WO2010003304A1 - 产脱氧紫色杆菌素的重组菌及其应用 - Google Patents

产脱氧紫色杆菌素的重组菌及其应用 Download PDF

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WO2010003304A1
WO2010003304A1 PCT/CN2009/000430 CN2009000430W WO2010003304A1 WO 2010003304 A1 WO2010003304 A1 WO 2010003304A1 CN 2009000430 W CN2009000430 W CN 2009000430W WO 2010003304 A1 WO2010003304 A1 WO 2010003304A1
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recombinant
concentration
bacillus
gene cluster
tryptophan
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PCT/CN2009/000430
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French (fr)
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邢新会
蒋培霞
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清华大学
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Priority claimed from CN2008101166013A external-priority patent/CN101319219B/zh
Priority claimed from CN2008102243591A external-priority patent/CN101368169B/zh
Application filed by 清华大学 filed Critical 清华大学
Priority to BRPI0910514A priority Critical patent/BRPI0910514A2/pt
Priority to US13/003,227 priority patent/US8778654B2/en
Priority to CN2009801258713A priority patent/CN102099477A/zh
Priority to JP2011516946A priority patent/JP5632370B2/ja
Priority to GB1100438.9A priority patent/GB2473401B/en
Publication of WO2010003304A1 publication Critical patent/WO2010003304A1/zh

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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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Definitions

  • the present invention relates to a recombinant strain producing deoxybacteria and its use.
  • Violacein is a secondary metabolite produced by microorganisms. It belongs to an anthracene derivative and is a water-insoluble blue-violet pigment which is formed by oxidative condensation of two tryptophan molecules. Since the discovery of the purple bacillus in the late 19th century, people have explored a lot of their biological functions. In recent years, with the deepening of research, zeocin exhibits important biological activity and can be used as a potential anti-tumor and anti-virus. Drugs and bio-dyes have broad application prospects in the fields of textile printing and dyeing, plant pathogenic fungi control, viruses and cancer treatment. Therefore, they are receiving more and more attention.
  • bacteriocin has the following biological activities: (1) It has a broad spectrum of antibacterial activity, such as anti-s taploylococcous aureus ⁇ Bacillus sp, streptococcus sp, myc obact eri urn, Neisserig, pseudomonas (Sanchez et al., Reevaluation) Of the Violacein Biosynthet ic Pathway and its Relat ionship to Indolocarbazole Biosynthesis. Journal 2006. 7, 1231-1240); (2) Antioxidant (Konzen et al., Antioxidant properties of violacein: possible relation on its biological funct ion. Journal 2006) . 14, 8307-8313); (3) Anti-tumor cells (de Carvalho et al.,
  • Deoxyribomycin is a structural analog that is one less oxygen atom than purple bacillus and is a by-product of the azoxystrobin synthesis pathway, usually accompanied by the production of purple bacillus. Since the proportion of deoxy violet bacillus in blue-purple is small, it is equivalent to one tenth of the amount of purple bacillus, and it is difficult to isolate enough to study its properties and functions. And technology is still rarely studied. Due to its low yield, it is difficult to isolate pure deoxypurinin. There are few studies on the properties and biological activities of deoxy violet bacillus. At present, in addition to reporting that it can inhibit protozoa (Matz, C et al. Marine Biofi lm
  • the recombinant strain producing deoxyi-biotin which is provided by the present invention is a recombinant strain obtained by introducing a gene cluster of deoxy violet bacillus synthesis into Escherichia coli BL21-CodonPlu S (DE3)-RIL or Pseudomonas putida.
  • the recombinant bacteria can produce deoxy violet bacillus by fermentation with tryptophan as a substrate.
  • the deoxyribomycin synthesis-related gene cluster is obtained by knocking out the oZ? gene in the purple bacillus synthesis gene cluster consisting of VioA, VioB, VioC, ioZ? and c ⁇ to obtain a recombinant gene cluster.
  • the gene cluster is specifically a gene as follows 1) or 2) or 3):
  • nucleotide sequence thereof is the DNA molecule of SEQ ID NO: 1 in the sequence listing;
  • the DNA molecule in the step 3) has more than 75% homology with the DNA molecule of 1).
  • the above stringent conditions can be carried out in a solution of 6XSSC, 0.5% SDS at 68 ° C, and then washed once with 2XSSC, 0.1% SDS and 1XSSC, 0.1% SDS.
  • the deoxyribomycin synthesis-related gene clusters are also within the scope of the present invention.
  • the gene cluster was introduced into the recombinant strain obtained by Escherichia coli BL21-CodonPlus(DE3)-RIL, and named as Escherichia coli BL-DV;
  • Escherichia coli BL-DV was deposited on June 25, 2008 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101), Deposit The number is CGMCC No. 2557.
  • the gene cluster was introduced into Pseudomonas sinensis as a recombinant strain obtained from mt-2 NCIMB 10432 and named as P. putida -VioADc
  • An expression cassette containing the gene cluster and a recombinant expression vector containing the gene cluster or the expression cassette are also within the scope of the present invention.
  • the method for producing deoxy violet bacillus in the present invention is to produce deoxy violet bacillus by fermentation using the recombinant Escherichia coli or the recombinant Pseudomonas putida using L-tryptophan as a substrate.
  • the concentration of the L-tryptophan when the fermentation produces deoxyviocin, the concentration of the L-tryptophan may be 0.3-0.5 g/L fermentation medium, specifically 0.4 g/L.
  • the fermentation temperature may be 10-37 ° C, specifically 20 ° C.
  • the Escherichia coli BL-DV CGMCC No. 2557 of the present invention can use IPTG as an inducer, and the concentration of IPTG can be 0 ⁇ ol/L ⁇ 0. 7-1. 3mmol / L, specifically 1. 0ramol / L.
  • the fermentation medium of the fermentation medium may be 0. 3-0. 5g / L fermentation medium, specific In the fermentation production of 0.4 g/L 0 , the fermentation medium may be any medium for providing the growth of Pseudomonas putida, specifically NaH 2 P0 4 ⁇ 2H 2 0 1. 0-2, 0 g / L, Na 2 HP0 4 ⁇ 12H 2 0 3. 0-4. 0 g/L, NH 4 C1 0. 5-1. 0 g/L, K2HPO4 ⁇ 3H 2 0 7.
