分泌β-甘露聚糖基因在构建的不同L-精氨酸高产菌中表达，使其能以魔芋粉和葡萄糖作为混合碳源发酵生产L-精氨酸。本发明构建的重组菌钝齿棒状杆菌CGMCC 0890/pMSPman优化后5L罐发酵96h L-精氨酸的产量为45±0.9g/L，发酵液中β-甘露聚糖酶的酶活为1505±5.7U/mL。(碳源添加方式：初始10g/L魔芋粉和50g/L葡萄糖，后期分批补加共计90g/L魔芋粉)。
(3)本发明在已构建的重组菌钝齿棒状杆菌CGMCC 0890/pMSPman的基础上，通过基因工程技术将精氨酸酶基因转入其中并表达，获得重组钝齿棒状杆菌CGMCC 0890/pMSPman-pargI。重组钝齿棒状杆菌CGMCC 0890/pMSPman-pargI能以魔芋粉和葡萄糖作为混合碳源发酵生产L-鸟氨酸。本发明所构建的重组钝齿棒状杆菌CGMCC 0890/pMSPman-pargI，经碳源添加优化5L罐发酵96h L-鸟氨酸的产量为23.5g/L，发酵液中β-甘露聚糖酶的酶活为1610±3.8U/mL。(碳源添加方式：初始10g/L魔芋粉和50g/L葡萄糖，后期分批补加共计90g/L魔芋粉)。
(4)本发明在已构建的重组菌钝齿棒状杆菌CGMCC 0890/pMSPman的基础上，通过基因工程技术将精氨酸脱亚胺酶基因转入其中并表达，获得重组钝齿棒状杆菌CGMCC 0890/pMSPman-padi。重组钝齿棒状杆菌CGMCC 0890/pMSPman-padi能以魔芋粉和葡萄糖作为混合碳源发酵生产L-瓜氨酸。本发明所构建的重组钝齿棒状杆菌CGMCC 0890/pMSPman-padi，经碳源添加优化5L罐发酵96h L-瓜氨酸的产量为26.3g/L，发酵液中β-甘露聚糖酶的酶活为1360±6.8U/mL。(碳源添加方式：初始10g/L魔芋粉和50g/L葡萄糖，后期分批补加共计90g/L魔芋粉)。
具体实施方式
材料与方法：
(1)引物：根据NCBI上公布的相关基因序列设计信号肽和目的基因融合引物。
(2)重组菌的构建：从相关菌种中抽提染色体DNA为模板，根据预先设计好的引物、PCR扩增条件和扩增体系进行PCR。采用凝胶回收试剂盒对PCR产物进行纯化和回收，琼脂糖核酸凝胶电泳检验回收产物的浓度。用相同的限制性内切酶相关表达载体和纯化后的PCR产物进行双酶切，琼脂糖核酸凝胶电泳检测酶切产物并用凝胶回收试剂盒对其回收，并用超微量分光光度计检测其浓度。将酶切后的载体和PCR产物按一定比例混合，在T4DNA连接酶的作用下过夜连接。将连接产物用CaCl2转化法转入E.coli BL21，获得含重组质粒的E.coli BL21。提取质粒，通过单双酶切及PCR验证后用电转化方法将重组质粒导入相关菌种中。最后，将含15％甘油的重组菌液保存-70℃冰箱。
(3)重组菌的产酸培养：将重组菌接种于新鲜的LB+0.5％Glucose液体培养基中进行活 化，接种量为1％。次日以1％转接于发酵培养基中(底物为魔芋粉和葡萄糖混合)，生长后期收集发酵液利用氨基酸自动分析仪测定其氨基酸含量(L-精氨酸及相关氨基酸等)。发酵培养基具备微生物生长所需的营养成分，并通过相关优化。
实施例1：信号肽引物与β-甘露聚糖酶串联的引物设计
根据NCBI上公布的相关基因序列和β-甘露聚糖酶基因序列设计信号肽和基因串联的大片段引物P1和P2(序列分别如SEQ ID NO:4和SEQ ID NO:5所示)。
P1:pMSPmanHindIIIF
5’-CCCAAGCTTATGTTCAAGAAGCACACCATCTCCCTGCTGATCATCTTCCTGCTGGCTTCCGCTGTTCTGGCTAAGCCAATCGAGGCTCATACTGTGTCGCCTGTGAATC-3’
P2:pMSPmanBamHIR
5’-CGCGGATCCTTACTCAACGATTGGCGTTA-3’
实施例2：β-甘露聚糖酶基因信号肽替换及其克隆
(1)B.subtilis CCTCC M 209200提取染色体作为模板DNA。