  • the solvent is water.
  • an inducer is added to the recombinant bacteria.
  • the inducing agent selected as necessary, 3. / Wii ⁇ -VioAD present invention can be used n-alkanes or more carbon atoms as an inducer of six, in particular n-octyl embankment, the concentration of n-octane is specifically 0.05 Ml/100 ml medium.
  • the fermentation temperature is specifically 20 °C.
  • Figure 1 shows the clusters of overlapping extension PCR recombinant deoxy violet bacillus synthesis.
  • Fig. 2 shows the results of PCR amplification of the fragments A, B, C and D of the purple bacillus synthesis-related gene cluster.
  • Fig. 3 is a diagram showing the results of high performance liquid chromatography identification of the pigment produced by the recombinant strain BL-DV.
  • Figure 4 is a graph showing the results of high performance liquid chromatography identification of the pigment produced by the recombinant strain P. putida-VioAD. The best way to implement the invention
  • Example 1 Recombinant bacteria producing deoxy violet bacillus
  • the genus Duganella sp. B2 CGMCC Ns2056 was transferred to a liquid medium (starch 15 g / L, ferrous sulfate 0. 03g / L, potassium nitrate lg / L, dipotassium hydrogen phosphate 0. 7g / L, Magnesium sulphate 0. 5g / L, tryptophan 0. 5g / L, pH adjustment of 7. 0), 25 °C, 200rpra culture for 36 hours, according to the Shanghai Biotech genomic DNA extraction kit instructions for the extraction of Rhodobacter B2 Genomic DNA.
  • the amplified fragments A and B use the PCR program I in Table 2, and the amplified fragment C uses the PCR program in Table 2
  • the PCR product fragments B and C were mixed in a volume ratio of 1:1 and then diluted 10-fold as a template for further PCR amplification.
  • the PCR reaction system was 50 L, and the above mixture containing the fragment C was 1. 5 L 2. 5 U TaKaRa Pfu DNA polymerase.
  • the PCR reaction procedure is PCR program III, when the second step is completed. The program was stopped, and 25 pmol of P3 and P6 primers were added to the reaction system, followed by steps 3 and 4 of PCR procedure III, and fragments B and C were ligated together to form fragment D (Fig. 1). Fragment D was purified by PCR product kit, and fragment D was cloned into pMD18-T vector to obtain
  • the PMD18-T-D vector, sequencing, and sequencing results showed that the nucleotide sequence of fragment D is in the sequence of sequence 1 from the 5' end of 3058-6198.
  • the pMD18-TD vector and the Nde I and Xhol I double-digestion expression vector pET30a were digested with e I and ⁇ 3 ⁇ 4 ⁇ I, and the 3057 bp fragment A, 3140 bp fragment D and pET30a vector enzyme were recovered. After the excision of the large fragment, the three fragments recovered above were ligated under the action of T4 DNA ligase to construct a recombinant expression vector pET30aVioAD.
  • the recombinant expression vector pET30aVioAD was transformed into E. coli DH5 ⁇ competent cells, and the transformed product was applied to LB plates containing ampicillin (100 g/ml).
  • the transformants were picked and cultured, and the plasmid was extracted by alkaline lysis.
  • the positive clones were sequenced, and the sequencing results showed that the nucleotide sequence of the VioAD fragment is shown in SEQ ID NO:1 in the sequence listing, and the sequence 1 is from the 5-1308 position at the 5' end to the VioA gene, which encodes VioA in the purple bacillus synthesis pathway.
  • the enzyme from the 5' end, at positions 1305-4322, is the VioB gene, which encodes the VioB enzyme in the purple bacillus synthesis pathway; the 4233-5612 from the 5' end is the VioC gene, which encodes the VioC enzyme in the purple bacillus synthesis pathway; From the 5' end, 5622-6197 is the VioE gene, which encodes the VioE enzyme in the purple bacillus synthesis pathway.
  • the VioD gene is not contained in the VioAD fragment.
  • the VioAD is a cluster of genes related to deoxy violet bacillus synthesis.
  • Double digestion of pMD18-T-D vector and I and 5k7 I double-digestion expression vector pCOMlO (Smits THM et al., New alkane-responsive expression) with se I and TVoi I double digestion fragment A, Not I and T/WI Plasmids for E. coli and Pseudomonas. Plasmid 2001. 46, 16 - 24. ) (Tsinghua University) Recover 3057 bp fragment A, 3140 bp fragment D and pCOMlO vector-digested large fragment, and the above recovered three fragments are The recombinant expression vector pC0M10VioAD was constructed by ligation of T4 DNA ligase.
  • the culture medium concentration at 0 °C was 0.77, and 0. ImM IPTG was added.
  • the correct expression of the four enzymes indicates that the deoxyribomycin synthesis gene cluster may contain rare codons, and the A coli BL21- CodonPlus (DE3) - RIL- VioAD producing deoxy violet bacillus is named Escherichia coli BL-DV;
  • the blue-purple ethanol solution of the BL-DV solution was dissolved in 100% methanol for high performance liquid chromatography analysis using an Agilent-1100 high performance liquid chromatograph.
  • the column was Agi lent Ecl ipse XDB-C18.
  • 150mm X 4 should be, 5 m ; column temperature 30 ° C; eluent is 70% by volume aqueous methanol solution; flow rate 1. 0 mL / min; detection wavelength: 570 nm.
  • Fig. 3 I: high performance liquid chromatogram of the pigment produced by Duganella sp. B2 CGMCC N22056, the first peak is purple bacillus, the second peak is deoxy violet bacillus; II: recombinant bacteria BL High-performance liquid chromatogram of crude pigment extracted from -DV.
  • E. coli BL-DV was deposited on June 25, 2008 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Datun Road, Chaoyang District, Beijing, China) Institute of Microbiology, National Academy of Sciences, Zip Code 100101), with accession number CGMCC No. 2557.