(2)根据NCBI网站上公布的β-甘露聚糖酶基因序列设计信号肽和基因串联的PCR引物。以B.subtilis CCTCC M 209200的基因组为模板利用大片段引物PCR，得到带有经密码优化的核苷酸序列如SEQ ID NO:1所示Mannase signalpeptide(简称MSP)的β-甘露聚糖酶基因，其中不带自身信号肽的β-甘露聚糖酶基因的核苷酸序列如SEQ ID NO:3所示。PCR扩增体系(50μL)：模板1μL，上下游引物各0.5μL，dNTP Mix 4μL，10×Ex Taq Buffer 5μL，灭菌ddH2O 38.5μL，Ex Taq DNA聚合酶0.5μL。PCR反应条件：94℃预变性，5min，一个循环；94℃变性，30s，56℃退火，30s，72℃延伸，1min30s，30个循环；72℃，10min，一个循环；4℃，10min，一个循环(其中退火温度和延伸时间根据不同引物和基因进行相对的调节)。采用凝胶回收试剂盒对PCR产物进行纯化和回收，电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中，-20℃冰箱保存备用。
实施例3：重组质粒pXMJ19-MSPman的构建
(1)将实施例2中PCR回收的产物与克隆载体pMD18-T连接，连接体系为solution I 5μL，目的基因4.8μL，pMD18-T质粒0.2μL，16℃过夜连接。连接接产物转化E.coilJM109，涂布于含100ug/mL氨苄青霉素的LB平板，经37℃培养过夜，挑取单菌落到含100ug/mL氨苄青霉素的10mL液体LB培养基中，37℃摇床过夜培养后提取质粒，命名为pMD18-T-MSPman，经PCR和酶切验证连接成功后，将菌液加入甘油于-70℃冰箱保藏。
(2)将(1)中提取的pMD18-T-MSPman质粒与表达载体pXMJ19分别进行HindIII与BamHI 的双酶切，利用凝胶回收试剂盒回收后进行连接，获得重组质粒pXMJ19-MSPman。上述重组质粒都通过PCR和酶切验证。
实施例4：重组质粒pXMJ19-MSPman电转化至钝齿棒状杆菌CGMCC 0890
挑取钝齿棒状杆菌CGMCC 0890分别接种于10mL LB液体培养基摇瓶中，30℃摇床培养过夜，取100μL过夜培养的菌液，接种于50mL的感受态培养基中，30℃摇床培养4h至OD562nm＝0.9左右，用无菌管离心去上清，再用10％的甘油悬浮菌体并离心，重复此步骤3次。最后分装于1.5mL的离心管，加入3uL重组质粒，于电压1800V，50mS条件下电转化。迅速向将电转化完成的菌体中加入800uL的新鲜LBG培养基(培养基组成：0.5％酵母提取物，1％蛋白胨，1％NaCl，0.5％葡萄糖)，并放置46℃的水浴锅中静置6min，之后在30℃摇床培养3h左右，离心涂布于含氯霉素抗性的平板。在30℃的恒温培养箱培养64h，挑取菌落形态正常的单克隆并进行培养。将培养后的单克隆进行质粒提取，酶切验证和PCR验证，得到阳性的重组菌株CGMCC 0890/pMSPman，并保藏于-70℃冰箱。
实施例5：重组钝齿棒状杆菌CGMCC 0890/pMSPman分泌的β-甘露聚糖酶酶活测定
在试管中加入2.0mL 5.0g/L刺槐豆胶底物溶液，65℃预热10min，加入2.0mL经过适当稀释的粗酶液，65℃精确反应15min。立即加入5.0mL DNS试剂，振荡摇匀，沸水浴中放置10min，置于冰水中冷却，然后加水补齐至10.0mL并摇匀。空白对照以2.0mL水代替适当稀释的粗酶液，540nm处测定吸光值。结合甘露糖标准曲线计算酶活力。酶活力单位定义：在上述测定条件下，以每1min产生1μmol还原糖的酶量定义为1个β-甘露聚糖酶活力单位(U)。
实施例6：重组钝齿棒状杆菌CGMCC 0890/pMSPman的发酵培养
将重组钝齿棒状杆菌CGMCC 0890/pMSPman种子液接种至魔芋粉培养基，在30℃，600r/min培养条件下进行发酵培养，96h L-精氨酸的产量为45±0.9g/L，发酵液中β-甘露聚糖酶的酶活为1505±5.7U/mL。
魔芋粉培养基(g/L)：
初始培养基中含有初始魔芋粉10，葡萄糖50，酵母粉8，(NH4)2SO420，K2HPO41.5，MgSO4·7H2O 0.5，FeSO4·7H2O 0.02，MnSO4·H2O 0.02，CaCO330。PH 7.0～7.2；发酵12h后开始分批补加魔芋粉，总补加量为90g/L。
对照：
(1)采用类似的方法，仅仅是将核苷酸序列如SEQ ID NO:1的信号肽，替换成β-甘露聚糖酶基因自身原有的信号肽(核苷酸序列如SEQ ID NO:2所示)，发酵显示：L-精氨酸的产量 为31±3.7g/L，发酵液中β-甘露聚糖酶的酶活为1050±3.8U/mL。
(2)采用类似的方法，仅仅是将核苷酸序列如SEQ ID NO:1的信号肽，替换成如：谷氨酸棒状杆菌来源的表层蛋白信号肽PS(核苷酸序列如SEQ ID NO:6所示)或者是枯草芽孢杆菌来源的α-淀粉酶信号肽AE(核苷酸序列如SEQ ID NO:7所示)，发酵显示：L-精氨酸的产量分别为26±1.5g/L和23±1.2g/L，发酵液中β-甘露聚糖酶的酶活分别为867±3.7U/mL和732±5.9U/mL。
由此可知，利用本发明的SEQ ID NO:1的信号肽，与其他的信号肽相比，能够显著提高重组菌在利用魔芋粉合成产物L-精氨酸的能力，产物提高量可达45.2％-96％，同时发酵液中β-甘露聚糖酶的酶活提高了43％-105％。
实施例7：重组钝齿棒状杆菌CGMCC 0890/pMSPman发酵L-精氨酸测定
发酵液中的L-精氨酸含量通过坂口试剂法初测。将发酵上清液稀释500倍，取1mL放入比色管中，加入4mL 0.375mol/L NaOH溶液，再加入1mL显色剂摇匀后30℃水浴20分钟，用分光光度计在520nm处测定每管吸光值，根据标准曲线计算发酵液中L-精氨酸含量。再经氨基酸自动分析仪准确测定。
实施例8：重组钝齿棒状杆菌CGMCC 0890/pMSPman-pargI构建的引物设计
根据NCBI上公布的相关基因序列和精氨酸酶基因序列设计的引物p1和p2(序列分别如SEQ ID NO:8和SEQ ID NO:9所示)
p1:pargIEcoRIF
5’-CGGAATTCATGAAAAAAGAAATCTCAGTTATTGG-3’
p2:pargISacIR
5’-CCGAGCTCTTATTTTAGTTTTTCACCGAATAAA-3’
实施例9：重组质粒pDXW10-argI的构建
以Bacillus cereus的基因组为模板，运用实施案例8所设计的引物，获得带EcoRI和SacI酶切位点的argI基因PCR产物，并经过试剂盒纯化。将纯化后的PCR产物和表达载体质粒pDXW10分别用EcoRI和SacI酶进行酶切，胶回收，16℃过夜连接。连接接产物转化E.coilJM109，涂布于含100ug/mL卡那霉素的LB平板，经37℃培养过夜，挑取单菌落到含100ug/mL卡那霉素霉素的10mL液体LB培养基中，37℃摇床过夜培养后提取质粒，命名为pDXW10-argI，经PCR和酶切验证连接成功后，将菌液加入甘油于-70℃冰箱保藏。
实施例10：重组质粒pDXW10-argI电转至钝齿棒状杆菌CGMCC 0890/pMSPman
所用电转化的出发菌种为钝齿棒状杆菌CGMCC 0890/pMSPman，筛选标记的抗生素为卡 那霉素。其余参考实施案例4得到的阳性重组菌株CGMCC 0890/pMSPman-pargI。
实施例11：重组钝齿棒状杆菌CGMCC 0890/pMSPman-pargI细胞内的精氨酸酶的酶活测定
配制0.2M底物L-精氨酸(0.2M碳酸盐缓冲液，pH 9.0)，取0.9ml底物溶液，加入0.1ml酶液，40℃反应10min。将酶反应液稀释相应的倍数，取1ml稀释后的反应液，用Chinard比色法测定反应液中L-鸟氨酸的含量。酶活定义单位为1min催化1umol L-精氨酸转化成L-鸟氨酸所需的酶量。(Chinard比色法：1ml标准液中依次加入1ml的冰醋酸，1ml混合酸(茚三酮溶液)，于沸水浴中反应1h，测定515nm下的吸光度值。)
实施例12：重组钝齿棒状杆菌CGMCC 0890/pMSPman-pargI的发酵培养基