  • CGMCC General Microbiology Center of China Microbial Culture Collection Management Committee
  • pCOMlOVioAD Transfer the recombinant vector pCOMlOVioAD to Pseudomonas putida mt-2 NCIMB 10432 to obtain recombinant corpse.
  • P. putida -VioAD and P. /?i/iii/a-pC0M10 were cultured in LB medium at 37 ° C for bacterial concentration 0D 6 , respectively . . 0 ⁇ 0. 0 ⁇ The percentage of the volume is 0. 05% ⁇ ⁇ , induced at 20 ° C for 30h. Centrifuge 50 mL of the fermentation broth at 7000 X g for 10 min, discard the supernatant, add the same volume of deionized water to the precipitate, mix it with a vortex mixer, centrifuge at 7000 X g for 10 min, then add none.
  • P. putida -VioAD blue ethanol solution was distilled under reduced pressure in 100% methanol for high performance liquid chromatography analysis using Agi lent-1100 high performance liquid chromatography, column % Agi lent Ecl ipse XDB- C18 , 150 mm X 4mra, 5 ⁇ m; column temperature 30 ° C; eluent is 70% by volume aqueous methanol solution; flow rate 1. 0 mL / min; detection wavelength: 570 nra.
  • the results of high performance liquid chromatography were as shown in Fig. 4.
  • the blue substance in the ethanol solution of the auxiliary P. putida-VioAD was consistent with the retention time of the deoxy violet bacillus of the Dugarwlla B2 purple bacillus byproduct. 9min), and only one peak.
  • the blue substance in the ethanol solution of put - VioAO is deoxy violet bacillus, recombinant Pseudomonas putida.
  • -VioAD can express VioA, VioB, VioC and VioE in the purple bacillus synthesis pathway, which can be synthesized. Deoxy violet bacillus.
  • Fig. 4 I: high performance liquid chromatogram of the pigment produced by Duganel la sp. B2 CGMCC Ns2056, the first peak is purple bacillus, the second peak is deoxy violet bacillus; II: A wiii a - High performance liquid chromatogram of the pigment produced by VioAD.
  • Example 2 Recombinant bacteria produce deoxy violet bacillus
  • Recombinant Bacterium BL-DV CGMCC No. 2557 produces deoxy violet bacillus
  • BL-DV CGMCC No. 2557 L-tryptophan, cell concentration (0D 6 value) when adding inducer (IPTG), amount of inducer, induction time 4 factors as test factors, deoxidation
  • IPTG inducer
  • induction time 4 factors as test factors
  • deoxidation The purple bacillus production was used as an indicator, and the above four factors were subjected to an orthogonal test of 4 factors and 3 levels, and the results are shown in Table 3.
  • the pigment was extracted in the same manner as in Example 1.
  • the pigment concentration was measured by measuring the absorbance of the pigment in ethanol solution at the maximum absorption wavelength.
  • the deoxy violet bacillus produced by Duganella sp B2 was measured at a wavelength of 562 nm, and anhydrous ethanol was used as a blank control.
  • the corresponding pigment concentration value is obtained by the absorbance value-pigment concentration standard curve, and each test is repeated 3 times, and the average value is obtained, and the obtained light absorption coefficient ⁇ is 9. 0955 1 ⁇ g- 1 ⁇ cm"O
  • the sulphate Duganella sp. B2 CGMCC NQ 2056 is added to the liquid medium (starch 15g / L, ferrous sulfate 0. 03g / L, potassium nitrate lg / L, dipotassium hydrogen phosphate 0. 7g / L, The magnesium sulfate was 0.5 g/L, the tryptophan was 0.5 g/L, the pH was 7.0 g, and the cells were cultured at 25 ° C for 30 hours, and the cells were collected by centrifugation, and the cells were extracted with ethanol to obtain a blue-violet substance.
  • B2 CGMCC No2056 produced deoxygenation of violet bacillus for quantitative analysis.
  • Quantitative analysis was carried out by measuring the absorbance of the blue-violet substance (pigment) in ethanol at the maximum absorption wavelength.
  • the deoxy violet bacillus produced by Duganel la sp B2 was measured at a wavelength of 562 nm, using anhydrous ethanol as a blank control, and the absorbance value was passed.
  • the pigment concentration standard curve obtains the corresponding pigment concentration value, and each experiment is repeated 3 times, and the average value is obtained, and the obtained absorption coefficient ⁇ is 14.
  • Deoxyribomycin is a by-product of the purple bacillus synthesis pathway, which usually has a low yield and is difficult to separate and cannot be scaled up and applied.
  • Escherichia coli BL-DV CGMCC No. 2557 and P. putida-VioAD of the present invention produce higher yield of deoxy violet bacillus; Escherichia coli BL-DV CGMCC No. 2557 can reach 0. 17g / L fermentation broth, P. put i da -VioAD can be reached in the usual shake flask culture to achieve 1. 5g / L fermentation broth, and easy to extract, separation and purification is simple.
  • the recombinant strain of the present invention is Escherichia coli or Pseudomonas putida, which is convenient to control and convenient for industrial production. _

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Description

产脱氧紫色杆菌素的重组菌及其应用 技术领域
本发明涉及产脱氧紫色杆菌素的重组菌及其应用。
Figure imgf000002_0001
紫色杆菌素 （violacein ) 是微生物产生的一种次级代谢产物。 它属于 吲哚衍生物， 是非水溶性的蓝紫色色素， 由两个色氨酸分子氧化缩合而 成。 自从 19世纪末紫色杆菌素被发现以来， 人们对其生物功能进行了大量 的探索， 近年来， 随着研究的深入， 紫色杆菌素展示出很重要的生物活 性， 可以作为潜在的抗肿瘤、 抗病毒药物及生物染料， 在纺织印染、 植物 病原真菌防治、 病毒和肿瘤治疗等医药领域有着广阔的应用前景， 因此， 越来越受到人们的关注。
大量研究证明， 紫色杆菌素具有下列生物活性： （1 ) 具有广谱的抗菌 活性， 如抗 s taploylococcous aureus^ Bacillus sp, streptococcus sp， myc obact eri urn, Neisserig, pseudomonas (Sanchez et al. , Reevaluat ion of the Violacein Biosynthet ic Pathway and its Relat ionship to Indolocarbazole Biosynthesis. Journal 2006. 7， 1231-1240)； ( 2 ) 抗氧化 (Konzen et al. , Antioxidant properties of violacein : possible relation on its biological funct ion. Journal 2006. 14, 8307-8313)； ( 3 ) 抗肿瘤细胞（de Carvalho et al.，
Cytotoxic act ivity of violacein in human colon cancer cells. Journal 2006. ) ; ( 4 ) 抗病毒性； （5 ) 抗原生动物； （6 ) 作为天然生 物染料加工各种材质布料（Akira SHIRATA, Isolat ion of Bacteria Producing Bluish-Purple Pigment and Use for Dyeing. Japan Agricultural Research Quarterly. 2000. 34 )， 总之， 紫色杆菌素具 有很高的医学价值及工业应用前景。
在已发现的产紫色杆菌素的菌株中， 对紫色色杆菌 C. violacewiT) 的 研究最为广泛。 2003年， C. violaceum全基因组测序完成， 为紫色杆菌 素合成途径解析及应用提供了保证。 但起初认为有 4个相关基因控制整个紫 色杆菌素的生物合成， 直到最近， 第 5个基因才得以发现， 整个代谢途径基 本明朗化。 紫色杆菌素的生物合成涉及一个基因簇， 长约 7. 3 k b， 包括 5个基因， 分别是 、 VioB、 VioE, ioZ？。
脱氧紫色杆菌素是是比紫色杆菌素少一个氧原子的结构类似物， 是紫 色杆菌素合成途径中的一种副产物， 通常伴随紫色杆菌素产生。 由于脱氧 紫色杆菌素在蓝紫色素中的比例较小， 相当于是紫色杆菌素合成量的十分 之一， 很难分离得到足够的量来研究其性质和功能， 目前国际上对其分离 的方法和技术还很少研究。 由于其产量很低， 分离出纯的脱氧紫色杆菌素 比较困难， 对脱氧紫色杆菌素性质及生物活性方面的研究较少， 目前， 除 了报道其可以抑制原生动物外 (Matz, C et al. · Marine Biofi lm
Bacteria Evade Eukaryotic Predat ion by Targeted Chemical Defense PLoS ONE, (2008) 3 (7)： e2744 ) ， 对其详细的其它功能尚不清楚。 我们 前期的研究发现脱氧紫色杆菌素具有和紫色杆菌素同样的染色和抗菌活 性， 因此， 脱氧紫色杆菌素很可能具有和紫色杆菌素同样的生物活性， 加 强对脱氧紫色杆菌素的基础和应用研究具有十分重要的科学和应用价值。 目前急需一种科学有效的方法来高效生产脱氧紫色杆菌素。 发明公开
本发明的目的是提供一种产脱氧紫色杆菌素的重组菌及其应用。
本发明所提供的产脱氧紫色杆菌素的重组菌， 是将脱氧紫色杆菌素合 成相关基因簇导入大肠杆菌 BL21- CodonPluS (DE3) - RIL 或恶臭假单孢菌中 获得的重组菌。 该重组菌能以色氨酸为底物发酵生产脱氧紫色杆菌素。
其中， 所述的脱氧紫色杆菌素合成相关基因簇， 是将由 VioA、 VioB, VioC, ioZ?和 c^组成的紫色杆菌素合成基因簇中的 oZ?基因敲除， 获 得重组基因簇。 其中， 所述基因簇具体为如下 1) 或 2) 或 3) 的基因：
1) 其核苷酸序列是序列表中序列 1的 DNA分子；
2) 在严格条件下可与序列表中序列 1限定的 DNA序列杂交且编码紫色杆 菌素合成途径中的 VioA、 VioB、 VioC和 VioE四种酶的 DNA分子；
3) 与 1) 的基因具有 75%以上的同源性， 且编码紫色杆菌素合成途径 中的 VioA、 VioB、 VioC和 VioE四种酶的 DNA分子。
所述步骤 3) 中的 DNA分子， 与 1) 的 DNA分子最好有 75%以上的同源 性。
上述严格条件可为在 6XSSC， 0.5% SDS的溶液中， 在 68°C下杂交，然 后用 2XSSC, 0.1% SDS和 1XSSC, 0.1% SDS各洗膜一次。
所述的脱氧紫色杆菌素合成相关基因簇也属于本发明的保护范围。 所述的基因簇导入大肠杆菌 BL21- CodonPlus(DE3)-RIL 获得的重组 菌， 命名为大肠杆菌 BL-DV;
大肠杆菌 BL-DV已于 2008年 06月 25日保藏于中国微生物菌种保藏管 理委员会普通微生物中心 （简称 CGMCC, 地址： 北京市朝阳区大屯路， 中国 科学院微生物研究所， 邮编 100101) ， 保藏编号为 CGMCC No.2557。