将重组钝齿棒状杆菌CGMCC 0890/pMSPman-pargI种子液接种至魔芋粉培养基，在30℃，600r/min培养条件下进行发酵培养，96h L-鸟氨酸的产量为23.5g/L，发酵液中β-甘露聚糖酶的酶活为1610±3.8U/mL。
魔芋粉培养基(g/L)：
初始培养基中含有初始魔芋粉10，葡萄糖50，酵母粉8，(NH4)2SO420，K2HPO41.5，MgSO4·7H2O 0.5，FeSO4·7H2O 0.02，MnSO4·H2O 0.02，CaCO330。PH 7.0～7.2；发酵12h后开始分批补加魔芋粉，总补加量为90g/L。
对照：
(1)采用类似的方法，仅仅是将核苷酸序列如SEQ ID NO:1的信号肽，替换成β-甘露聚糖酶基因自身原有的信号肽(核苷酸序列如SEQ ID NO:2所示)，发酵显示：L-鸟氨酸的产量为21±0.8g/L，发酵液中β-甘露聚糖酶的酶活为1025±5.8U/mL。
(2)采用类似的方法，仅仅是将核苷酸序列如SEQ ID NO:1的信号肽，替换成如：谷氨酸棒状杆菌来源的表层蛋白信号肽PS(核苷酸序列如SEQ ID NO:6所示)或者是枯草芽孢杆菌来源的α-淀粉酶信号肽AE(核苷酸序列如SEQ ID NO:7所示)，发酵显示：L-鸟氨酸的产量分别为17±1.5g/L和15±1.3g/L，发酵液中β-甘露聚糖酶的酶活分别为865±6.7U/mL和752±3.9U/mL。
由此可知，利用本发明的SEQ ID NO:1的信号肽，与其他的信号肽相比，能够显著提高重组菌在利用魔芋粉合成产物L-鸟氨酸的能力。
实施例13：重组钝齿棒状杆菌CGMCC 0890/pMSPman-pargI发酵液中L-鸟氨酸测定
发酵液中的L-鸟氨酸含量采用Chinard比色法初步测定，同时相关样品通过氨基酸分析仪精确测定。
实施例14：重组钝齿棒状杆菌CGMCC 0890/pMSPman-padi构建的引物设计
根据NCBI上公布的相关基因序列和精氨酸酶基因序列设计的引物P3/P4，序列分别如SEQ ID NO:10和SEQ ID NO:11所示。
P3:padiSacIF
5’-CCGAGCTCATGAAGCATCCGATACATGTT-3’
P4:psdiHindIIIR
5’-CCCAAGCTTTTAAATATCTTTACGAACAATTG-3’
实施例15：重组质粒pDXW10-adi的构建
所用引物参见实施案例14，其余操作参见实施例9。
实施例16：重组质粒pDXW10-adi电转至钝齿棒状杆菌CGMCC 0890/pMSPman
所用电转化的出发菌种为钝齿棒状杆菌CGMCC 0890/pMSPman，筛选标记的抗生素为卡那霉素，重组质粒为pDXW10-adi。其余参考实施例4得到的阳性重组菌株CGMCC 0890/pMSPman-padi。
实施例17：重组钝钝齿棒状杆菌CGMCC 0890/pMSPman-padi细胞内的精氨酸酶的酶活测定
配制0.2M底物L-精氨酸(0.2M碳酸盐缓冲液，pH 9.0)，取0.9ml底物溶液，加入0.1ml酶液，40℃反应10min。将酶反应液稀释相应的倍数，取1ml稀释后的反应液，用氨基酸测定仪反应液中L-瓜氨酸的含量。酶活定义单位为1min催化1umol L-精氨酸转化成L-瓜氨酸所需的酶量。
实施例18：重组钝齿棒状杆菌CGMCC 0890/pMSPman-padi的发酵培养基
将重组钝齿棒状杆菌CGMCC 0890/pMSPman-padi种子液接种至魔芋粉培养基，在30℃，600r/min培养条件下进行发酵培养，96h L-瓜氨酸的产量为26.3g/L，发酵液中β-甘露聚糖酶的酶活为1360±6.8U/mL。
初始培养基中含有初始魔芋粉10，葡萄糖50，酵母粉8，(NH4)2SO420，K2HPO41.5，MgSO4·7H2O 0.5，FeSO4·7H2O 0.02，MnSO4·H2O 0.02，CaCO330。PH 7.0～7.2；发酵12h后开始分批补加魔芋粉总补加量为90g/L。
对照：