所述的基因簇导入恶臭假单孢菌 Pseudo隱 as putida) mt-2 NCIMB 10432获得的重组菌命名为 P. putida -VioADc
含有所述基因簇的表达盒和含有所述基因簇或所述表达盒的重组表达 载体也属于本发明的保护范围。
本发明的又一个目的是提供一种生产脱氧紫色杆菌素的方法。
本发明所提供的生产脱氧紫色杆菌素的方法是以 L-色氨酸为底物利用 所述的重组大肠杆菌或所述的重组恶臭假单孢菌发酵生产脱氧紫色杆菌 素。
以大肠杆菌 BL-DV为例， 其发酵生产脱氧紫色杆菌素时， 所述所述 L- 色氨酸的浓度可为 0.3- 0.5g/L发酵培养基， 具体可为 0.4g/L。 所述发酵 温度可为 10-37°C， 具体可为 20°C。 所述方法中， 还包括在重组菌的细胞 浓度为 0D600=0.6- 1.0时， 向所述重组菌中加入诱导剂。 最好在所述细胞 浓度为 0D600=0.8， 加入诱导剂， 所述诱导剂根据需要选择， 本发明的大 肠杆菌 BL- DV CGMCC No.2557可使用 IPTG作为诱导剂， IPTG的浓度可为 0. 7-1. 3mmol/L， 具体可为 1. 0ramol/L。
以重组恶臭假单孢菌尸. ^ί - VioAD为例， 其发酵生产脱氧紫色杆 菌素时， 所述 L-色氨酸的浓度可为 0. 3- 0. 5g/L发酵培养基， 具体可为 0. 4g/L0 所述发酵生产中， 发酵培养基可为任何提供恶臭假胞菌生长的培 养基， 具体可为 NaH2P04 · 2H20 1. 0-2, 0 g/L、 Na2HP04 · 12H20 3. 0-4. 0 g/L、 NH4C1 0. 5-1. 0 g/L、 K2HPO4 · 3H20 7. 0-8· 0 g/L、 lOOmM MgS04 · 7H20 10-15 mL/L、 甘油 3-4 mL/L和酵母提取物 0. 5- 1. 5 g/L， 溶剂为水。 所 述方法中， 还包括在重组菌的细胞浓度为 0D600=1. 0时， 向所述重组菌中 加入诱导剂。 所述诱导剂根据需要选择， 本发明的 3. /wii^ -VioAD可使 用碳原子数大于六的正烷烃作为诱导剂， 具体为正辛垸， 所述正辛烷的浓 度具体为 0. 05 ml/100ml培养基。 所述发酵温度具体为 20°C。
附图说明
图 1为重叠延伸 PCR重组脱氧紫色杆菌素合成相关基因簇。
图 2为 PCR扩增获得紫色杆菌素合成相关基因簇片段 A、 B、 C和 D的 结果。
图 3为重组菌 BL-DV所产色素高效液相谱鉴定结果图。
图 4为重组菌 P. putida -VioAD所产色素高效液相谱鉴定结果图。 实施发明的最佳方式
下述实施例中的实验方法， 如无特别说明， 均为常规方法
实施例 1、 产脱氧紫色杆菌素的重组菌
1 ) 脱氧紫色杆菌素合成相关基因簇
将杜擀氏菌属 Duganella sp. ) B2 CGMCC Ns2056转接入液体培养基 (淀粉 15g/L、 硫酸亚铁 0. 03g/L、 硝酸钾 lg/L、 磷酸氢二钾 0. 7g/L、 硫酸镁 0. 5g/L、 色氨酸 0. 5g/L， 调节 pH 为 7. 0 ) ， 25 °C , 200rpra 培养 36小时， 按上海生工基因组 DNA提取试剂盒说明书提取杜擀氏 B2菌基因 组 DNA。
根据紫色杆菌素基因簇序列， 采用 Ol igo 7. 10 软件设计 3 对引物， 引物序列如表 1， 其中 Pl、 P2扩增 vioA及部分 vioB基因部分序列， 扩增 产物命名为片段 A; P3、 P4扩增部分 vioB及 vioC基因， 扩增产物命名为 片段 B; P5、 P6扩增 vioE基因， 扩增产物命名为片段 C; P4和 P5两条引 物之间有 48bp的重复序列 （图 1 )
PCR引物设计
引物 酶切 引物序列
编号 位点
P1 5 - -GGATcATTAATGACAAATTATTCTGACATTTGCATAG-3' Ase I
P2 5' - -AAGAGTGGACTTGGCGGCCGCTTCGACCTG-3' Not I
P3 5 - -TATAAGCGGCCGCCAAGTCCAC-3' Not I
5' - -TGGCGTGCGGTGGCATGGCGTCTCCTTAGTTTACCCTTCCAAGTTT
P4
GTACC-3'
5' - -GGTACAAACTTGGAAGGGTAAACTAAGGAGACGCCATGCCACCGCA
P5 _
CG- -3
P6 5' - -GGAATGTCCTCGAGTTCCGACACGAAAACGCTGGC-3' Xhol l 分别使用 PI和 P2 P3和 P4及 P5 ε和 P6引物及高保真 Pfu DNA聚合 酶对杜擀氏 B2菌基因组 DNA进行 PCR 扩增， PCR 反应体系为 50 μ L, DNA 模板为 0. 5 μ g，上下游引物各 25 pmol, 2. 5U Pfu DNA 聚合酶。
扩增片段 A和 B采用表 2中的 PCR 程序 I， 扩增片段 C采用表 2中的 PCR 程序 II
表 2. PCR扩增程序
PCR 步 循 温度设定及时间
程序 骤 环次数
1 1 94 C 3 rain
I 2 30 94° C C 1 min, 72 C 3 rain
3 1 72 ° C 10 min
1 1 94° C 3 min
II 2 30 94 C 1 min, 68° C 1 min , 72° C 1 min
3 1 72° C 10 min
III 1 1 94° C 3 min
2 2 94 C 1 rain , 50° C 1 min, 72 ° C 5 rain
3 30 94° C 1 rain , 50 ° C 1 min, 72 ° C 5 rain
4 1 72 ° C 10 min
PCR产物片段 B和 C按 1 : 1 的体积比混合， 然后稀释 10倍， 作为进 一步 PCR扩增的模板。
PCR反应体系为 50 L，上述含有片段^ C的混合物 1. 5 L 2. 5U TaKaRa Pfu DNA 聚合酶。 PCR反应程序为 PCR程序 III， 当运行完第 2步时 停止程序， 向反应体系中加入 P3、 P6 引物各 25 pmol后接着运行 PCR程 序 III中的 3、 4步骤， 将片段 B和 C连接在一起形成片段 D (图 1 ) 。 用 PCR产物试剂盒纯化片段 D， 将片段 D克隆到 pMD18-T载体中， 获得
PMD18-T-D载体， 测序， 测序结果表明片段 D的核苷酸序列为序列表中序 列 1 自 5 ' 末端第 3058- 6198位所示。
PCR 扩增获得紫色杆菌素基因簇片段 A、 B、 C和 D的结果如图 2所 示。
用 e I和 Λ¾ί I双酶切片段 A、 Not I和 I双酶切 pMD18-T-D载体 及 Nde I和 Xhol I双酶切表达载体 pET30a， 回收 3057bp的片段 A、 3140bp的 片段 D和 pET30a载体酶切后的大片段， 将上述回收的三个片段在 T4 DNA 连 接酶的作用下连接构建重组表达载体 pET30aVioAD。 将重组表达载体 pET30aVioAD转化入大肠杆菌 DH5 α感受态细胞， 转化产物涂于含氨苄青霉 素 （lOO g/ml ) 的 LB平板上， 挑取转化子培养后采用碱裂解法提取质粒， 筛选含插入片断的阳性克隆， 进行测序， 测序结果表明 VioAD片段的核苷 酸序列如序列表中序列 1所示， 序列 1自 5 ' 末端第 1-1308位为 VioA基因， 编 码紫色杆菌素合成途径中的 VioA酶； 自 5 ' 末端第 1305-4322位为 VioB基 因， 编码紫色杆菌素合成途径中的 VioB酶； 自 5 ' 末端第 4323-5612位为 VioC基因， 编码紫色杆菌素合成途径中的 VioC酶； 自 5 ' 末端第 5622-6197 为 VioE基因， 编码紫色杆菌素合成途径中的 VioE酶。 VioAD片段中不含有 紫色杆菌素基因簇中的 VioD基因。 该 VioAD即为脱氧紫色杆菌素合成相关 基因簇。
用 se I和 TVoi I双酶切片段 A、 Not I和 T/W I双酶切 pMD18- T- D载体 及 I和 5k7 I双酶切表达载体 pCOMlO (Smits T. H. M. et al.， New alkane-responsive expression vectors for E. coli and Pseudomonas. Plasmid 2001. 46, 16 - 24. ) (清华大学） 回收 3057bp的片段 A、 3140bp 的片段 D和 pCOMlO载体酶切后的大片段， 将上述回收的三个片段在 T4 DNA 连接酶的作用下连接构建重组表达载体 pC0M10VioAD。
2 ) 表达宿主的选择
a) 将重组载体 PET30aVioAD分别转入£ BL21及 A coli , BL21- CodonPlus (DE3) - RIL， 获得重组菌 BL21- VioAD及 coli BL21- CodonPlus (DE3) - RIL- VioAD, 以 pET30a载体分别转入 Ε· co i BL21及 coli BL21-CodonPlus (DE3) -RIL, 获得的重组菌^ coli BL21-pET30a和 E. coli BL21- CodonPlus (DE3) - RIL- pET30a为对 昭、、、。
在 LB培养基中 37°C培养菌体浓度 0D600为 0. 7， 加入 0. ImM IPTG，
20°C诱导 30h。 将 50mL发酵液在 7000 X g下离心 10min， 弃上清液， 在沉 淀物中加入无水乙醇 5mL， 用漩涡混合器将其混匀， 然后在 200W超声波清 洗器中振荡 0. 5h， 将振荡液 9000 X g离心 5min， 保留乙醇溶液。
在£ coli BL21-pET30a P Ξ. coli BL21-CodonPlus (DE3) -RIL- pET30a中没有得到蓝色物质； 在 7i BL21- VioAD中也没有得到蓝色 物质， 而在 A co i BL21-CodonPlus (DE3) - RIL- VioAD中能合成蓝色 素。 表明重组表达载体 pET30aVioAD在 A coli BL21中合成脱氧紫色杆 菌素的 4个酶没有完全正确表达或者某些酶表达量很低， 而在 £ coli BL21- CodonPlus (DE3) - RIL中合成脱氧紫色杆菌素的 4个酶正确表达， 说 明脱氧紫色杆菌素合成基因簇中可能含有稀有密码子， 将产脱氧紫色杆菌 素的 A coli BL21- CodonPlus (DE3) - RIL- VioAD命名为大肠杆菌 BL-DV; 重组菌 BL- DV的蓝紫色的乙醇溶液经减压蒸馏获得的物质溶于 100%甲 醇中进行高效液相色谱分析， 使用 Agilent-1100高效液相色谱仪， 色谱柱 为 Agi lent Ecl ipse XDB- C18， 150mm X 4應， 5 m; 柱温 30°C ； 洗脱剂 为体积比为 70%的甲醇水溶液； 流速 1. 0mL/min ； 检测波长： 570nm。
高效液相色谱检测结果如图 3所示， 由重组菌 BL- DV CGMCC No. 2557 合成的色素与杜擀氏菌属 Duganella B2紫色杆菌素副产物——脱氧紫色杆 菌素保留时间一致 （4. 9min ) ， 并且只有一个峰值。 上述实验结果表明， 构建的载体 pET30aVioAD在菌株 E. coli BL21-CodonPlus (DE3) -RIL成功 表达合成脱氧紫色杆菌素相关的酶， 并在体内催化合成脱氧紫色杆菌素。
图 3中， I： 杜擀氏菌属 （Duganella sp. ) B2 CGMCC N22056所产色 素高效液相色谱图， 第一峰为紫色杆菌素， 第二峰为脱氧紫色杆菌素； II： 重组菌 BL-DV所产色素粗提物色高效液相谱图。
大肠杆菌 BL- DV已于 2008年 06月 25日保藏于中国微生物菌种保藏管 理委员会普通微生物中心 （简称 CGMCC, 地址： 北京市朝阳区大屯路， 中 国科学院微生物研究所， 邮编 100101 ) ， 保藏编号为 CGMCC No. 2557。 b ) 将重组载体 pCOMlOVioAD转入恶臭假单孢菌 (Pseudomonas putida) mt-2 NCIMB 10432 , 获得重组菌尸 . wiic¾ -VioAD, 以 pCOMlO
Pseudomonas putida mt-2 NCIMB 10432 , 获得的重组菌 尸. wiio¾" C0M10为对照。
P. putida -VioAD和 P. /?i/iii/a-pC0M10分别在 LB培养基中 37°C培养 菌体浓度 0D6。。为 0. 7， 加入体积百分含量为 0. 05%正辛垸， 20°C诱导 30h。 将 50mL发酵液在 7000 X g下离心 lOmin, 弃上清液， 在沉淀物中加入同体 积的去离子水后用漩涡混合器将其混匀洗涤， 在 7000 X g下离心 lOmin, 然后加入无水乙醇 50mL， 用漩涡混合器将其充分混匀， 将洗涤液 7000 X g 离心 10min， 上清转移到另外的干净的容器中， 重复加入无水乙醇溶液， 如上步骤直到菌体呈现灰白色为止， 合并所有的乙醇提取溶液。
在 P.