(1)采用类似的方法，仅仅是将核苷酸序列如SEQ ID NO:1的信号肽，替换成β-甘露聚糖酶基因自身原有的信号肽(核苷酸序列如SEQ ID NO:2所示)，发酵显示：L-瓜氨酸的产量为22±0.9g/L，发酵液中β-甘露聚糖酶的酶活为1002±4.7U/mL。
(2)采用类似的方法，仅仅是将核苷酸序列如SEQ ID NO:1的信号肽，替换成如：谷氨酸棒状杆菌来源的表层蛋白信号肽PS(核苷酸序列如SEQ ID NO:6所示)或者是枯草芽孢杆 菌来源的α-淀粉酶信号肽AE(核苷酸序列如SEQ ID NO:7所示)，发酵显示：L-瓜氨酸的产量分别为19±1.3g/L和16±0.7g/L，发酵液中β-甘露聚糖酶的酶活分别为832±7.7U/mL和763±6.9U/mL。
由此可知，利用本发明的SEQ ID NO:1的信号肽，与其他的信号肽相比，能够显著提高重组菌在利用魔芋粉合成产物L-瓜氨酸的能力。
实施例19：重组钝齿棒状杆菌CGMCC 0890/pMSPman-padi发酵液中L-瓜氨酸测定
发酵液中的L-瓜氨酸含量通过氨基酸分析仪精确测定。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2015095286-appb-000001
Figure PCTCN2015095286-appb-000002
Figure PCTCN2015095286-appb-000003
Figure PCTCN2015095286-appb-000004
Figure PCTCN2015095286-appb-000005

Claims (10)

  1. 一种提高微生物利用魔芋粉合成产物的方法，其特征在于，所述方法是将信号肽与β-甘露聚糖酶基因融合，然后将融合片段连接到表达载体上并转化到宿主微生物内得到重组微生物，利用重组微生物在以魔芋粉为主要碳源的培养基中发酵生产产物；
    所述信号肽的核苷酸序列是(a)～(d)中任意一项：
    (a)SEQ ID NO:1所示的序列，
    (b)包含SEQ ID NO:1所示的碱基序列、并且具有信号肽活性的DNA，
    (c)包含在SEQ ID NO:1所示的碱基序列中缺失、取代或添加至少1个碱基所得的碱基序列、并且具有信号肽活性的DNA，
    (d)与SEQ ID NO:1所示的碱基序列在严紧条件下杂交、并且具有信号肽活性/来源于枯草芽孢杆菌属细菌的DNA。
  2. 根据权利要求1所述的方法，其特征在于，所述β-甘露聚糖酶基因来源于枯草芽孢杆菌属，但不限于枯草芽孢杆菌属。
  3. 根据权利要求1所述的方法，其特征在于，所述产物为L-精氨酸、L-鸟氨酸或L-瓜氨酸。
  4. 根据权利要求3所述的方法，其特征在于，所述微生物为钝齿棒状杆菌，但不限于钝齿棒状杆菌属。
  5. 根据权利要求1所述的方法，其特征在于，所述表达载体为pXMJ19，但不限于pXMJ19。
  6. 根据权利要求3所述的方法，其特征在于，所述微生物还表达精氨酸酶基因，合成产物为L-鸟氨酸。
  7. 根据权利要求3所述的方法，其特征在于，所述微生物还表达精氨酸脱亚胺酶基因，合成产物为L-瓜氨酸。
  8. 根据权利要求1所述的方法，其特征在于，所述培养基中初始含有10g/L魔芋粉和50g/L葡萄糖，在发酵后期通过分批补料方法补加共计90g/L魔芋粉。
  9. 一种重组钝齿棒状杆菌，其特征在于，所述重组钝齿棒状杆菌表达与SEQ ID NO:1的信号肽融合的β-甘露聚糖酶基因；所述信号肽与β-甘露聚糖酶基因融合后，连接到表达载体上并转化到宿主菌内。
  10. 一种信号肽，其核苷酸序列是(a)～(d)中任意一项:
    (a)SEQ ID NO:1所示的序列；
    (b)包含SEQ ID NO:1所示的碱基序列、并且具有信号肽活性的DNA；
    (c)包含在SEQ ID NO:1所示的碱基序列中缺失、取代或添加至少1个碱基所得的碱基序列、并且具有信号肽活性的DNA；
    (d)与SEQ ID NO:1所示的碱基序列在严紧条件下杂交、并且具有信号肽活性/来源于枯草芽孢杆菌属细菌的DNA。
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