Figure imgf000009_0001
的乙醇溶液中没有得到蓝色物质； 在 P. putida - VioAD的乙醇溶液中得到蓝色物质。
P. putida -VioAD的蓝色乙醇溶液减压蒸馏获得的物质溶于 100%甲 醇中进行高效液相色谱分析， 使用 Agi lent-1100高效液相色谱仪， 色谱柱 % Agi lent Ecl ipse XDB- C18， 150mm X 4mra, 5 μ m; 柱温 30 °C ； 洗脱剂 为体积比为 70%的甲醇水溶液； 流速 1. 0mL/min ； 检测波长： 570nra。
高效液相色谱检测结果如图 4所示， 輔 P. putida - VioAD的乙醇溶 液中的蓝色物质与杜擀氏菌属 Dugarwlla B2紫色杆菌素副产物——脱氧紫 色杆菌素保留时间一致 （4. 9min ) ， 并且只有一个峰值。 说明 put - VioAO的乙醇溶液中的蓝色物质为脱氧紫色杆菌素， 重组恶臭假单孢菌 尸. -VioAD能表达紫色杆菌素合成途径中的 VioA、 VioB、 VioC和 VioE 4种酶， 可以合成脱氧紫色杆菌素。
图 4中， I： 杜擀氏菌属 （Duganel la sp. ) B2 CGMCC Ns2056所产色 素高效液相色谱图， 第一峰为紫色杆菌素， 第二峰为脱氧紫色杆菌素； II： A wiii a - VioAD所产色素高效液相谱图。 实例 2、 重组菌生产脱氧紫色杆菌素
1 ) 重组菌 BL-DV CGMCC No. 2557生产脱氧紫色杆菌素 对重组菌 BL-DV CGMCC No. 2557以 L-色氨酸、 加入诱导剂 ( IPTG ) 时 的细胞浓度 （0D6。。值） 、 诱导剂的量、 诱导时间 4因素为试验因素， 以脱 氧紫色杆菌素产量为指标， 对上述四因素进行 4因子 3水平的正交试验， 其结果见表 3。
色素的提取同实施例 1 ; 色素浓度的测定是通过测定色素的乙醇溶液在 最大吸收波长的吸光度值来衡量， Duganella sp B2所产脱氧紫色杆菌素测 定波长为 562nm， 以无水乙醇作空白对照， 通过吸光度值——色素浓度标准 曲线得到相对应的色素浓度值， 每个试验重复 3次， 取平均值， 得到的吸光 系数 ε为 9. 0955 1 · g—1 · cm"O
表 3.正交试验设计及结果
Figure imgf000010_0001
表 3结果表明， 在这 4因素中， 影响脱氧紫色杆菌素产量的主次顺序 为细胞浓度〉 L-色氨酸〉 诱导时间〉诱导剂的量， 根据计算结果可知最佳组 合应为在 LB培养基中添加 L-色氨酸 0. 4g/L， 诱导剂 （IPTG ) 的量 1. 0 ramol/L, 加诱导剂时的细胞浓度为 OD600=0. 8， 20°C诱导 30h。
在上述最佳条件下， 进行三批验证试验， 脱氧紫色杆菌素产量分别为
0. 183 g/L、 0. 165 g/L和 0. 153 g/L ， 平均为 0. 167 g/L。
2 ) 重组菌 A /wii a - VioAD生产脱氧紫色杆菌素
将 P. putida -VioAD接入添加色氨酸的 E2液体培养基
(NaH2P04 · 2H20 1. 3 g/L, Na2HP04 · 12H20 3, 0 g/L, NH4C1 0. 9 g/L, K2HP04 · 3H20 7. 5 g/L, lOOmM MgSO, · 7H20 10 mL/L ,甘油 3 mL/L, 酵母提取物 1. 0 g/L, pH为 7. 0 ) ， 色氨酸的终浓度为 0. 4g/L， 30°C， 200rpm振荡培养过夜。 次日按 lOml/lOOml的接种量， 接种至新鲜的含卡 那霉素 （50 g/ral ) 的添加色氨酸 （终浓度 0. 4g/L ) 的 E2液体培养基中 30Ό继续发酵培养 3-4 h至 0D6。。为 1. 0时加诱导剂正辛垸 （0. 05 ral/100ml ) 进行诱导， 20°C诱导培养 30 h， 离心收集菌体， 菌体用乙醇进 行提取， 得到蓝紫色物质。
将杜擀氏菌属 Duganella sp. ) B2 CGMCC NQ 2056接入液体培养基 (淀粉 15g/L、 硫酸亚铁 0. 03g/L、 硝酸钾 lg/L、 磷酸氢二钾 0. 7g/L、 硫酸镁 0. 5g/L、 色氨酸 0. 5g/L， pH为 7. 0 ) ， 25 °C， 200rpm培养 36小 时， 离心收集菌体， 菌体用乙醇进行提取， 得到蓝紫色物质。
对上述两种蓝紫色物质分别进行高效液相色谱检测， 方法同实施例 1。 P. putida -VioAD的蓝紫色物质的高效液相色谱检测结果表明该蓝紫色 物质为脱氧紫色杆菌素。 杜擀氏菌属 Duganella sp. ) B2 CGMCC Na2056的 蓝紫色物质的高效液相色谱检测结果表明该蓝紫色物质为紫色杆菌素和脱氧 紫色杆菌素的混合物。
%P. putida - VioAD和杜擀氏菌属 i Duganella sp. ) B2 CGMCC No2056 生产的脱氧紫色杆菌素进行定量分析。
定量分析是通过测定蓝紫色物质 （色素） 的乙醇溶液在最大吸收波长的 吸光度值来衡量， Duganel la sp B2所产脱氧紫色杆菌素测定波长为 562nm， 以无水乙醇作空白对照， 通过吸光度值——色素浓度标准曲线得到相对应的 色素浓度值， 每个试验重复 3次， 取平均值， 得到的吸光系数 ε为 14. 852
1 1 -1
1 · g · cm 。
定量分析结果表明， P. putida -VioAD生产的脱氧紫色杆菌素的量较 高， 最终产量达 1. 5g/L (平均值） ，远高于杜擀氏菌属 i Duganella sp. ) B2 CGMCC No2056的脱氧紫色杆菌素的合成量 （0. 16g/L ) 。
工业应用
脱氧紫色杆菌素是紫色杆菌素合成途径的副产物， 通常产量很低， 分 离困难， 无法进行规模化生产及应用。 本发明的大肠杆菌 BL-DV CGMCC No. 2557和 P. putida -VioAD生产脱氧紫色杆菌素的产率较高； 大肠杆菌 BL-DV CGMCC No. 2557可以达到 0. 17g/L发酵液， P. put i da -VioAD通常 的摇瓶培养就可以达到 1. 5g/L发酵液， 且提取方便， 分离纯化简单。 本发 明的重组菌是大肠杆菌或恶臭假单胞菌， 方便控制， 便于工业化生产。 _

Claims

权利要求
I、 一种脱氧紫色杆菌素合成相关基因簇， 是将由 VioA、 VioB、 VioC、 VioD和 VioE组成的紫色杆菌素合成基因簇中的 VioD基因敲除， 获 得重组基因簇。
2、 根据权利要求 1所述的基因簇， 其特征在于： 所述基因簇为如下
1 ) 或 2 ) 或 3 ) 的基因：
1 ) 其核苷酸序列是序列表中序列 1的 DNA分子；
2 ) 在严格条件下可与序列表中序列 1限定的 DNA序列杂交且编码紫色杆 菌素合成途径中的 VioA、 VioB、 VioC和 VioE四种酶的 DNA分子；
3 ) 与 1 ) 的基因具有 75 %以上的同源性， 且编码紫色杆菌素合成途径 中的 VioA、 VioB、 VioC和 VioE四种酶的 DNA分子。
3、 一种重组大肠杆菌， 是将权利要求 1或 2所述的基因簇导入大肠杆 菌 BL21-CodonPluS (DE3) -RIL中获得的产脱氧紫色杆菌素的重组菌。
4、 根据权利要求 3所述的重组大肠杆菌， 其特征在于： 所述重组大肠 杆菌为大肠杆菌 BL- DV CGMCC No. 2557。
5、 一种重组恶臭假单孢菌， 是将权利要求 1或 2所述的基因簇导入恶 臭假单孢菌中获得的重组菌， 获得的产脱氧紫色杆菌素的重组菌。
6、 根据权利要求 5所述的重组恶臭假单孢菌， 其特征在于： 所述恶臭 假单孢菌为恶臭假单孢菌 Pseudomonas put i da) mt-2 NCIMB 10432。
7、 含有权利要求 1或 2所述基因簇的表达盒。
8、 含有权利要求 1或 2所述基因簇或权利要求 7所述的表达盒的重组 表达载体。
9、 一种生产脱氧紫色杆菌素的方法， 是以 L-色氨酸为底物利用权利要 求 3或 4所述的重组大肠杆菌发酵生产脱氧紫色杆菌素。
10、 根据权利要求 9所述的方法， 其特征在于： 所述 L-色氨酸的浓度 为 0. 3-0. 5g/L。
I I、 根据权利要求 9或 10所述的方法， 其特征在于： 所述 L-色氨酸 的浓度为 0. 4g/L。
12、 根据权利要求 11所述的方法， 其特征在于： 所述发酵温度 10 - 37°C o
13、 根据权利要求 12所述的方法， 其特征在于： 所述发酵温度为 20
V。
14、 根据权利要求 13所述的方法， 其特征在于： 所述方法中， 还包括 在重组菌的细胞浓度为 OD6 =0. 6-1. 0时， 向所述重组菌中加入诱导剂。
15、 根据权利要求 14所述的方法， 其特征在于： 所述细胞浓度为 OD600=0. 8
16、 根据权利要求 14或 15所述的方法， 其特征在于： 所述诱导剂为 IPTG, 所述 IPTG的浓度为 0. 7-1. 3 ol/L
17、 根据权利要求 16所述的方法， 其特征在于： 所述诱导剂的浓度为 1. 0mmol/L
18、 一种生产脱氧紫色杆菌素的方法， 是以 L-色氨酸为底物利用权利 要求 5或 6所述的重组恶臭假单孢菌发酵生产脱氧紫色杆菌素。
19、 根据权利要求 18所述的方法， 其特征在于： 所述 L-色氨酸的浓度 为 0· 3-0. 5g/L
20、 根据权利要求 18或 19所述的方法， 其特征在于： 所述 L-色氨酸的 浓度为 0. 4g/L
21、 根据权利要求 20所述的方法， 其特征在于： 所述发酵生产中， 发 酵培养基为 NaH2P04 · 2H20 1. 0-2. 0 g/L Na2HP04 · 12H20 3. 0-4. 0 g/L
NH4C1 0. 5-1. 0 g/L K2HP04 · 3H20 7. 0-8. 0 g/L lOOmM MgS04 · 7H20 10- 15 mL/L、 甘油 3-4 mL/L和酵母提取物 0. 5- 1. 5 g/L， 溶剂为水。
22、 根据权利要求 21所述的方法， 其特征在于： 所述方法中， 还包括在 重组菌的细胞浓度为 0D6 =L 0时， 向所述重组菌中加入诱导剂。
23、 根据权利要求 22所述的方法， 其特征在于： 所述诱导剂为正辛垸， 所述正辛垸的浓度为 0. 05 ml/lOOml培养基。
24、 根据权利要求 23所述的方法， 其特征在于： 所述发酵温度为 20 (：。
PCT/CN2009/000430 2008-07-11 2009-04-22 产脱氧紫色杆菌素的重组菌及其应用 WO2010003304A1 (zh)

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